Enzyme activities
Enzymes are among the most remarkable bio-
molecules because they show extraordinary
specificity in catalysing biological reactions.
They have usually been named by adding the
suffix "-ase" to the name of the substrate, the
molecule on which the enzyme exerts its cata-
lytic action. For example, urease catalyses the
hydrolysis of urea to ammonia and CO2, phos-
phatase catalyses the hydrolysis of phosphate
esters to orthophosphate and the correspond-
ing ester. However, this nomenclature has not
always been practical and therefore a system-
atic classification of enzymes has been
adopted on the recommendation of an Inter-
national Enzyme Commission. The new sys-
tem divides enzymes into six major classes,
which are subdivided further into subclasses,
according to the type of reaction catalysed. A
recommended name, a systematic name and a
classification number is given to each enzyme.
For example, the systematic name for phos-
phodiesterase is phosphoric diester hydrolase,
while its classification number is EC 3.1.4.
Research into soil enzymes has increased
steadily over the last 30 years; new theoretical
approaches and methods have been intro-
duced, and a wealth of information on various
enzyme reactions in soil has been collected.
Various activities associated with biotic and
abiotic components contribute to the overall
activity of soil enzymes. According to Burns
(1982), an enzyme may be associated physi-
cally with proliferating animal, microbial and
plant cells, and it may be located in the cyto-
plasm, in the periplasm of gram-negative
bacteria or attached to the outer surface of
cells. The enzyme may be present in non-
proliferating cells (microbial spores or proto-
zoan cysts), in entirely dead cells or in cell
debris. The enzyme may also be present as
an extracellular soluble molecule, temporarily
MethcxJs in Applied Soil Microbiology and Biochemistry
ISBN 0-12-513840-7
associated in enzyme-substrate complexes,
adsorbed to clay minerals or associated with
humic colloids. Some of these categories may
represent various stages in the life of an
enzyme; an intracellular enzyme in a viable
cell may still function after the cell dies and
thus it becomes associated with cell debris; it
may be released in the aqueous phase as cell
membranes are broken and eventually be
adsorbed by soil colloids that are still active.
Usually enzyme-clay and enzyme-organic
polymer complexes show a remarkable resis-
tance to proteolytic and thermal denaturation
(Sarkar et al 1980, 1989; Burns 1982, 1986;
Trasar-Cepeda and Gil-Sotres 1987, 1988;
Nannipieri et al 1988).
Using current methods, it is impossible to
decide which combination of activities in the
categories above has been determined experi-
mentally (Burns 1982; Nannipieri et al 1990;
Nannipieri 1994). Both chemical and physical
bacteriostatic agents have been used to inhibit
enzyme production and assimiliation of reac-
tion products by growing populations of
microorganisms. Toluene, one of the most fre-
quently used bacteriostatic agents, can serve
as a carbon source for fungi and bacteria; in
addition, it may have a plasmolytic action and
cause the release of intracellular enzymes. The
effect of toluene depends on soil type, assay
conditions and the enzyme assayed (Ladd
1978). Sterilization of soils by high-energy
electron beams or gamma irradiation has fre-
quently been used to inactivate soil micro-
organisms in order to determine the level of
enzyme activity that is not associated with
living and active microbial cells (Beck and
Poschenrider 1963; Powlson and Jenkinson
1976; Skujins 1978). However, this approach
suffers from some of the objections regarding
Copyright© 1995 Academic Press Ltd
Allrightsof reproduction in any form reserved
Enzyme activities
the use of toluene (Ladd 1978; Nannipieri
1994).
Measurements of soil enzyme activities have
to be interpreted with caution. These measure-
ments represent the maximum potential rather
than the actual enzyme activity because the
incubation conditions of enzyme assays are
chosen to ensure optimum rates of catalysis.
The concentration of substrate is in excess
and optimal value of pH and temperature are
selected so as to permit the highest rate of
enzyme activity, and the volume of the reac-
tion mixture is such that it allows free diffusion
of substrate.
Problems arising from the interpretation of
measured soil enzyme activity have often led
to the conclusion that soil enzyme assays have
312
no meaning in ecological and agricultural
terms (Nannipieri 1994). It is noteworthy that
other experimental methods in soil micro-
biology (for example, the measurement of
actual nitrification in situ) are based on the
use of unrealistic conditions. In addition,
enzyme measurements answer qualitative
questions about specific metabolic processes
and, in combination with other measurements
(ATP, AEC, CO2 evolution, etc.), may increase
our understanding of the effect of agrochemi-
cals, cultivation practices, and environmental
and climatic factors on the microbiological
activity of soil (Skujins 1978; Nannipieri 1994).
Current methods for the determination of
enzyme activities of soil are presented in this
chapter.
Chapter 7

Protease activity
(Ladd and Butler 1972)
Proteins are the most abundant organic mole-
cules in cells, constituting 50% or more of their
dry weight (Bremner 1967; Alexander 1977;
Burns 1978; Warman and rsnor 1989). Pro-
tease activities, detected in microorganisms,
plants and animals, catalyse the hydrolysis of
proteins to polypeptides, and oligopeptides to
amino acids. Because of the high molecular
weight of proteins, the first enzymatic step in
protein d^radation occurs outside microbial
cells. Compounds with low molecular weight
such as amino acids can then be transported
into the ceHs by specific transport systems and
deaminated there (Burns 1978; Ladd 1978;
Ladd and Jackson 1982; Warman and Bishop
1987; Lahdesmaki and Piispanen 1989).
Nearly all microorganisms in soils are cap-
able of protein degradation, which is generally
linked to ammonium release (Alexander 1977;
Alef and Kleiner 1986; Morra and Freeborn
1989). In soil, proteases are present in living
and active cells, in dead cells, as free
enzymes, and adsorbed to organic, inorganic
or organomineral particles (Sarkar et al 1980,
1989; Loll and Bollag 1983). The protease
activity of soil has a pH optimum of eight
and a temperature optimum of about 55°C.
At temperatures above 60°C, the enzyme is
denaturated (Hoffmann and Teicher 1957;
Ladd 1972; Ladd and Butler 1972; Mayaudon
et al 1975).
Air drying or storage of different soils at - 8 ,
4 or 22°C significantly decreases the protease
activity (Ladd 1972; Speir and Ross 1975;
Speir et al 1981; Beck 1986). In the field,
protease activity varied with the season; it is
not correlated with changes in microbial popu-
lations (Ladd 1978). Nevertheless, significant
correlations are mostly found t)etween the
protease activity and several microbial para-
meters estimated under laboratory conditions
like the arginine ammonification, the substrate-
Enzyme activities
Protease activity
K. Alef
R Nannipieri
induced respiration, heat output, nitrogen
mineralization and ATP (Beck 1984a, 1984b;
Holz 1986a, 1986b; Schulz-Berendt 1986;
Alef et al 1988; Suttner and Alef 1988). It
decreased with the depth and increased after
soil treatment with organic compounds such
as straw (Holz 1986a; Kandeler 1986; Loll and
Bollag 1983). Protease can be extracted from
soils and the activity estimated in crude
extracts (Burns 1978; Ladd 1978; Nannipieri
et al 1980, 1982; Loll and Bollag 1983).
Casein-hydrolysing enzymes, as any other
extracellular enzymes acting on substrates
with high molecular weight, are supposed to
be short lived in soil (Ladd 1978; Nannipieri et
al 1980); indeed any protection of the enzyme
against proteolytic degradation by soil colloids
may render it inaccessible to the high molecular
weight substrate-
Several assays differing in the type of sub-
strate and procedures used for determining
products and incubation conditions are avail-
able to estimate protease activity in soils.
Casein, azocasein, gelatin, peptides and albu-
mins have been used as substrates, while both
short and long incubation times (between 2
and 16 h) have been employed (Hoffman and
Teicher 1957; Ladd and Butler 1972; Beck
1973; Ross etal 1975).
The method of Ladd and Butler (1972) is
presented and discussed.
313
Protease activity
Materials aiKi appmrntu^
Spectrophotometa'
ShaWng water batti ladjiisted^ja to SffO)
CenWfyge and cenWfuge tubes (25 mi)
Folded Alter papi^
G\^^ tube^ With screw c ^ ^
Ci'^iiiioals and aolutton^
Tris buffer (50 rroi, pH 8*1)
Dissolve 6.05 g Trts piydroxy methyl
amino methane in 7(K) mi distiiied water,
adjust the pH to 8,1 wth HCI and dilute
to 1{K}0 ml With distill€KJ wat^.
Sodium casemate (2%)
Sysf^dTKi 10 Q sodium oa^ein^e in w^rm
drstiR^ water (5(rC) an^ i^ng i ^ wMi
dlstiii^ci wat^r to 500 wi (use a sfirn^. If
onl^ cs^^in Is av^iisMe, dl^c^e 10 g in
Ttte buffi^, adjust ttm p^ 1k> &1 iMtti
NaOH soiutlon and f^^gu^ to sm mi
with Tife buff^ (SO imA, pH 8.1)
Trichlopc^au:^*:^ i ^ d (15%)
IMssotve 75 9 mMmmc^mmM (TCPil
in abmit WQ wi dRsUHed w a ^ imid dMirte
to 500 mt iMtti cBstBed %i^€r.
AB<aBne reagait
Dilute 60 mt of N ^ ( 1 M) wttfi distnied
w t f ^ bafcHe di^KiMi^ 1 ^ g Ns^CK^
(wM^ fte# ki tto sdurii^ mi M ^ up
Ols^ive 0.5 g O%0[>4^SM^ IniftsHied
wat€r m^ cflfute ^ tCXI nM I K ^ dIsUNed
waien
Dli^ofve 1 9 potas^um acKlhim ^ u ^ t e
(C4H4KNa0e4H20) In disUlied water and
diuta to ItX) mt iMfh dnsUfod water.
Mix 1000 ml of N a O H / r ^ ^ solution
witti 20 ml of CySO4 scMHim a%tl 20 ml
of polba^wf 8CK£um tertnBAd ^AIHGHI.
Dilute 167 ml of Fcrfhi re^^g^t to 500 ml
with distilted ws&m.
314
Tyrosine sts^cfeird solution (5C» fig mi^ 1
Dii^soh^ m mg tyrosine in Tns buffer
and dilute to 100 ml with Trts tniffen
Prooediim
Plaoe 1 g of moist, sieved soli (2 mm) in a
c^ntriftige tudbe, add 5 ml Tris tniffer and 5
ml sodium oa^lnstte solutton, Stoj^er t»^
tub^, mm ttie ocmt^nts and incubate for 2
h at SO^C cm a shal^tng water bath. At the
mid dl Imnibadton, add 5 ml of TCA solution
a i d mix ttie contents thoroughly To
perfomfi the controls, add 5 ml of Na
oesainate solution at the Btid of the
incubiMcm and immedtatety before ^ding
U ^ TCA sduttoa Centrifuge the resulting
sc* m;^)H9nstens (10,0C»-12,CX)0 rev min"^\
10 nrtm). Fif^ttoSml of tli^d^rsiyfjemat^t
fcito titf)es, mix wrftti 7.5 ml of the alkaline
r e a g ^ ^ ami bu^^ulmte fc^ 15 mi n at monri
j ^ n p ^ t u i e . After adcfing 5 ml of ttie Folin
fflt^ M o ^mss tab€^ and mes^jre t t ^
1 h at 700 nm
tf^ mmmmdMMim becxKnes oM'^feant);
CaM»riritoi msrve
Fip^eO^ 1 , It, d,4aiid 5 iri of thet^x^ne
solution Mto 00^ tob^, ackl 5 wi Na
cas^r^dle m^ t^lhg lip to 10 mi vMth Tns
tHiffar. Thw mki 5 n* TCA SCAJBCHI aanri
ps^fONrm tf^ ii^isiiRiments as desc^r^^
CcKiec^ ttie m e a i ^ r ^ absorbam^ for the
i^34^b Mid oadteu^
CX15
(7*1)
vi^^eiAfi^btt^tiry vi^ightof 1 g of nK>^
scrit, 15 Is tt^^ad ^Dlums of scMtons
Chapter 7
 Protease activity

md^Smitott^soRftiVm assay mdCh^

to % i ll^wirirt,'^^1^1^^

of TCAi^iiribla l^^t^rte denVatives t ^ tha

nWi^KJ^^ b^icM^ i i ^ ; 1Y»3 diluted Fotin-

C^K^iHi f i s ^ ^ ^KNJd ba s^Hf^ at 4''C.
11^ pH andtempera&imcomiitioits are
ttiose raported ^y Ladd arKt BiMm (1972). It
liiacciiwifi^cbdltti8ritIlia aottM%
nm^s^immm^ iiimM ba carriad out at

c»fid^ to ftid Via i^rnisd t^t^dlttc^is f ^ 1|ia

mM to M ai!^t^S64 Dp^wrt: aolla r r ^
fkmmAfii^mm^t^^ tmt ttub^ te$t^
••:!t^^iimK:iim6^-i9^'mm%

Enzyme activities 315

Urease activity
Urease
activity
The enzyme urease catalyses the hydrolysis of
urea to CO2 and NH3 with a reaction mechan-
ism based on the formation of carbamate as
an intermediate (Tabatabai 1982).
H2NCONH2 + H2O -^ 2NH3 + CO2 (7.2)
This enzyme is very widely distributed in nat-
ure, being present in microbial, plant and ani-
mal cells. It also catalyses the hydrolysis of
hydroxyurea, dihydroxyurea and semicarba-
zid; it contains nickel and its molecular weight
may range from 151,000 to 480,000 Da (Brem-
ner and Mulvaney 1978; Blakeley and Zerner
1984).
A variety of methods has been used to assay
the urease activity of soil (Bremner and
Mulvaney 1978; Gosewinkel and Broadbent
1984; Simpson et al 1984, 1985; Kandeler
and Gerber 1988; McCarty et al 1989). Most
of these methods involve determination of
ammonia liberated on the incubation of
toluene-treated soil with buffered urea solu-
tion. Other methods involve the estimation of
the rate of urea hydrolysis in soils by determin-
ing the residual urea or the '''^002 liberated
after incubation (Skujins and McLaren 1969;
Douglas and Bremner 1970, 1971; Bremner
and Mulvaney 1978; Mulvaney and Bremner
1979). Other commonly adopted methods
have not involved the use of buffer to control
pH or addition of toluene to inhibit microbial
proliferation (Hoffman and Schmidt 1953;
Galstyan 1965; Tabatabai and Bremner 1972;
Zantua and Bremner 1975a, 1975b; Franken-
berger and Johanson 1986; Kandeler and
Gerber 1988).
There are controversial reports regarding the
pH optimum of urease activity in soil: both
neutral, pH 6-7 (Hoffman and Schmidt 1953),
and alkaline, pH 8.8-10 (Tabatabai and Bremner
1972; May and Douglas 1976; Kandeler and
316
K. Alef
P. Nannipieri
Gerber 1988; Perez-Mateos and Gonzalez-
Garcedo 1988) values have been observed.
Urease in soils is tightly bound to soil
organic matter and soil minerals and Km
values have been found to range between 1.3
to 213 mM (Gosewinkel and Broadbent 1984;
Bonmati et al, 1985; Kandeler and Gerber,
1988; Gianfreda et al 1992; Lai and Tabatabai
1992).
A temperature optimum as high as 60°C has
been observed and the urease is usually dena-
turated at 70°G. The incubation temperature of
assays ranged from 15 to 42°C. However, soils
are generally incubated at 30°C (Sumner 1951;
Zantua and Bremner 1977; Bremner and
Mulvaney 1978; Kissel and Cabrera 1988;
Moyoet al 1989).
Ureases extracted from soil have been found
to be resistant to thermal and proteolytic dena-
turation (Burns et al 1972; Nannipieri et al
1974). Enzyme-organomineral complexes
with high molecular weights have been found
to be more resistant than complexes with
lower molecular weights (Nannipieri et al
1978a). According to Burns et al (1972)
urease-organic complexes of high molecular
weight are likely to possess molecular arrange-
ments that permit the movement of substrates
and products toward the enzyme, but not that
of large molecules, such as proteases.
The urease activity in soils is very stable and
rarely influenced by air drying, irradiation or
storage at temperatures between - 6 0 and
22°C (McLaren 1969; Pancholy and Rice
1973; Zantua and Bremner 1975b; Kandeler
and Gerber 1988; Fenn et al 1992).
Urease activity was not significantly con^e-
lated with microbial biomass and was affected
differently by heavy metals, oxygen concentra-
tions and nitrogen availability in different types
of soils (Tabatabai 1977; Nor 1982; Doelman
and Haanstra 1986; Cochran et al 1989;
Chapter 7
 Urease activity

McCarty and Bremner 1991; McCarty et al
1992).
Urease inhibitors have been used frequently
to prevent a rapid hydrolysis of urea in agricul-
tural soils (Fillery et al 1984, 1986; Simpson et
al 1984,1985; Haare and White 1985; Cai et al
1989; McCarty et al 1989; Zhengping et al
1991;Xiaoyanetal 1992).
The methods involving the estimation of
ammonia released have been evaluated
thoroughly and are therefore presented here.
Rea^g^ls im detwi^tatton <rf NH4^ f
Estimation of urease
activity
(Tabatabai and Bremner 1972)
' 'P^patta 40.mj^of h i # c ^ ^
ttie same buffer. Pm^m a fmsh i^rtiittcm
dsBly and stone at 4*tX ; vJi^
Dissolve 1(K} iTig of r^g^rit gracl^
Ag2^4 In 7C» irt of dfettled ni^tan ?
dissolve 188 g of r^igent §imla Kdi ^
ttie s»>lutiori and dituta to ItXX) ml i^tti
cfefllted visiter; s^; '
H2SO4 (O.CKB M|
Indicator solutton
Dissoiva 0.66 9 bt>nioari^<ri preen m4
QJ3Q g of rr^tt^l n ^ in athmioi ^%)wki
imt^s tip to 1CXX) ml wItt rthstfibt; ^ ^
8 ^ ac^^fcricMca^

flask f l ft. Mix 4D 9 <* t>&ite^^ ;

oTImtoftNa^Alite
fMaksi^m^ anpEUf^ri^

^NsfcjBib to 2tt» irt 1 ^ i ^ ^ ^ L ^,,.^..

i ^ B | K j ; i a j , i a » , 2000 m
;;fh@K«ahaa*,
\flat|W'i,:da^5€atei4!:^;^^*i;^

W^ fiycN>xy rri6tiiyO mnim m^ane buffer t wafir andiMiJ^ito1^(^Mi%Mi

p j ff^, IKW OJ^ frtl #1

:tyfer, mk ttm.oo«^*^c^^,^ .,.,..„, .^,,^

fejaft SIX. t ^ ^ ttia frK^yft)ilH9ii aldl

Enzyme activities 317

Urease activity

approximately 35 ml of K C I - ^ S 0 4
soiutlcmt swM Vtmflasklor a %w aec^^nctet Estimation of urease
m^ sdfow ti^ flask to stesiiKj mM tha
ccHit^ite iwm celled to roomfmmpmwimm activity
{dkmA 5 mln). fting 145 iBie c^mtoil^ to 50 (Kandeler and Gerber 1988)
ml by addition of KCM^2S04 s«^uton and
mix the contents thortnighiy* To pmfcmn
controls, follow the procedure tks^b&A
for assay of urease activity, bull make Hie Principie of the mettiod
addition of 1 mi of 0.2 M urmsofyticm after CotorimeMc determinaticm of released
the addittofi of 35 ml of Ka*nA^04 amnKmia aft^ ttie inoyibatlo^ of i ^ l
soiutioi. It h reoKiim^^Kted ttiat at least smrif^^ with urea soii^on fw 2 h a t 3 r € .
three replications are caatied out. The
ruction shows a linear time course up to 5
h. Materfate and apparatus
Estimation of released ammonia Inoibator a^lJustaUle to 3 F 0
Pip^e 5 ml of borte acM tadio^or
solution Into m &ienm^^flask ar^ {Kit %ecta^hotom^^
It in M wpmM plac^ ^m C t i a p ^ 3, Afotumetrto ftisks (100^ SOO, 1000,2000 n ^
Bremw afid Bcfe^ards 1^^)* f ^ ^ e 20 Bimmm^Mwsk^ p5,1CK) irt) x f
riM of ftie i^^^iiMrig 1 ^ i y s p ^ f i ^ n M o a
disWIa^m flask (too fi4, m^ 0 ^ g fe^O
m^ i^sgm cK^liAe tt^ oof^mi^ u ^ l 30
irri of ctt^KIa^ are i:^4h3c^6^^^ta^ 1
& ^ m ^ ^ flask. TMiBto tfi^^Metfe^
vritti OJSOS M H y ^ 4 p k ^ p v ^ j ^ ^^ tls^^cri%91;.4 9 urea hi/
Keeney 1 9 ^ ; 1 mt^Mm4pM&^W
ecp^^a*wtt Ip 3tl im N H 4 ^ i} ^

t^#8 ^tjn^4iM^^^^ 'mj^i^ms^

<fert X 5 (7^3)

wtiere C is t i e meamir^ NH4-N Skxfarw'^Beq^fete s d u ^ " ' \ ; '''4;;: ?'?'•'>:'

msi^m^m% dmt is tt^ Ay m^Uis^f 1 ^

mM m^f $ Is tt^ K i ^ ^ g ^ ^ i j ^ 1^1fenthe
lB$t aKi ^ Is Hm toM vcAiKne 1^ tt^ ^ H
siispcHf^^mi.

wHiv i ^ 13« i^i^KBmJl i l a i ^

318 Chapter 7
 Estimation of urease activity

of cB6i»ed Sft^^^E^ ii*^^^tfc^

cbnlitocA^ioy^lde hi 1(K> mi disHHed

^^;m0 the ^urilon ^^icMtly
Dissolve 56.^ Q disodlum t^tralximte,
mISQg disodium iieMbmsie (wat<^ fre^)
In li^X) mi wmm distiifed wat^« Aft^
ocM^hi^, iK^u^ to |4^ 10 imtti ftoCM
wiMm ^0%) and bring up to 2(XXiwi
with distilted waten
jteimonriim standard solution
Sduftiont
CNs^Kriva $.f& QrnnmrniumohkHld^ In
# ^ 1 ^ mAm m\4 Mng up witfi disttfied
wmt to 106D ml (1000 m NH4-N mi^^).
1}m scA^lcm is ^^^:^ t r seiii^rai weet^
' d^n^tyatf^-ra^ A ^ ^^^;;JVri:^':^ 4!'
F^rooedura fcr tto biMf^aied mettiod
PlaoB S g of n^jst sc* m^p^ to
Grto^T^^^ aa^<1C@ n% add 2Mmi
[ mAMon ^md 201
d^^bedfa* tNif mm^s^mid i
^ftfthttie addttionof ao mIKa s«>!utlon at
ttie errf of IncubatkHi.
F%)6tte 1 nil o l t t ^ Isyraiioitfum i^i^rNJteyid
^^Aitttm Nfcijg^la^ 1iibl«i, «MUb ifMi$ M

Sc*Jti<m B
' ' -'' , '.. '>{>'^''',\''* i- a-^fi,,^/ \''V^'^.'^">,f^'^^--'^''^c..'''''" ,
F%»«» 0.0,1A 1*5» 2 A 2*5 mi of ' . ;'••"' "C;: ' /* , I , ','•" V^"'*'^ i""'^"^-. ."'-," M'

sdu^n t Into ^liimeM::flaslos(KX) mQ

«pd l»ft^ lipto1(X} ml ^h KD sduQcm.
'•I: S''

f^^oq^K|yN^f0'1^

j^^UilQ^ 11N»i^c^^tt¥pfias^ and

di^solbedatove but iiMi 2J ntf ^ " - '-• "•"' •''' ••'"^'-'•• '''-''-'Ipi^^^}"''
Wat€r, ^ acUttiews^ soiiitton at the

iNMfo^ 4(^ is^Wdn. It 1$ iB)(^NmfmK^

idufc:/^,, , .-tS:

: (ISO n^, tiiefi add 9 n^

Enzyme activities 319

Urease activity
T»^KC»-^fea04 si^uftoa must be
{M^^paiBd i ^ tSs^ioMiig KCI into ttie Ag2S04
sc^utic^ bi^Dause Ag2S04 does not
dissoive m KOI.
Tim i^dHion <rf KCM^S04 stqps the
m^zymid^ t i n ^ hydfOi)^$ aid the
s^yispi&nsicm am stetKl ft»r2 h befcH'ethe
d^c^nJnattcNi of ammonium, because no
addMonel e^nmonium is retrased.
The inhibitor Is tmt used in the method of
Ksmde^ and 6erb^ (1988). Ttie sample
dttutkHi by KCS may oonsiderabty sTow
dtomi ttie^izymittte ^::tMty mtmi towev^,
ttfues fcr amnmcmium ^K:fractei and
fOlrat^ iMiouhf not dffer mmmg different
m8^isyD6^tienlB«
| % < ^ m ^ ^ i k i bora^ has b ^ USCKJ as
idfi IrthWto- fw w ^ ^ acBWty to soil
y^ie^4M^^ arkf C^or^srier-^Cat^o
1 ^ . .
320
The fiRri^on of solJ simpenslor^ should be
carried o^A wRh ntlTc^pfvfiieefitterpaper to
avoid ntfrcHien otmlani^naaon of fh^ flttrate.
The ocriour developed In ihe ammonium
meiy^B (method of Kand^a^ and Gabm*
1988) is s t ^ i e for at l^aist 8 h at room
TTie methckt itosed on oslprim^c
detemndn^Mon of uma m not proposed
b ^ ^ u ^ it has omtaln defioienol^ mjch as
the inksfeitffy of sckne regents or ttie
diromogen wmrKiunds, tow
reproducitHtity, taok of saisiflvfty and tack
of linearity atio# urea cdhcentratiohs; in
addition, ttie procedure is ttme ccmsimtlns
(Tabatabai 1982),
to
cstbn^e Hm m^^BB m^si^in s<^ls
fPer^^Maiec^ m$ i3oitzaiez^<]iaiiDedo
Chapter 7
 Amidase activity in soils

Amidase activity
in soils W.T Frankenberger, Jr
(Frankenberger and Tabatabai 1980a) M.A. Tabatabai

Amidase (acylamide amidohydrolase, EC activity increases ranging from 3 to 16%

3.5.1.4) is the enzyme that catalyses the (average 9%). Heating of field-moist and air-
hydrolysis of amides, and produces ammonia dried samples for 2 h before assaying amidase
and the corresponding carboxylic acid activity showed that this enzyme is inactivated
at temperatures above 50''C.
R:C0NH2 + H2O -^ NH3 + R:COOH (7.5)
Studies of the distribution of amidase in
Amidase acts on C-N bonds other than pep- soils showed that it is concentrated in surface
tide bonds in linear amides. It is specific for soils and decreases with soil depth (Franken-
aliphatic amides, and aryl amides cannot act berger and Tabatabai 1981a). Statistical analy-
as substrates (Kelly and Clarke 1962; Florkin sis indicated that the activity of this enzyme is
and Stotz 1964). This enzyme is widely distri- significantly correlated with organic carbon in
buted in nature. It has been detected in ani- surface soils (r = 0.74***) and in soil profiles
mals and microorganisms (Bray et al 1949a; (r = 0.89**). Amidase activity also was signifi-
Clarke 1970). Amidase is also present in the cantly correlated with total nitrogen (r =
leaves of corn (Zea mays L.), sorghum (Sorghum 0.74***), percentage of clay (r = 0.69***) and
bicolor L. Moench), alfalfa (Medicago sativa L.) urease activity (r = 0.73***) in the 21 surface
and soybeans {Glycine max L.) (Frankenberger soil samples studied. There was no significant
and Tabatabai 1982). Microorganisms shown relationship between amidase activity and soil
to possess amidase activity include bacteria pH, or percentage sand. Amidase activity and
(Clarke 1970; Frankenberger and Tabatabai microbial counts obtained with acetamide or
1985), yeast (Joshi and Handler 1962) and propionamide as a substrate in the absence
fungi (Hynes 1970. 1975). of toluene indicated that these substrates
Information concerning the distribution, Induced production of this enzyme by soil
specificity and kinetic properties of amidase microorganisms.
in soils is needed because its substrates
(amides) are potential nitrogen fertilizers
(Frankenberger and Tabatabai 1981c). Among
the various nitrogen compounds that can
serve as substrates for amidase, the sparingly This ri^thod involves detemfiinaHon of flia
soluble oxamide and formamide have been NH4-N fBteased by mtmi&SB adHvtty when
tested as nitrogen fertilizers (Beaton et al son is IrtoMbated wWi buffered p.1 M Trts
1967). ^ d t o x y meth]^ ^emifio mettiane (THM^,
Air-drying field-moist samples results in a pH 8-5) amkte ^AJWOH and Wyene art 3 7 X .
decrease of amidase activity ranging from 14 T l ^ imm<Hi{i«?i r e u s e d is cM^ii^bi^ b^ ^
to 33% (average 21%) (Frankenberger and 1 ^ ^ piooedi»B ki\^^in^to^^ftrn^^of llm
Tabatabai 1981a). Freezing of field-moist immtMlmi son ssmf^le ^fth ZJ& u K O
samples at -20°C for 3 months resulted in

P< 0.01; ***P< 0.001.

Enzyme activities 321

Amidase activity in soils
m^Me) and s t ^ m distitiatlon of m eUqmt
of t t e i^esiM^ suspertsicm mVn M ^ for
3.3 tirin. ITie p t K ^ u r a g i v ^ quantitetive
iBcc^^^ry of NHU-N addad to soite amj d o ^
tmt mum d iwilcal t i ^ m l y ^ of »ie
Materials and aiiqiafatiia
bK^ybalor PFC) or temperatore-controli^
DfstiiiaBim flask (1(X} mQ
Steam distlltatlon apparatus (see Keaney
m^ H^wcm, 1982)
Chamicala and aoiiilioi^
Toti^f^: BsNr-c^Wed m ^ ^ fisher
SotonMb Co., Ctites^o, HJ
Tite-milj*t^te ?^d iHiWr {OJ M, pH 8-5)
PssoJve 12*2 g <rf Tiia ^ydroxym^^ttr^
mrkm m^imm f^ibh^HcartMed re^^nt)
hn a i K ^ ^ ari crfiAi^m; aj^ist tti^ pH to
8.Sfc^M^sMtm wWh jsAKHit 0.1 M H2SO4
mni dilulall^eoiytoiviMi water to 11.
iMcte scMic^W (0JS «i)
AM 2*0 irtl, 2 J 5 8» cr 3.65 Q of
fcmnarMcte CMdMS) o ^ M ^ , ac^tamlde
P p m i ^ M a # t orf^t^hm^mda
(AMrfdh oeMadI, respective, tmo a 1(X}
l^dbk^sMm dtM^ ^M MHHis^yt acetate
p.a»M)aoh^n
DfescftreUS g <rf reagwt-^fade
UCfetC2H^O^:2H^ in alwit 7{» ml of
1 {'Mil mmr ^sM mbc tlK^iy^My.
dmmmi^mt ^t^m^mn oxid^ bote acid
jtag^)«A^ S(^Ui:io, G.CMI2S «i Ha^4)
322
Pmt^m m t!iBsa\bmi t>y K^^ay am)
Nelsor!(1982).
PiiKmfcire
Pt^a 5 g of soit {< 2mm) In a 50 ml
volumetric flask, add 0.2 mt of toluene ^ d
9 ml of Tris buffer, swM the flask for a few
TOConds to mix the contents, add 1 ml of
0.5 M amkje solution and swiri the flask
again for a fi^i/ seconds. Tlien sto(^>^ the
ftosk and jf^ace It In 2^ incubatcH* at 37^C.
Hie incubator time vales v^th the $(^oiflc
substmte ajded. Hydrolysis of fonfnamide
is r^ativeiy rapki ami am be m^tsured
quantttatlv^ aftw 2 h of inort^aticKi. The
rates of hydtolysls of aoetamide and
popionamde dm somev^at skmrn 9ian
ttie nsfte of hydroli^s <rf fomiamide, and
they can tm cNtenntined quaitt^ively aft^
24 h of fncut»srtion.
A f t ^ hnc^baUon, i^^nov^ iSrm stopp^» adkl
ipjm>xlmate 35 ml >c* KCI-
LK}2(C2HaQ^:2H20 sokitton, s>iriri ttie
f i a ^ l w a few s^oorKis, and dtow the flask
to s^ml unfll ttb c ^ n t ^ ^ have cooted to
room temp^a&iiB ^jsribmit 5 min}, iim%
dHutettte i^^Ajmato 50 nri t^theadcMJon of
ttie flask aid h t v ^ tt seveiti ikm^ to mix
ttieconl^fe.
To detemwi^ HH4-H m tt^ f^^^jMng woM
md pipsm mWttA mo^ ^ ttie
mi^Bpafiskm M o a iQO nA cKsMtaito flask
si^mn disMlt^on of ttiis wjIkg^MSi IAMN 0«2 g
of MgOft>r3.3 min m dasolbed l ^
Bren%n^ (196Sa).
ttidt ^ ncft danl^^^ft^tmi0 ^ m^&b ttirm^
anrrida^ac^l^. To pmfmmmm^nt
Mfik&se i»:^ih%, te^ make ^m m$M0n^
1 iiy of OJ M amkle button i^E^tt^
adcMton of KCHiO^C^H^Q^t^s^
Chapter 7

b^ifona mm HVfi^mntu^m^ dikmmi to
j^lMKl i f i M i ^ ^ M ^ , KCI mil prBoiptete
^ c ^ mA <3l Itm i^hrttoi) wMi tbrr^. ^ ^
fHiipio^af adfcftng Hm KCNiiwi^ acetate
i^iiittcm to the wit sam^0 affc^ iacul>atloti
Is to In^cttva^ anMa^MS^ aiHl to allow
qiiartttative tkimmimikm of the NH4-N
i#eissedl p ^ d b y ami FredMcks 1954). The
i$(M mj^pm^m mwifs^sS "k^ HH4-H must
be mm^ l^^mmj^^ ftimedrat^ befme
sipfAn^ 1m HH444 mnal^^.
TNl ^ t t r m t r i jfM dbmnfed for amtdlase
mM^ ^ tJ^ frmkmim^ and
Tst^Edat:^ 1 9 ^ ^ Is^^^^me^n^^ M g f ^ t h ^
tfiat itepcNtect for )aimA^i$e at^Ml^ of
l^^^uidto^K^iiKS^ I t ^ ^ i ^ ^ ^ ^ Iscrfat^d 1t<m a
iW^.%t^ ifeifjitfc^ fe *ie pH o|*nrium for
diMclgyseacU^
tiie |tf^ is|MfiM df ^mi^tti^ fri j ^ M u l ^ ^ are
l A ^ 2 { ^ lyri^ t{wi tti^ sama
c ^ ^ ^ i ^ f i t t ^ f % l ^ In i ^ pri^s^^K^ of Trte
cxmi^MiiMem Mrts^;Mttri|p[the ma^dikm
i0ca^PM » % a « ^ i ^ » ^ ^ Icr ttie
1980b).
Enzyme activities
Amidase activity in soils
Enz^irie-oatalysed re^K^ions usually show
tinsayr iBlationships b e t w ^ t t ^ amount of
l^xKtuct fcm^ m^ tfme of tmnibatiom
Fimnatkm of NM4^ withfoiTr^mlde&& a
mitetiate follows zmo-ott^ Mnetic^ for at
hBdst 4 h ^ranti^fiberger and Tabatabai
19%}a). V%^i ac^sMtilde arudi
(m>picmanide em i^ed as substrates, the
ol:^erved r^ationship existed up to 80 and
36 h, ie^)ectively. Hydmlyste of aoet^mide
Mid pro^cmamfcle m oonsider^^ siow^
than Ihe l i y d r o ^ s of fomiamlde- The
former two substrates have to be incubated
for 24 h to measure diff^ences In amidase
activity quantttatively In sdls. I n t a f ^ ^ c e
by miorDbM ac&h/lty cfe^s not seem to
oc^ur durir^ ttm 24 h inaibatiar) time as
emient by ttie lir^sar r^tkmship beta^een
tto artKHint of HH4-H n^^ised m¥i the time
erf mc^yibaUon in t t e |:Hfi^enca of toluene.
Tolume ^ould t^e added to soft samites
befcre adcHrig 9 mi of biJf^ b^sau^^ tests
Indicated ttiat it U ^ s effective In
sTrisl
gi^s^nia^c^te^Si^ iBSK^ions pticeed at a
sK^ibattdn itfrithlria c^tEdn Mr^parafajre
t a i ^ as k^ mMmm$i:ftm^$^:B^
retaiiis Ite ftri ac^tt^v At a teittp^nsibire
^bW0 V^ mne§B, itm a ^ l t y faNS^s to
d^^^a^i^ t^ec^tu^^ of mwfi^m ii^a^Vj^km.
Stu^^te of If^ eildot of timiperai^ cHn
4if!fMa^ acUy% In scite^ ^ N ^
Si;aiiii(i^^ftti^<m bnaciyit^ mtMsem £Pd
i^^ifc^ i r f f m m M ^ d e ^ K l fqhmi0
iftBN^^l^i^lf^ ac^\%. iMfiidasels ftrKfii^^ In
ttie pies€Ni<^ of ttie sui:^traft€^ viMiout
toly^e. Among v ^ o u s c h ^ c a l saRs,
N ^ i m l NaAsC^ have jm kitdMory ^ e c t
rnimi^^^ a c t t ^ . Hie W^lrffidrt tiy F^
mpk^t^Bmm et al fiSS^ *^y»^ « ^
of #iidtese even ilKH^h ttM gfOiiiB ^ € m
I of tti^ active
323
Amidase activity in soils

i»i25^€k Tito eff^?t$ erf i ^ v ^ t^^ancJSO/^0=^raiikeiiber8erand

.^tec^toippvldea'i^^ Tab^s^^:^ 1 ^ ^ * &^z;^iia Mn^tlra

imtiKMh f B ^ 3 ^ to ttia Mai^r^ fofis: IC, wm mm-comfm&SNfB Miibitors

Na% NHU% NOs^, f«^% Na"^, Ct^, CN^, f^ranl^nb^^ i»td Tabatoai 1981 b).

324 Chapter 7

L-Asparaginase activity
of soils W.T. Frankenberger, Jr
(Frankenberger and Tabatabai 1991a, 1991b)
The enzyme, i-asparaginase (L-asparagine
amidohydrolase, EC 3.5.1.1) has an important
role in nitrogen mineralization of soils. The
chemical nature of nitrogen in soils is such
that a large proportion (15-25%) of the total
soil nitrogen is often released as NH4 by acid
hydrolysis (6 M HCI). Some evidence suggests
that a portion of the released NH4 comes from
the hydrolysis of amide (asparagine and gluta-
mine) residues in soil organic matter (Sowden
1958). Bremner (1955) reported that after acid
hydrolysis of humic preparations, 7.3-12.6%
of the total nitrogen was in the form of
amide-N. L-Asparaginase activity was first
detected in soils by Drobni'k (1956). This
enzyme catalyses the hydrolysis of L-aspara-
gine, producing L-aspartic acid and ammonia
as shown below:
NHo
L-asparaginase | (r = 0.78**) in 26 surface soil samples exam-
• H — C — COOH + NH«
I ^
CHo
I percentage of clay or sand. There was, how-
CO COOH
I ever, a significant correlation between L-
NHo (7.6)
The enzyme is widely distributed in nature. It
has been detected in both plants and micro-
organisms (Wriston 1971). Plants may con-
tribute substantial amounts of L-asparagine
to soils and by the action of soil L-asparagi-
nase, release N H / to the inorganic nitrogen
pool. Soils have been tested for L-asparagi-
nase activity by Beck and Poschenrieder
(1963), simply by adding L-asparagine to soils
p < 0.01
Enzyme activities
L-Asparaginase activity of soils
M.A. Tabatabai
and monitoring the NH4"^ released, but the
actual assay was not tested thoroughly using
systematic studies of factors affecting the
release of NH4"^. Frankenberger and Tabatabai
(1991a) developed a simple and sensitive
method for the assay of L-asparaginase activ-
ity in soils and determined various parameters
that affected the observed activity. The method
developed involves determination of the NH4"^-
N released by L-asparaginase activity when soil
is incubated with buffered (0.1 M Tris, pH 10) L-
asparagine solution and toluene at 37°C. Stu-
dies on the distribution of L-asparaginase in soil
profile samples revealed that its activity gener-
ally decreases with sample depth and is
accompanied by a decrease in organic C con-
tent (Frankenberger and Tabatabai 1991b).
Statistical analyses indicated that L-asparagi-
nase activity was significantly correlated (**p
< 0.01) with organic C (r = 0.86**) and total N
ined. There was no significant relationship
between L-asparaginase activity and the
asparaginase activity and amidase (r =
0.82**) and urease (r = 0.79**) activities in
the surface samples studied (Frankenberger
and Tabatabai 1991b).
325
L-Asparaginase activity of soils
actlvfty in soils, Thte method uses s t ^ m
distiliation to detarniir^ ttie NH4 produced
by t-asparaglnase aotiv^ y^tm% soil Is
ifmxtmt&i wHh buff^Bd (0,2 M Trfe, pH 10)
t - a s p ^ ^ i r ^ solution and toluene at 37**C
for 2 h. The pt)cedyre dev^oped givas
quantitative mcovery of NH4-^N added to
softs and cto^ not cause diemto^
hydmiysis of t-asp^aglne.
Malteriais and aM^^iatiis
Volumetrio flasks (50 mQ
Incubator {ZT'C) or temperature-controfled
w^erbath
C^tilfation flask (100 m^
Steam distiiiatlon apparatus (see Ke^ey
and Neison 1982)
OiKmiicals ami ^ i i t t o i ^
Toluene
Rsh^-oertifled reag^t i f i s h ^ Scientlfto
Co., CM:^^Pt i^*
Tris iHifte- 0)J lyi, pH 10)fefsmpm^ tjy
dit^olvir^ 12,2 g of Tris pytkoTsy mettiyl)
amino mettiamie i^ynm<^^W^ r^agmt) In
about fXXi ml of waten ad|i^ng tt^ pH to
10 by tKration wiljh 0«1 u Ns£>H enti dfluting
the scNi^n vwtti wa*^ to 1 1
t*Mf^raglrm Glutton (0.5 M) is pn^^ared by
dti^soMing U S g trmfmrnsirm p^ma
Otmtfk^ 0>.t St Loi^s, **0) into a 25 ml
vctor^M^fti^k. 71^ vohmmm^lmtmi by
mMf^ Tils tHiffer. The ocMiri^ts am rtmBdi
yMe ftiming lK>t tap i ^ € r over tt^ fl^k.
Pdta^iym chloride ^£ M)HsiNver sulfriiiate
(1(K> pat$ 1 0 ^ solution fs prepared by
dissolving 100 nng reagent grade AsbS04 In
abmit 700 ml wat^, d l s ^ M i ^ 188 g
i ^ g ^ t p^de KCS In M s sc^utlcm ami
diluMf^ ttie sdiuticm to 1 L
fto^p^il^ 1:^ the d€timmlnistt^ of
irormiftium < n ^ i ^ k 0 f i oxiNdle, borto add-
mdk^aS^ t^uUcm, Q.&3& H S U ^ I M I C maU^ is
pi^m^ m cfe^scrit:^ by Kearney m<i
N^sm (198^.
326
Pr<K^6dure
A S g soR ssHfTQrie (< 2 mm) in a ^ rrri
vc^umeWc flask Is treats witti 0J ml
toluene afid 9 mf Tns tnilM Ttm fl^k Is
swirled for a few sec^^ds to mix ttte
contents; 1 ml 0,5 M tnaspars^ine solirtion
is then added a i d tim flask Is swirted again
for a few seconds. The I t e ^ is sto^i^^^ieKl
a i d placed in &n hiaut)ator a t 3 7 ^ f o r 2 h.
Aft^ trK^ubation, the stopper is removed
and approximately 35 ml KCI-^2S04
solution is added. TheflaskIs swided fc^^ a
imff seconds and allowed to ^m^ until the
contents have ccK>led to room t^mp^ature
^bcNut 5 min). Itmt ttie volume is itfiuted to
SO ml by ttie SKiditbn of iCCMg2^4
solution, mui 13m l&a^ is stc^^>ered and
Inverted sevc^ U m ^ to mix ttie cxNfit^^ite.
To d^ermJTO NH4-NI In the r^5y«*ig soil
sii^pension, the flask is Invated msmsi
times ^Kl a 20 nri tik|i^ of t r ^ mi^pN^mlcm
Is pi(^tted Into a KK) tti dMIMion ftas^
a i d tt^ NH4-N n^ic^ised h d^^mnlr^ by
stesm dB^RMon of thm ^Iquot viMt 0^3! g
WgO fcr 3 ^ nrtn, as described by Kmm^
In tt^ OEHttrols, tti^ pnDcedwe d^NC^ibed for
nm ^^^lay of t-a^^a^^ina^e mol&v^ k
laihm^ t^ the 1 ml 0.S M L-^n^gmB^m
SQfcitkm h adcted afl€r ttm atidMim dfthe
alcftf^ Urn K : C M 6 J ^ 4 scMsm to#is sdi
s a n f ^ aft€r kicdb^cm U totaK^ivaftet-
s^^pssmg^mse mid ^ifciw a tp3^ifeaate%
ctet^tnlni^tort of tlie NH4-JSI i ^ ^ ^ s ^ .
Discai^on
10.0 0^mnkm\mpa&t m^ JdtodS^atm 1 ^ 1 ^ .
MtticHJ^htti^^eh slightly HKS^ lu^Mty at
^ i ^ Ificw pH 8 to 12,
V i ^ r ^ Vie sufa^toBte Ci^i^r^y^gir^)
coTK^in&aticm in ttie m(alhod ti^ai^o^
Chapter 7

^ t o w ^ that ttie com&fitm^cm adopted (50
nm) Is $atisfaot<^ for ttm ^ $ ^ y of
irm$m^ipmB a^tiv% m isolis
^rm\kmb&tgs^ and Tabari^d^ 1991^. At
this ooncento^icm, t-a^i^rs^inasa te
I t-aspafj^me and the
Mr^tlc^ Hie is^p^mmt i^ oonsleyit
mmB^ BA nm m nine soils
fmnkmim^ ami Tal^atgrt:^ 11^1 a). The
o - l ^ n i ^ of m^fm^itm U ^so hydroi^s^
fri Ji^^ biA tt^ aottvtty ^ oriiy i^KHit 16% of
the actMty of ttie t*isomer at a saturating
subsfrate coitoc^tratlon.
vA^:^imginas0 m^ify can tie det^tnined
i;^fn^ 1 ^ s d i ¥toMm\fm^ tat tinear relatkNnsliip
b f t w ^ttH^iN^nmintof ^ i a»^ the ^^TKiunt
^i^^ i^rtiased stowed ttiat S g of
lioV^M^s^lcto^f^
t-ispaiagbme lEK^Mfy 0=fa5tcaib(^9^ m^
, IfUicating eith^
bec^n*^ ^ HnMk]^ factor cir t i i ^ oie of the
pi^^i^K^f^ or i-ai^p^airiio a : ^ \
aott\%*
of M H (darfv^ fDcrni
lb 4^Mbr to tti^ iipcited for airricis^^
Enzyme activities
L-Asparaginase activity of soils
aizyme t-asparaginase appear to be
slBUe to freezing and thawif^, mfi to
stCHage in the ftozim statefi(>rpcrkHJs of up
to 6 riKinths or ledger ^UBm and Wriston
1971). The ei^^igiy of ^::th^tk>n <rf the
reaction catalysed by t-^asii^Hragina^ in
nine soils (exi^i^sed in kJ n^l^^) mr^ied
frmn 20,2 to 34J Cavang^e 26.6 kJ mol""^)
f ^ r ^ k m t ^ g ^ and T^^al^A^ 1 ^ 1 ^ . The
temp^atum coeffloients for t-^
activ% in ^ h t SCHIS, for 1
betwei^ 1 0 1 ^ 50^C, mr^^ ftiwi 1,12 to
1*70, virith an average of 1,39
fl^mnkenb^ger and Tatetebad 1991 a).
S^^mt stmll^tlon is to itmMvtaie
soft enzr^es, tnit and
Tat^alD^ ( l ^ l a ) ftniiKl of
t-^aspar^hn^ase acuity tti^lii^dbnent
This if¥E^t^i^m mo^Mtjf k\ the
pi^ence of t<^u^^ nky be due to a
c h a n ^ in Itie p&m^&M^ tif fStm mk^rcbM
a^t ftmnk^me to miriNs^tes wmi enpryme
(1967)^
^Kiitivtty,
1 t ^ e»ec*5 of l-feCabt P"
iu:^ki on i-mf3^ms^fi^smwslS>tM
tttat a firee m^l^rf^r^ im^ety Is i
to niiHiil^n t t o as^ftire missjptm*lim effect
of c ^ m i ^ 1 ^ lamki^m # K M ^ t t ^ solt
vt^lwi tt^ JM iHrfNr 1 ^ ^ ^ f^t^^aed to
o o n ^ n 5 nm yM% r^fmit to flto fc)rih:^ng
ions: r . Nan Mn^\ a % NCJ^^PO/^ ^ i d
8 0 / ^ ^ r m k m b ^ 9 ^ and Tabat£tt:ii^
iWi^. Tr^towit of i^tisuMi MariF ftrMbtts
i^i^:^^f^lna$e ^ l ^ « t l ^ taMMHi^
i^&m^ to be lelated to ttm t M l k ^ o l l ^ ^
lec^ir^ fcH* s^Dth^Mcm of t i ^ ^i2gffim.lBto^
C^^^ and i ^ ^ activate 1^
a^Hvit^ in sc4.
327
L-Glutaminase activity of soils
L-GlutamJnase activity
of soils WT. Frankenberger, Jr
(Frankenberger and Tabatabai 1991c, 1991d)
L-Glutaminase is among the amidohydrolases
that supplies available nitrogen to plants. This
hydrolase is specific and acts on C-N bonds
other than peptides. i-Glutaminase (i-gluta-
mine amidohydrolase, EC 3.5.1.2) activity in
soils was first detected by Galstyan and
Saakyan (1973). The reaction catalysed by this
enzyme involves the hydrolysis of L-glutamine
yielding L-glutamic acid and NH3:
COOH
I (r = 0.76**). There was no significant relation-
NHg — C — H COOH
I L-glutaminase I
I ^
CHo CHo
I taminase activity and amidase (r = 0.82**),
CO CH,
I
NHo COOH
{7J)
L-Glutaminase is widely distributed in nature
and has been detected in several animals
(Sayre and Roberts 1958), plants (Bidwell
1974) and microorganisms (Imada et al 1973).
Microorganisms that have shown to contain L-
glutaminase activity include bacteria, yeasts
and fungi. Plants and microorganisms are
probable sources of i-glutaminase activity in
soils, however, the main source is believed to
be microbial in nature. Among the bacteria,
very high levels of L-glutaminase activity have
been reported in Achromobacteraceae soil
isolates (Roberts et al 1972). Fungal species
that are known to produce L-glutaminase
include Tilachlidium humicola, Verticillium
p < 0.01
328
M.A. Tabatabai
malthousei and Penicillium urticae (Imada et
al 1973).
L-Glutaminase activity in soil profile samples
generally decrease with sample depth (Fran-
kenberger and Tabatabai 1991d). The relation-
ship between L-glutaminase activity and
organic C, using pooled data (33 samples)
from five soil profiles, was highly correlated
(r = 0.92**). The soil properties that related to
L-glutaminase activities in 25 surface soils
included organic C (r = 0.79**) and total N
ship between L-glutaminase activity and pH,
percentage of clay or sand. There was, how-
ever, a significant correlation between L-glu-
urease (r = 0.78**) and L-asparaginase (r =
0.92**) activities in surface soil samples stu-
died. In the presence of trace elements (5
|imor^ g"^ soil), the average inhibition of L-
glutaminase in three soils showed that Ag(l),
Hg(ll), Sn(ll), Gr(lll), Ti(IV) and W(VI) were the
most effective inhibitors (average >25%)
(Frankenberger and Tabatabai 1991b).
Prbiciple of the ineltK^d
L^hiteyminase catalyses the hycirolyBis of
t-ffutamlne to produce ammonra and t-
giutamic mM. A drnpia, precise, mpid and
s^i^iUve mettiod to assa^ttsactivity was
of NK4 feiaasad by t-0iutam)nase a^ivity
Chapter 7
 L-Glutaminase activity of soils

ifiAm% f to t-ghitgrtnine, THAM

\ a l 3 r G l o r 2 h The NH4-
11:^to^toi^iNitof
iv^2J&mkC\ Qontmnmg a
\ ctf m aiiqiiiDt ofttieii^yltfr^ soil
S^MHii MsMiiwi a^^saratus (see Keeney
wmm^ M, pH 10)
^1^^^^ 12J2 g df Tflte
fliiadMUt
r^ a # ^ f t ^ the pH to 10
Prer^Bd as ite^ffi^d t>y K e ^ w ^ d
Nelson (1982).
Prooeifcira
A 5 g soil sample <<2 nw) In a 50 ml
volumeMc flask is treats v^^ ^3L ml
toluwe ami 9 ml TrtsterffenThe ftesk Is
^m^i^A for a few seconds to mix ttie
omtents; 1 mi 0.5 M i^lutaminasa ^iution
isiSKlded and ttie flask n mia^ again for a
imi seo^nds. T ! ^fla^kIs stc^pered and
k0ptat37^Cfor2h.
Aft^ incubation, the stopper is removed
a i d ^(m>dmate{y 35 ml KCM^2S04
^hitton Is added* Ottier stei:^ Involved in
ttHs {m)oedure me ctes^bed by
Frankeit^frpr m^ li&^sS^&M (1i^1#. The
I # l 4 ^ r ^ s i s ^ Isctete^nAnedi^sdte^bed
1 ^ Keeney 1 ^ H^simm {19S25. To p^ltmn
controls, tt^ |2«cK::a(!hi^ fcr tN^as^y of
t-gliAamrnase ^ foMc^ed, e x o i ^ t l ^ 1 mt
<tf 0.S M tiilnteniiMi^ soliMcHi is JNided after
ttm acMtlcm ctf tt^ K 0 ^ g ^ ^ 4 r^tgwt A
second o m ^ 1^ bic^uded, but ifirfttKHit soil
lo acoenint A^ partial c ^ m o d hydtol^s of

i.-Qiutamtr)e ^ mM) decxm^^ic^ses iipc^

^ai^icfl^ii^^ 1.t2 g of
i i g ^ i t t p ^ |^gp?m C ^ m k ^ C>>«, St
l3dW^|04ii^f:M1O^^
ani i t i i ^ ^nMIe ninraig t¥^ lap ^mlm
i^>^e^^m d i l c ^ e 0^.5 M H H ^ ^ $uiphate
(1(» mg imr^) sduHon
PNKC^ 1(X) 11^ of raag^t grada
^ ^ ^ > 4 luiiKHit 700 irri of watsfy
ii»$<ri^ 1 M g <H^ r^i^Bierit g r ^ ^ KCIifi
0 ^ scAM:^ and itfhito tt^ sotutori to 11
Rai^iente for ctefe^tmii^icm of amiT%)filyni
(fra^m^iym oxhto, iaorfc acid Imlicator
soluticmp 0.00s M suif^uric acid)
i^sdHitg ki tiie presence of Trie t^iffi^
|0«1 Mt { ^ 1(9 The iBte of dNMntk:»9d
l^^tlmty^ feM0^^ an es^x^r^iM^ omre
wNti bK^reasAi^ l€Nnip^^iAuBfe iSMi fanges
iPCNfH 0*5 to WL% itt 30^ and WQ^
r^^pec^ivcfy ^mrkmc^cmQm laid Tiri^afasri:^!
Il^lc}. Decc^r^xmKton of t-glirtamir^ is
a i ^ concentnaikm cl^>^Kl^it in buffer
^utlons* L-Qlutamine does n^t
dea>mpo$e In wati^, howe^^, in ttie
|M'€^ence of Trts^ its cteccmiposltion
ino^as^ \Mth mito^^easein concentration
mNdk time, ^(^roximate^ 2 % <rf t^lu&miine
(50 xps4 ctocompc^es in2 h in t ) ^ presmce
of Trie boffer, v«^€»Ba^ 18% Is deoMtiposed
in 24 h at 3 r c . "^xvl^ r^uMto "mm
lepmted by & ^ et at (1949b), wNo found
ttmt 28% of Li^utamlne {20 mM) wis^s

Enzyme activities 329

L-Glutaminase activity of soils
decomposed in 20 h in the presence of
phc^phate buffer but only 6% in a
phc^phate-free (veron^ buffet
Frs^kenb^er and Tatotabaii ( 1 ^ 1 c)
observed ca. 2% deoon^)csKion of
L-gtutamine during th^ 2 h as^iy m\6
t^tipmBture of 37^0, tnit ttiis was
accounts for by using the proper controls,
L-Glut^sminase activity in soils exhibite high
activity within a remarlcably tKoad range of
pH. Jtm optimum pH of t-gluteminase
activity In soil t$ 10 ^rank^fc^ger mtd
Tabatabal 1991o), The apparait pH
optimum for clay-adsorbed enzj^es
generally i$ disi^aced or^ m two pH units
to more alkaline values. This shift in pH
optimum to higher valu<^ occurs because
SK^idity at tto clay surface is significantly
greats thai in tnitk solution P o ^ and
Mortland 1990),
Frankenbeiigi^ and Tatm^rt^ (1991c)
showed that 5Q rrm of t-gtutamine is
sa^factory for the ssssay of tigflutemln^^e
activity in smls. M thm cx>r»san1^tion,
t^tutamina^ t)ecomes satoat€ti with
t^fuftamtf^ arKi Hm nsfte ^ ^ ^ t l M l y fdbws
z^o-mic^ Wmtlcs. The I H ^ ^ H ^ of
glutmirine Is ^so hydre^sed in soils but at
only 7% of ^ e activity of ^tm irmmmr at
sabimttng cxmc^Arattons of ttie sutetrate
^ mM), Ihe activity of t^lu^minase Is
{:^portior^ to flte ammint of scrii u^ad in
the assay. A lin^r r^a&Hiship has b&^
i^tabli^ed between ttm ^izyme adUvity
m¥i the aumint of smi i^ed, i^p to S g of
soil V^rani^nbiergi^ aiKt Tab^teris^ 11^1 c).
The amount of NHi-N i^^ased dteviat^
from linearity wh^i greata^ amounite of soil
(>5 g) Bm i^ad, Indic^ng tt^ eitf^r ttie
subsbBte concentration is llmitir^ ttie
r^wotion rate m <me of the fHoducts ^H4 or
t*giutamto add) is InhRHOi^ t^tutiEUfninase
activity, the criss^ved deviation from
linearity of the ammint of NHI |»oduc^
Witt) Increasing tim mtmM of $^i afY}e£B^
to t>e due to a substtiarte limitation
0Frarri(ent)erE^ mvi IdtmiBfm 1991c).
AMK>ugh VmfomiatiiKiof NH4 In st^ls
shows zero-order reactions fCH* at les^ 4 h.
330
ttie reactk>n r a t ^ deviate Arcnn linearity with
|:»t>longed inoiti^rtlcNr). Maxlm^d
i-glutamlns^e acM\dty ki i^ll ocmirs at 5ff*C
under ttie ccMidltions of ms&jf. The
aotivaticm mmg^ c# Vtm m^c^tm catiriysc^
t ^ L-glutemlnase of nine ^ I s nsur^ed from
20.3 to 39.9 kJ mor^ {m^^^ 324)
0=^rmk^berg(^ and Tabatabsrt 1991c). The
sut)strate cx>ncentration activity cnirves of
M i ^ ^ i Mnetk^ 1 : ^ ^ . The Mk^a^is
coi^tante (JQ rmiged from 8 ^ to ^ , 6 rm
f^rankenb^g^ and Taftsatatidi 1%1^« The
Qto values of the reactkm catalysed by
t-gfutamins^e in nine soils ex(K>sed to
temperatures of 10-60% rar^ed ffmn 1.19
to 1.85 (av^age 1,49) fl^raikentwg^ and
Tat)atabsd l l ^ l c ) . Btemn st^ltzaMsn
( l a r c , 1 h) Irmcth^ates t^lutamrtna^ tot
after tti^ tr^rbmerA. ir43Samt^mm acMv^
Is g r ^ e r ki %:rfu^r«^tr^dted soHs flian In
tt^ abs^io@ of tirfu^e. t-Glytaminase in
i^ts Is sbongiy InhMted f ^ severs!
sulphydryl group r ^ s ^ t s , Iha dffedts of
tH^Cbf iH<^kKtcmi^^cHjry>6iiBait^ mM, and
iodbac^tk^ a€^ i^j^^S^^^ttiafta free
i ^ p f q ^ ^ nK:ri^% n e o e ^ ^ lo n^itein
t i ^ ac^fe «^i^pim. inhii»9^ ite f u M ^
$»iqi:^piHtod 1:^ ttie ftr^ii^
te not <mty kMbited by t ^ ^ l a ^ 1^
rr«EHmmt)€ra[oate, tnit afe^ i^ Ag^, Wb^*^
a i d Ci^^ at ^EHw^rti^ ti^gitm
€^ri^:)€^r^^raMc^m |ttartmsgi 1971)* i^rfie^f^ ttie
d^mtoai sattSt mily N ^ tia^ an MM^my
^lect cm sm! t^u^^Hi^^^^^ adlh%*
WitWttcm t^ F^ a^gHifesls I N * t-
gliAsMitfimse recptoes U^^m C ^ ^ to t)e
ac^e. At 5 mM, tKitti Ca?^ m^ Mfif*
adKfated t-giutemlnase a^:tMty 1 ^ IM1
averse of 4 ami 12%, r^pec^My
0^rg^kenb6»g^ m^ M^iiBk!^ 1991o), fiteo,
t-^iutenrrins^^ ^ad^Hy in isxMs Incr^^es. i -
GlutarMm^e ac^Nrity m iKst ^BiSmA^d H0^m%
tt^ Trts toffcr used o^nt^ns S tmn wtti
respect to tt^ frtk»^r^ mm K\ fste^
Mn^% C r , NOs^, P O / ^ or S O / ^ ,
Chapter 7

L-Histidine ammonia
lyase activity WT. Frankenberger, Jr
(Frankenberger and Johanson 1982)
Histidine ammonia-lyase (EC 4.3.1.3) is the
enzyme that catalyses the irreversible non-
oxidative deamination of L-histidine to urocan-
ate and ammonia:
CH2CH(NH2)CCX>- C=C-
% ^ "
ri '
L-histidine Urocanate
The enzyme, histidase (i-histidine NHa-lyase)
was first discovered in 1926 by Gyorgy and
Rothler (1926) and Edibacher (1926) who
observed an increase in NH4"^ when L-histi-
dine was added to aqueous liver extracts. In
1930, Edibacher et al (1930) later observed
that glutamic acid, NH3 and a reduced acid
(formic acid) were the reaction products:
L-Histidine + 4H2O -^
Glutamic acid + NH3 formic acid
Since then, the enzyme that catalyses the de-
amination of L-histidine to urocanate has been
studied in both mammalian and microbial sys-
tems and was referred to as "histidase",
"histidinase", and "histidine-a-deaminase".
The International Enzyme Commission has
now named this enzyme "histidine NH3"
lyase" (Florkin and Stotz 1964).
The amino acid, L-histidine, was first
detected in soils by Schreiner and Shorey
*** p _
Enzyme activities
L-Histidine ammonia lyase activity
M.A. Tabatabai
(1910). It comprises approximately 10% of
the total basic amino acids found in soil hydro-
lysates (6 M HCI) (Bremner 1965a). Histidine is
unique in that it bears a weakly basic imidazo-
lium group, and it is the only amino acid whose
R group has a pK near 7.0. The imidazole
CCX)- group of L-histidine can function as a nucleo-
+NH«
philic group in the binding of substrates in
various enzymatic reactions.
L-Histidine is widely distributed in nature. In
Ammonia
plants, animals and microorganisms it can
(7.8) account for <1 to 8% of the amino acids
found in proteins (Tabor 1954). Among the
microorganisms, L-histidine NHs-lyase pre-
parations have been purified from Aerobacter,
Bacillus, Klebsiella, Pseudomonas and Sal-
monella species (Lessie and Neidhardt 1967).
The distribution of L-histidine ammonia lyase
activity in soils is correlated with organic car-
bon (r = 0.70***) and total N (r = 0.55*) in the
topsoil (Frankenberger and Johanson 1983a).
There was no significant correlation between
L-histidine NHs-lyase activity and soil pH,
(7.9) cation exchange capacity, percentage of
clay and percentage of sand. The activity of
this enzyme is concentrated in surface soils
and decreases with profile depth. Air-drying of
field-moist soil samples increased L-histidine
NHs-lyase activity by an average of 18%.
••'.'^=•- ' ' J ^ - ' - <';>'V'','
331
L-Histidine ammonia lyase activity
acUvify wi^n sod is incubated with bufferBd
(0*1 M Tris, pH 9.0) t-histidlne solutjon a i d
toluwe at 3Tt), TTie NH4 missed is
dateimln^ by a procedure Involving
tn^tment of the Incubated soil sample with
23 M KCI containing a i-hlstidine NH3 lyase
inhii:ritpr {umnyl acetate) and steam
dtetlHatioi of an sJiquot of the n^sulting
suspension with MgO. This proc^uns
gives quantitative recovery of NH4-N
released In soils and does not cause
chemic^ hydrolysis of the $ul:^trate.
Materials amt am^aratiis
\toiumetrtc flasks (50 mi)
Incubator 0TC) or temperature-controlled
Deflation flasks {100 mi)
Ste^sm d^tillatlon apparatus (see Keeney
ma N^son, 1982)
Chemicals and soiiittons
Tolu€^ie
ffeNri:aKtifl^ reagent pii^^HT ^lenlif to
Q>,, Chicago, IL),
TifeH^t|rf«jrte ackl buffer (0.1 M, pH 9-0)
£>i^$$olve 12-2 g of Trie 0iy<feoxy methyl)
amlr^oi rr^haiefl=ish^-<>&rtlfi^reag^^^
In about 8{K> ml of wat^, mij/u^ tto pH to
9.01^ttb^Wcmififlft 0.1 M H2SO4 aid
dNuteH^ ^lotlofi witti wat^to 1 1
t-MW*rte twMUon (6.S %^
JWd 5 i 2 g of iK+)-histldine
h^tebc^Imldte mon<%dmte (Wdri^^
tmtWi^ into a $0 ml volumetric flask.
Make up t i e volume by s«Jdlng Tris
t»jfferfitfidmix the contents. Store the
^s«:^ijion In a refrig^aton
l^ot^^um c#*k:»1de (2,5 iyj>Hirany! acetate
Ol^olve 2.12 g of reagent gmde UO^-
(CaMgO^^H^O in about 700 ml of
waiM, dssolve 188 g of r^Nagent grade
1051 In this soliition, dilute the solution to
11 wift water and mix tN:m5ugWy.
332
Prepare M s solution Immediately t)efore
use.
Reagents for i l ^ determinatton of
annrm)nium (magnesium oxkie, borte ^ i d
indicator solution, QXX&5 M sulphuric aclcQ
Prepare as descdt>^ by K^ney and
Nelson (1982),
Proc^ure
Place 5 g of soil (< 2 mm) in a a3 ml
volumetric flask, add 0,2 ml of toluene and
9 ml of Tris buffer, swiri the flask for a few
seconds to mix the contents, add 1 ml of
0.5 M t-hlstidir^ sotutbn mxdi swiri ttie flask
again im a few s^>onds. Then stopper the
f l ^ k s^d frface It In €MI Incubator at 3 r C tw
48 h.
Mti&t iiK^bation, remove the stop|)ar^ add
approximately 35 ml of «te KCI-
UOaCQaHaO^b aoiutlon, swirt the flask for a
fma sa;x>ncte ar^ji allow the flask to s t a ^
until t t e contents have cooled to room
tempmature (afcKiyt 5 min). Jtmn cHlute flie
volume to ^ ml l ^ ttie addition of KCt-
U02(CtHa05fe J^luttoi, stopper «ie flask
and invert it sev^^^ t t m ^ to mix itm
con^ilB.
To detenniw NH4-N in t i e re^uWt^ soH
si^^p^ision^ lnv€^ the fli^ sev^id tirn%
and ppette a 20 ml tilquot of ttve
sust^nsion I r ^ a 1(K} iri (d^sttllatiaii fla^
mtd dmtmtAm ^&m NH^M i ^ e a s ^ t ^
^eam <flstiteon df tfiis aitcpKft wItti 0.2 f
of M ^ for 3JS fi4n ^ d ^ c ^ t t ] ^ fayKe^fW
a i d N^sw (1982).
Go^trote ^ o u l d be p^ormed In i^«*
s ^ e s of and]^ses to account f w t i e NH4-N
not derived Ircra t-hlstldlne thrmigh
i-hmtjdine NHa-lyiK^ actwty. To pcrtomi
l^ntn^ls, firilow t i e proc€NJure ctesoribed
for t i ^ as^y of t^hl^Wlne NHs-^^a^se
activist biA tmim t i e i^Milton of 1 ml ^ 0.5
M t-MsMine s c ^ i ^ n Mter ttia mk^m of
KCMJC^^H^Oj^ f B i ^ » ^ Ttw p^pdse of
acfeTH^ t i e K O N r e ^ ms^^ s^Mkm to
t i e soil sample aftm* IncaibatlcHi ibto
Chapter 7

hmc^ate tti^ enz^e, t-^Mstidbie NH3-
tMmti^k^^^ Urn MHr^ n^^ased. Ttm
wcM mj^i^ksmt mwfysod for NH4-N infist
j^^fYij^iri^ for NH4-'N w ^
fmmdm&^ iipMrmii &c^^ of L^hlslicHne
iHifl^lFi^^f^rger and Johans(»i 11^2).
theirf^ optimym of adi t-Nstldina NH3-
Ijf&smWjn SKKid la^pre^erri; ^tti the value
->' as i^^x^akJ t ^ Raohier {1959)*
rioted iille^»iei»»H» i^
;i^,;T^-
Enzyme activities
L-Histidine ammonia lyase activity
ff^"»tf^*«9» »id Jcrtiamon iaS2).
A»»ar fs^ation^fa^we«nth« amourt of
sc^i SHKi NH4-N r^£»ied iraJBc^fted t M $ g
of s<« was «*s««a<wy fw i « ^ Q ^
L-WsOcBm NH^ lyaee acjM^
ff«rtcei*^gpM' md ^rfiamcm 19825.
However, theanoimt i^ NH4-N
<Je^^a«5fr(»nibnesHtiy «rih^.
«nounte of so« a » used, hwBcsWng eWier
tt»attt»sutKSbate c»»ic«ii^i^^xr) b a
KrnWng faaw cr ofte of »e product
released ^ H * * or uiocanate) hWtsits the
Under the stdndard condWons descrtaed,
the «»JiiiiHMbfi of l«lr-N dertved imm
< ^ * 188 hfffai*ef#e»ger«BdJirf^nson
1 9 ^ Irtorfeienoe ^irtcrobW ac*h%
<to^ 4Krt ;»»«ti1» ooc»r dittftnsttie48 h
amount <rf I#i4-N itfiased »Kl »»e <rf
friaA)attan IntfieI
P*<1 The ^eay of ^^^Ust^ie te^»e
twIOwSTtJ
\'. ^ -^''""''"
|Nw4e»4:^igpM- «rkt ^MiBKKKm 1^1^;
iHi^tirdryl mde^ teiieoe^»^ totnaktMn
333
L-Histidine ammonia lyase activity

s ^ s ^€«*adl to fMO^rtcfo a vsBtety of * ^ M t#*s^Bfc# l#fe%€^e A ^ t i ^ 11m

m« v ^ Inspect to tN^ fe^wkig k>ii^: K% t^tlcft^ NNb ^ ^ i ^ a ^ % ^

Na^, a ^ , NOa^. PO/^ and SO/^

334 Chapter 7

Phosphatase
activity
Phosphatases catalyse the hydrolysis of phos-
phate esters and are enzymes with relatively
broad specificity, capable of acting on a num-
ber of different structurally related substrates,
but at widely different rates. They have
received trivial names according to their sub-
strates; phytase, nucleotidases, sugar phos-
phatases and glycerophosphatase belong
to the group of phosphoric monoester
hydrolases (EC 3.1.3), whose mechanism of
reaction is reported in
o microorganisms and animals (Beck 1973;
II
R - O - P - O® H^ HO - P +
Nucleases, which catalyse hydrolysis of ribo-
and deoxyribonucleic acids to their individual
nucleotides, and phospholipases, which cata-
lyse the hydrolysis of phospholipids, belong to
the group of phosphoric diester hydrolases
(EC 3.1.4), whose mechanism of reaction is
reported in
OH OH
I
^n — p
R'O P == O Hp HO - P = O + R^OH
R^ R%
where R^ and R^ are alcohol, phenol or nucleo-
tide groups.
The other three groups of enzymes, phos-
phoric triester hydrolases (EC 3.1.5), enzymes
acting on phosphoryl-containing anhydrides
(EC 3.6.1) and enzymes acting on P-N bonds
(EC 3.9), such as phosphoamidases (EC
3.9.1.1), are also called phosphatases (Florkin
and Stotz 1964). According to Malcolm (1983),
the temi phosphatases also includes enzymes
Enzyme activities
Phosphatase activity
K. Alef
R Nannipieri
C. Trazar-Cepeda
hydrolysing pyrophosphates, metaphosphates
and inorganic polyphosphates.
Phosphomonoesterases have been exten-
sively studied in soil because they catalyse
the hydrolysis of organic phosphomonoester
to inorganic phosphorus which can be taken
up by plants. According to their optimum pH,
phosphomonoesterases are classified as acid,
neutral and alkaline phosphatases; the first
two enzymes have been detected in animal,
microbial and plant cells. On the other hand
alkaline enzymes have been found only in
R - OH Burns 1978; Chonkar and Tarafdar 1981; Dick
and Tabatabai 1983). Both acid and alkaline
phosphomonoesterases are supposed to play
(7.10)
an important role in plant nutrition because
their activity in the ectorhizosphere is higher
than in bulk soil ffarafdar and Jungk 1987),
while the content of organic phosphorus
shows an opposite trend.
Both acid (4-6.5 as pH optimum) and alka-
line phosphatase (9-10 as pH optimum) have
been found in soil (Speir and Ross 1978).
According to Eivazi and Tabatabai (1977),
acid phosphatase is predominant in acid
soils, while alkaline phosphatase prevails in
alkaline soils. Usually assays are carried out
near neutral pH (6.5-7.0) (Speir and Ross
1978; Nannipieri et al 1988). The optimum
(7.11)
temperature has been found to range from 40
to 60''C and assays are usually carried out at
37**C (Hoffmann 1967; Tabatabai and Bremner
1969; Nannipieri et al 1988; Doelman and
Haanstra 1989).
Since the early 1960s, artificial substrates
have replaced natural ones in assays for deter-
mining phosphatase activity of soil (Speir and
Ross 1978). Artificial substrates such as phe-
nyl phosphate or p-nitrophenyl phosphate are
low molecular weight esters that are more
rapidly hydrolysed than natural substrates.
335
Phosphatase activity

and they contain an organic moiety which is studied extensively in soil (Juma and Tabata-
easily determined; methods using natural bai 1977; Mathur and Sanderson 1978; Speir
substrates are based on the determination and Ross 1978; Mathe and Kovacs 1980; Beck
of inorganic P and present the problem that 1984a; Gadkari 1984; Nakas et al 1987; Rastin
phosphate can not be recovered quantita- et al, 1988; Wilke 1988; Cochran et al 1989;
tively from soil (Speir and Ross 1978). Doelman and Haanstra 1989). Usually ortho-
Michaelis constants of phosphomonoes- phosphate inhibits phosphatase activity in soil
terases have been determined in soil (Dick (Kiss et al 1974; Nannipieri et al 1978b; Speir
and Tabatabai 1984; Trasar-Cepeda and and Ross 1978; Spiers and McGill 1979; Appiah
Gil-Sortes 1987, 1988; Juma and Tabatabai et al 1985; Lopez-Hernandez et al 1989).
1988). According to Cervelli et al (1973),
Michaelis-Menten kinetics cannot be applied
without a correction factor that takes into
consideration the adsorption of the substrate
p-nitrophenyl phosphate by soil. Kinetic para-
Phosphomonoesterase
meters of phosphomonoesterases extracted activity
from soil have also been determined (Nanni-
pieri et al 1988; Kandeler 1990); humus-phos- Assay of
phatase complexes extracted by sodium
pyrophosphate and fractionated by ultrafiltra-
phosphomonoesterase activity
tion and gel chromatography present at least (Hoffman 1967, modified by Beck 1984a)
two enzymes (or two forms of the same
enzyme) catalysing the same reaction and
characterized by markedly different K^ and
Vmax values (Nannipieri et al 1988). Humus- :^lf;''"" /'%'< //'-''•'',',' I"',' '-P", ''''^y' '^'-y'y'''^^^' 'H-'^'ifk^?'^'/.. » '- -'"
phosphomonoesterases complexes with
higher molecular weight are more resistant to
proteolytic and thermal denaturation than com-
plexes with lower molecular weight (Nannipieri
etal1988).
The activity of phosphomonoesterases is
strongly influenced not only by pH and temp-
erature values, but also by the organic matter
content, soil moisture and anaerobiosis. Due
to these effects, phosphomonoesterase activ-
ity in soil varies with the season (Speir and
Ross 1978; Beck 1984a; Sparling et al 1986;
Pulford and Tabatabai 1988; Rastin et al 1988).
Air-drying decreases the phosphomonoes-
terase activity of soil (Speir and Ross 1978;
Sparling et al 1986); enzyme activity increases
after the remoistening of air-dried soil. The
phosphomonoesterase decreases with soil
depth (Speir and Ross 1978; Beck 1984a;
Cochran et al 1989) and does not correlate to
bacterial number (Nannipieri et al 1978b; Speir
and Ross 1978). The influence of pollutants,
soil treatment and cultivation on enzyme activ-
ity and phosphate mineralization has been

336 Chapter 7
 Phosphomonoesterase activity

I^KHiMttmi'dinii

C^mBOt «ia r^^^^Ms for ttie blarrtcs JSMICI

W^rK»{ ^ g g^^ dWt h^')»

, C X 100
^A2)
dM^xfxlO

;^ m^^^^^^^^fmj ^S^^yi^^^m |«[iPS9^pNr«aa«E^ ^ ^*

%8irrt8ii'^diA" l^it^ll^^ W7B; lBmik li^Mla;

oftNifte;
'<^^^:^^^^fK^^^^r^

Enzyme activities 337

Phosphatase activity

Phosphomonoesterase activity
CTabatabai and Bremner 1969; Eivazi and
Tabatabai 1977)
^^:
W^Mi'4k;-W^^'-}:'t '•
MhiiC^^ Of ttm i i i e i h ^
^l»h%1^^«Htt^Mled ws^to 1000
of |Ghrtbt^:rf^iol rrt^said after 1 ^

338 Chapter 7
 Phosphomonoesterase activity

l^ust ttit vc>hinNS^ to S M by adhdWion of

tlie acWWcm of C^fe pcmm^ cS^^erslon

^^^fWM^ ^Mc^isi yt^ui^^ps^tocDdiBct

C^e is mqylredtodhoo^hf^ a iHiff^,

CNrioiMHcKi because $oH i:4K^plm^$0^^N%^
JfiNI:Medl b^ aootate and i^tead^'i:rfic^NA0
c^Ac^tettm;iHdbqf^h^ jm miUHitiB of tHiffais, wMl0 fiud^^se m Mdblteci t^ both

Fk^s 1978). H ^ M4bHim t ^ cifialeh

{3l^c^3Ma lm0mW iirttrit^My n^^ed to the
|H«&pj*«i¥rf g""^ dhart h^^) ^
^ffadt of f^lK^phad^ p^ocrim 1983).
incomplete extraction of p-nitrc^henol was
obj^vadfcisoils yrilh high orp^te rmttar

<pariM30va i^eki^o^tB^ r^^isad

, 1 ) ^ ^mettiod^cl l ^ M M : : | n d -ih^^w^

k ^ ^ b ^ f In INal^^^^^^ Mia
fcHM^ ccm^e^rift^^^ lo 7>S iiiM^
1 ^ ^ ba€»i tL^^ 1^: ^sM^ #l#r^3Mi 11^:^.

|pi|Oii^|^iM^. Is. ^

Phosphodiesterase
activity
(Browman and Tabatabai 1978)
"••• ^'•'•"' :mmm
Phosphodiesterase has been detected in
microorganisms, plants and animals (Brow-
man and Tabatabai 1978). Usually the activity
of phosphodiesterase is much lower than the
phosphomonoesterase in soils (Eivazi and
Tabatabai 1977). Air-drying did not affect
phosphodiesterase activity, while steam steri-
lization for 20 h at 120°C inactivated it (Eivazi
and tabatabai 1977). On the other hand

Enzyme activities 339

Phosphatase activity
Sparling et al (1986) observed an inactivation
of the enzyme activity after air-drying. Ortho-
phosphate inhibited the enzyme activity in a
competitive way (Browman and Tabatabai
1978). The enzyme activity is significantly cor-
related with the organic C content of soils
(Browman and Tabatabai 1978). Phosphodies-
terase has been extracted from a forest soil
and chromatographically fractionated into
seven fractions (Hayano 1987); 2,4-cyclic-
nucleotide-2-phosphodiesterase (EC 3.1.4.1.6)
or 2,3-cyclic-nucleotide-3-phosphodiesterase
(EC 3.1.4.3.7) seem to be enzymes of one of
these fractions.
l%lm^rie of ttie iiietfiod
1 1 ^ f i i ^ l ^ based m\ttiet^ksSmrnkwAUm
:i^ i ^ i ^ i i e ^ g ^ p r f M ^ ^ after tt^
; i^i^^ far 1 h I t
y^' -iy^. V-'-^)'''
:; f f i f e ^ ^ W S 1 1 0 ^ socftim Ws-p-
; V: f l ^ in 80 ml of Tife
«tl*l
340
Dis^lve tZ*2 g of Trte fli^ifoxy tneth^)
mwm xm^mm In 800 ml of d^fitlM
iimei, miliM ttm pH to 12 v^dth HaOH
(0*5 ^ and l^r^ up to 1000 m} wtth
Tris BOluMon (0.1 u, pH 10)
Dissolve 12.2 g of Trts piydroxy methyl
mrAtm m^Hiar^ in 800 mi disttttod water
Bmi difcileto1CXK) mt vifth dmVlled water.
Stamta^d p-nftrojrtienol solution
See estlmatkm of
Procedure
f%ce 1 0 c r f s b i ^ ^ mm), nfK^ist soli in aii
e ^ r m y ^ l l ^ k p } mO, acki Oi! ntf
\iMmm^AMlM\»MmwA 1 iM socfltim
\ii^rp^^i^im^m^ p N ) 6 p M e mA^^te« Mbc
fn€^yi:Nad^far1 ti aft 37"^). Altir tt^
I M ^ D i ^ a l/Vfi^^ 1 ^ I c M ^ fftt^
ioT
ttiH^ e i A r ^ i ^ ^ ^ori^i^ ami MnrtocN^^
b€^c»e Ab^ttcm of fiH» sbR »y^&$>^ii^ M
' s ^ esttf^ADii of {tic^NiNmifiQ^
Cidtetriattmi
vsse ^%iitfc»i of |:rtio^p4K^^
Chapter 7

^^mobk^ im^bation of soH samples
1 ^ inoBase m a decrease in
^ ^ t t % ^itond myd Tabaiabai 1988).
Htm c^lce of buffer is important because
ortN:ptosphate, EDTA and cli^te (5 mM)
sipnMcsMy Mttiits the p tosphodiesterase
i^vtty of soil 0rowman and TabaM>^
1978)-
J ^ vatii^ betvirec^ 1.3 and 2 mM for the
^^(tfi^rate d^i^nttrc^)henyl (^osphate have
bem foynd prowman and Tabatabai 1978).
Phosphotriesterase
activity
(Eivazi and Tabatabai 1977)
Paraoxanase, the enzyme responsible for cat-
alysing the cleavage of diethyl-p-nitrophenyl
phosphate (paraoxon) into p-nitrophenol and
diethyl phosphate is indicated as a phospho-
triesterase. However, according to Schmidt
and Laskowski (1961), enzymes like paraoxa-
nase should be classified as a phosphoric
anhydride hydrolase rather than phosphatase
because of acidic properties of nitro-substi-
tuted phenols. Since the method for assaying
the activity of these enzymes in soil is based
on the use of Tris-p-nitrophenyl phosphate,
the term "phosphotriesterase" may not be a
suitable name for identifying these enzymes.
Phosphotriesterase activity in soil shows an
optimum pH around 10; it is increased by air-
drying and inactivated by steam sterilization
(Eivazi and Tabatabai 1977).
Mtai&iAft bif fits iiii^tt^id
r^MspMrptt:^^ feleased
Enzyme activities
Phosphodiesterase activity
Materials ami apparatus
The same materials and apparatus
described in the eaill^r sectkin on
phosphomorK>estera^ activity are required
as welf as lOO^-m^h glass beads.
Ch^nieals and solutioi^
Toluene
Modified universal buffer stock solution
Prepare as describe in the section on
phosphomonoesterase activity.
MUBpH10.0
Tl^ate 200 ml of MUB stock sofutton to
pH 10-0 uniiBr continuous stfning v^th
0.1 M NaOH and then dilute to 1009 ml
with distiiied water.
&>dlym Trls^p-nitrophenylphosphate In
solid fomi,
CaOia 0.5 M solution
Pr^i^m as d i ^ c ^ b ^ in Vm $ecti<m on
{[^hosphomonoestera^ s^i^JMty.
NaOH 0«S M solution
See ^tlmatic^ of
NaOH 0.1 M TOfution
See ^^htiattc^ of
S t a n d i p-nftmphmml solutton
Pmp^m as Indicted in ttie ^^oHon on
ptosphomorK3estefase adtrvity.
Procedure
nace 23 mg of scNdium Tris-j>
nttropti^yti^osf^te (nscrfuble In watei) in
a 3Q ml Erfenmeypr flask, mixed wtti 1 g of
rm>ist sieved ^ trnn) mM m^i m^mmd wWi
2 g of lOO-^n^sh gtg^s beacfe. ttimi add
0,2S irri of Ibokmm (added tkas^i^^i^m to ttw
m^ao^ of ttie jg^a^ beaels) a i d 5 rrrt cf
MUB, pH 10.0. l^of^^ theflasics,mbc flie
341
Phosphatase activity

orthophophate, but also because the sub-

strate (pyrophosphate) may continue to be
hydrolysed abiotically (for example, at low
pH) and the presence of pyrophosphate may
inhibit the measurement of orthophosphate.
A pH optimum of 8 has been observed in soil
(Dick and Tabatabai 1978) while heating of
moist soils over 60°C inactivates enzyme
activity (Tabatabai and Dick 1979). Toluene
and EDTA have no effect on the pyrophospha-
tase activity, while inhibition is observed when
soil is steam-sterilized or treated with formal-
dehyde, fluoride, oxalate and carbonate (Dick
and Tabatabai 1978). The activity decreased
with depth, and it is positively correlated with
organic C and clay content. The pyrophospha-
tase activity of soil is negatively correlated with
the CaCOs content of soil (Tabatabai and Dick
1979). The best method for preserving enzyme
activity is to store the field-moist soil for 2
months at 5°C (Tabatabai and Dick 1979).

Pyrophosphatase activity
(Dick and Tabatabai 1978)

The enzyme catalysing the hydrolysis of pyro-

phosphate to orthophosphate is the inorganic
pyrophosphatase (pyrophosphate phospho-
hydrolase, EC 3.6.1.1) and has been extracted
from bacteria, animals, and plants (Dick and
Tabatabai 1978). Interest in this enzyme
activity in soils derives from the fact that
polyphosphates are used as fertilizers.
It is difficult to determine pyrophosphatase
activity in soil not only due to problems con-
cerning the extraction and determination of

342 Chapter 7
 Pyrophosphatase activity

%&m arKi ad|im tti^ Virfuit^ to.SQO mi

g itf scKiumigBanftain liK^ytiK%f ml of

II^ANKJ W ^ ^ afKi adp^ Ihe voltymo^ fo

MA w^.l)il&^$#ticm^ iin
T%'

''4^^^^i^;^:^f^MM'B^^^:^^}~

Enzyme activities 343

Phosphatase activity

;-, '"ii-

^fcp>fe 9I % %0, j S , ^0 aiKl 2$ f^ is^ tfie { ^ r o f ^ i ^ s i ^ ^ oqKWb^aiScm above ^ ii»^

ftitp 106 mt votufii^Mo % i ^ , #€i|ust ttie <^in l^e dh^ t^ t y ^ laictc^^: (i) MtfeMcHm of
tfie pyiic^lio^]fpteise # ^ aft Mgh

acUvattoii of p p o p ^ s f ^ ^ iMiKfing
^iqiiofe of the dliwe s l a r ^ ^ s t ^ tte tf^ substrata to ih# $oU ani^iii^ may not ba
s i i f l k ^ t at hi^h pyrc^hosi>hata

a n ^ ^ of | M ^ c ^ ^ flbc»f:Mod

4i^fcfeitt|^^g^j^^ ViMfi tkfia|ri tlM pra^^nca

l^r!c^:Masi^^ ppwi^it W^ wM

344 Chapter 7

Cellulase
activity
Cellulose is the most abundant structural poly-
saccharide of plant cell walls. Microbial degra-
dation of cellulose is therefore an important
process in the degradation of plant debris
(Rai and Srivastava 1983; Sinsabaugh and
Linkins 1988), Cellulose is a linear polymer of
D-glucose with p(1-4) glucosidic linkages. The
minimum molecular weight of cellulose from
different sources has been estimated to vary
from about 50,000 to 250,000 in different
species, equivalent to 300-15,000 glucose
moieties. Although cellulose has a high affinity
for water, it is completely insoluble in it.
Cellulase catalyses the hydrolysis of cellu-
lose to D-glucose and consists of at least three
enzymes: endo-p-1,4-glucanases (EC 3.2.1.4);
exo-p-1,4-glucanases (EC 3.2.1.91); and p-
glucosidases (EC 3.2.1.22) (Lee and Fan
1980; Eriksson and Wood 1985; Sinsabaugh
and Linkins 1989). Exocellulases bind to crys-
talline cellulose and cleave celluloligosaccha-
rides from the non-reducing ends of cellulose
molecules while endocellulases randomly
cleave glucosidic linkages along non-crystal-
line parts of cellulose; p-glucosidases release
glucose from celluloligosaccharides and aryl-
p-glucosides.
The most important properties affecting the
susceptibility of the cellulose to enzymatic
hydrolysis are the degree of crystallinity, the
nature of the associated substances and the
surface area (Marsden and Gray 1986).
The degradation of cellulose in soils is a
slow process and depends on the concentra-
tion, location, and mobility of cellulases
(Hayano 1986). Type of litter, substrate con-
centrations, pH, temperature and water con-
tent significantly affect cellulose degradation
(Hunt 1977; Schroder and Gewehr 1977;
Schroder and Urban 1985; Sinsabaugh and
Linkins 1988; Tateno 1988; Kshattriya et al
1992). A differential adsorption of the various
Enzyme activities
Cellulase activity
K. Alef
R Nannipieri
cellulases on the lignocellulosic material has
been hypothesized. According to Sinsabaugh
and Linkins (1988), exocellulase is largely
associated with the cellulose component,
while endocellulase is adsorbed by the lignin
component. Cellulose is the first component
that is hydrolysed and the ratio of endocellu-
lase to exocellulase activity increases during
the decomposition of deciduous leaf litters.
The cellulase activity of soil generally has
optimum activity at pH 5-6 and at a tempera-
ture ranging from 30 to 50°C (Benefield 1971;
Pancholy and Rice 1973; Hope and Burns
1987). Air-drying strongly inactivates cellulase
activity in soil (Speir and Ross 1981). An
increase in cellulase activity occurs in rhizo-
sphere soil, as compared to the activity of
non-rhizosphere soil. The effect of vegeta-
tion, season and type of agricultural manage-
ments on cellulase activity of soil has been
studied extensivelly (Kiss et al 1978).
Soil cellulases are mainly produced by fungi
(Clark and Stone 1965; Yamana et al 1970;
Hayano 1986; Rhee et al 1987). Only a few
species of Actinomyces are able to grow on
cellulose as their only carbon source (Stutzen-
berger 1972). Clostridium seems to be the main
cellulolytic microorganism under anaerobic
conditions (Gottschalk et al 1981; Joliff et al
1989).
Several methods are available to estimate
cellulase activity of soil. They are based on
the determination of either released reducing
sugars or evolved ^^C02. Cotton strips, radio-
isotope-labelled cellulose and carboxy methyl
cellulose (CMC) are used as the substrate for
the enzyme (Benefield 1971; Pancholy and
Rice 1973; Latter and Howson 1977; Ibister
et al 1980; Sato 1981; Schinner and von Mersi
1990).
345
Cellulase activity
Assay of cellulase activity
(Schinner and Von Mersi 1990)
l%fcic#le of ttie fiieihad
The fi^tKKJ Is b^mi <m the ttetwnlnaUon
of ff^^sed lediK^ing sugars aft^ the
inoubatfcm of ^ i t ^^ampies witt) C^C for 24
h at SO^. Urn acivitics of mdc^lyomiase
and p^hiK)C^diase am mt^ be astimaitBd.
MiAerlals ami anpaiatim
%iedta^^
liii^bafe^ acji^teWe to 1K^
mim^^ a#^$laA3to to I C ^
,'\f
\ -y './
iiti9#ib be i^€»i^ * ^ f 1 ^ ^
:Ji8g|taC¥ariAtt» jn iJhsMM iM^A^igii^ idMMe
346
to 1(XX) it4 Witt) disliiled water.Store fat
tX^Nre 1 •§ s of l^ite ammcmhim
^l^haN^, 1 g of sodiuin dodeo^
m ^ ^ t ^ ^iti 4J2 mi of o<ma^ti#ed
H ^ 4 ^ dtetilled wat^ (StrC), ami bring
up to 1Q00 ml with distifled watw«
<3ai^c^ rr¥^;K^i^:bBte glutton
^^ mg dCX» irt"^ dfsMII^ wrter.
B ^ r # fl | a i ^ # or 5 9 (toiBsQ of tnofts*
w^i% fki^m^^/^hdSmand IS ml of
€ ^ i^item; 1 ^ ^ ^ ^ ^ i s ^ arKil^ioyibate
^betoe
^,;}'':-:
i f ^ ; 1 f ' I r t of the Pi#0'w»h / ;>
:*ii^Nf^^^^^
Chapter 7
 Assay of cellulase activity

GfcK:?!^ i^ulv«toit |tg Q^^ dwt 24 h"^^) =

i^etwe^ ttie o^hi^^e i^^^^yi^s Mid other
CX¥Xf fiilaicA:^ pai^amet^B M3C3tfs 9%s ^ at
|7,14)
mfx d^
^ e i B 0 b ttie maamired gliKx^se {[Mspc^^i of ttm qi^tiirideH[^3rit^^rMrig
^liitJcKm o ^ tie perfcmned Ir^ oxMi^on
the vi^iime of t t ^ test ^spenskm^n tfiis wWi H2O2 at ait alteilne pH ^

agrioAij^ mid ^ W f O B ^ solls)» SMT te

Iti^ w ^ h t tif the i t K ^ s(M ii^s^
aid dut ^ tliH^ w i ^ M of 1 9 mol^ soiL Assay of cellulase activity
(Hope and Burns 1987)

%3^1S^it^^nM^^
iNiitnot
ttie ac^^^^lb^

tNi^ if j ^ (Wti^oti^^ Idf IcM'^s^ ^E^« Itie

^a^TO?K««*.'';: A'-'^'": :r;? ^' ,5:;; ^-^^ ^ -

p(]f^^v::;>;;v^-;^j:; <

IjN^'fM'^^ o m l a M ^
0^dzici#

Enzyme activities 347

Cellulase activity
Reagents for mie determination of r^uoing
sugars: ^
Coppi^ regent:
Solution I
Dissotve 15 s Na-K4artamte and 30 g
NaaCOa in about 3(K) mi distifled wat^.
Then dissolve 20 g NaHCOs in the
sdiMon.
Glutton II
Dissolve 180 g Na2S04 in SOO ml
distilled water, bofl the soltrtion to
remove dJssoiv^ air. Allow the solution
to st^^d at room temperature for
OMling.
Glutton III
Mix ^fytions t and II, m^ diiute to 1(XK3
ml witti distilled water.
Solution W
Dissolve 5 g CUSO4JH2O aid 45 g
Na2i^4 in dlstliled wsri^ ami bring up to
2a) ml with distilled v^t^*
Solution V
ShorHy befc»B use, mix m ^ volume of
j^ytion IN arKi one vcriumeof solutk^n IVl
^senate^noVbdate ^ I U I ^ K I
Dissolve 2S g ammonlum-HKNybdate In
450 wi distilted wat^, mM 21 ml ttf
<K>r»c€aifrated H2SD4 wder oc^nuous
s M r ^ . FInidfy add 25 ml df
ls^HAs^4.7HaO (3 g 25 wT^ wmm) and
mixttiCHmighly*ABow the j^ution to
staiKl for 2 days at 37X^ aid flit In brown
bottle* ^ o r t ^ before use, dilute tmn
volume of this sK)lution with tim volume
ofH2»34(0J5*«|.
(Suoose-monohyck^ate staicteud ^ u t i ( ^
79 mg 1000 mP^ disUMed water.
f%5e 1 g of moist soil in an Blwrni^cr
fljs^ {25 mQ, mid S ml of acdbaite buff^ rnidi
348
03 g A\dlcel. Cap the flasks and incubate
for 16 h at 4XfC bi a shaking water bath«
Finally, stc^ the reaction by oentrihigatlon
i2S0Og, 10 min). To prepare thie control,
make the addlHon of Avicei after flie
hicubation period and immediately before
o^ntrifuga^^. All measurements should be
o a n i ^ out at least in duplicate, Detemiine
ttie reducing sugsrs in the suji^matanls
accxirding to the method of Nelson and
&>mog]^ (in Spiro 1966); pipette 1 ml of the
supernatant into test tubes, add 1 mi
solution % cap tiie tubes with glass balls
and boil in a water bath for 20 min. After
cooling, add 1 ml of the diluted arsenate-
molybdate solution and mix welL Rnatly
dilute the mixtiro with 3 ml of distilled
watar arui m^^ure ttie colour opttc^
der^i^ at 520 nm-
CalHbraltoii ctinre
See assay of cellulase activity.
C^ricmlaami
€km&ci the msjEu^r^fnents for tte blank
<^^K5C«^ ^ g g^^ dwt 16 h^^) =
Cx V
(7.15)
dwt
w h ^ ^ O Is fite m€^tswed s^ucc^^
icxmc^rtbalkm^ mr^ s^pematSHfi^, y Is
IN» vtriuHTie of tii^ mM si^p&nslCHfi Qn this
t^^s^mn, 5.5 ml) luid dwt ^ Urn dry i i ^ g h t d
1 g tmhA mil
Hm ^fizpf^tic reaoticm Is linear for 16 h
^^im and Burr^ 1987).
trK:^ti^1lkm of atril suspension with azide
pmm^ ttie asslmiterth>n of glucose by
sc^l mtoocHii^sms. ¥m tt^se re^^^ir^ it h
mm^&sH^ that inhtt^^:^ are u^^d 9*iope
Mid & M ^ 11^7). Sinc^ a2:kie ik>m mA
aiwa>s M i l t ^ miciobtal min^aiizaMon of
Chapter 7
 Assay of cellulase activity

ji^aM^t ciayfoim pm !effic^eiid^ of the

Enzyme activities 349

p-Glucosidase activity

p-Glucosidase K. Alef
activity R Nannipieri

P-Glucosidase (P-o-glucoside glucohydrolase,

EC 3.2.1.21) is the rate limiting enzyme in the Assay of the p-
microbial degradation of cellulose to glucose. glucosidase activity
The enzyme catalyses the hydrolysis of gluco-
sides according to the following reaction: (Tabatabai 1982; Eivazi and Tabatabai 1988)

Principle of the method

The metN>d i$ based on the deteimination
of the released p-nifropheool after ttie
Incubation of ^ 1 with j>nH:rophenyl
glucosrcte solution for 1 h at 37''C:
(7.16)

It has been detected in microorganisms, plants

7 2
and animals (Bahl and Agrawal 1972; Dey and
Pridham 1972; Wallenfels and Weil 1972). This
enzyme is included in the category of glucosi-
2 OH
dases that hydrolyse disaccharides. a-Gluco-
sidase (called maltase EC 3.2.1.20), which (7.17)
catalyses the hydrolysis of a-o-glucopyrano-
side, is also included among glucosidases.
Other glucosidases are a-galactosidase (EC
3.2.1.2) and ^-galactosidase (called lactase
EC 3.2.1). p-Glucosidase is more prominent Marlertals aiKl ^ p a r a b i s
in soil than a-giucosidase and a and p galac- S|^^:^bt:f^tomrt^
tosidases fTabatabai 1982). p-Nitrophenyl-p-o-
glucoside has been used as a substrate to Wekm ^hsi^m^ bath adjystable to 3 7 X
estimate p-glucosidase activity in soils. The Rltt^ pai^r (Whatmm 2v)
Km values ranged from 1.3 to 2.4 mM for this
substrate (Eivazi and Tabatabai 1988). The p-
glucosidase was inactivated at 70°C and shows Chemicals and eotutions
a significant correlation with the organic matter
of soil (Eivazi and Tabatabai 1988). Modffied univ^isai b u f f ^ ^ U B ) , pH 8.0
See ^sttimHcHi of

350 Chapter 7
 Assay of the p-glucosidase activity

; 1^iip€Hfahiif# ofj r o i^ i^ad lor t i ^ m^

'^:ph^a2j ami T a l ^ ^ ;-"^^'

wa$ii^:^e$say ft>rari4^c^6^t!^^ erf

IHittrc^rtiemit Irtmi sdBs*

fungi

Assay of p-glucosidase
activity
(Hoffmann and Dedeken 1965)

Enzyme activities 351

p-Glucosidase activity
9,6 wfth N ^ H (20%) arri dilute wWi
d i a l e d wati^ to 20(X) mL
^bromo quhione ohicmmlde sbhition
200 mg 100 mF^ ^teiol.
^b$t^t0 solution {2305 9 j^-glucc^kte-
Dissolve 2.3C^ g p^iucosicte-satignhi in
lalbo^ 85 m{ of dfetWed wat^ and bring
yp to tlK> ml with distilled wat^«
Saligiiin stamiard solution
0*1 mg mV^ disHBed water.
Trtuene
F^yocectare
V^^^o&JS g^^df itipist, alev^ 0 nun) k»l into a
lift^ismtrfc fl0k ( ^ mO, iKli 1 irt toluwe
lyid sdlow to stknd tofiS mmwi tts^
ti^f^Htoe* n ^ add tflfWof mibsfeate
biNt^jd^on iMMBUm o c ^ e r ^ pfWUml witii
^(ffstHed m^m, f^Mmoi0A^m^ Mm
idtbm^l^i^^ ot]fteiW<teJMifli^;
j ^ mto inl^siim V^ 0|^^ at 578
pfiif^i^rf^ iirt of
; 'Of 19^ $M^^^ ',:-"/,
352
scMution. M^isuremente ^ e cs^ed out In
Mplicate wHth one coMrot.
CaRbraUoii miive
Pipette 0,1» 3« S and 10 ml of ttie standard
soiution into vtHiimetric fla^ C^ ml)^ acfci
20 ml act^te buffer* 2 ml bor^e buff^, 0 ^
of dilHon^ qylnone diic^mlde sotutkHi,
and cMute to £@ mi wMi cKstilied wat^. After
^ mbi/mes^ijre the optica dcNnsity at 578
Caiculattoii
Ccm^ct for the control idx>A calculate briefly
SaBdn^fg^dwt3h^^)c;
(7,18)
dmK5
^i^ie^ O ^ ttm measured ^Icin
K:mmdtt^^kml^ irt^ ilbate), cM i$ ttia
diy ifi^eN^ <rf 1 0 mofet ^ ^ S9 fe tt^ totti
voitiitm ofjMe r^e^aiclkm ws^t^ md 5 M ttie
i i ^ i ^ ofj t t ^ i i ^ ^ ^ ^nains sl^ta
fi^^^i^se o f j ^ shc»t I r i ^ ^
tbe m^^md^"^^
Chapter 7
 Saccharase activity

Saccharase K. Alef
activity R Nannipieri

Maltose, lactose and sucrose are the most the ratio saccharase to amylase activity was
common water-soluble disaccharides, which greatest in the less developed soils (Ross
consist of two monosaccharides joined by a 1975).
glycosidic linkage. One of the most abundant
sugars in plants is sucrose, a disaccharide of
glucose and fructose [O-p-o-fructofuranosyl- Assay of saccharase
(2,1)-a-D-glucopyranoside]. Unlike most dis-
accharides and oligosaccharides, sucrose activity
contains no free anomeric carbon atoms. For
(Schinner and Von Mersi 1990)
this reason, sucrose does not undergo mut-
arotation and does not act as a reducing
sugar. It is much more readily hydrolysed
than other disaccharides. IHtiolpte af file iiiettioit
Saccharase or invertase (p-o-fructofurano-
side fructohydrolase EC 3.2.1.26) catalyses of iB^sed riendltidiigi sugars ^ w t ^
the hydrolysis of sucrose to o-glucose and D-
fructose, and is widely distributed in micro-
organisms, animals and plants. In soil it has
optimum activity at pH 5.0-5.6 and tempera-
ture of 50''C (Roberge 1978; Frankenberger
and Johanson 1983c). The Km values of sac- See ^thtiaOoii ts/t i^eMy^g^ acjtf^^
charase in soils have been found to range from
16.3 to 42.1 mM (Frankenberger and Johanson
1983c).
ChiHTiidk^ts and w/^Okxm :
Air-drying decreases saccharase activity of
soil (Hoffmann 1959; Ross 1965, 1968; Fran-
kenberger and Johanson 1983b), while activity
is very stable at 4°C and decreased with soil
depth (Hoffman and Elias-Azar 1965; Dutzler-
Franz 1977; Holz 1986a, 1986b). Toluene and Dissolve 12 9 i^f ^K^m^ to i ^ s ^ e
sterilization with gamma rays had no influence fadl^« sttr at 45"^ 1 ^ 2 h m^ dtiite to
on the enzyme activity (Voets et al 1965; 1CXJO till with acetate tH4ff^;TMi^utta«i
Roberge 1978). Hoffmann and Pfitscher <aBi t ^ stored fm% w e ^ at 4^*0*
(1982) found significant correlations between
saccharase activity and the organic C in soil. In B^^a^erds fc^ ttie iM^rmf^^
contrast, Dutzler-Franz (1977) found no signif- radudr^ sugars:
icant correlations (n = 54).
Saccharase activity in soil was the only car-
bohydratase activity significantly correlated See ^matlcm of iMMmB adlh%.
with the carbohydrate:lignin ratio of added Ri^entB
plant material (Rice and Mollik 1977). In a
climosequence of soils in tussock grassland. See ^timsAlcHi of cdtuie^ ^ ^ ^ 0 ^ .

Enzyme activities 353

Saccharase activity

mQ, #te tto iniitkm f£^:tor jN^mi 40), S is tf^

w ^ ^ t of tt^im^i^ tised in gr^^f^^^td
See i^fi^Mcm 0f cM* Js^Jte cfcy y ^ h t of 1 9 of mafet acMl

erf actfvOy.
V^ ii^x»wni^ t l ^ method by & ^
m^ Von l^ltersl (1^0) for tt^ cfeb^mlnaUon
of C^Oc^iyiaee ^ : t i \ % In sc^l, beoat^ it
Pl€»^ $Qci nf^iet wme4 ecM j ^ mii$ In an tt^ metiKKt p i ^ ^ Frar4i^rrib€»g^
B^mtm^ f ^ k (icm m^t iK:lkl IS rM ctf
micros^ scdtytiofi iml 15 flit ctf aciN^M^
buffet stor^er the ftestc amifcioytoi*e1m 3
tl at SO^'C. After the iiK^bation Mm the
fie^iWnS aoll $ij^|>^^skm; tii ^ ocKittol, the Assay of saccharase
mri^fin^e <1S M irf siK:^i^ adupoui) is
adti^ at tiia ^ of tfie tF^4:)iti^ activity
tmiwlted^ t><#Dro of (Hoffmann and Pallauf 1965)
tt^ filial^ to ^ frit ^
Itnlofi

'.?Vv^^^/^-^ft / ,'^

v*"'"'' ' -' '•• " " • • ' • ' ' '

1 ^ e^Hnf^M df

arKi ca!(^ate ^^ccmilm*^ the l^oiMr^

^3fc«:^^iig S"^^*^ 24:W^'k ;?:^'!'^''5'\' ^

Bxdm " ^^-'".i' '

^ttie
rttie
¥^me^dfttiB

354 Chapter 7

fMiCO^^mim^^ arid 2QQ g ^02804
Iji^w #0^^ oipoitm^ and
;Mb(%^ of BiriySon 1 syiHd 25
\r^^,^mMMm^ms^mmiwf3^mui Mr^ up \^Mt
ISmiftThen
of
Enzyme activities
Assay of saccharase activity
theflItrsrUcm,A sea)nd ocmferod wthout soil
stouid be chanted out, ft is r^c^c^iimmd^
0mt ^1 mm^mjmtmi^ mm i^^ pvkin
Mplicate. Finite S mlflltiBlBMo a
volumeMo fl^k C1CX) ii#, 1 ^ 4 mi <rf
€^1:^3^ r^gfs^nt arKi bcril bi a waf^ batfn for
25 mln. Aft^ aKrfing, ^iM 2 ml of N%HK>4
solution and 5 ml of mol^^Ktete ^iutfon 3,
mbittioroyghty,ami allow io stafKl for 60
ftm. RnaJiy, dtlute to 100 ml with cSsflried
¥^m ami alter 15 mln ms^amMm the optroari
demlty at 578 nm.
Calibratioii mirve
Pipem 0 , 5 , 1 0 , 1 5 BM 20 mt of ttie
standard ^ohittcm into ^umeMc fl^ks
(100 m% adkl 10 ml c^ lEsceMataiflPsraiid
tSkAB to fWm¥^!(MKMm
C^tcHfrntoe ttm i«cl^
ttfiai sugar ocmoantoaaon^ i ^ (^ 1^, 1CK);
«200|lslrt:^\;;;^^
C?^)
lA^
MttiM y g ^ s ^ ^yi^ «%fN^ li^
355
Xylanase activity

Xylanase activity^ K. Alef

(Schinner and Von Mersi 1990) R Nannipieri

Xylans, plant homopolysaccharides, consist- iBducin9 sugars

ing of xylose, arabinose, glucose, mannose
and galactose, can be degraded and used as
Recent A
carbon sources by soil microorganisms. Xyla-
nases (3.2.1.8 xylan 4-xylanhydrolase) include See ^^tirrmtiofi of celluiai^ acfiv%.
endoxylanases, which catalyse the hydrolysis
Reagent B
of xylans to oligosaccharides, and exoxyla-
nases, which catalyse the hydrolysis of oligo- See i^timatioii of c^Uase activity.
saccharides to reduced monomers. Like the
R^ient C
cellulases, the estimation of xylanase activity
in soils is based on the determination of redu- See astfrmtkm of oeRiiia^ idttvH^.
cing sugars (Hashimoto et al 1971; Schinner
dliK^o^ fiKmc%dlmi0 soltitt^ '
and Von Mersi 1990). The pH optimum of
xylanase activity has been shown to range l^e ^^kimtkm of oMtute^E^ activH^^^
from 5 to 7, while its temperature optimum is
generally around 50°C (Schinner and von Mersi
Pm&^k^m ^ '
1990).

bsth t l O O ^ for ^ c i c % | S ^ ^ ^

i!fypsM&A out hi jbf|pflaile«

'^ns n'"^ ';-*,"' •

Soe (KS^tttiiAon of

356 Chapter 7
 Xylanase activity

of ^^dchK^r^ m^aiB is
ftcNTttiet^ntt)! ^ IsH of j ^ soiutiG^ )ia^ <o be
l%Nr ttiiai 10.5 ^ a r aiding r^^gfwte A
^THJ B, and Ic^^ar than 2
MM:^:^^^ Q^^ dvirt 2 4 h"^^) ^

'MXVkf:
w^n #10^1^ a n^giatMi ocmiriteWon w i ^ soR
lycftrpQCNn^^NB^ pnc^l^^i^ and
^a«*Mty*
High ocHfio<^traUcms of Sjsdte» anitKmiuiii

oftha ftic^idie^ dlsbj^ the mdiK^i^ i

0 « | ^ 19^ Iflfl^ ^ ttie

# o h M ^ c^^ be p^HffcM^ t ^ file

Enzyme activities 357

Lipase activity

Lipase activity K. Alef

(Cooper and Morgan 1981, modified by Schinner et al 1991) R Nannipieri

Lipids are a heterogeneous group of com-

pounds, having in common the property of
insolubility in water but solubility in non-polar P^ftiH^le D^flmmetficHl
solvents such as chloroform, hydrocarbons or
alcohols. They rarely exist in an organism in Hm ffimmmmmii of ttie fliK:Nri^sceiit
the "free" state but are typically combined with 4^etti^ ymbeltlfemna (4-MU) i^eased
proteins or carbohydrates as lipoproteins or
Idft^ VhelmiA^m of soN mth 4zff¥^t0
lipopolysaccharides (see Chapter 9). A signifi-
cant proportion of lipids enter soil in the form
of triacylglycerols, the primary storage fat in
plant and animal tissue. Therefore the initial
degradation step involves the enzyme lipase
(triacylglycerol acylhydrolase, EC 3.1.1.3),
with the liberation of fatty acids and glycerol.
The reaction can also stop at the monoglycer-
ide stage (Desnuelle 1972).
Fatty acids are long chain carboxylic acids.
The parent molecules are the long, straight-
chain saturated acids, but there may be
many modifications or substitutions in the
chain that produce branched, unsaturated,
hydroxy-, keto-, epoxy- or cyclic acids. The 4 ^ " ' " ' ^ '""
most abundant natural fatty acids are the cis-
monounsaturated or polyunsaturated deriva-
tives. Under aerobic conditions, fatty acids
are degraded by a stepwise cleavage of 2-
carbon fragments (p-oxidation) with final pro-
duction of CO2 and H2O; acetyl-CoA produced
by this type of degradation is further metabo-
lized to yield ATP (Mahler and Cordes 1971).
Other oxidation mechanisms (a-oxidation, a-
oxidation) do not completely break down the
fatty acid molecule, but result in the formation
of oxygenated or dicarboxylic acids. Under
anaerobic conditions the fatty acids are often
accumulated in the environment. Several
methods are available to estimate the lipase
activity in soils (Pokorna 1964; Pancholy and ^^^^$y^, 'm^^^
Lynd 1972; Cooper and Morgan 1981).
Because of the short incubation time, the
method of Cooper and Morgan (1981), modi-
fied by Schinner et al (1991), is presented and
discussed.

358 Chapter 7
 Lipase activity

: - . - ; ; ! - F : : - - = . .r- • • ' . • ' • •

dfc '<A^iK ^^IkJi^^S w^te^afedMk ,JahJKdM^.^ik S L l k J ^ "^ . ^

r^N-spilSKiitneiaR •:•

^^0^^

Enzyme activities 359

Chitinase activity

Chitinase activity K. Alef

(Rodriguez-Kabana et al 1983, modified by Rossner 1991) R Nannipieri

Chitin, a homopolymer of N-acetyl-glucosa-

mine in P(1,4) linkages is the major organic
element in the exoskeleton of insects, crus-
taceans, and many species of fungi and other Chith suspar^fon (5% w M
organisms. Enzymatic hydrolysis of chitin to
15 g of chWn me mmea wWi 2 ( » iiil of
acetyl glucosamine is mediated by two hydro-
cono^itrated HCi and sttnrad im 3 h at
lases, chitinase (EC 3.2.1.14) and chitobiase.
mom t^tipemture. T 1 ^ susfwdacl^
Chitinases are common in nature and are
cWBn Is tl^n fHt^Bdl thimish Itisi ;H':
produced by bacteria, fungi, plants and the
digestive glands of animals that consume
chitin-containing materials. In plants this
enzyme is induced and accumulated in
response to microbial infections (Boiler 1985).
Chitinase released by rhizobacteria is believed sypeitialiirit ^ Ifi^fi f i ^ ^
to play an active role in the biological control of
plant pathogenic fungi. Soil chitinase activity
decreases with increasing soil depth. Signifi-
cant correlation between chitinase activity and
nitrogen content has also been found (Veon et
al 1990).

1Y^ m^tiNi m imma mVm kK^ft^cm of

Miflb^is ate ai^ratim

C^trtftiin^ and c^Mft^Mfem tubes
C^^fylost <«a^^ tube

f^^^^jff^^^ t ^ l # ( t m ^ ^CK^ TOium)

Si»ari<6r

360 Chapter 7

4-Pfn0tt^ rnnirmybmm ahMiyde stock
sdutkHi ^MW^
G^^oim to g DMBA In a mixtym of B7J&
fi4 of a^oentrntecl aoetto acid m^ 12.5
M wm^GT^lmted HOI and ^ a « at 4^C.
MHx one ti^iymt of DMBA stodk soiutiCHi
\iMh f o r vcrtymes of ocmo^tr^ed s^^^o
wM. Ptapam a fr€^ scrfiMon daily.
soliitlcm (45 mu)
Dlssohro 0 ^ g of l^acet^ ghicosamnino
ki ^ ill) of disfltled iiyater afid taing up
to 50 rrt* f^ie^^HTO a
^ ^ 9 ^ ; ^ JO g of imrist ^ In an ^temnN^^
1 ^ PK^ 5 ii4 of {:^K9q[4i^ite tnifl^.
iOl5iWirftt^#Wn
1 aft 5 ^ m ^(M^n SO iMfi* M
nr^ist \m canted out at i^^t ki
Enzyme activities
Chitinase activity
{^pette 0,0«S, 1.0 and 2 ^ ml of the
N - a c ^ l gluoc^amine ^ i ^ i c m Mo
wlyirii^'c flasl^ p 3 mQ, i^fd 12.5 iM of
{:riK^(Aate i^iff^^ 25 ml of KOI soiuMon and
taing up vi^h di^Hled water to 1 ^ ml.
PlF^tte 0«5 ml of eadhi aoltjMon Into g l ^ ^
taA>i^ and p6ifmm ttie deton^mtfon erf H-
m^^ gliicc^^mrrtne las d e s ^ b ^ above.
11^
iN^aoetyl giuco^mlr^ conb^ritratfof^ In ttie
caittH^on curve are 0,50, KX), 150,200
and 250 |jig ml^^
Oofft^ tt^ itefta 1c^ 9ie bl^f^ aiKi c a l o ^ ^
gdwt:"^ieh:^^)«
1?^
if4we 0 % tt^ i m ^ g ^ j ^ l^N^^yl
v% 11^ f M ^ w i e iN^
% #¥^ cfcy w ^ g ^ €# 1 f ^mmCw^
t4ai^24}i
€fc:^iAc ^ a i m c M c a ^ erf tt^ i^solbed
1 ^ Relsas^ M €i|1955). ^ ^^ ^^^^^ ^^^^^7
t o i k y ^ W M M : ^ mNm^:^ a d ^ ^
361
Catalase activity

Catalase activity K. Alef

(Beck 1971) R Nannipieri

The enzyme catalase (hydrogen peroxidase
oxidoreductase, EC 1.11.1.6) has a detoxify-
ing function in cells by catalysing the following
reaction:
H202-^ H2O+JO2 (7.24)
Induced hydrogen peroxide is poisonous for
cells, oxidizing the SH-groups of proteins.
Partial reduction products of oxygen like the
superoxide anion (62)" and hydrogen peroxide
may be formed during electron transport to
molecular oxygen via the aerobic respiration,
as well as in various hydroxylation and oxy-
genation reactions. These products are extre-
mely reactive and capable of irreversibly
damaging various biomolecules.
Aerobic cells generally contain the enzyme
superoxide dismutase, which converts super-
oxide into hydrogen peroxide and molecular
oxygen:
anaerobic bacteria. Catalase was the first
enzyme studied in soil (Konig et al 1906,
1907; May and Gile 1909). Its estimation is
based on the determination of released O2
(Weetall et al 1965; Beck 1971; Kuprevich
and Shcherbakova 1971a; Trevors 1984; Holz
1986b). Catalase activity is detected in plant
and animal ceHs. This interferes with the esti-
mation of microbial catalase activity in soil
(Beck 1971; Ladd 1978). In addition, Mn and
Fe, as well as organic substances in soils
catalyse the liberation of O2 from H2O2
(Baeyens and Livens 1936; Beck 1971;
Kuprevich and Shcherbakova 1971b). Cata-
lase activity is very stable in soil. It shows
significant correlation with the content of
organic carbon and decreases with the soil
depth (Beck 1971; Ladd 1978). No relation
has been found between the catalase activity
and soil fertility (Skujins 1978).

2O2" + 2H^ -> HoOp + O- (7.25)

Enzyme systems like the L- and o-amino-acid
oxidases contain tightly bound FMN or FAD as
prosthetic groups. These enzymes catalyse
the oxidative deamination of amino acids:
L-Amino acid + H2O + E-FMN ->
a-Keto acid + NH3 + E-FMNH2 (7.26)

D-Amino acid + H2O + E-FAD -^

a-Keto acid + NH3 + E-FADH2 (7.27)
The reduced forms of the L- and o-amino
acid oxidases can be reoxidized directly with
molecular oxygen to form hydrogen peroxide:
E-FMNH2 + 0 2 - ^ E-FMN + H2O2 (7.28)

E-FADH2 + 0 2 ^ E-FAD + H2O2 (7.29)

All aerobic and most of the facultative anaero-
bic bacteria contain catalase activity. This
enzyme has not been detected in obligate

362 Chapter 7
 Catalase activity

Fcr t i ^ daterminatiori d o x ^ t ^ fwmedt

a f p M to |M16.8 vritti H a lastd dilule to
1000 rrt with dlstllteci
&)«lhmi cartKmtfte sedation (tO%)
I^^^D0 OJIg of M i ^ ie»id 20 ml of sodium
cmtom^B BoiiAkm (10%) instead of $cril in
mi Brlmm^y^ flask, i»dd 10 mi ctf H2Q2
(3%) ami i B ^ ttm dNar^0 In Urn gas
voiim^ in ttm Scheibler appa^tus (c^*
U^glass tube). The o)q^gan formed Is
s the 100%.

tolCWOml X 100 (7.30)

DmXdwt
;\5*¥^|SI^.. wh^e 0^ ^ t t e Oa dev^ped bi t t o
'\,^:: irfbS6^f)€^ of a23de ID mBRtilr^, DA IS tt^ O2
dm^^A^pt^ Iri t t e p^a^^fK^ of aiJcl^ m
i:\u^fes^the O2 c ^ ^ k ^ p ^ to ttie
^of Mn ill miBHttres and cAi^ b ttie

^f.

j ^ n ^ i e ^ bean 4 i i ^ to d a k ^ t a jtti^
^ i « 6 ^ jMyO^ t ^ ^ t J M ^ $^K^ 1871;^ UM

i; j ^ 11^ aMI f ^ 2£k ii4 of

,''-'-'-'' ' \."';. / ,''-"' 'IS-'^^-'/'

\ can elttN^ M M t cr a ^ ^ e the

Enzynfie activities 363

Arylsulphatase activity

Arylsulphatase
activity K. Alef
(Tabatabai and Bremner 1970a) P. Nannipieri

Sulphatases catalyse the hydrolysis of organic present in the form of organic sulphates
sulphate esters and have been classified (Tabatabai 1982). However, Jarvis et al (1987)
according to the type of the ester in arylsul- found no correlation between arylsulphatase
phatases: alkylsulphatases, steroid sulpha- activity and sulphur mineralization. Another
tases, glucosulphatases, chondrosulphatases problem may develop because p-nitrophenyl
and myrosulphatases (Tabatabai 1982). sulphate, which was probably used as the sub-
Arylsulphatase (EC 3.1.6.1) catalyses the strate, does not occur in nature.
in'eversible reaction: Fitzgerald et al (1985) expressed doubts
about the importance of the p-nitrophenyl
R.OSOa" + H2O -> R.OH + H^ + (804)^" sulphate dependent-activities in soils and
(7.31) suggested the use of p^SJtyrosine sulphate
and has been detected in microorganisms, as a substrate.
plants and animals (Nicholls and Roy 1971).
The enzyme also catalyses the hydrolysis of
p-nitrophenyl sulphate, potassium phenyl sul-
phate, potassium nitrocatechol sulphate and f^mfrib of fte iiiettiod
potassium phenolphthalein sulphate.
The Km values were found to range from 0.2
of p-rttot$*^Kril mlaasad Bi^isk th^
to 0.95 mM, when p-nitrophenyl sulphate was
used as the substrate, and were dependent on si^^i^iafe fcr 1 h at 37^.
the soil type (Perucci and Scarponi 1984).
Arylsulphatase activity decreased with soil
depth and showed significant congelations Msti^ki aiKi Bi^fmnslw
with the content of soil organic carbon, total ^^h\
S9# esttrm^ttw #f phc^syrticNnrmK^^
nitrogen and cation exchange capacity (Taba-
tabai and Bremner 1970b; King and Klug 1980;
Appiah and Ahenkorah 1989). Inorganic sul- C^tiiNiiik^ls and s<AittDiisi ;v ;
phur [(S04)^~, S(IV) and S(VI)] inhibit arylsupha-
tase activity of soil (Dodgson and Rose 1976;
Fitzgerald 1976; Jarvis et al 1987). Strong
inhibition has also been detected with I3^i^oiy0 ^ 9 of sodltm a ^ t a ^
(P04)^", (As04)^", (Mo04)^" and (^NO^f' but
not with NOsT, NOi" and CI" (Al-Khafaji and
Tabatabai 1979; Tabatabai and Bremner
1970a). The role of arylsulphatase in sulphur
mineralization in soils is not clear. The impor-
tance of this enzyme activity in the sulphur
mineralization is derived from the finding that
most of the total S found in surface soils is

364 Chapter 7
 Arylsulphatase activity

buffer and ttm soliAlon to

SO ml wth biiff^«
CaCfe (0-S u) See ^Umation of pho^homcmi^sterase
See asttmaMm of acUvity,

NaOH(Q<SM)
p-Nitro{:rfierK>l stsoidard solution
Dissolve 1.0 9 p^*ro|]*ienol in «4H>ut 70
mi dotted w a l ^ mv:i mtM to 1(KX} ml
Stwe at 4^C.
f ^ c e 1 9 of m^i m&>ifed C2 mm) soil m
B^tm^^mk (50 m^, add 0.25 ml
t(Ai^r^ 4 ml iMf aoeiHte iHjffeHr, 1 ml of
IM4l^i^^Km^ milfMte solirtlon aiKJ rmc tt^
ocMfMtoMs^ tk^ the IMtshB ami tfioAiate fc^
:400rmTo
. fiiial<^ ttw ^ ^ M c p of
Atr^diyir^ iBudly increased ttie
ay^!|34iatase aottvlty (Tal^at^af ami
Bremn^ 1970a^«
it tia^ be^i observed that ac^itate biiff^
gives higher activities than other types of
bAiffms (Tabatabal 1982),
The i^t^sfrate c^mi^cNni^tion In the assay Is
eqiat to 5 ptm; tHmmm^ in ^ i s wMi higher
efizyiiM activitt^, NghcH* |>nHiqphefiyi
s i i l r ^ e oom^ntoatk^^ sNKitd be ysed
{Titea^Ai£» i p d & e i m i ^ 197Cltt;
S^naitK^tandm m^ Fenott 1981).
T t ^ iNWWcm of tohiei^ (0*1-^1*0 «3f^
Mi^Njr §mi R a p ^ i t (197?) a ^

of {:^K^{4KmioiK^ers^e

Enzyme activities 365

References
References
Agrawal KML, Bahl OP (1968) Giucosidase of
Phaseolus vulgaris. J Biol Chem 234: 103-111.
Alef K, Kleiner D (1986) Arginine ammonlfication in
soil samples. Veroff Landwirtsch-Chem Bunde-
sanstalt Linz/Donau 18: 163-168.
Alef K, Beck Th, Zelles L, Kleiner D (1988) A conn-
parison of methods to estimate microbial biomass
and N-mlneralization in agricultural and grassland
soil. Soil Biol Biochem 20: 561-565.
Alexander M (1977) Soil Microbiology. Wiley, New
York.
Al-Khafaji AA, Tabatabai MA (1979) Effects of trace
elements on arylsulfatase activity in soils. Soil Sci
127: 129-133.
Appiah MR, Ahenkorah Y (1989) Arylsulphatase
activity of different latosol soils of Ghana
cropped to cocoa {Theobroma cacao) and coffee
(Coffea conephora var. robusta). Biol Fertil Soils 7:
186-190.
Appiah MR, Halm BJ, Ahenkorah Y (1985) Phos-
phatase activity of soil as affected by cocoa pod
ash. Soil Biol Biochem 17: 823-826.
Baeyens J, Livens J (1936) Catalytic power of a soil
and fertility. Agricoltura 30: 145-155.
Bahl OP, Agrawal KML (1972) Alfa-Galactosidase,
and p-glucosidase, and p-N-acetylglucosa-
mlnldase from Aspergillus r\iger. In: Methods in
Enzymology, vol 28. Ginsburg V (ed.). Academic
Press, New York, pp. 728-734.
Beaton JD, Hubbard WA, Speer RC (1967) Coated
urea, thiourea, urea, urea-fomialdehyde, hex-
amine, oxamide, glycoluril, and oxidized nitrogen-
enriched coal as slowly available sources of
nitrogen for orchardgrass. Agron J 59: 127-133.
Beck Th (1971) Die Messung der Katalaseaktivitat
von Boden. Z Pflanzenernahr Bodenkd 130:
68-81.
Beck Th (1973) Uber die Eignung von Modellversu-
chen bei der Messung der biologischen Aktivitat
von Boden. Bayer Landw Jb 50: 270-288.
Beck Th (1984a) Mikrobiologische und bio-
chemische Charakterisierung landwirtschaflich
genutzter Boden. I. Mitteilung: Die Ermittlung
einer bodenmikrobiologischen Kennzahl. Z
Pflanzenernahr Bodenkd 147: 456-466.
Beck Th (1984b) Mikrobiologische und biochem-
ische Charakterisierung landwirtschaflich genutz-
ter Boden. II. Mitteilung: Beziehung zum
Humusgehalt. Z Pflanzenernahr Bodenkd 147:
467-475.
Beck Th (1986) Aussagekraft und Bedeutung enzy-
matischer und mikrobiologischer Methoden bei
der Charakterisierung des Bodenlebens von land-
366
wirtschaflichen Boden. Veroff Landwirtsch-chem
Bundesanstalt Linz/Donau 18: 75-100.
Beck Th, Poschenrleder H (1963) Experiments on
the effect of toluene on the soil microflora. Plant
Soil 18: 346-357.
Benefield CB (1971) A rapid method for measuring
celluiase activity in soils. Soil Biol Biochem 3:
325-329.
Bidwell RGS (1974) Plant Physiology. Macmillan,
NewYork, pp. 173-206.
Blakeiey RL, Zerner B (1984) Jack bean urease: the
first nickel enzyme. J Mol Catal 23: 263-292.
Boiler T (1985) Induction of hydrolases as a defense
reaction against pathogens. IN: Cellular and
Molecular Biology of Plant Stress. Key JL,
Kosuge T (eds). Liss, New York, pp. 247-262.
Bonmati M, Pujola M, Sana J, Soliva M, Felipo MT,
Garau M, Ceccanti B, Nannipieri P (1985) Chem-
ical properties, populations of nitrite oxidizers,
urease and phosphatase activities in sewage
sludge amended soils. Plant Soil 84: 79-91.
Boyd SA, Mortland MM (1990) Enzyme interactions
with clays and clay-organic matter complexes. In:
Soil Biochemistry, vol 6. Bollag J-M, Stotzky G
(eds). Dekker, New York, pp. 1-28.
Bray HG, James SP, Raffan IM, Ryman BE, Thorpe
WV (1949a) The fate of certain organic acids and
amides in the rabbit. Biochem J 44: 618-625.
Bray HG, James SP, Raffan IM, Thorpe WV (1949b)
The enzymic hydrolysis of glutamine and Its
spontaneous decomposition in buffer solutions.
Biochem J 44: 625-627.
Bremner JM (1955) Studies on soil humic acids: I.
The chemical nature of humic nitrogen. J Agric
Sci 46: 247-256.
Bremner JM (1965a) Inorganic forms of nitrogen. In:
Methods of Soil Analysis, Part 2. Black CA et al
(eds). Agronomy 9. American Society of Agronomy,
Madison, Wl. pp. 1179-1237.
Bremner JM (1965b) Organic nitrogen in soils. In:
Soil Nitrogen. Bartholomew WV, Black CA, Evans
DD, Ersminger LE, White JL, Clark FE (eds).
Agronomy 10. American Society of Agronomy,
Madison, Wl, pp. 93-132.
Bremner JM (1967) Nitrogenous compounds. In:
Soil Bochemlstry. McLaren AD, Peterson GH
(eds). Dekker, New York, pp. 19-66.
Bremner JM, Edwards AP (1965) Determination and
isotope-analysis of different forms of nitrogen in
soils: I. Apparatus and procedure for distillation
and determination of ammonium. Soil Sci Soc Am
Proc 29: 504-507.
Bremner JM, Keeney DR (1966) Determination and
isotope-ratio analysis of different forms of nitro-
Chapter 7

gen in soils. 3. Exchangable ammonium, nitrate
and nitrite by extraction-distillation methods. Soil
Sci Soc Am Proc 30: 577-582.
Bremner JM, Mulvaney RL (1978) Urease activity in
soils. In: Soil Enzymes. Bums RG (ed.). Academic
Press, New York, pp. 149-196.
Browman MG, Tabatabai MA (1978) Phosphodi-
esterase activity of soils. Soil Sci Soc Am J 42:
284-290.
Brown PR, Smyth MJ, Clarice PH, Rosemeyer MA
(1973) The subunit structure of the aliphatic
amidase from Pseudomonas aeruginosa. Eur J
Biochem 34: 177-187.
Burns RG (1978) Enzyme activity in soil, some theo-
retical and practical consideration. In: Soil
Enzymes. Burns RG (ed.). Academic Press, New
York, London, pp. 73-75.
Bums RG (1982) Enzyme activity in soil: Location
and possible role in microbial ecology. Soil Biol
Biochem 14: 423-427.
Bums RG (1986) Interaction of enzymes with soil
minerals and organic colloids, in: Interactions of
Soil Minerals with Natural Organics and Microbes.
Huang PM, Schnitzer M (eds). Soil Science
Society of America, Special Publication Number
17, Madison, Wl, pp. 429-451.
Burns RG, Pukite AH, McLaren AD (1972) Concern-
ing the location and persistence of soil urease.
Soil Sci Soc Am Proc 36: 308-315.
Butler LG (1971) Yeast and other inorganic pyro-
phosphatases. In: The Enzymes, vol 4. Boyer PD
(ed.). Academic Press, New Yoric, pp. 529-541.
Cai GX, Freney JR, Muiriiead WA, Simpson JR,
Chen DL, Trevitt ACF (1989) The evaluation of
urease inhibitors to improve the efficiency of
urea as N-source for flooded rice. Soil Biol
Biochem 21: 137-145.
Cervelli S, Nannipieri P, Ceccanti B, Sequi P (1973)
Michaeles constant of soil acid phosphatase. Soil
Biol Biochem 5: 841-645.
Chonkar PK, Tarafdar JC (1981) Characteristics and
location of phosphatases in soil-plant systems. J
Indian Soc Soil Sci 29: 215-219.
Claris AE, Stone BA (1965) Properties of a p-1,4-
glucan hydrolase from Aspergillus niger. Biochem Edibacher S, Kraus J, Scheurich N (1930) A com-
J 96: 802-807.
Clarke PH (1970) The aliphatic amidases of Pseu-
domonas aeruginosa. Adv Microb Physiol 4:
179-222.
Cochran VL, Elliott LP, Lewis CE (1989) Soil micro-
biol biomass and enzyme activity in subarctic
agricultural and forest soils. Biol Fertil Soils 7:
283-288.
Cooper AB, Morgan HW (1981) Improved fluoro-
metric method to assay for soil lipase activity.
Soil Biol Biochem 13: 307-311.
Cosgrove DJ (1977) Microbial transformations in the
phosphorous cycle. Adv Microb Ecol 1: 95-134.
Desnuelle P (1972) The lipase. In: The Enzymes.
Enzyme activities
References
Bayer PC (ed.). Academic Press, London, pp.
575-615.
Dey PM, Pridham JB (1972) Biochemistry of alfa-
galactosidases. In: Advances in Enzymology, vol
36. Meister A (ed.). Wiley, New Yori<, pp. 91-130.
Dick WA, Tabatal MA (1977) Determination of ortho-
phosphate in aqueous solutions containing labile
organic and inorganic phosphorus compounds. J.
Environ Qual 6: 82-85.
Dick WA, Tabatabai MA (1978) Inorganic pyro-
phosphatase activity of soils. Soil Biol Biochem
10: 59-65.
Dick WA, Tabatabai MA (1983) Effects of soils on
acid phosphatases and inorganic pyrophospha-
tase of corn roots. Soil Sci 136: 19-25.
Dick WA, Tabatabai MA (1984) Kinetic parameters
of phosphatases in soils and organic waste
materials. Soil Sci 137: 7-15.
Dodgson KS, Rose FA (1976) Sulfohydrolases. In:
Metatx)lism of Sulfur Compounds, vol 7, Meta-
bolic Pathways. Greenberg DM (ed.). Academic
Press, New Yoric, pp. 359-431.
Doelman P, Haanstra L (1986) Short- and long-term
effects of heavy metals on urease activity in soils.
Biol Fertil Soils 2: 213-218.
Doelman P, Haanstra L (1989) Short- and long-term
effects of heavy metals on phosphatase activity in
soils: an ecological dose-response model
approach. Biol Fertil Soils 8: 235-241.
Douglas LA, Bremner JM (1970) Extraction and
colorimetric determination of urea. Soil Sci Soc
Am Proc 34: 859-868.
Douglas LA, Bremner JM (1971) A rapid method of
evaluating different compounds as inhibitors of
urease in soils. Soil Biol Biochem 3: 309-315.
Drobnl'k J (1956) Degradation of asparagine by the
soil enzyme complex. Cesk Mikrobiol 1: 47.
Dutzler-Franz G (1977) Einflup einiger chemischer
und physikalischer Bodenmerkmale auf die
Enzymaktivitat verschiedener Bodentypen. Z
Pflanzenemahr Bodenkd 140: 329-350.
Edibacher S (1926) A communication on intermedi-
ates of histidine metabolism. Part I. Z Physiol
Chem157: 106-114.
munication on intermediates of histidine metabo-
lism. Part II. Z Physiol Chem 191: 225-242.
Eivazi F, Tabatabai MA (1977) Phosphatases in soils.
Soil Biol Biochem 9: 167-172.
Eivazi F, Tabatabai MA (1988) Glucosidases and
galactosidases in soils. Soil Biol Biochem 20:
601-606.
Eriksson KE, Wood TM (1985) Biodegradation of
cellulose, in: Biosynthesis and Biodegradation of
Wood Components. HiguchI T (ed.). Academic
Press, London, pp. 469-503.
Fenn LB, Tipton JL, Tatum G (1992) Urease activity
in two cultivated and non-cultivated arid soils.
Biol Fertil Soils 13: 152-154.
367
References
Fillery IRR Simpson JR, De Datta SK (1984) Influ-
ence of field environment and fertilizer manage-
ment on ammonia loss from flooded rice. Soil Sci
Soc Am J 48: 914-920.
Fillery IRR De Datta SK, Craswell ET (1986) Effect of
phenyl phosphorodiamidate on the fate of urea
applied to wetland rice fields. Fertilizer Res 9:
251-263.
Fitzgerald JW (1976) Sulfate ester fonnation and
hydrolysis: a potentially important yet often
Ignored aspect of the sulfur cycle of aerobic
soils. Bacteriol Rev 40: 698-721.
Fitzgerald JW, Watwood ME, Rose FA (1985) Forest
floor and soil arylsulphatase: hydrolysis of tyrosin
sulphate, an environmental relevant substrate for
the enzyme. Soil Biol Biochem 17: 885-887.
Florkin M, Stotz EH (1964) Comprehensive Bio-
chemistry, 13. Elsevier, Amsterdam, pp. 126-134.
Frankenberger WT Jr (1983) Kinetic properties of
L-histidine ammonia-lyase activity in soils. Soil
Sci Soc Am J 47: 71-74.
Frankenberger WT Jr, Johanson JB (1982) L-Histi-
dine ammonia-lyase activity in soils. Soil Sci Soc
Am J 46: 943-948.
Frankenberger WT Jr, Johanson JB (1983a) Distribu-
tion of L-histidine ammonia-lyase activity in soils.
Soil Sci Soc Am J 136: 347-353.
Frankenberger WT, Johanson JB (1983b) Method of
measuring invertase activity of soils. Plant Soil 74:
301-311.
Frankenberger WT, Johanson JB (1983c) Factors
affecting invertase activity in soils. Plant Soil 74:
313-323.
Frankenberger JR, Johanson JB (1986) Use of
piasmolytic agents and antiseptics in soil
enzyme assays. Soil Biol Biochem 18: 209-213.
Frankenberger WT Jr, Tabatabai MA (1980a) Ami-
dase activity in soils: I. Method of assay. Soil
Sci Soc Am J 44: 282-287.
Frankenberger WT Jr, Tabatabai MA (1980b) Ami-
dase activity in soils: II. Kinetic parameters. Soil
Sci Soc Am J 44: 532-536.
Frankenberger WT Jr, Tabatabai MA (1981a) Ami-
dase activity in soils: III. Stability and distribution.
Soil Sci Soc Am J 45: 333-338.
Frankenberger WT Jr, Tabatabai MA (1981b) Ami-
dase activity in soils: IV. Effects of trace elements
and pesticides. Soil Sci Soc Am J 45:1120-1124.
Frankenberger WT Jr, Tabatabai MA (1981c) Fate of
amide nitrogen added to soils. Agricul Food
Chem 29: 152-155.
Frankenberger WT Jr, Tabatabai MA (1982) Amidase
and urease activity in plants. Plant Soil 64:
153-166.
Frankenberger WT Jr, Tabatabai MA (1985) Char-
acteristics of an amidase isolated from a soil
bacteria. Soil Biol Biochem 17: 303-308.
Frankenberger WT Jr, Tabatabai MA (1991a) L-
368
Asparaginase activity of soils. Biol Fertil Soils
11:6-12.
Frankenberger WT Jr, Tabatabai MA (1991b) Factors
affecting L-asparaginase activity in soils. Biol Fer-
til Soils 11: 1-5.
Frankenberger WT Jr, Tabatabai MA (1991c) L-
Glutaminase activity of soils. Soil Biol Biochem
23: 869-874.
Frankenberger WT Jr, Tabatabai MA (1991d) Factors
affecting L-Glutaminase activity of soils. Soil Biol
Biochem 23: 875-879.
Gadkari D (1984) Influence of the herbicide goltix
on extracellular urease and phosphatase in
suspended soil. Zbl Mikrobiol 139: 415-424.
Galstyan ASH (1965) A method of determining the
activity of hydrolytic enzymes in soil. Soviet Soil
Sci 2: 170-175.
Galstyan ASH, Saakyan EG (1973) Determination of
soil glutaminase activity. Doklady Acad Nauk
SSSR209: 1201-1202.
Gianfreda L, Rao MA, Violante A (1992) Adsorption,
activity and kinetic properties of urease on mont-
morillonite, aluminium hydroxide, and Al (OH)x-
montmorillonite complexes. Soil Biol Biochem
24: 51-58.
Gosewinkel U, Broadbent FE (1984) Conduc-
tometric determination of soil urease activity.
Commun Soil Sci Plant Anal 15:1377-1389.
GottschalkG, Andressen JR, Hippe H (1981) The
Prokaiyotes, vol II. Stan- MR Stolp H, Truper HG,
Balows A, Schlegel HG (eds). Springer Verlag,
Berlin, pp. 1767-1803.
Gyorgy R Rothler H (1926) Conditions for the auto-
lytic formation of ammonia in nature. Series II.
Determination of ammonia derived from amino
acids and nitrogen containing substances. Bio-
chem Z 173: 334-347.
Haanstra L, Doelman P (1991) An ecological dose-
response model approach to short- and long-
term effects of heavy metals on arylsulfatase
activity in soil. Biol Fertil Soils 11: 18-23.
Haare EA, White WC (1985) Fertilizer market profile.
In: Fertilizer Technology and Use. Engelstad OP
(ed.). American Society of Agronomy, Madison,
Wl, pp. 1-24.
Hagedorn HC, Jensen BN (1923) Zur Mikrobestim-
mung des Blutzuckers mittels Ferrizyanid. Bio-
chem Z 135, 46-58.
Hamson AF (1979) Variation of four phosphorus
properties in woodland soils. Soil Biol Biochem
11:393-403.
Hartman SC (1971) Glutaminase and y-glutamyl-
transferases. In: The Enzymes, vol 4. Boyer PD
(ed.) Academic Press, New York, pp. 79-100.
Hashimoto S, Muramatsu T, Fumatsu M (1971)
Studies on xylanase from Trichoderma viride.
Agr Biol Chem 35: 501-508.
Hayano K (1986) Cellulase complex in tomato field
Chapter 7

soil: induction, localization and some properties.
Soil Biol Biochem 18: 215-219.
Hayano K (1987) Characterization of a phospho-
diesterase component in a forest soil extract.
Biol Fertil Soils 3: 159-164.
Hayano K, Tubakil (1985) Origin and properties of
P-glucosidase activity of tomato-field soil. Soil
Biol Biochem 17: 553-557.
Hoffmann E, Schmidt W (1953) Uber das Enzym-
system unserer KulturkxJden. II. Urease. Biochem
Z323: 125-127.
Hoffmann G (1959) Distribution and origin of some
enzymes in soil. Z Pflanzenernahr Dung Bodenkd
77:243-251.
Hoffmann G (1967) Eine photometrische Methode
zur Bestimmung der Phosphatase-Aktivitat in
Boden. Z Pflanzenernaehr Bodenkd 118:161-172.
Hoffmann G, Dedken M (1965) Eine Methode zur
Kolorimetrischen Bestimmung der p-Glucosi-
dase-Aktivitat im Boden. Z Pflanzenernach
Budenk108: 193-198.
Hoffmann G, Elias-Azar K (1965) Verschiedene
Faktoren der Bodenfruchtbarkeit nordiranischer
Boden und Ihre Beziehung zur Aktivitat hydroly-
tischer Enzyme. Z Pflanzenernahr Dung Bodenkd
108: 199-217.
Hoffmann G, Pallauf J (1965) Eine kilorimetrische
Methode zur Bestimmung der Saccharaseaktivi-
tat in Boden. Z Pflanzenernahr Dung Bodenkd
110:193-201.
Hoffmann G, Pfitscher A (1982) Korrelationen von
Enzymaktivitaten Im Boden. Z Pflanzenernahr
Bodenkd 145: 36-41.
Hoffmann G, Teicher K (1957) Das Enzymsystem
unserer Kulturboden VII. Protease II. Z Pflanzener-
nahr Dung Bodenkd 3: 243-251.
Holz F (1980) Automatisierte, enzymatlsche-photo-
metrische Lysinbestimmung und ihre Anwendung
als Screeningsmethode in Zuchtungsprogram-
men. Landwirtsch Forschung 33: 272-289.
Holz F (1986a) Automatische photometrische Bes-
timmung der Aktivitat von Bodenenzymen durch
Anwendung (enzymatisch-)oxydativer Kupplung-
sreaktionen im Durchflup. II Mitteilung: Die Bes-
timmung der Saccharaseaktivitat. Landwirtsch
Forschung 39: 245-259.
Holz F (1986b) Automatisierte, photometrische Bes-
timmung der Aktivitat von Bodenenzymen durch
Anwendung (enzymatisch-)oxydativer Kupplung-
sreaktionen im Durchflup. I. Mitteilung: Die Bes-
timmung der Katalaseaktivitat. Landwirtsch
Forschung 39: 139-153.
Hope CFA, Burns RG (1987) Activity, origins and
location of ceilulase in a silt loam soil. Biol Fertil
Soils 5: 164-170.
Hunt H (1977) A simulation model for decomposition
in grasslands. Ecology 58: 469-484.
Hynes MJ (1970) Induction and repression of aml-
Enzyme activities
References
dase enzymes in Aspergillus nidulans. J Bacterid
103:482-487.
Hynes MJ (1975) Amide utilization in Aspergillus
r^idulans: Evidence for a third amidase enzyme.
J Gen Microbiol 91: 99-109.
Ibister JD, Shippen RS, Caplan J (1980) A new
method for measuring cellulose and starch degra-
dation in soils. Bull Environ Contam Toxicol 24:
570-574.
Imada A, Igarasi S, Nakahama K, Isono M (1973)
Asparaginase and glutaminase activities of micro-
organisms. J Gen Microbiol 76: 85-89.
Jakoby WB, Fredericks J (1964) Reactions cata-
lyzed by amidases. Acetamidase. J Biol Chem
239: 1978-1982.
Jarvis BW, Lang GE, Wieder RK (1987) Arylsulphat-
ase activity in peat exposed to acid precipitation.
Soil Biol Biochem 19: 107-109.
Joliff G, Edelman A, Klier A, Rapoport G (1989)
Inducible secretion of a ceilulase from Clostri-
dium thermocellum in Bacillus subtilis. AppI
Environ Microbiol 55: 2739-2744.
Joshi JG, Handler P (1962) Purification and prop-
erties of nicotinamidase from Torula cremoris. J
Biol Chem 237: 929-935.
Juma NG, Tabatai MA (1977) Effects of trace ele-
ments on phosphatase activity in soils. Soil Sci
Soc Am J 41: 343-346.
Juma NG, Tabatabai MA (1988) Comparison of
kinetic and thermodynamic parameters of
phosphomonoesterases of soils and of corn and
soybean. Soil Biol Biochem 20: 533-539.
Kandeler E (1986) Aktivitat von Proteasen in Boden
und Ihre Bestimmungsmoglichkeiten. VDLUFA-
Schriftenreihe 20: 829-847.
Kandeler E (1990) Characterization of free and
adsorbed phosphatases in soils. Biol Fertil Soils
8: 199-202.
Kandeler E, Gerber H (1988) Short-term assay of soil
urease activity using colorimetric determination of
ammonium. Biol Fertil Soils 6: 68-72.
Keeney DR, Nelson DW (1982) Nitrogen Inorganic
forms. In: Methods of Soil Analysis, Part 2, 2nd
edn. Page AL, Miller RH, Keeney DR (eds).
American Society of Agronomy, Madison, Wl,
pp. 643-698.
Kelly M, Clarke PH (1962) An inducible amidase
produced by a strain of Pseudomonas aeru-
gir)osa. J Gen Microbiol 27: 305-316.
King GM, Klug MJ (1980) Sulfohydrolase activity
in sedhnent of Wistergreen Lake, Kalamazoo
Country, Michigan. AppI Environ Microbiol 29:
950-956.
Kiss S, Stefanic G, Dragan-Bularda M (1974) Soil
enzymology in Romania (Part I). Contrib Bot
. Unlv Babes-Bolyia Cluj pp. 207-209.
Kiss S, Dragan-Bularda M, Radulescu D (1978) Soil
polysaccharidases: activity and agricultural
369
References
importance. In: Soil Enzymes. Bums RG (ed.).
Academic Press, New York, pp. 117-147.
Kissel DE, Cabrera ML (1988) Factors affecting
urease activity. In: Ammonia Volatilization from
Urea Fertilizer. Bock BR, Kissel DE (eds). TVA,
National Fertilizer Development Center, Muscle
Shoals, pp. 53-66.
Konig J, Hasenbaumer J, Coppenrath E (1906)
Several new properties of cultivated soils.
Landw Versuchs-Stationen 63: 471-478.
Konig J, Hasenbaumer J, Coppenrath E (1907)
Relationships between the properties of soil and
the nutrient uptake by plants. Landw Versuchs-
Stationen 66:401-461.
Kshattriya S, Sharma GD, Mishra RR (1992) Enzyme
activities related to litter decomposition in the
forests of different age and altitude in North
East India. Soil Biol Biochem 24: 265-270.
Kuprevich VF, Shcherbakova TA (1971a) Soil
Enzymes. US Department of Commerce,
National Technical Infonnation Service, Spring-
field, Va. (Pochvennaya Enzimologiya Nauka
tekh. Minsk, 1966; translated from Russian.)
Kuprevich VF, Shcherbakova TA (1971b) Compara-
tive enzymatic activity in diverse types of soil. In:
Soil Biochemistry, vol 2. McLaren AD, Skujins J J
(eds). Marcel Dekker, New York, pp. 167-201.
Ladd JN (1972) Properties of proteolytic enzymes
extracted from soils. Soil Biol Biochem 4: 227-
237.
Ladd JN (1978) Origin and range of enzymes in soil.
In: Soil Enzymes. Burns RG (ed.). Academic
Press, New York, pp. 51-96.
Ladd JN, Butler JHA (1972) Short-term assays of
soil proteolytic enzyme activities using proteins
and dipeptlde derivatives as substrates. Soil Biol
Biochem 4: 19-30.
Ladd JN, Jackson RB (1982) Biochemistry of
ammonification. In: Nitrogen in Agricultural Soils.
Stevenson FJ (ed.). Agronomy Monograph 22,
American Society of Agronomy, Madison, Wl,
pp. 173-228.
Lahdesmaki P, Pilspanen R (1989) Changes in con-
centrations of free amino acids during humiflca-
tlon of spruce and aspen leaf litter. Soil Biol
Biochem 21: 975-978.
Lai CM, Tabatai MA (1992) Kinetic Parameters of
immobilized urease. Soil Biol Biochem 24: 225-
228.
Latter PM, Howson G (1977) The use of cotton
strips to indicate cellulose decomposition in the
field. Pedobiologia 17: 145-155.
Law AS, Wriston JC Jr (1971) Purification and prop-
erties of Bacillus coagulans L-asparaginase. Arch
Biochem Blophys 147: 744-752.
Lee YH, Fan LT (1980) Properties and mode of
action of cellulase. Adv Biochem Eng 17:101-129.
Lessie TG, Neidhardt FC (1967) Formation and
operation of the histidine-degrading pathway in
370
Pseudomonas aeruginosa. J Bacterid 93: 1800-
1810.
Loll MJ, Bollag JM (1983) Protein transformation in
soil. Adv Agron 36: 351-382.
Lopez-Hernandez D, Nino M, Nanniplerl P, Fardeau
JC (1989) Phosphatase activity in Nasutitermes
ephratae termite nests. Biol Fertil Soils 7:134-137.
Mahler RH, Cordes EH (1971) Biological Chemistry.
Harper & Row, New York, London.
Malcolm RE (1983) Assessment of phosphatase
activity in soils. Soil Biol Biochem 15: 403-408.
Marsden WL, Gray PP (1986) Enzymatic hydrolysis
of cellulose in lignocellulose materials. CRC Crit
Rev Microbiol 3: 235-276.
Mathe P, Kovacs G (1980) Effect of Mn and Zn on
the activity of phosphate in soil: I. Phosphatase
activity of a calcareous chernozen soil under
maize. Agrokem Talajtan 29: 441-446.
Mathur SP, Sanderson RB (1978) Relationship
between copper contents, rates of soil respira-
tion and phosphatase activities of some hlsto-
zols in an area of Southwestern Quebec in the
summer and the fall. Con J Soil Sci 58: 125-134.
Mathur SP, Rayment AF (1977) Influence of trace
element fertilization on the decomposition rate
and phosphatase activity of a mesic fibrlsol. Can
J Soil Sci 57: 397-408.
May DW, Glle PL (1909) The catalase of soils. Puerto
Rico Agr Exp Sta Circular no. 9: 3-13.
May PB, Douglas LA (1976) Assay for soil urease
activity. Plant Soil 45: 301-305.
Mayaudon J, Batistic L, Sarkar JM (1975) Properties
of proteolytically active extracts from fresh soils.
Soil Biol Biochem 7: 281-286.
McCarty GW, Bremner JM (1991) Production of
urease by microbial activity in soils under aerobic
and anaerobic conditions. Biol Fertil Soils 11:
228-230.
McCarty GW, Bremner JM, Chac HS (1989) Effects
of N-(n-butyl)thiophosphorictriamlde on hydro-
lysis of urea by plant, microbial, and soil urease.
Biol Fertil Soils 8: 123-127.
McCarty GW, Shogern DR, Bremner JM (1992)
Regulation of urease production In soil by micro-
bial assimilation of nitrogen. Biol Fertil Soils 12:
261-264.
McLaren AD (1969) Radiation as a technique In soil
biology and biochemistry. Soil Biol Biochem 1:
63-73.
McLaren AD, Estermann EF (1957) Influence of pH
on the activity of chymotrypsin at the solid-liquid
interface. Arch Biochem Blophys 68: 157-160.
Mon'a MJ, Freeborn LL (1989) Catalysis of amino
acid deaminatlon In soils by pyridoxal-5-phos-
phate. Soil Biol Biochem 21: 645-650.
Moyo CC, Kissel DE, Cabrera ML (1989) Tempera-
ture effects on soil urease activity. Soil Biol Bio-
chem 21: 935-938.
Mulvaney RL, Bremner JM (1979) A modified dia-
Chapter 7

cetyl monoxime method for colorimetric determi-
nation of urea in soil extracts. Commun Soil Sci
Plant Anal 10: 1163-1170.
Nakas JP, Gould WD, Klein DA (1987) Origin and
expression of phosphatase activity in a semi-
arid grassland soil. Soil Biol Biochem 19: 13-18.
Nannipieri P (1994) Productivity, Sustainability and
Pollution. In : Soil Biota. Management in Sustain-
able Fanning Systems (Parkhurst CE, Doube BM,
Gupta W, Grace PR, eds), CSIRO, Adelaide, Aus-
tralia, pp. 238-244.
Nannipieri P, Ceccanti B, Cervelli S, Sequi P (1974)
Use of 0.1 M pyrophosphate to extract urease
from a podzol. Soil Biol Biochem 6: 359-362.
Nannipieri P, Ceccanti B, Cervelli S, Sequi P (1978a)
Stability and kinetic properties of humus-urease
complexes. Soil Biol Biochem 10: 143-147.
Nannipieri P, Johnson PL, Paul EA (1978b) Criteria
for measurement of microbial growth and activity
in soil. Soil Biol Biochem 10: 223-229.
Nannipieri P, Ceccanti B, Cervelli S, Matarese E
(1980) Extraction of phosphatase, urease, pro-
tease, organic carbon and nitrogen from soil.
Soil Sci Soc Am J 44: 1011-1016.
Nannipieri P, Cecconti B, Conti C, Bianchi D (1982)
Hydrolases extracted from soil: their properties
and activities. Soil Biol Biochem 14: 257-263.
Nannipieri P, Ceccanti B, Bianchi B (1988) Charac-
terization of humus-phosphate complexes
extracted from soil. Soil Biol Biochem 20: 683-
691.
Nannipieri P, Grego S, Ceccanti B (1990) Ecological
significance of the biological activity in soil. In:
Soil Biochemistry, vol 6. Bollag J-M, Stotzky G
(eds). Marcel Dekker, New York, Basel, pp.
293-355.
Nicholls RG, Roy AR (1971) Arylsulfatase. In: The
Enzymes, vol 5, 3rd edn. Boyer PD (ed.). Aca-
demic Press, New York, pp. 21-41.
Nor YM (1982) Soil urease activity and kinetics. Soil
Biol Biochem 14: 63-65.
Pancholy SK, Lynd JQ (1972) Quantitative fluores-
cence analysis of soil lipase activity. Soil Biol
Biochem 4: 257-259.
Pancholy SK, Rice EL (1973) Soil enzymes in rela-
tion to old field succession: amylase, cellulase,
invertase, dehydrogenase and urease. Soil Sci
Soc Am Proc 37: 47-50.
Pari< JT, Johnson MJ (1949) A submicrodetermina-
tion of glucose. J Biol Chem 181: 149-151.
Pere^-Mateos M, Gonzalez-Carcedo S (1988)
Assay of urease activity in soil columns. Soil
Biol Biochem 20: 567-572.
Perucci P, Scarponi L (1984) Arylsulphatase activity
in soils amended with crop residues: kinetics and
thermodynamic parameters. Soil Biol Biochem
16: 605-608.
Pokorna V (1964) Method of determining the lypo-
Enzyme activities
References
lytic activity of upland and lowland peats and
mucks. Pochvovedenle 106: 85-87.
Powlson DS, Jenkinson DS (1976) The effects of
biocidal treatments on metabolism in soil. II.
Gamma in'adiation, autoclaving, air-drying and
fumigation. Soil Biol Biochem 8: 179-188.
Press MC, Henderson J, Lee JA (1985) Arylsulpha-
tase activity in peat in relation to acidic deposi-
tion. Soil Biol Biochem 17: 99-103.
Pulford ID, Tabatabai MA (1988) Effect of water-
logging on enzyme activities in soils. Soil Biol
Biochem 20: 215-219.
Rai B, Srivastava AK (1983) Decomposition and
competitive colonization of leaf litter by fungi.
Soil Biol Biochem 15: 115-117.
Rastin N, Rosenplanter K, Huttermann A (1988)
Seasonal variation of enzyme activity and their
dependence on certain soil factors In beech
forest soil. Soil Biol Biochem 20: 637-642.
Rechler MM (1969) The purification and charac-
terization of L-histidine ammonia-lyase (Pseudo-
monas). J Biol Chem 244: 551-559.
Reissig Jl, Strominger JL, Leioir LF (1955) A mod-
ified method for the estimation of N-acetylamine
sugars. J Biol Chem 217: 959-966.
Reuter G (1976) Gelande- und Laborpraktikum der
Bodenkunde. VEB Veriag, Beriin.
Rhee YH, Hah YC, Hong SW (1987) Relative con-
tributions of fungi and bacteria to soil carboxy-
methylcellulase activity. Soil Biol Biochem 19:
479-481.
Rice L, Mollik MAB (1977) Causes of decreases in
residual carbohydrase activity in soil during old-
field succession. Ecology 58: 1297-1309.
Roberge MR (1978) Methodology of soil enzyme
measurement and extraction. In: Soil Enzymes.
Burns RG (ed.) Academic Press, New York, pp.
341-370.
Roberts J, Holcenberg JS, Dolowy WC (1972)
Isolation, crystallization, and properties of
Achromobacteraceae glutaminase-asparaginase
with antitumor activity. J Biol Chem 247: 84-90.
Rodriguez-Kabana R, Godoy G, Morgan-Jones G,
Shelby RA (1983) The determination of soil chit-
inase activity; conditions for assay and ecological
studies. Plant Soil 75: 95-106.
Ross DJ (1965) Effects of air-dry, refrigerated, and
frozen storage on activities of enzymes hydrolys-
ing sucrose and starch. J Soil Sci 16: 86-94.
Ross DJ (1968) Some observations on the oxidation
of glucose by enzymes in soil in the presence of
toluene. Plant Soil 28: 1-11.
Ross DJ (1974) Glucose oxidase activity in soil and
its possible interference in assay of cellulase
activity. Soil Biol Biochem 6: 303-306.
Ross DJ (1975) Studies on a climosequence of soils
in tussock grasslands. Invertase and amylase
activities of topsoils and their relationship with
371
References
other properties. N Zealand J Soil Sci 18: 5 1 1 -
518.
Ross DJ, Speir TW, Giltrap DJ, McNellly BA, Molloy
LF (1975) A principal components analysis of
some biochemical activities in climosequence of
soils. Soil Biol Biochem 7: 349-355.
Rossner H (1991) Bestimmung der Chitinase-Akti-
vitat. In: Bodenbiologlsche Arbeitsmethoden.
Schinner F, Ohiinger R, Kandeler E (eds).
Springer Verlag, Berlin, pp. 66-70.
Sarathchandra SU, Pen-ott KW (1981) Determination
of phosphatase and arylsulphatase activities in
soils. Soil Biol Biochem 13: 543-545. •
Sarkar JM, Batistic L, Mayaudon J (1980) Les hydro-
lases du sol et leur association avec les hydrates
de carbone. Soil Biol Biochem 12: 325-328.
Sarkar JM, Leonowicz A, Bolag JM (1989) Immo-
bilization of enzymes on clays and soils. Soil Biol
Biochem 21: 223-230.
Sato K (1981) Relations between soil microflora and
CO2 evolution upon decomposition of cellulose.
Plant Soil 61: 251-258.
Sayre FW, Roberts E (1958) Preparation and some
properties of a phosphate-activated glutaminase
from kidneys. J Biol Chem 233:1128-1134.
Schinner F, Von Mersi W (1990) Xylanase-, CM-
celiulase- and invertase activity in soil: an
Improved method. Soil Biol Biochem 22:511-515.
Schinner F, Ohiinger R, Kandeler E (1991) Bodenbio-
loglsche Ark)eitesmethoden. Springer Verlag, Ber-
lin, New York.
Schmidt G, Laskowsky M Sr. (1961) Phosphate
ester cleavage (survey). In: The Enzymes, 2nd
edn. Boyer PD, Lardy H, Myrbach K (eds). Aca-
demic Press, New York, pp. 3-35.
Schreiner O, Shorey EG (1910) The presence of
arginine and histidine in soils. J Biol Chem 8:
381-384.
Schroder D, Gewehr H (1977) Stroh- und Zellulo-
seabbau in verschiedenen Bodentypen. Z Pflan-
zenernahr Bodenkd 140: 273-284.
Schroder D, Urban B (1985) Bodenatmung, Cellulo-
seabbau und Dehydrogenaseaktivitat in verschie-
denen Boden und ihre Beziehungen zur
organischen Substanz sowie Bodeneigenschaf-
ten. Landwirtsch Forschung 38: 166-172.
Schulz-Berendt V (1986) Der Stickstoff-Haushalt
eines Ruderalstandortes als Grundlage der Beur-
teilung von Okosystem-Veranderungen. Disserta-
tion, University of Bremen.
Simpson JR, Freney JR, Westelaar R, Muirhead WA,
Leuning R, Denmead OT (1984) Transformations
and losses of urea nitrogen after application to
flooded rice. Aust J Agric Res 35:189-200.
Simpson LR, Freney JR, Muirhead WA, Leuning R
(1985) Effects of phenylphosphodiamidate and
dicyandiamide on nitrogen loss from flooded
rice. Soil Sci Soc Am J 49: 1426-1431.
Sinsabaugh RL, Linkins AE (1988) Adsorption of
372
cellulase components by leaf litter. Soil Biol
Biochem 20: 927-931.
Sinsabaugh RL, Linkins AE (1989) Cellulase mobility
in decomposing leaf litter. Soil Biol Biochem 21:
205-209.
Skujins JJ (1967) Enzymes In soil. In: Soil Biochem-
istry, vol 1. McLaren AD, Peterson GH (eds).
Marcel Dekker, New York, pp. 371-414.
Skujins J (1978) History of abiotic soil enzyme
research. In: Soil Enzymes. Burns RG (ed.). Aca-
demic Press, London, pp. 1-49.
Skujins J J, McLaren AD (1969) Assay of urease
activity """^C-Urea in stored, geogically preserved,
and in irradiated soils. Soil Biol Biochem 1:89-99.
Sowden FJ (1958) The forms of nitrogen In the
organic matter of different horizons of soil pro-
files. Can J Soil Sci 38: 147-154.
Sparling GP, Speir TW, Whale KN (1986) Changes in
microbial biomass C, ATP content, soil phospho-
monoesterase and phospho-diesterase activity
following air-drying of soils. Soil Biol Biochem
18: 363-370.
Speir R, Lee R, Pansier EA, Cairns A (1981) A
comparison of sulphatase, urease and protease
activities in planted and in fallow soils. Soil Biol
Biochem 12: 281-291.
Speir TW, Ross DJ (1975) Effects of storage on the
activities of protease, urease, phosphatase, and
sulphatase in three soils under pasture. N Zealand
Sci 18: 231-237.
Speir TW, Ross DJ (1978) Soil phosphatase and
sulphatase. In: Soil Enzymes. Burns RG (ed.).
Academic Press, London, pp. 197-250.
Speir TW, Ross DJ (1981) A comparison of the
effects of air-drying and acetone dehydration
on soil enzyme activities. Soil Biol Biochem 13:
225-229.
Spiers GA, McGill WB (1979) Effects of phosphorus
addition and energy supply on phosphatase pro-
duction and activity in soils. Soil Biol Biochem 11:
3-8.
Spiro RG (1966) Analysis of sugars found in glyco-
proteins. In: Methods In Enzymology, vol 8. Neu-
feld EF, Ginsberg V (eds). Academic Press,
London, pp. 326.
Stefanic G, Beck Th, Schwimmer J, Hartmann F,
Varbanciu A (1984) Apparatus for measuring the
soil catalase activity. Fifth Symposium on Soil
Biology, Romania lASI, pp. 47-50.
Stutzenberger FG (1972) CellulolytIc activity of Ther-
momonospora curbata: optimal assay conditions,
partial purification and product of the cellulase.
AppI Microbiol 24: 83-90.
Sumner JB (1951) Urease. In: The Enzymes, vol 1.
Sumner JB (ed.). Academic Press, New York, pp.
873-892.
SuttnerT, Alef K (1988) Con-elation between arginine
ammonification, enzyme activities, microbial
Chapter 7

biomass, physical and chemical properties of
different soils. Zentralbl MIkroblol 143: 569-573.
Tabatabai MA (1977) Effects of trace elements on
urease activity In soils. Soil Biol Blochem 9: 9-13.
Tabatabai MA (1982) Soil enzymes. In: Methods of
Soil Analysis, Part 2, Chemical and Microbiologi-
cal Properties. Page AL, Miller EM, Keeney DR
(eds). American Society of Agronomy, Madison,
Wl, pp. 903-947.
Tabatabai MA, Bremner JM (1969) Use of p-nltro-
phenyl phosphate for assay of soil phosphatase
activity. Soil Biol Blochem 1: 301-307.
Tabatabai MA, Bremner JM (1970a) Arylsulpha-
tase activity of soils. Soil Scl Soc Am Proc
34: 225-229.
Tabatabai MA, Bremner JM (1970b) Factors affect-
ing soil arylsulfatase activity. Soil Scl Soc Am
Proc 34: 427-429.
Tabatabai MA, Bremner JM (1972) Assay of urease
activity In soil. Soil Biol Blochem 4: 479-487.
Tabatabai MA, Dick WA (1979) Distribution and
stability of pyrophosphatase In soil. Soil Biol
Blochem 11: 655-659.
Tabor H (1954) Metabolic studies on histldlne, his-
tamine, and related Imidazoles. Phannacol Rev 6:
229-343.
Tarafdar JC, Jungk A (1987) Phosphatase activity In
the rhizosphere and Its relation to the depletion
of soil organic phosphorous. Biol Fertil Soils 3:
199-204.
Tateno M (1988) Limitation of available substrates
for the expression of cellulase and protease
activities in soil. Soil Biol Blochem 20: 117-118.
Trasar-Cepeda MC, Gil-Sotres F (1987) Phospha-
tase activity in add high organic matter soils in
Galicia (NW Spain). Soil Biol Blochem 19: 2 8 1 -
287.
Trasar-Cepeda MC, Gil-Sotres F (1988) Kinetics of
acid phosphatase activity In various soils of
Galicia (NW Spain). Soil Biol Blochem 20:275-280.
Trevors JT (1984) Rapid gas chromatographic
method to measure H2O2 oxidoreductase (cata-
lase) activity in soil. Soil Biol Blochem 16: 525-
526.
Veon M, MIkyeshlte K, Sawado Y, Obe Y (1990)
Assay of chltlnase and /V/-acetylglucosamidase
activity in forest soils with 4-methylumbelliferyl
Enzyme activities
References
derivatives. Z Pflanzenernahr Bodenkd 154:
171-175.
Voets JP, Dedeken M, Besseme E (1965) The beha-
viour of some amino acids in gamma Irradiated
soils. Naturwissenschaften 52: 476.
Wallenfels K, Weil R (1972) p-Galactosldase. In:
The Enzymes, vol 7, 3rd edn. Boyer PD (ed.).
Academic Press, New York, pp. 617-663.
Warman PR, Bishop C (1987) Amino-N compounds
found In soil organic matter hydrolysates of a
loamy sand using an Immobilized protease reac-
tor column. Biol Fertil Soils 5: 219-224.
Warman PR, Isnor RA (1989) Evidence of peptides in
low-molecular-weight fractions of soil organic
matter. Biol Fertil Soils 8: 25-28.
Weetall HH, Wellky N, Vango SP (1965) Detection of
microorganisms In soil by their catalytic activity.
Nature 205: 1019-1021.
Wilke B-M (1986) Einflup verschledener potentieller
anorganlscher Schadstoffe auf die mikrobielle
Aktivltat von Waldhumusformen unterschledllcher
Pufferkapazitat. Bayreuther Geowissenschaftllche
Arbelten, Band 8, University of Bayreuth.
Wilke BM (1988) Langzeltwirkungen potentieller
anorganlscher Shadstoffe auf die mikrobielle Akti-
vltat einer sandigen Braunerde. Z Pflanzenernahr
Bodenkd 151: 131-136.
Wriston JC Jr (1971) i-Asparaginase. In: The
Enzymes, vol 4. Boyer PD (ed.). Academic
Press, New York, pp. 101-121.
Xiaoyan Z, LIkai Z, Guanyun Wu (1992) Urea hydro-
lysis in a brown soil: effect of hydroquinone. Soil
Biol Blochem 24: 165-170
Yamana K, Suzukki H, NIshizawa K (1970) Purifica-
tion and properties of extracellular and cellbound
cellulase components of Pseudomonas fluores-
cens van cellulosa. J Blochem 67: 19-35.
Zantua Ml, Bremner JM (1975a) Comparison of
methods of assaying urease activity in soils. Soil
Biol Blochem 7: 291-295.
Zantua Ml, Bremner JM (1975b) Preservation of soil
samples for assay of urease activity. Soil Biol
Blochem 7: 297-299.
Zantua Ml, Bremner (1977) Stability of urease in
soils. Soil Biol Blochem 9: 135-140.
Zhengping W, Van Cleemput O, Demeyer P (1991)
Effect of urease inhibitors on urea hydrolysis and
ammonia volatilization. Biol Fertil Soils 11: 43-47.
373