298 J. hist. Brew.. September-October, 1984, Vol. 90, pp.

298-302

EFFECT OF pH, TEMPERATURE, AND CALCIUM IONS ON BARLEY MALTct-AMYLASE

ISOENZYMES

By E. Blrtoft, C. Anotiolk andS-E. Kulp

(Department ofBiochemistry and Pharmacy, Abo Akademi, Porthansgatan 3, SF- 20500 Abo 50. Finland)

Received 31 January 1984

In this study we have detected the a-amylase isoenzymes in the two barley varieties 'Ingrid' and 'Porno'. We
further report on the effect of different pH and temperatures on the activities of the a-amylase I and II (following
the nomenclature of MacGregor & Daussant18) in 'Porno' malt, as well as the stabilising effect of Ca2* under
such different conditions. The importance of enzyme concentration and the inhibition of Hg2* is also considered.

Key words: pH. calcium, a-amylase. isoenzymes.
Introduction
Since the work of Frydenberg and Nielsen7 on 98 varieties,
it has been known that the a-amylase system in barley is
composed of isoenzymes. Reports on the number and
amounts of these isoenzymes vary in the literature possibly
due to differences between barley varieties7 and to the broad
spectrum of separation and detection methods used.12162026
The isoenzymes are synthesised at different stages under
the'development5-21 and germination1417 of the seed, which
has to be considered when researching the enzyme systems
in barley. Because of such differences, and of the great
number of nomenclature systems used, it is rather difficult
to compare the results from different laboratories.
By immunological methods the a-amylase isoenzymes are
divided into two main groups.610" The a-amylases synthe
sised in the aleurone layer are dependent on the plant
hormone gibbcrellic acid, whereas those synthesised in the
scutellum are not.8
The effect of different conditions on the stability of barley
a-amylase isoenzymes has only been partly established.
Differences between the isoenzymes are obtained at high
temperatures and low pH,712 •>•"«• and with respect to their
calcium ion requirements and sensibility to inhibitors.712
Experimental
Purification ofa-amylase bycompetitive affinity chromato-
graphy.—Two commercial barley varieties, 'Porno' and
'Ingrid', and malted barley of the same batches, were used
for isolation of a-amylase by the method of Kruger &
Tkachuk.13 Purification by competitive affinity chromato
graphy on glycogen-AH-Sepharose 4B was performed as
described by Tkachuk.27 To eluated fractions (4-5 ml) con
taining a-amylase, 45 ul 01 m calcium acetate, pH 5-5, was
added, and they were left over night at 20°C to hydrolyse the
glycogen.
Preparative separation of a-amylase isoenzyme I and II
by ion exchange chromatography.—a-Amylase was isolated
from 'Porno' malt by the method of Bathgatc & Palmer.2
After heat treatment at 70°C for 15 min denatured protein
was centrifuged off and the a-amylasc was precipitated with
60% (NH4)2SO4, dialysed against water and finally freezc-
dried. This preparation will be referred to as the 'crude
extract'.
Separation of the isoenzymes was performed by ion
exchange chromatography essentially as described by
MacGregor et a/,20 though we used CM-Scpharose CL-6B
(2-5xl4-5cm column). About 100mg (corresponding to
6800iu) of the freeze-dried a-amylasc dissolved in 005 m
sodium acetate buffer pH 50 was applied to the column.
Elution was initially performed with the acetate buffer and
fractions at 4-8 ml were collected for detection of a-amylasc
I. The elution medium was then changed to acetate buffer
containing 0-3 m NaCI to eluate a-amylase II. The two iso-
enzymc preparates were concentrated with Diaflo-Ultrafilter
UM-10 (Amicon) and rechromatography was then per
formed. The purified isoenzyme preparations were finally
concentrated and freeze-dried.
a-Amylase activity measurements.—a-amylasc activity in
isolated preparations was determined by the Phadebas
method24 at 37°C (0-2 m acetate buffer, pH 5-4).
The activity in fractions from column chromatography
was measured with an iodine reagent (0015% l2 and 015
& Kl in water) as follows: 100 mg soluble starch (Merck) was
suspended in distilled water and boiled. 10ml 2 m sodium
acetate buffer (pH 5-4) and 011 g CaCl2 was added, and the
solution diluted to 100 ml with distilled water. To 1 ml of
the starch solution (37°C) 0-1 ml of the test solution was
added and incubated for 15 min at 37°C. 10 ml iodide re
agent was added and the absorbancc measured at 620 nm
against buffer. A standard calibration curve was made with
isolated 'crude extract' of known activity (determined by the
Phadebas method). The curve was linear in the range 0-25
to 2-Oiu/ml. If necessary the test solution was diluted to
correspond to this range.
In stability experiments a-amylase activity was measured
as follows: As substrate, a freshly prepared solution of gelati
nised 'Porno' barley large starch granules (5 mg/ml) was used
in 0-5 M sodium acetate buffer, ph 5-4. The starch granules
had been prepared by the technique of Bathgatc at a/1 To
09 ml of the starch solution 0' 1 ml enzyme solution from
the stability experiment was added and incubated for 10 min
at 30°C. The reaction was stopped by adding 0-1 ml of 3%
NaOH and the reducing power measured by the Nelson
method.21 Any residual starch was*centrifuged off before
absorbancc was read at 540 nm against a blank without
enzyme. Results were expressed as a percentage of the
activity of an untreated enzyme.
Separation of a-amylase isoenzymes by isoelectric focus
ing.—Separation of isoenzymes by isoelectric focusing on
polyacrylamid gel plates was performed with the LKB
Ampholinc PAG plates. pH gradient 3-5—9-5, according to
the maker's instructions. The focusing time was 90 min. The
pH gradient in the gel was measured with a surface electrode.
a-Amylase isoenzymes were detected by activity staining
by incubation of the PAG plates in 1% soluble starch in
0-2 m sodium acetate, pH 5-4, containing 001 m CaCI2, at
37°C for 20 min. Residual starch was developed in a 004%
of I, in 0-4% KI solution.
pH-optimum.—Starch solutions (gelatinised large starch
granules, 5 mg/ml) with different pHs were incubated with
a-amylase I (0-25 mg/ml) or a-amylase II (005 mg/ml) for
10 min at 30°C and the reducing power was then measured
according to Nelson.23 For pH below 5-6 a 005 m sodium
acetate buffer was used, and over pH 5-6 005 M sodium
phosphate buffer was used.
Temperature optimum.—Gelatinised large starch granules
(5 mg/ml, 005 M acetate buffer, pH 5-4) were incubated with
Vol. 90, 1984] BERTOFT ETAL: O-AMYLASB 1SOBNZYMHS 299

1 1 2 3 4
Fig. 1. a-Amylase isoenzymes separated by isoelectric focusing in a pH 3-5-9-5 gradient. The two main groups
of a-amylases arc designated a-1 and ct-II. (a) Isocnzymc pattern in preparations purified by competitive affinity
chromatography. I. Porno malt (two samples); 2. Porno barley; 3. Ingrid malt; 4. Ingrid barley, (b) Isoenzyme
pattern of a-amylases prepared from Porno malt. 5. Freeze-dried preparation of a-amylasc I after ion exchange
chromatography; 6. Freeze-dried preparation of a-amylasc II after ion exchange chromatography; 7. Freeze-
dried crude enzyme extract; 8. Crude enzyme extract heat treated at 60"C for 100 min.

TABLE I. Some Characteristics of a-Amylases in Malted Porno pH 5-4 containing different concentrations of CaCl2. To
Barley 0-9 ml enzyme solution 01ml 10-4m HgCl2 was added.
After 10 min at 30°C the activity was measured as described
Isoelectric Temperature above.
Sample point pH optimum optimum (°C)

a-Amylasc I 4-1-4-3 3-0-5-5 70

a-Amylase II 5-3-6-1 5-0-5-4 65
a-amylase I (0-15mg/ml) or a-amylase II (0-05 mg/ml) at
different temperatures for 1 min and the reducing power was
then measured."
Temperature stability.—Different concentrations of the
isoenzymes in 005 M acetate buffer were held at 45°, 60°, or
70°C. At suitable time intervals 0-1 -ml aliquots were taken
for activity measurement at 30°C.
The effect of ethylenediaminetetraacetate (EDTA) at dif
ferent temperatures were measured as follows: To 0-9 ml of
the enzyme solution (a-amylase 1 1-6 mg/ml or a-amylase II
0-6 mg/ml) was added 0-1 ml 100 him EDTA solution and
incubated for 10 min at the respective temperatures. Activity
was then measured as described above, with the exception
that the used gelatinised starch solution contained 10 mM
EDTA.
To measure the effect of high temperature at different pH
a 0-01 M acetate buffer was used at pH 5 and 6, and 0-01 m
barbital-acetate buffer at pH 7 and 8. The stability was also
tested in the presence of 001 m CaCl2. After heat treatment
for 5 min at 70°C the pH was adjusted to 5-4 and the activity
measured at 30°C.
Stability at low pH and i(TC.—To 1 ml enzyme solution
(2-1 mg/ml of a-amylase I or 0-75 mg/ml of a-amylase 11)
was added 0-5 ml 003 m acetate buffer pH 3-5 (giving a final
pH of 3-6). At suitable time intervals, 0-1 ml aliquots were
taken for activity measurements. The stability of a-amylase
II was also tested in the presence of bovine serum albumin
(BSA) and CaCl2.
Inhibition by mercury chloride.—a-Amy\ase 1(1-6 mg/ml)
or a-amylase II (0-6 mg/ml) was dissolved in acetate buffer
Results
Competitive affinity chromatography on glycogen-AH-
Sepharose 4B gave very pure a-amylase preparations as
shown by isoelectric focusing, though the yield from the
purification was poor, especially in the case of barley malt
samples. This was probably due to a limited capacity of the
column used. Isoenzyme compositions in the tested varieties
after separation by isoclectric focusing arc shown in Fig. l(a).
The isoamylases constituted two clearly distinct groups.
a-Amylase I was the main component in the barleys and was
composed of one or two strong and one weaker band. Malt
contained only small amounts of this isoenzyme group and
it was not always detectable on the gels. a-Amylase II was
composed of one dominating band and two or three weaker
bands. Isoelectric point (IP) values of the two isoenzyme
groups are given in Table I. A very weak band (IP 6-9)
showing amylase activity was found in the two barley var
ieties, but not in the malts. This band possibly represents
a-amylase III.14
Preparative separation of 'Porno' malt a-amylase isoen
zymes on CM-Sepharose CL-6B gave a yield of 75% of the
total applied activity. The two eluated peaks did not how
ever contain completely separated isoenzymes. A second ion
exchange chromatography of the respective peaks gave a
pure a-amylase II preparation. The a-amylase I contained
hardly detectable amounts of a-amylase II [Fig. l(b)], and
was considered as pure a-amylase I in the following exper
iments. The a-amylase I preparation had an activity of
5 iu/mg and a-amylase II 33-5 iu/mg.
Table I gives the pH- and temperature optima for the two
purified isoamylases. In Fig. 2 the effect of different tem
peratures on the stability of the enzymes, without and in the
presence of EDTA, is shown.
a-Amylase I is sensitive to high temperatures. A 10 min rest
at 60°C inactivates the enzyme almost completely, whereas
a-amylase II still is very active EDTA has a marked effect
300 BHRTOFT/;7"/l/.: a-AMYLASE 1SOI-NZYMES [J. InSt. BrCW.

TABLE 11. Half Times for Heat Inaclivalion of Malt a-Amylases

a-Amylaso I a-Amylase II
100

Sample
concentration Temperature t}
Sample (mg/ml) (°C) (mm)

a-Amylasc 1 1-50 45 7600

1-50 60 2-33
2-25 60 6-90
300 60 8-63
50 1-50 70 0-69
a-Amylasc 11 0-50 45 29000
0-25 60 20-29
0-50 60 25-56
100 60 6900
0-50 70 9-48
a-Amylase 1 + 2-25 +
60 2300*
a-Amylase II 0-25

fL. *tifor the initial 50 min of heat inactivation.

30 60 30 60

Temperature CO

Fig. 2. Stability of barley malt a-amylascs at different temperatures.

The enzymes were treated at the respective temperature for
10 min. The stability is expressed as a percentage of the origin a-Amylase I a-Amylase II
activity at 30°C. D=Without EDTA; ■ in the presence of 100
lOmM EDTA.

100

50

S 50
r
pH

Fig. 4. Stability of barley malt a-amylascs at 70°C, 5 min, at different

pH. The stability is expressed as a percentage of the original
activity at 308C, pH 5-4. D. Without CaCL; ■, in the presence
oflOmMCaCL.

60 90
100
Time (min)

Fig. 3. Stability of barley a-amylascs at 60°C. The stability is

expressed as a percentage of the original activity at 30°C. •,
a-Amylase I, 1-50 mg/ml; ■, a-amylase I, 2-25 mg/ml; O,
a-Amylase II, 0-25 mg/ml; D, a-Amylasc II, 0-50 mg/ml; A, a
mixture of a-amylase I (2-25 mg/ml) and a-amylase II
(0-25 mg/ml).

on the stability of a-amylase I at all temperatures, a-Amy

lase II is sensitive to EDTA only at high temperatures (60°C).
In such circumstances it is, however, completely inactivated.
The stability of the enzymes was further tested at 60°C as
a function of incubation time and with different enzyme con
centrations (Fig. 3). a-Amylase I is inactivated considerably
faster than a-amylase II despite a much higher concen
tration. A concentration of 0-5 mg/ml a-amylase II makes
the enzyme stable up to 5 min, whereas a three-fold concen
tration of a-amylase I looses about 80% of its activity in this
period. The heat inactivation of the enzymes is a first order
reaction process. Thus the half times for heat inactivation
with different concentrations at 60°C can be calculated
(Table II). Half times at different temperatures are also given Fig. 5. Stability of barley malt a-amylascs at pH 3-6 and 30°C. The
stability is expressed as a percentage of the original activity at
in the table.
pH 5-4. •, a-Amylasc I; O, a-amylase II; A, a-amylasc II in the
The results seemed to show a possibility of preparing an presence of 1-33 mg BSA/ml; D, a-amylase II in the presence
a-amylase II preparation free from a-amylasc I activity by of 6-67 mM CaCI,.
Vol.90, 1984] BERTOFT ETALl a-AMYI.ASK ISOENZYMI-S
100
so
5 10 100
CaCI, concentration (mM|
Fig. 6. Inhibition of barley malt a-amylases by 10"s m HgCI2 in the
presence of CaCI,. The stability is expressed as a percentage of
the original activity in the absence of the inhibitor. ■. a-Amy-
lasc I; D. m a-amylasc II.
simple heat inactivation of the latter. Fig. 3 and Table II
therefore also show the results of a combination of the two
isoenzymes (corresponding to 11-2S iu/ml of a-amylase I
and 8-37 iu/ml of a-amylase II) with a total protein concen
tration of 25 mg/ml. This mixture gave a first order reaction
of inactivation up to 50 min, whereafter the reaction order
no longer was the same. A control with isoelectric focusing
of the mixture after 60 min at 60°C showed no difference in
isoenzyme pattern as compared with untreated enzymes. A
heat treatment ofthe freeze-dried 'crude enzyme' extract (0-5
and 2-5 mg/ml) up to 100 min was also tested with the same
result; a-amylase I was not inactivated more than a-amylasc
II [Fig. l(b)].
Figure 4 shows the stability ofthe isoenzymes after a 5 min
heat treatment at 70°C at different pH, without and in the
presence of 001 M CaCl2 The effect of Ca2+ ions is very
marked at all tested pHs especially a-Amylase I is stabilised
by Ca2\ and at higher pH a-amylase II is also completely
stabilised. At pH 5 the effect of Ca2+ on this latter enzyme
is however not quite so pronounced.
The stability of the isoenzymes at pH 3-6 is illustrated
in Fig. 5 as a function of time. a-Amylase I is very stable
at this pH. Even after 5 h the enzyme retained 85% of its
activity, whereas a-amylase II was totally inactivated after
5 min. The stabilising effect of an inert protein (BSA) and
of CaCI2 on a-amylase II was also tested. BSA had only a
minor effect on the enzyme, as compared to the very strong
effect of Ca2+.
A treatment of a dialysed 'crude enzyme' extract at pH 3-6
would thus be expected to give an enzyme preparation free
of a-amylase II activity. To test this hypotheses two concen
trations ofthe 'crude extract' were treated at pH 3-6 and the
resulting isoenzyme composition controlled by isoelectric
focusing. With a concentration of 2 mg/ml the amylasc
activity in the 'crude extract' decreased to 4% within 15 min,
and was then stable at this level even after I h. This treat
ment gave a preparation totally free from a-amylasc II
activity. A concentration of 10 mg/ml, however, stabilised
301
this latter isoenzymc, so it still had a clearly detectable
activity after 1 h.
Inhibition of a-amylasc I and II by I0~5M HgCl2 was inves
tigated with a different Ca;* concentrations (Fig. 6). In the
absence of Ca2* both isoenzymes were sensitive to the inhibi
tor. Addition of Ca2* stabilised a-amylasc II. At 01 m CaCI2
90% of the activity remained. With this Ca2+ concentration
80% of the a-amylase II activity remained even after a 50
times increased inhibitor concentration to 5 x 10"4 M HgCI2.
Contrary to a-amylase II the a-amylase I isoenzyme was not
influenced by Ca2f to any noteworthy extent. Its activity
remained at about 35-40% regardless of the calcium ion
concentration.
Discussion
The isoenzyme composition in barley was slightly differ
ent from that in malted barley as shown in Fig. l(a). Barley
contained comparatively large amounts of a-amylase I,
whereas a-amylase II was the dominating enzyme in malt.
MacGrcgor et a/21 found only a-amylase I in immature
'Conquest' barley. However, in 'Porno' barley, the same var
iety as was used in our work, MacGrcgor et a/19 also found
isoenzymes of the 11—111 group, which they believed
depended on germination ofthe barley. We found a-amylase
II in both 'Porno' and 'Ingrid' barley and it is therefore poss
ible that different varieties contain either a-amylase I or both
isoenzyme groups.
The compositions of individual bands in the two isoen
zyme groups were slightly different in 'Porno' and 'Ingrid'
varieties. However, the amount of enzyme applied on the
polyacrylamide gel and the separation method used are very
critical for the obtained result. We also tried clcctrophoresis
on agar and agarose gels without separation ofthe isoenzyme
groups.
The obtained IP values for the two isoenzyme groups
(Table I) are rather similar to those in 'Himalaya' barley10
but are lower than those reported by MacGregor14 in 'Con
quest1 barley. This probably also reflects differences between
barley varieties.
We found only trace amounts of a-amylase 111 in barley,
and in malt this isoenzyme was completely absent. a-Amy
lase III has been reported9 as a very sensitive isoenzyme that
is converted to a-amylase II upon heat treatment.18 The
malts had been kilned and the isolation ofthe enzymes con
tained a treatment at 70°C for 15 min, which could be the
reason for the absence of a-amylase III in this work. Ion
exchange separation of the isoenyzmes gave a very similar
isoelectrophoretic pattern as the very pure amylase prep
aration obtained by affinity chromatography (Fig. I), and
was thus regarded as a suitable method for preparative
isolation.
The obtained pH-optimum for a-amylase II (Table I) is
in good agreement with the optimum for isoenzyme A
reported by Tanaka & Akazawa.26 a-Amylase I had a very
broad pH-optimum, which also has been found for a-amy
lase components earlier,12-26 although it was lower than
previously reported. Our results are instead very similar to
the pH-optimum of 3-6-S-75 for a-amylase isoenzymes in
immature wheat.22 MacGregor15 reported a rather sharp
pH-optimum of 5-5 for barley a-amylase I. Again, the isoen
zymes in different varieites probably have slightly different
properties.
Earlier reported temperature optima for mall a-amylases
915 arc lower than our result (Table 1). However, at higher
temperatures the enzymes are inactivated and the obtained
results will be dependent on the enzyme concentration and
incubation time, as shown in Fig. 3. Under the conditions
ofthe assay (pH 5-4, incubation time 1 min) a-amylase 1 has
a higher temperature optimum than a-amylase II, despite
the fact that the former enzyme is much more sensitive to
high temperatures. It is also known that starch hydrolysis
proceeds at a very high rate under mashing conditions.34
302 BI-RTOKTfcT/l/L: a-AMYI.ASE ISOHNZYMI3
The reported results on heal stability of a-amylase isocn-
zymes have been somewhat controversial. According to
MacGregor" a-amylase 1 is very sensitive at 70°C. which was
also the result in this work (Fig. 3, Table II). Tanaka &
Akazawa26 obtained 30% inactivation of isoenzyme B and
even a heat activation of isoenzymc A. According to
Frydenberg & Nielsen' the isoenzyme group A, B was quite
heat stable, and the E, F, G group was converted into C. D
enzymes. The stability is strongly dependent on enzyme con
centration, and a mixture of the enzymes stabilises both
a-amylascs (Fig. 3). Calcium ions also have a very strong
effect on the heat stability of both isoenzymes (Fig. 4).
When investigating the inhibition of a-amylases by
EDTA, the condition of the assay is of great importance. The
calcium binding capacity is probably dependent on the con
figuration of the native enzyme, and strong binding would
be achieved only under optimal conditions. There seem to
be important differences in the calcium ion binding of the
isoenzymes. a-Amylase II looses Ca2+ easily only in unfa
vourable conditions. Thus, at 60°C a-amylase II was strongly
inhibited by EDTA, but not at lower temperatures (Fig. 2).
On the other hand EDTA did affect the activity of a-amylase
I even at lower temperatures. In the presence of a surplus
of Ca2+ the stabilising effect on a-amylase I is very strong
(Fig. 4).
a-Amylasc I was very stable at pH 3-6 (30°C), whereas
a-amylasc II was rapidly inactivated (Fig. 5). This is in good
agreement with earlier results.71215 However, Ca2+ was
shown to affect the results, making a-amylasc II quite stable
at this low pH.
HgCl, is commonly used as an enzyme inhibitor when iso
lating starch from cereal grains. It is interesting to notice that
Ca2+ can neutralise the inhibition by HgCI2 of a-amylase II,
but it has no effect on a-amylase I (Fig. 6). Possibly the stabi
lising effect of Ca2+ on the configuration of a-amylase II is
so strong that Hg2+ no longer affects the activity. It is also
possible that Ca2+ and Hg2+ competitively bind to the same
site in the enzyme, so that Hg2+ is not complcxed at higher
Ca2* concentrations. The binding of Hg2+ to a-amylase I is
either so strong (irreversible?) or the binding of Ca2* so weak
that high Ca2+ concentrations have no effect. The enzyme
was composed of two isoenzyme bands [Fig. l(b)], and it is
possible that only one of these was inhibited by HgCl,,
because 35% of the activity of a-amylase I remained at all
calcium ion levels.
The stability and inhibition tests were done to find an easy
way to get an isoamylasc preparation free from activity of
the other isoenzyme. A treatment for 15 min at pH 3-6 of
a dialysed crude enzyme extract (2 mg/ml) gave a prep
aration containing only a-amylase I activity. According to
the great difference in heat stability of the two isoamylases
(Figs 3 and 4), it would have been possible to get a prep
aration containing only a-amylase II activity, though the
heat treatment would cause a considerable loss of this ac
tivity too. Surprisingly, a-amylase I was very stable in a
mixture of the isoenzymes, so that a preparation free of its
activity was impossible to achieve. It seems that the isoen
zymes stabilise each other. Scheifer el aP* have suggested an
(J. Inst. Brew.
interesting theory about the starch synthesising enzymes,
where starch synthetase and branching enzyme form a
functional complex giving rise to the branched amylopectin
molecules, while the free synthctase only synthetises straight
polysaccharide chains. We therefore believe it could be
possible that the starch degrading a-amylase isoenzymes also
form a functional complex possibility affecting the starch
hydrolysis. At pH 3-6 (and moderate protein concentration)
this complex would be separate into its components. Isoelec-
tric focusing possibly has the same effect. If this hypothesis
is true it will be of great importance when discussing starch
hydrolysis in the germinating barley.
Acknowledgements.—We are grateful to Dr H. Maunula,
Raision Tchtaat. Raisio (Finland), for kindly providing the
barley and barley malt samples.
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