Palm, D., H. W Klein, R. Schinzel, M . Buehner, and E. J. M . Helm-
Thiemt Analysis of oligosaccharides
reich: The role of pyridoxal 5'-phosphate in glycogen phosphory-
lase catalysis. Biochemistry 29 (1990). 1099-1107.
Ziegast, G.,and B. Pfannemuller: Phosphorolytic syntheses with
di-, oligo- and multifunctional primers. Carbohydr. Res. 160
(l987), 185-204.
Fiske, C. H., and Y Subbarow: The colorimetric determination of
phosphorus. J. Biol. Chem. 66 (1925), 375-400.
Rick, W ,and H. P. Stegbauer: a-Amylase -Messung der reduzier-
enden Gruppen, in: Methoden der enzymatischen Analyse, Bd.
1. Ed. U. Bergmeyer,Verlag Chemie, Weinheim 1974, pp 918-923.
French, D., and G.M. Wild: Primer specificity of potato
phosphorylase. J. Am. Chem. SOC.75 (1953), 4490-4492.
Suganurna. T., J. Kitazono, K. Yoshinaga, S. Fujimoto, and T. Naga-
hama: Determination of kinetic parameters for maltotriose and
higher malto-oligosaccharides in the reaction catalyzed by a-D-
glucan phosphorylase from potato. Carbohydr. Res. 21 7 (1991),
213-20.
Evers, B., P. Mischnick, and J. Thiem: Synthesis of 2-deoxy-a-D-
arabino-hexopyranosyl phosphate and 2-deoxy-maltooligosac-
charides with phosphorylase. Carbohydr. Res. 262 (1994), 335-
341.
Glucoamylase Research: An Overview
This paper reviews the main features of glucoamylase research de-
scribing essentially more recent developments on microorganisms,
production and properties of glucoamylases. It is an important indus-
trial enzyme and is widely used in starch saccharitication, brewing
and distilling industry. Glucoamylase can be derived from a wide vari-
ety of sources including plants, animals and microorganisms. Most of
the production techniques essentially, however, are based on micro-
bial sources. It is an extracellular enzyme and has got second place
(next to proteases) in world's distribution and sales among industrial
enzymes. Most of the production of glucoamylase is carried out using
liquid fermentation process. Technique of solid state fermentation,
however, have also been employed for its production and recently
this technique is gaining renewed interest from researchers for its
production. The paper also describes aspects related with the purifica-
tion and characterization of glucoamylase enzyme.
1 Introduction
The amylase family of enzyme have been well characterized
through the study of various microorganisms. Two major clas-
ses of starch degrading enzymes have been identified in the
microorganisms viz. alpha amylae (endo-1,4-a-D-glucan glu-
cohydrolase, EC 3.2.1.1) which randomly clevages the 1,4-a-D-
glucosidic links between adjacent glucose units in linear amy-
lose chains and glucoamylose (synomym amyloglucosidase
and also referred as glucogenic enzyme, starch glucogenase,
gamma amylase; exo-1,4-a-D-glucan-glucanohydrolase,EC
3.2.1.3) which hydrolyses single glucose residues from the
non-reducing ends of amylose and amylopectin in a step-wise
manner. Unlike the alpha amylases, most glucoamylases are
also able to hydrolyse the 1,6-a-linkages in the branching
points of amylopectins, although at a slower rate than the 1,4-
a linkages. Both of these enzymes are produced by different
~~ ~ ~
sluchlstnrke 47 (1995) Nr 1 1 S 139-445 @ VCH Verlngsgesellschaft mbH, D-69451 Weinheim. 1995
[12] Mischnick, P., B. Evers, and 1.
containing 2-deoxy-a-D-arabino-hexosylresidues by the reduc-
tive cleavage method. Carbohydr. Res. 264 (1994), 293-304.
[I31 Mischnick, P.: unpublished result.
[14] McCready, R. M., and W 2. Hassid: Preparation of a-D-glucose-1-
phosphate by means of potato phosphorylase. Meth. Enzymol. 3
(1957), 137-143.
[lS] Saenger, W , C. Niemann, R . Herbst, W Hinrichs, and 7: Steiner:
Chrystal structures of linear and cyclic oligosaccharides: An
overview. Pure Appl. Chem. 65 (1993), 809-817.
[16] St.-Jacques, M., P. R . Sundararajan, K. J. Taylor, and R . H. Mar-
chessault: Nuclear magnetic resonance and conformational stud-
ies on amylose and model compounds in dimcthyl sufoxide so-
lution. J. Am. Chem. SOC.98 (1976), 4386-4391.
Address of authors: Dip1.-Chem. Britia Evers, and Professor Dr. Joa-
chim Thiem. Institut fur Organische Chemie, Universitat Hamburg.
Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany.
(Received: June 13, 1995).
Ashok Pandey, Trivandrum (India)
Glucoamylase-Forschung: €in Uberblick. Diese Arbeit umfaBt die
hauptsachlichsten Ergebnisse der Glucoamylase-Forschung und be-
schreibt im wesentlichen jungere Entwicklungen uber Mikroorganis-
men, Herstellung und Eigenschaften der Glucoamylasen. Die Glu-
coamylase ist ein wichtiges industrielles Enzym und wird weit ver-
breitet in der Stirkeverzuckerung angewendet sowie in der Brauerei-
und in der Brennereiindustrie. Glucoamylase kann gewonnen wer-
den aus einer groBen Vielfalt von Quellen einschliel3lich Pflanzen,
Tieren und Mikroorganismen. Die meisten Produktionsverfahren ba-
sieren jedoch auf mikrobiellen Quellen. Es handelt sich um ein extra-
cellulares Enzym und steht an zweiter Stelle hinter den Proteasen in
der weltweiten Verteilung und im Verkauf unter den industriellen En-
zymen. Der groBte Teil der Glucoamylaseproduktion wird im flussi-
gen FermentationsprozeB ausgefuhrt. Jedoch wurde die Feststofffer-
mentation angewendet, und dieses Verfahren gewann kurzlich erneu-
tes Interesse von Forschungsgruppen zu ihrer Herstellung. Die vorlie-
gende Arbeit beschreibt auch Aspekte zur Reinigung und Charakteri-
sierung des Glucoamylaseenzyms.
microorganisms but sometimes both can be produced by the
same organism [l].
Glucoamylase enzyme has got wide applications in the food
and fermentation industries viz. in the processes of starch sac-
charification, brewing and distilling. It has got 14% distribu-
tion and sales (second position) on worldwide distribution
and sales of industrial enzymes [2].
Fleming [3] classified glucoamylases into two groups, one of
which converts starch and P-limit dextrins completely into
glucose and the other which converts starch 80% and /3-limit
dextrins 40% into glucose. Panose and a-limit dextrins, how-
ever, are completely hydrolysed to glucose by the both groups
[3]. The molecular size and the structure of the substrate play
an important role in determining the rate of hydrolysis by glu-
coamylases [4]. The affinity of the enzyme for the substrate is
linearly related to the degree of polymerization within the
range 2-5 units. The position and nature of bonds in the se-
quence also affect the rate of hydrolysis.
0038-9056/95/11110439505 00+ 2510 43 9
2 Substrates
A number of substrates, essentially starchy or related nature,
have been reported to be hydrolysed by glucoamylases. This
includes starches, glycogens, amylose, amylopectin, dextrins,
maltodextrins, maltose, isomaltose, dextran, panosan and
oligo-, di- and polysaccharides etc. Sometimes, pretreatment
of substrate becomes necessary for glucoamylase action:
alternatively pre-treatment (i.e., liquefaction/gelatinization)
helps in better hydrolysis rate and improved product yields.
Originally, it was thought that glucoamylases did not degrade
the raw starch [3]. It was Manners [S] who first reported degra-
dation of raw starch by glucoamylase. Subsequently, a number
of reports have appeared on hydrolysis of raw starch [6-151.
3 Production of Glucoamylase
The production of glucoamylase for converting starchy sub-
strates to dextrose has been well elaborated in a number of pa-
pers and patents [16-301. An important factor in the produc-
tion of this enzyme is the simultaneous production of
transglucosidase and/or alpha amylase. Transglucosidase ca-
talyses the formation of unfermentable dextrose polymers, for
example isomaltose and oligosaccharides, which ultimately
lower the yields of dextrose. Thus, the formation of transglu-
cosidase is an undesirable phenomenon and should be con-
trolled.The formation of alpha amylase, however, may be a de-
sirable one as it acts on starch to catalyse the formation of sac-
charides of lower moelecular weight which are broken down
to dextrose with a relative ease by glucoamylase. If, however,
relatively large amounts of alpha amylase are produced, it may
be detrimental to the production of dextrose as it may pro-
duce saccharides which may polymerized to unfermentable
dextrose polymers by transglucosidase.
Commercial production of glucoamylase is carried out in vari-
ous steps, essentially because of the environmental factors re-
quired for the optimum growth of the microorganism being
employed for the production may differ with those required
for the production of enzyme. These parameters include nutri-
ents supplementation, pH of medium, osmotic relationship,
degree of aeration, temperature and control of contamina-
tions during fermentation. Maintaining the purity of the me-
dium also is a very important factor, especially when the fer-
mentation is carried out under aerobic conditions.
3.1 Production techniques
Glucoamylase is known to be prepared by fermentation pro-
cesses involving certain microorganisms. Takarnine [31,32], as
early as in 1914 reported the production of moldy bran (con-
taining enzyme preparation from Aspergillus oryzae) in solid
state fermentation (SSF). Underkofler et al. [33] and Boyerand
Underkofler [34] reported production of moldy bran in shallow
trays in SSF. Traditionally, glucoamylase, however, has been
produced by liquid or submerged fermentation (SmF) and
one-way process in solution has been well developed. Most of
the commercial production, thus, is carried out using this tech-
nique.
Technique of solid state fermentation has gained renewed in-
terest from researchers for production of enzyme in view of its
several economical and engineering advantages [35-401. A
number of reports have appeared recently on production of
glucoamylase enzyme by SSF [41-471. Few attempts have also
been made for the production of glucoamylase from immobi-
lized microorganisms [48]. Because the enzyme industry is
highly competitive, reliable estimates of the extent to which
~~
440
different production techniques are used are not easy to estab-
lish. Although specific fermentation process adopted by differ-
ent manufacturers varies degree to degree, there remains
these two methods i.e., SmF or SSF for the production of glu-
coamylase enzyme [48].
4 Sources of Glucoamylases
Glucoamylases have been mostly widely reported to occur in
microorganisms [49] and also in animals [SO, 511 and plants
I.52). A large number of microorganisms, including bacteria,
yeast and fungi are capable of producing glucoamylases. Fi-
lamentous fungi, however, constitute the major source among
all microorganisms [53]. Strains of two genus viz. Aspergillus
and Rhizopus are mainly used for commercial production of
glucoamylases.
In past few years, a variety of aerobic as well as anaerobic mi-
croorganisms have been investigated for their ability for pro-
ducing glucoamylases. A few aerobic bacteria, such as Bacillus
stearothermophillus (Srivastava, [54]), Flavobacterium sp. (Ben-
der, [55]) and Halobacterium sodamense (Oken, [56]) have been
reported to produce glucamylase. A few anaerobic-bacteria,
such as Closrridirrm sp. (Tuchiya et al., [57], Ohnishi et al. [%I),
C. thermosaccharolytictrm (Specka et al. [59]), C. acetobutylicum
(Chojecki and Blaschek, [60], Ensley et al. [61], Hockenhull and
Herbert [62]) and C. thermohydrosri~uricum(Hynn and Zeikrrs
[63, 641) have also been reported to produce glucoamylase.
Frikzii and Nikrini [6S] produced glucoamylase from a yeast
strain of Enclomyces sp. Everrova (661) reported a glucoamylase
preparation from Endomycopsis capsularies. Occurance of glu-
coamylase in Schwanniomyces occidentalis and in Saccharomy-
ces diastaticrrs was reported by Gellissen et al. [67] and Yama-
shita et al. (681. respectively.
Several strains of Aspergillus and Rhizopus have most com-
monly be exploited for the synthesis of glucoamylase (cf. Ta-
ble 1). Considerable efforts have been made to synthesis it in
Aspergillrrs sp. (Manjrrnath et al. [72], Tan; et al. [73]),A. awam-
ori (Hayashida, [74], Atria and Ali [7S], Srniley et al. [76], Haya-
shida et al. [77] and [79],Facciotti et al. [81]),A. candidus [86],A.
foetidus (87-901, A . niger (Bartoszweick [91], Mitra and Sane
[93], Li et al. [95], Barton et al. [99], A . oryzae [46, 1121, A. phoe-
nicrrs [48, 1161 and A . saitri [I 171. It is clearly evident from Ta-
ble 1 that A . niger is mainly used for glucoamylase production.
Rhbopus sp. are also known to produce glucoamylases. It is
known that glucoamylase of Rhizopus sp. is generally pro-
duced by solid cultures consisting of wheat bran and produc-
tivities are better when compared with liquid cultures. Nishise
et al. [lS] reported a liquid culture system for the production
of glucoamylase using Rhizopus sp. Production of raw starch
digestive glucoamylases showing different behaviour (towards
raw starch) were reported by several workers using Rhizopus
sp. (Takahashi et al. [12] and Tani et al. [14]). Aspergillus sp.
(Medda et al. [13]), R. niveus (Saha and Ueda, [154]) and A.
awamori var. kawachi (Hayashida and FIor [78]). Some workers
also investigated the behaviour of the multiple forms of the
glucoamylases towards raw starch from other sources as A . ory-
zae (Miah and Ueda [114]), Mucor rouxianus (Yamazaki et al.
[ll]) and Monascus kaoliang (Zizuka and Mineki [134, 1361.
Few thermophilic fungi have also been used for the produc-
tion of glucoamylases which is expected to be thermostable.
Ruo et al. [160, 1631 reported thermostable glucoamylase from
the culture filtrate of Therrnornyces lanuginosus. Subraharnan-
yam et al. [69] described a thermostable glucoamylase prepara-
tion from Iorulu therrnophilia. Tayloret al. [133] reported a ther-
mophilic culture of Humicola lanuginosa which produced ther-
mostable glucoamylase.
starch/starke 4 7 (1995) Nr. 11 S 439-445
Table 1 . Microorganisms Producing Glucoamylases. 5 Characterization of Glucoamylase
Strains References
A number of reports have appeared on purification and charac-
Bacillus stearothermophillus WI terization of glucoamylases from different sources. Table 2
Flavobacterium sp. I551 cites some of such examples. It is evident that glucoamylases
Halobacterium sodamense [561 are generally active on acidic side of neutrality with optimum
Clostridium sp. P 7 , 581 values around 4.5-5.O.The glucoamylases I and I1 from a ther-
C. Thermosaccharolyticum 1591 mophilic fungus Humicola Ianuginosa showed the pH optima
C. acetobutylicum [60-621
C. thermohydrosulSuricum
at 4.9 and 6.6, respectively (Tayloret al. [133]). Glucoamylase I1
[63, 641
Endomysis sp. [651 so produced is stable up to pH 11.0. The glucoamylases from
Endomycopsis capsularis [661 C. cerebella (King, [130]). and C. rolfsii (Kaji et al. [166]) was sta-
Schwanniomyces occidentalis [671 ble up to pH 9.0 while that of M. rouxianus showed pH stabil-
Saccharomyces diastaticus [681 ity up to 8.0 (Yamasaki et al. [ll]). The optimum temperature
Tomla th erm o p hilia (691 for glucoamylases from most of the microorganisms is be-
Acremonium zonatum [701 tween 40’-60’C (cf. Table 2).
Amylomyces rouxii [711
Aspergillus sp. [13, 72-74] A bacterial glucoamylase was purified and characterized from
A. awamori [7, 75-85] the anaerobic thermophilic bacterium Clostridium thermosac-
A. candidus 1861 charolyticum by Specka et al. [59]. The enzyme which consisted
A. foetidus [87-901 of a single subunit has an apparent molecular weight of75,OOO.
A. niger [42-45, 84, 91-1111 The purified enzyme attacked a-1,4- as well as a-1,6-glycosidic
A. oryzae [46, 112-1151 linkages in various a-glucans. The enzyme showed the pH op-
A . phoenicus [48, 1161 tima at 5.0 and temperature optima at 7OoC and attacked poly-
A. saitri 11171 sacharides preferentially. Medda et al. [ 131 studied the raw
A . terreus [47, 113, 118-1261
starch adsorption and elution behaviour of glucoamylase I of
Cephalosporium eichhorniac ~ 7 1
C. charticola black Aspergillus sp. The enzyme was completely adsorbed
[I281
Collectotrichum gloesporordes ~291 onto raw starch at pH 3.4 which was the isoelectric point of the
Coniop h o ra cerebella ~301 enzyme.
Fusidium sp. [131, 1321 Glucoamylase of A. awamori var. kawachi was found to consist
Humicola lanuginosa WI of three types ( Yoshino and Hayashida, [23]), GA I, GA I’ and
Monscus kaoliang 1134, 1361 GA 11, with molecular weigths 90.000, 83.000 and 57.000 re-
Mucor rouxianus [ l l , 1351
iM. javanicus
spectively. GA I and G A I’ were optimally active at pH 3.8
[I371
Neurospora crassa [I381
while GA I1 at pH 4.0 and the pH stability range was 2-10,2.5-
N. sitophila [ 1391 6.5 and 4-7, respectively, for these. The isoelectric points were
Paecilotnyces globosus [I401 3.55, 3.45 and 3.28 and at 60°C,GA I was stable while GA I
P. varioti ~411 and GA I1 were unstable. Similarly, the former could hydro-
Penicillium oxalicum [142, 143, 1441 lyse raw starch while other two could not. The N-terminal
Piricularia oryzae [I451 amino acids were alanine for G A I, alanine plus isoleucine for
Rhizopus sp. [12, 14, 15, 146, 1471 GA I’ and isoleucine for GA 11. Hayashida and Flor [78] re-
R. delemer [ 148- 1511 ported production of high amount of raw starch digestive glu-
R. javanicus [154, 1531
R. niveus [154, 155, 156-1581
coamylase from a mutant starin of A . awamori var. kawachi
Schizophyllum commune [I591
which has reduced protease activity.
Therrnomyces lanuginosus V601 A commercial preparation of glucoamylase from A . niger con-
Trichoderma viride [161, 1621 sisted of at least six species (Ono et al. [ 170, 171]),five forms of
which were fully characterized. They were apparently different
in molecular weight (Table 2). The optimum pH varied be-
tween 3.5-5.0 and when compared with enzymological
There are only few reports which deal with the production of characteristics, one of the species was peculier and quite differ-
amylases or glucoamylases using Basidiomycetes. Kawai [ 1641 ent from others. Pazur et al. [98] reported two forms of glu-
reported occurence of amylolytic activity in Basidiomycetes coamylase from A. niger. The molecular weight of glucoamy-
strains. Shimazaki et al. [ 1591isolated an extracellular sacchari- lase I was 99,000 and that of glucoamylase I1 was 1,12,000.
fying amylase from Schizophyllum commune which was identi- Both forms of glucoamylases contain covalently linked carbo-
fied to be glucoamylase. Schellart et al. [161,162] reported glu- hydrate (containing D-mannose, D-glucose and D-galactose
coamylolytic activities in the strains of Trichoderma viride. residues) and are therefore glycoenzymes. Glucoamylase I
Similarly, there are few reports which describe glucoamy- and I1 showed identical amino acid composition. However,
lolytic activities in yeast strains such as in Endomyces sp. the two glycoenzymes differ in carbohydrate content, glu-
( F u k u i and Nikuni, [65]), Endomycopsis capsularis (Evertova, coamylase I1 containing as much as double carbohydrate units
[66]), Schwanniomyces occidentalis (Gellissen et al. [67]) and per molecule as glucoamylase I. The carbohydrate-protein
Saccharomyces diastaticus (Yamashita et al. [68]). There is yet linkage in the glycoenzyme is essentially glycosidic to the hy-
another class of microorganism viz. phytopathogenic fungus droxyl group of L-serine and L-threonine residues.
which secrets starch degrading enzymes. Though, there are at There are several reports which deal with the characterization
least three phytopathogenic fungi, Rhizoctonia solani, Cla- of two forms of glucoamylase from A . niger (Fleming and Stone,
dosporoium cladosporioides and Collectotrichum capsici which [172]; Lineback and Aira, [173]; Lineback et al. [89]; Azuret al.
have been reported to have amylolytic activities in culture fil- [98, [I741 and Saito [175]). Glucoamylase I was shown to con-
trates (Charya et al. [ 1651. Collectotrichum gloeosporoides prob- tain 13% carbohydrate and had a molecular weight of 74,900
ably, is the only one phytopathogenic fungus which produce while glucoamylase I1 had a molecular weight of 54,300 and
glucoamylases also (Kruuse et al. [129]). contained 18% carbohydrate [ 1731. Mannose, glucose and

starch/stlrke 47 (1995) Nr. 11 S 439-445 44 1

Table 2. Characterization of Glucoamylase.
Source Forms Mol.Wt. Opt. p H Opt. Temp. Raw Starch Ref.
(“C) Digestion
Aspergillus awamori 1 83,700-90,000 4.5 60 +
A. awamori var. kawachi 3 57,000-90,000 - - +
A . awamori var. kabachi 3 90,000 3.8 - +
83,000 3.8 - -
57,000 4.0 - -
A . awarnori var. kawachi 2 90,000 4.5-5.0 - +
A . niger 6 99,500 3.5-4.0 - 0
83,200 4.0-4.5 0
58,400 5.0 - 0
80,800 4.0-4.5 - 0
78,500 4.0 - 0
A: niger 2 99,000 4.5-5.0 - 0
1,12,000
A. niger 1 90,000 4.5 50 0
A. niger - - 4.0 60 0
A . otyzae 3 76,000 4.5 60 +
38,000 4.5 50 0
38,000 4.5 40 0
A. niger 3 80,000 3.5-4.0 50-60 0
90,000 0
70,000
A. phoenicus - - 4.5 60 0
A. saitri - 90,000 4.5 - 0
A. lerreus - - 4.0 60 0
A . terreus - 70,000 5.0 60 0
Cephalosporiura
chart ico la - 54 60 0
Coniophora cerebella - 4.0-4.5 0
Corticum rolfsii - - 4.5 40-50 0
Clostridium
thermosaccharolyticum 1 75,000 5.0 70 +
Ciosrridium sp. 1 77,000 4.5 65 0
Endomyces sp. - 55,000 - 0
Endomycopsis capsularis - 4.5 40-50 0
Endomycopsis sp. - 53.000 -
Hum icola la n ugin 0s 2 - 4.9 65 0
6.6 70 0
Monascus kaoligang 2 48,000 4.5 50 0
68,000 4,7 60 0
Mucor rouxianus 2 59,000 4.6 55 +
49,000 5.0 55 +
Paecilomyces varioti 1 69,000 4.5 - 0
Penicillium oxalicum 2 84,000 5.0 55-60 +
86,000 4.5 60 +
Piricularia oryzae 1 94,000 6.5 50-55 0
Rhizopus sp. 3 74,000 4.5-5.0 - +
58,600 +
61,400 +
Rhizopus sp. 2 - - +
R. delemer 1,00,000 4.5 40 0
R. niveus 5 4.5-6.0 - +
Schizophyllum commune 1 66,000 5.0 40 +
Thermomyces lanuginosus 1 57,000 70 0

galactose were the sugars present. The carbohydrate moieties
in both were same and were a-glucosidically linked through
mannose to hydroxyl groups of serine and threonine in the
polypeptide chain. The amino acid composition of glucoamy-
lases also varied from species to species. The difference be-
tween the basic and acidic amino acids residues ofA. nigerglu-
coamylase I and 11, is 25 and 14, respectively [173]. The ab-
sence of methionine distinguishes the glucoarnylases from En-
domyces sp ( F u k u i and N i k u n i , [65])and Endomycopsis s p . (Gra-
cheva et al. [167]). Glucoamylase from A.phoenicis did not con-

442 starch/stlrke 4 7 (1995) Nr. 11 S 439-445

tain any tryptophan and only one unit of methionine was de-
tected in it. It did not contain any amino sugars (Lineback and
Baumann [21]. Ono et al. [169] purified the glucoamylase of A.
oryzae by extraction with 1% NaCl solution, precipitation with
ethanol and acarbose affinity chromatography. The purified
enzyme was homogeneous and was a glycoprotein containing
about 4.8% glucosamine and 7.8% neutral saccharides.
A major homogeneous glucoamylase was purified from A . sai-
tri (Takahashi et al. [117]) which was glycoprotein containing
18% neutral sugars and 0.77O/o glucosamine. Its molecular
weight was estimated to be 90.000 and the N-terminal amino
acid was identified as alanine. The enzyme was stable between
pH 2.5 and 7.5 (optimum pH 4.5) and it retained full activity at
temperature up to 5OOC.
Ali and Hossain [118] studied the characteristics of glucoamy-
lase from A . terreus. The enzyme was a glycoprotein with an
isoelectric point at 3.4 and was optimally active at pH 4.0 and
6OOC. It was remarkably stable over a pH range of 3.0-7.0. The
enzyme exhibited specificity for substrate containing a-1,4-
glucosidic linkages. Gosh et al. [120] reported purification and
characterization of glucoamylase of A . terreus NA-170 mutant
which was shown to be homogeneous. The purified enzmye
showed the pH and temperature optima at 5.0 and 60°C with
stability range between pH 3.0-7.0 and 3Oo-75OC, respectively.
Ueda and Kano [146] purified the glucoamylase system of Rhi-
zopus sp. By fractionation, two kinds of glucoamylases were
obtained; one has a strong debran,ching activity while the
other has a weak debranching activity. Morbam et al. [I551
studied the influence of dielectric constants and ligand bind-
ing on thermostability of glucoamylase from R. niveus. Investi-
gations were carried out mainly at 60°C (pH 4.5) in relation to
kinetics both in the presence and in absence of various li-
gands. With substrate analogues, such as glucose, lactose and
gluconolactones and with glycerol, the thermostability of the
enzyme greatly increased where as with dioxane, ethanol and
glycol, it decreased. From the model equations, binding con-
stants of the ligands were estimated in order to confirm the va-
lidity of the assumptions. Saha and Uedu [I541 studied the raw
starch adsorption, elution and digestion behaviour of glu-
coamylase of R. niveus. The preparation consisted of five
forms which were separated by preparative isoelectric focus-
ing. All of them were strong in debranching activity, raw starch
adsorption and digestion. Among them, two were major, glu-
coamylase C and D having isoelectric points 8.45 and 9.1 and
carbohydrate contents 14.9 and 12.7%, respectively.
Takahashi et al. [12] reported three kinds of glucoamylase
from Rhizopus sp., Gluc 1, Gluc 2 and Gluc 3 which have simi-
lar pH optima and their molecular weight was estimated to be
74,000,58,600 and 61,400, respectively. The pH optima for raw
starch digestion were different and all the enzymes digested
raw starch. Two forms of glucoamylases were isolated and pu-
rified from the culture ofMonascus kaoliang nov. sp. F-I which
were homogeneous in nature. The G A I and G A I1 exhibited a
pH optima at 4.5 and 4.7, respectively. The molecular weight of
GA I was 48,000 and that of GA I1 68,000 (lizuka and Mineki,
[134]). Takeda et al. [141] studied the purification and substrate
specificity of glucoamylase of Paecilomyces variori AHU 9417.
The enzyme was purified by precipitation with ethanol, chro-
matography on DEAE-Sepharose CL-6B, gel filtration and
preparative disc electrophoresis. The enzyme was homogene-
ous. The molecular weight was estimated to be 69,000 and pH
optima at 4.5. The enzyme preferentially hydrolyses a-1,4-glu-
cosidic linkages in a series of maltooligosaccharides and is ca-
pable of slowly hydrolysing isomaltose, panose and nigerose.
Piricularia oryzae, the pathogenic fungus of rice blast disease,
produced a glucoamylase which was an optimum pH at 6.5
and optimum temperature between 5Oo-60"C. The molecular
starch/stlrke 47 (1995) Nr. 11 S 439-445
weight was 94,000 (Yuhki et al. [145]). The glucoamylase of 7h-
ermomyces lanuginosus established to be homogenous [160].
The enzyme was a glycoprotein with an average molecular
weight of about 57,000 and a carbohydrate content of 10-12'Yo.
The enzyme hydrolysed successive glucose residues from the
non-reducing ends of starch molecule. It did not exhibit any
glucosyltransferase activity. The enzyme was unable to hydro-
lyse 1,6-a-glucosidic linkages of isomaltose and dextran. It
was optimally active at 7OoC and showed remarkable resis-
tance towards chemical and thermal denaturization.
Shimazaki et al. [159] reported an extracellular saccharifying
amylase preparation from Schizophyllum cotnrnune which was
identified to be glucoamylase. The enzyme was purified by
ammonium sulfate precipiation, acid-clay-treatment and Se-
phadex G-100 gel filtration which gave a single band.
Acknowledgements
The author is grateful to Dr. A. D. Damodaran, Director and Dr. Vijay
Nair, Head, Chemical and Life Science Division, for their interest in
the project on glucoamylase production. He thanks Mr. P. Selvakumar
for his help in the preparation of the manuscript. A research grant
from the Department of Biotechnology, Govt. India, New Delhi, is
gratefully acknowledged.
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Address of author: Dr. Ashok Pandey, Scientist. Regional Research
Laboratory, Trivandrum.
Trivandrum-695019. India.
(Received: April 21, 1993).
445