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Korean Biochem. J.(1985). Vol. 18. No.3, pp. 222-232

Purification and Characterization

of Glucoamylase from Aspergillus shirousamii*

Byung Gook N at and Chul- Hak Yang''

Dept. of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151, Korea t Jinro Research Institute, Bucheon

(Received July 3, 1985)

Abstract: Glucoamylase (a -1,4 glucan glucohydrolase, EC 3.2.1.3) of Aspergillus shirousamii was purified using ammonium sulfate fractionation, DEAE-Sephatel chromatography, affinity chromatography on Concanavalin-A Sepharose 4B and isoelectric focusing. The purification achieved was 19.8 fold from crude extract with a yield of 47.5%. Its molecular weight was estimated to be about 89,000 by Sephadex G-200 gel filtration and SDS-polyacrylamide gel electrophoresis. The pI value of the enzyme was 3.4. The enzyme was stable below 55°C and retained almost full activity after 1 h. But, it was very labile over 70°C. At this temperature, it lost almost all activity witin 10 min. Maximum hydrolysis was occured at 65°C for 15 min and 60°C for 60 min, respectively. The enzyme was most stable at pH 4.5, and was more stable in the acidic region than in neutral region. The pH optimum of the enzyme was 4.5 with p-nitrophenyl a -D-glucopyranoside as a substrate. The enzyme activity was inhibited by Hg++. Ca++ markedly increased the heat stability of the enzyme, but it did not affect on the enzyme activity. Most of the anions tested showed the inhibition effect on the enzyme activity. Among these, B407 -2, As02- and S03 -2 inhibited remarkably up to 94%, 95% and 88%, respectively. In kinetic studies, the enzyme hydrolyzed maltotriose more than twice of the rate for maltose. Isomaltose and p-nitrophenyl a - D-glucopyranoside showed much lower relative rate of hydrolysis than maltose, 0.15 and 0.066, respectively. For the highmolecular-weight substrates, amylopectin showed the highest and dextrin showed the lowest relative rate of hydrolysis, respectively.

Glucoamylase (EC 3.2.1.3, a -1,4 glucan glucohydrolase, r -amylase, amyloglucosidase) is an exo-acting enzyme which hydrolyzes a-l,4 glucan links in polysaccharides so as to remove succesive glucose units from the non-reducing ends of the chains. It is an extracellular enzyme and is excreted to the culture medium. The existence of glucoamylase was reported for the first time by Kitahara et al. (1949). They found the existence of a new saccharifying enzyme in the culture medium of Aspergillus

§ Reprint request to Chul-Hak Yang.

* This work was supported in part by a grant from the Korea Science and Engineering Foundation (1984).

awamorii and named it r -amylase. Thereafter, it has been purified and characterized from various fungi by many investigators: from Aspergillus sp. (Mahmoud et al., 1978; Takahasi et al., 1981; Razzaque et al., 1981; Inokuchi et al., 1978; Manjunanth et al., 1981), from Rhizopus sp. (Takahasi et al., 1981; Handa et al., 1976; Tanaka et al., 1983; Hou and Chung, 1984a,b), from Mucor sp. (Yamasaki et al., 1977) and from Piricularia sp. (Yuhki et al., 1977). Glucoamylases occur almost exculusively in fungi and the enzyme used commercially originate from strains of either Aspergillus sp. or Rhizopus sp. Recently it also has been found in some bacteria (Fla-

222

Glucoamylase of A, shirousamii

vobacterium sp., Bender, 1981) and some yeast (Saccharomyces sp., Yamashita ei al., 1984; Endomyces sp., Fukui and Nikuni, 1969).

It has also been Known that glucoamylase hydrolyzes a -1,6 and a -1,3 linkages rather slowly than the a-I ,4 linkage (Pazur and Kleppe, 1962). Fleming (1968) classified glucoamylase into two groups, one yielding total hydrolysis of starch and f1-limit dextrins, and the second yielding 80% hydrolysis of starch and 40% conversion of f1-limit dextrins to glucose. The former type can hydrolyze a -1,6 linkages. Recently, it was found that two or more types of glucoamylases are excreted from some strains of fungi. Razzaque et al. (1978) prepared three forms of glucoamylases from Asp. oryzae. Takahashi et al. (1978) purified three forms of glucoamylase from Rhizopus species. But, these isozymes were very similar in enzymatic properties and showed common antigenicity. They suggested that the multiplicity of glucoamylase may be a modification of an original enzyme by certain proteolytic enzymes.

Becuse of its industrial usefulness, glucoamylase has received considerable attention. It has many biotechnical applications, e.g. in the production of glucose, high-conversion syrups and fermented ethanol. Here, we report the purification and characterization of the glucoamylase from Aspergillus shirousamii which has been used widely in fermented ethanol industries.

Materials and Methods

Materials

Wheat bran koji (a commercial preparation of glucoamylase from Aspergillus shirousamiii was kindly supplied by the distillery of jin Ro Ltd. Soluble starch was purchased from Shinyo Pure chemicals Co., Ltd. p-Nitrophenyl a -Dvglucopyranoside, amylose, amylopectin, glycogen, dextrin, maltotriose, isomaltose and marker proteins were obtained from Sigma Chemical Co. DEAE-Sephacel, Sephadex G-200, Concanavalin A -Sepharose 4B were purchased from Pharmacia Fine Chemicals. Ammonium sulfate and maltose were obtained from E. Merck. Isoelectric focusing column #8101 and Ampholine buffer (pH 2.5-4.0, pH 3.5-5.0) were purchased from L.K.B.

223

Assay methods

Glucoamylase activity was determined by the method of Leisola et al. (1980) with some modification. The reaction mixture consisted of 50 1'-1 of 0.2% p-nitrophenyl a - D-glucopyranoside solution in 0.02 M acetate buffer (pH 4.5) and 50 1'-1 of enzyme solution. After incubation at 60°C for 15 min, the reaction was stopped by adding 1 ml of 1 % sodium carbonate solution. The released p-nitrophenol was measured at 420 nm with Varian DMS 80 Spectrophotometer. The amount of p-nitrophenol formed was determined from the standard curve. One unit of the enzyme activity was defined as the amount of enzyme that release 1 I'- -mole of p-nitrophenol from p-nitrophenyl a -D-glucopyranos ide per min under the specified condition. Protein contents were estimated by the method of Lowry et al. (1951) with bovine serum albumin as a standard.

Enzyme purification

The purification procedure was as follows. All operations were performed at 0-4°C, unless otherwise mentioned.

Step 1. Preparation of crude extract: 50 g of wheat bran koji were added to 500 ml distilled water. The mixture was stirred for 1 h at room temperature, and the insoluble materials were removed by centrifugation at 10,000 g for 15 min. The supernatant was used as a starting material for purification of the glucoamylase.

Step 2. Ammonium sulfate fractionation: In ammonium sulfate fractionation, pre-test was performed to determine a proper concentration of ammonium sulfate. Various concentrations ranging from 40% to 95% for complete saturation were used. The salt was added gradually with stirring on ice bath. After stirring on ice bath for 1 h, the sample was left to stand at 4°C overnight, then the precipitate was separated by centrifugation at 10,000 g for 15 min. The glucoamylase activities of the precipitate and the supernatant were determined by the assay method. To the crude extract, solid ammonium sulfate was added to 50% saturation. After stirring for 1 h, the precipitate was removed by centrifugation at 10,000 g for 15 min. To the supernatant fluid, ammonium sulfate was further added to 95% saturation and the mixture was centrifuged af-

Korean Biochem. J. (1985), Vol. 18(3)

224

B. G. Na and C.-H. Yang

ter standing overnight. The precipitate was dissolved in a minimum volume of distilled water and dialyzed on a 0.02 M acetate buffer (pH 4.5). After dialysis, the enzyme solution was centrifuged to removed any insoluble residue.

Step 3. Column chromatography on DEAE-Sephacel: 10 ml of the supernatant solution from step 2 was applied on a column (l.8X 12 cm) of DEAE-Sephacel equilibrated with 0.02 M acetate buffer (pH 4.5). The column was washed with 150 ml of the same buffer and eluted with 300 ml of a linear gradient of 0-l.0 M N aCI in the buffer. Each fraction of 5 ml was collected at a flow rate 20 ml/h. The fractions that contained the enzyme activity were pooled and concentrated with Diaflo PM 10 ultrafilter.

Step 4. Concanavalin A-Sepharose 4B affinity chromatography: The concentrate was applied to Concanavalin A-Sepharose 4B column (2.2X7 ern) equilibrated with 0.02 M acetate buffer, pH 4.5, containing 0.5 M NaCI, l.0 mM csci, and l.0 mM MnClz. The column was washed with a sufficient volumn of the buffer and then eluted with 300 ml of 0-0.5 M linear gradient a -methyl-D-mannoside in 0.02 M acetate buffer (pH 4.5) containing 0.5 M NaCI. The enzyme active fractions were pooled and concentrated with Diaflo PM 10 ultrafilter.

Step 5. Preparative isoelectric focusing: The concentrate from step 4 was dialyzed against a l.0% solution of glycine. Isoelectric focusing with carrier ampholites (pH 2.5-5.0) was conducted in a gradient of sucrose (0-50% w/v). Electrophoresis was done in a 110 ml column (LKB #8101) at 4°C for 48 hrs with final voltage of 500 V. After termination of electrophoresis, 3.5 ml fractions were collected and the pH values were measured at room temperature. Fractions of the enzyme activities were pooled and dialyzed against a 0.02 M acetate buffer (pH 4.5) and used for all further studies.

Estimation of molecular weight

I. gel filtration method. The molecular weight of the enzyme was estimated by gel filtration on a Sephadex G-200 column (2.2x80 ern) by the method of Andrews (1965) with 0.02 M acetate buffer, pH 4.5. The void volume of the column was measured by Blue Dextran 2,000. Reference proteins were catalase (beef liver, M.W. 250,000), alkaline phosphatase (calf intestine, M.W. 100,000),

Korean Biochem. J. (1985), Vol. 18(3)

albumin (bovine serum, M.W. 68,000) and trypsin (beef pancreas, M.W. 23.800).

II. SDS-polyacrylamide gel electrophoresis. The method of Weber and Osborn (1969) was adapted with some modification. Continuous gel of 5% polyacrylamide in 0.1 M sodium phosphate buffer (pH 7.0) containing 0.2% SDS was used. Running buffer was composed of 0.05 M sodium phosphate and 0.1% SDS, pH 7.0. Denaturated proteins were prepared by incubating proteins in boiling water for 5 min in a solution which contained 0.01 M sodium phosphate, 1% SDS, 1% f3 -mercaptoethanol, 0.015% bromophenol blue and 36% urea, pH 7.0. Electrophoresis was performed at room temperature for 4 hrs at 120 V of constant charge. After an electrophoresis, the proteins in the gel were detected by 0.1% Coomassie Brilliant Blue R-250 in 10% acetic acid and 47.5% ethanol. Bovine albumin (cross-linked, monomer, M.W. 66,000; dimer, M.W. 132,000; trimer, M.W. 198,000; tetramer, M.W. 264,000) was used for marker protein.

Thermostability studies

Aliquots of the enzyme solution were heated at various temperatures (55-70°C) for 0.5-60 min with appropriate time intervals. After heating, immediately the remaining enzyme activities were determined under the standard assay conditions. To determine the Ca + + effect on the enzyme thermostability, the enzyme solutions containing 10 mM CaClz were treated with the same method as described above.

Temperature optimum

Temperature optimum was measured with p-nitrophenyl a - D-glucopyranoside as substrate. The reaction mixture was prepared with the same composition as the standard assay method. The mixtures were incubated at various temperatures for 15 min and 60 min. After incubation, the reaction was stopped by adding 1 ml of N aZC03 solution. The released p-nitrophenol was measured at 420 nm.

pH stability studies

The enzyme solution was dialyzed against distilled water. Buffers at various pH were prepared with 0.05 M citrate buffer (pH 3.0-4.0), with 0.05 M acetate buffer (pH 4.0-5.5), and with 0.05 M phosphate buffer (pH 5.5-7.5). The enzyme solution and each

Glucoamylase of A, shirousamii

buffer solution were mixed with 1:1 ratio and incubated at room temperature for 1 h. After incubation, the enzyme activities were determined under the standard assay condition.

pH optimum

The enzyme solution was dialyzed on distilled water. 0.02 mg/ml solutions of p-nitrophenyl a - D-glucopyranoside in 0.05 M citrate buffer (pH 3.0-4.0), 0.05 M acetate buffer (pH 4.0-5.5) and 0.05 M phosphate buffer (pH 5.5-7.5) were used as substrate. The other procedures are same as shown in the standard assay method.

Effects of metal ions and anions

The effects of metal ions and anions were studied in 0.02 M acetate buffer (pH 4.5) with 10 mM concentrations of metal ions and with 25 mM concentrations of anions, respectively. After preincubation for 60 min at room temperature, the effect!'; were examined under the standard assay condition.

Determination of kinetic parameters

Kinetic parameters, Km and Vrnax, were determined from Lineweaver-Burk plots (1934)_ The following substrates of different sizes were used; soluble starch, amylose, amylopectin, glycogen, isomaltose, dextrin, maltotriose, maltose and p-nitrophenyl a -D-glucopyranoside. The assay mixture consisted of 1 ml of substrate solutions and 0.1 ml of enzyme solution. The concentration ranges of the substrates were as follows: the high-molecular-weight substrates from soluble starch to dextrin,

225

0.05-l.0%; the others, 5-40 m M. Glucose liberated was determinded by the 3,5-dinitrosalicylic acid (DNS) reagent method of Bernfelt (1955). After incubation at 60°C for 15 min, 2 ml of DNS solution was added to the assay mixture. The tube containing this mixture was heated for 5 min in boilling water. After cooling in running tap water, add 18 ml of water and measured the liberated glucose at 575 nm with spectrophotometer. A blank was prepared in the same manner without enzyme. A calibration curve established with standard glucose solutions (O.l-LO mg in 1 ml of H20).

Results

Purification of glucoamylase

A summary of the purification of glucoamylase from Aspergillus shirousamii is given in table l. The specific activity of glucoamylase was increased from 0.14 U/mg protein to 2_77 U/mg protein, indicating purification factor of 19.8 times. The yield was 47.5%_ To determine the proper concentrations of ammonium sulfate for fractionating the enzyme, pretests were performed. The result is shown in table 2. As the result of the pretest, the fraction from 50% to 95% saturation was collected. This step provided l.7 fold increase in enzyme activity.

In DEAE-Sephacel column, a minor peak was detected at the washing step. But the main peak was eluted at 0.26-0.32 M NaCI with a main protein peak (Fig. 1). Only the main fractions were further purified in the following steps. Glucoamy-

Table 1_ Summary of the purificationof glucoamylase. A unit of enzyme activity was defined as the amount liberating 1 f1 mole of p-nitrophenol per min at 60°C under the standard assay condition described in the text. Protein contents were estimated by the method of Lowry et aL (1951).

Purification Total protein Total activity Specific activity Yield
procedure (mg) (units) (units/mg protein) (%)
C rude extract 312 44.0 0.14 100
(NH4hS04
fractionation 153.8 36.9 0.24 83.9
(0.5-0.95 S)
DEAE-Sephacel 65.6 29.5 0.45 67.0
Concanavalin - A 25.6 26.1 1.02 59.3
Sepharose 4B
Isoelectrofocusing 7.55 20.9 2_77 47.5
(pH 2.5-5.0) Relative purity

1.7

3.2

7.3

19.8

Korean Biochem. J_ (1985), Vol. 18(3)

226

B. G. Na and C.-H. Yang

Table 2. Precipitation of glucoamylase by ammonium sulfate. Various concentrations ranging from 40% to 95% for complete saturation were used. The salt was added gradually with stirring on ice bath. After stirring for 1 h, the samples left to stand at" 4'C overnight. The precipitate was separated by centrifugation at 10,000 g for 15 min. The glucoamylase activities of precipitate and supernatant were determined by assay method described in the text.

(NH4lzS04 concentration (% saturation)

Glucoamylase activity (% of original)

Precipitate

Supernatant

40 50 60 70 90 95

94.3 90.0 84.6 70.8 20.3

l.2

l.0 2.9 7.3

2l.5 7l.6 90.5

3,0

~ 2,0 «

o

~ 1,0 «

~ 0,5 ~

:z

FRACTION NUMBER

Fig. 1. Column chromatography on DEAE-Sephacel. Enzyme activity was measured at 420 nm for p-riitrophenyl released (A420 0--0) and proteins (.--.).

lase binded to Concanavalin-A and it was eluted at 0.05-0.25 M a -methyl-D-mannoside as a single peak with a little tailing (Fig. 2). This step provided about 2.3 fold increase in enzyme activity. In isoelectric focusing, the enzyme active fractions were showed single peak at pH 3.35-3.47 (Fig. 3). From this fact, it is concluded that the isoelectric point of the glucoamylase from Aspergillus shirousamii is about pH 3.4. This isoelectric focusing step provided about 2.7 fold increase in enzyme activity.

Estimation of molecular weight

From the gel filtration on Sephadex G-200 column (2.2X80 cm, l't=300 ml, Vo=107 ml), the molecu-

Korean Biochem. J. (1985), Vol. 18(3)

3,0

I

~C> 2,0

N ~

<r

~ 1,0 -er

w

9

Vl o :z :z

,5 ~

,

o

-' >:r >UJ :E

ls

10

20

30

40

50

60

FRACTION NUMBFR

Fig. 2. Concanavalin A-Sepharose 4H atnnity Chromatography. The column (2.2 X 7 em) was washed with sufficient volume of 0.02 M acetate bufer, pH 4.5, containing 0.5 M NaCl, l.0 mM CaCl2 and l.0 mM MnCI2. The Sample was eluted with 0-0.5 M linear gradient a -methyl-D-mannoside in 0.2 M acetate buffer (pH 4.5) containing 0.5 M NaCI. .--., Protein concentration A280; o~o, Enzyme activity, A420; , a - Methyl- D-mannoside.

lar weight of the enzyme was estimated to be approximately 89,000 daltons (Fig. 4). The result of SDS-polyacrylamide gel electrophoresis of the enzyme was shown in Fig. 5a and the migration patterns of the enzyme and reference proteins were in Fig. 5b. The molecular weight of glucoamylase was estimated to be approximated 88,000 daltons from the curve. From those results, it is concluded that the enzyme consisted of a single polypeptide chain.

Glucoamylase of A, shirousamii

3,0

30

7,0

2,0

I

c>

~

« 1,0

/

25

1 0 15 20 FRACTION NUMBER

Fig. 3. Isoelectric focusing. Electrophoresis was done in a 110 ml column (LkB ;I: 8101) with carrier ampholite (pH 2.5-5.0) in a gradient of sucrose (0-50%) at 4°C for 48 h with final voltage of 500 V. 3.5 ml fractions were col-

lected. , pH gradient.

I{'
C>
....
"
~ 3,0 CATALASE
:c
l!:)
i_g 2,0
0::
:s 1,0 ALKALINE FHm-fAT~
=> GLUCOAMYLASE
l:d BSA
c5 0,6
1:
0,2 TRYPSIN
0,5 1,0 15 Knv

Fig. 4. Molecular weight determination by gel filtration on Sephadex G-200 column (2.2 X80 ern). Eluting buffer was 0.02 M acetate buffer (pH 4.5). Void volume was determined by Blue Dextran 2,000. Reference proteins were catalase (beef liver, M.W. 25,000), alkaline phosphatate (calf intestine, M. W. 100,000), serum albumin (bovine, M.W. 68,000) and trypsin (beef pancreas. M.W. 23,800).

Tbermostability studies

Fig. 6 showed heat inactivation of the enzyme at various temperature from 55°C to 70°C in 0.02 M sodium acetate buffer (pH 4.5). This enzyme was very stable below 55°C. At 55°C it had retained 92% of original activity during incubation for 60

227

ABC

Fig. Sa. SDS-polyacrylamide gel electrophoresis in 5% continuous polyacrylamide gel. Electrophoresis was performed at room temperature for 4 hrs at 120 V of constant charge. Lane A; reference, Lane B; purified enzyme, Lane C; crued extract.

'" TRIMER
'~ 1.9
)(
~ 1.3 DIMER
:I:
l!:)
UJ
~ 1,0 GLU [QAMY LASE
0::
«
-'
§j 0,7 WINE ALBUMIN
-' (MON(J1ERJ
0
2,0 L
0,5 0.5 RELATIVE MOBILITY

1,0

Fig. 5b. Molecular weight determination by SDS-polyacrylamide gel electrophoresis. Continuous gel of 5% polyacrylamide was used. Reference was bovine serum albumin (cross-linked, monomer, M. W. 66,000; dimer, M. W. 132,000; trimer, M. W. 198,000; tetramer, M. W. 264,000).

min. But over 65°C, the lability was rapidly increased. At 70°C, it had lost almost all activity within 10 min. To produce 50% enzyme inactiva-

Korean Biochem. J. (1985), Vol. 18(3)

228

B. G. Na and C.-H. Yang

20

7(f[

60

TIME (MIN)

Fig. 6. Thermostability of glucoamylase. Enzyme solulions were heated at various temperature (55-70°C) for 5-60 min. After heating, the remaining enzyme activities were determined immeadiately under the standard assay conditions.

tion, it required 15 min at 65°C and 2 min at 70°C, respectively.

Ca + + has increased the thermostability of the enzyme (Fig. 7). When 10 mM of Ca++ was added, about 50% of activity was remained after incubation for 10 min at 70°C. After incubation for 60 min at 70°C, about 20% of original activity was still remained.

Temperature optimum

The effect of temperature on hydrolyzing activity of enzyme was determined at various temperature for 15 min and 60 min. Maximum hydrolysis was obtained at 65°C for 15 min and at 60°C for 60 min, respectively (Fig. 8).

pH stability

To test the pH stability, glucoamylase was incubated for 1 h at room temperature in the buffers from pH 3.0 to pH 7.5 The enzyme was the most stable at pH 4.5 and was more stable in the acidic region than in the neutral region (Fig. 9). It retained about 70% of activity at pH 3.0, but it lost 85% of activity at pH 6.5.

pH optimum

The effect of the pH on the hydrolyzing activity of the enzyme was determined at 60°C as a function

Korean Biochem. J. (1985), Vol. 18(3)

00 ••••••• -c STD • .....__. CaCh 10mM

..... >

~

~ 60

1,.0

\

\

~\ .....

".

20

5 10

20

3

TIME(MINl

Fig. 7. Ca++ Effect on the thermostability. The enzyme solutions containing 10 mM CaCl2 were treated for 5-60 min at 70°C. After heat treatment, the remaining activities were determined under the standard assay condition. e --e, 10 mM CaCl2 were added; 0--0, standard.

0,5
0
N
...
:{ 0,1.
0,3
0,2
0,1 . ...... 0 ••••• 11··

•••.• o:

o·

;/~:

/'

~\ ...••.

\ -,

'"

__ 15min 0 •••••• -0. 60min

30

40

50

60

70

80

TEMPERATURE n)

Fig. 8. Temperature optimum. The reaction mixture was as shown in the text, and incubated at various temperature for 15 min and 60 min.

of pH. As shown in Fig. 10. It also had a similar curve to the pH stability curve. Maximum hydrolysis occurred at pH 4.5 and the relative hydrolysis ratio was higher at lower pH region than at neutral region.

Effects of metal ions on glucoamylase

The effects of various metal ions on the enzymatic activity were determined at a concentration of 10 mM at pH 4.5. The activity was inhibited 31% by Hg + +. Other cations, such as Ca + +, Zn + + ,

Glucoamylase of A, shirousamii

0,5

~ 0,4 <:(

0,3

0,2

0,1

3,0

4,0

6,0

7.0

5,0

pH

Fig. 9. pH stability of glucoamylase. The enzyme solution was dialysed against distilled water. Buffers at various pH were 0.05 M citrate buffer (pH 3.0-4.0), 0.05 M acetate buffer (pH 4.0-5.5) and 0.05 M phosphate buffer (pH 5.5-7.5). The enzyme solution and each buffer solution were mixed with 1: 1 ratio and incubated at room tempera, ture for 1 h. After incubation, The enzyme activities were

• determined under the standard assay codition.

0,5

0,3

fJ

:::(

0,2

0,1

pH

Fig. 10. pH optimum. 0.02 mg/ml solution of p-nitrophenyl a -D-glucopyranoside in 0.05 M citrate buffer (pH 3.0-4.0), 0.05 M acetate buffer (pH 4.0-5.5) and 0.05 M phosphate buffer (pH 5.5-7.0) were used as substrate solutions. The enzyme solution was dialysed against distilled water. The enzyme activities were measured under the standard assay method.

229

Mg++, Sn" ", Na ", K+ as well as EDTA had little effect on the activity (Table 3).

Table 3. Effects of Metal Ions. The effects of metal ions were studied in 0.02 M acetate buffer (pH 4.5) with 10 mM concentrations of metal ions.

Metal ion

Relative activity (%)

Concentration

Ca++ Zn++ Mg++ Hg++ Sn++ Na+ K+ EDTA

10 mM

102 98 97 69 90 96 94 92

Effects of anions on glucoamylase

Anion effect on glucoamylase activity was examined at a concentration of 25 mM. Most of anions tested showed inhibition effect. Among these B407-Z, AsOz- and S03-2inhibited remarkably up to 94%, 95% and 88%, respectively. Table 4 shows the tendency of the divalent anions have more inhibition effect than the monovalent anions (Table 4).

Table 4. Effects of Anions. The effects of anions were studied in 0.02 M acetate buffer (pH 4.5) with 25 mM concentration of anions.

Anion Concentration Relative
activity (%)
ci- 25 mM 83
F- 65
C03-2 39
N03-2 82
CH3COO- 57
B407-2 6
As02- 5
S03-2 12 Kinetic studies

The kinetic parameters, K; and Vmax, were determined at pH 4.5 at 60°C. The results for the highmolecular-weight substrates were given in Table 5. Amylopectin showed the highest Km and Vmax value. Amylose showed the lowest Km value, and dextrin showed the lowest Vmax value. For the relative value of Vmaxl Km, amylose showed the highest value and dextrin showed the lowest value. On the other hand, relative rate of hydrolysis, amylopectin

Korean Biochem. J. (1985), Vol. 18(3)

230

B. G. Na and C.-H. Yang

Table 5. Kinetic parameters and relative rates of hydrolysis of glucoamylase for high molecular weight substrates. The values of Km and Vmax at pH 4.5 at 60°C were determined from Lineweaver-Burk's plot. The concentration range of the substrate was 0.1-1.0%. The relative rate of hydrolysis was measured at the concentration of 1.0%.

Substrate s; (%) Vmax Vmax/ s; Relative rate
(units/mg protein) of hydrolysis
Soluble starch 1.05 6.4 6.10(1.00) 1.00
Amylopectin 1.67 15.2 9.10(1.49) 1.59
Amylose 0.63 7.7 12.22(2.00) 1.28
Glycogen 1.33 12.9 9.70(1.59) 1.57
Dextran 1.25 4.4 3.25(0.58) 0.64 Table 6. Kinetic parameters and relative rates of hydrolysis of glucoamylase for low molecular weight substrates. The values of Km and Vmax at pH 4.5 at 60°C were determined from Lineweaver-Burk's plot. The concentration range of the substrate was 5-40 mM. The relative rate of hydrolysis was measured at the concentration of 40 mM.

Substrate s; (mM) Vmax Vrnru/Km Relative rate
(units/mg protein) of hydrolysis
Maltose 21.7 6.55 0.302(1.00) 1.00
Maltotriose 17.8 10.38 0.583(1.93) 2.12
Isomaltose 35.7 1.64 0.046(0.15) 0.15
p-Nitrophenyl- 12.8 0.33 0.026(0.086) 0.066
D-glucopyranoside showed the highest value and dextrin showed the lowest value. The kinetic values for the low-molecular-weight substrates were as shown in Table 6. Maltotriose was higher than maltose in Vmaxl Km value. Isomaltose and p-nitrophenyl a -Di-glucopyranoside were pyranoside were much lower than maltose in Vmaxl Km value. For the relative rate of hydrolysis, maltotriose showed twice than that of maltose. Isomaltose and P-nitrophenyl a - Dvglucopyranoside showed much smaller rate than maltose, 0.15 and 0.066, respectively.

Discussion

Mahmoud et at. (1978) studied the pattern of the precipitation of glucoamylase from Aspergillus foetidus by ammonium sulfate. They reported that 92.3% of the original glucoamylase was precipitated at the ammonium sulfate concentration of 0.79 saturation. But in this study, only 7l.6% of the original glucoamylase was precipitated at the concentration of 0.9 saturation. From this fact, we have found the glucoamylase of Aspergillus shirousamii is very stable at high concentration of ammonium sulfate.

The minor peak shown in DEAE-Sephacel Chromatography suggested the possibility of the exsi-

Korean Biochem. J. (1985), Vol. 18(3)

tence of minor enzyme. Inokuchi et at. (1981) isolated minor glucoamylase from Aspergillus saitoi, and suggested that the minor enzyme had different molecular weight and specific activity compared with the major enzyme. But, they had very similar isoelectric points (pH 3.86 and pH 3.85). From our results the minor peak shown at the washing step represented that the minor enzyme has the different isoelectric point from the major enzyme. Lineback et at. (1978) reported the two type of glucoamylase from Asp. niger which had different isoelectric point (pH 3.4 and pH 4.0). Razzaque et at. (1978) revealed the existence of three forms of glucoamylase I, II and III which had isoelectric points of 4.0, 3.9 and 3.6, respectively.

All fungal glucoamylases are composed of glycoprotein containing from 5 to 20% carbohydrate, and generally contain glucose, glucosamine, mannose and galactose (Forgarty et al., 1980). The carbohydrate fractions linked glycosidically to amino acid residues and bind easily to Concanavalin-A. So, we used Concanavalin-A Sepharose 4B for affinity chromagraphy and achieved 2.3 fold increase of purity. The isoelectric points of g lucoamylases of Aspergillus species ranged from pH 3.4 to 5.6. In this study, the detected pI value of the enzyme (pH 3.4) showed the lowest level among the

Glucoamylase of A, shirousamii

Aspergillus species.

In general, glucoamylases were shown to be stable up to 50°C (Takahashi et al., 1978, 1981; Handa et al., 1976). However, glucoamylase from Piricularia oryzae was stable only below 40CC and lost its activity completely at 60'C within 15 min (Yuhki et al., 1977), while the glucoamylases from Endomyces species lost a half of the original activity at 55cC in 10 min (Fukui and Nikuni, 1969). Glucoamylase from Aspergillus shirousamii showed some higher heat stability than the enzymes from other strains. It was very stable at 55cC and retained about 60% of original activity at pH 4.5 for 60 min at 60°C.

Fukui nad Nikuni (1969) reported that Ca" " displayed no protecting effect on the glucoamylase from Endomyces species but accelerated the inactivation. But, in this study, Ca + + acted as a good protector against the heat inactivation of glucoamylase from Asp. shirousamii. In the absence of Ca + +, it lost it's activity completely at 70cC within 10 min. By the addition of 10 mM of Ca" ", it retained about 50% of activity at the same conditions. This was coincident with the result of Bender (1981) which shown in the effect of Ca++ on a cyclodextrin -degrading glucoamylase from Flavobacterium species.

The temperature optima of most glucoamylase were in the range of 40-60cC (Forgerty, 1983). The glucoamylase from Aspergillus shirousammi showed a little higher optimum temperature, 65°C for 15 min. The result was in agreement with its higher thermostability. When the reaction time was prolonged to 60 min, temperature optimum was shifted down to 60°C as the result of partial inactivation of the enzyme.

Glucoamylaes were reported very stable up to pH 7.0 (Ueda et aI., 1974; Hayashida et aI., 1976; Takahashi et al., 1981; Handa et al., 1976). The pH optimum of this enzyme showed generally in the range pll 4.5-5.0. The glucoamylase from Aspergillus shirousamii was stable up to pH 5.5 and its pH optimum was pH 4.5. Whereas no difference was observed in the optimum pH of this enzyme as compared with other fungal enzymes, its pH stability was quite different. It lost more than 80% of its activity at pH 6.5 at room temperature within 60 min.

Takahashi et al. (1981) reported the reversible inhibition effect of Hg++ on the activity of glu-

281

coamylase from Asp. saitoi. The same result was obtained with the glucoamylase from Asp. shirousamii.

Rate of substrat hydrolysis by glucoamylase are aitected both by molecular size and structure, and also by the next bond in sequence (Abdullah et al., 1963). Thus the rate of hydrolysis of a -1,4 bonds increases linealy with the molecular weight of the substrate at least up to maltopentose (Fukui and Nikuni, 1969). Pazur and Kleppe (1962) reported that maltose was hydrolyzed at about 30 times the rate for isomaltose. In kinetic studies, the glucoamylase from Asp. shirousamii hydrolyzed maltotriose more than twice the rate for maltose. Isomaltose and p-nitrophenyl a -D-glucopyranoside showed much smaller relative rate of hydrolysis than maltose, 0.15 and 0.066, respectively. This results were in agreement with the result of the above investigators. For high -molecular-weight substrates, the order of Vmaxl Km was not in agreement with the order of relative rate of hydrolysis, exclusively for amylopectin. It seems to be that this disagreement occurs from the fact that the concentration for determining relative rate of hydrolysis is higher than Km value of amylose.

References

Abdullah, M., Fleming, 1. D., Taylor, P. M. and Whelan,

W. J. (1963) Biochem. J. 89, 35.

Andrews, P. (1965) Biochem. J. 96, 595. Bender, H. (1981) Eur. J. Biochem. 115, 287.

BernfeJt, P. (1955) Methods in Enzymology, Vol I (Colowick, S. P. and Kaplan, N. 0., ed.), Academic Press Inc., New York, p149.

Fleming, 1. D. (1968) Starch and Its Derivatives (Radley, J A., ed.), Chapman and Hall Ltd., London, p498.

Fogarty, W. M. and Kely, C. T. (1980) Econ. Microbio!' (Ire) 115.

Fogarty, W. M. (1983) Microbial Enzyme and Biotechnology (Forgarty, W. M., ed), Applied Science Publishers, London, 1.

Fukui, T. and Nikuni, Z. (1969) Agri. Bioi. Chern. 33, 884. Handa, T., Mineki, S. and Iizuka, H. (1976) Eiyo to Sokyuryo 29, 439.

Hayashida, S., Noruma, T., Yoshino, E. and Hongo, M. (1976) Agri. Bioi. Chem. 40, 141.

Hou, W. Y. and Chung, M. J. (1984a) Korean J. Food Sci.

Techno!. 322.

Korean Biochem. J. (1985), Vol. 18(3)

232

B. G. Na and C.-H. Yang

Hou, W. Y. and Chung, M. J. (1984b) Korean J. Food Sci.

Technol. 392.

Inokuchi, N., Takahashi, T. and Irie, M. (1981) J. Biochem. 90, 1055.

Kitahara, K. and Kurushima, M. (1949) J. Ferm. Technol. 27, 254.

Laemmli, U. K. (1970) Nature (London) 227, 680 -. Leisola, M., Ojamo, H. and Kauppinen, V. (1980) Enzyme Microb. Technol. 2, 121.

Lineback, D. R., Russell, I.]. and Rasmussen, C. (1969) Arch. Biochem. Biophysics 134, 539.

Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Soc. 56, 658.

Lowry, O. R., Rosebrough, N.]., Farr, A. L. and Randall, R. J. (1951) J. BioI Chem. 193, 265

Manjunath, P. and Raghavendra Rao, M. R. (1981) J.

Biosci. 3, 333.

Mahmoud, S. A. Z., Mashhoor, W. A., El-Haddad, M. E. and El-Gamal, S. E. (1978) Appl. Biochem. Microbial.

14, 369.

Pazur, J. H. and Kleppe, K. (1962) J. BioI. Chem. 237, 1002.

Razzaque, A. and Ueda, S. (1978) J. Ferment. Technol. 56, 296.

Tanaka, A., Fukuchi, Y., Ohnishi, M., Hiromi, K., Aibara, S. and Morita, Y. (1983) Agri. BioI. Chem. 47, 573.

Takahashi, T., Tsucida, Y. and Irie, M. (1978) J. Biochem. 84, usa.

Takahashi, T., Inokuchi, N. and Irie, M. (1981) J. Biochem.

89, 125.

Weber, K. and Osborn, M. (1969) J. BioI. Chem. 244,4406. Whelan, W.]. (1963) Biochem. J. 89, 35.

Yamasaki, Y., Tsuboi, A. an Suzuki, Y. (1977) Agri. Bioi.

Chem. 41, 2189.

Yamashita, I., Matano, T. and Fukui, S. (1984) Agri. BioI.

Chem. 48, 161l.

Yuhki, A., Watanabe, T. and Matsuda, K. (1977) Die Starke 8,265.

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Korean Biochem. J. (1985), Vol. 18(3)