Kinetic patterns of glucoamylase isozymes isolated from Aspergillus species

ARCHIVES

OF

BIOCHEMISTRY

AND

Kinetic

Patterns Isolated

K. L. SMILEY,

144, 694-699

BIOPHYSICS

of from

Regional

Received

Research

January

Glucoamylase

lsozymes

Aspergillus

D. E. HENSLEY,

Northern

(1971)

Species

M. J. SMILEY, Laboratory,’

21, 1971;

accepted

Peoria, March

AND

H. J. GASDORF

Illinois

61604

4, 1971

The existence of multiple forms of glucoamylase has been known for some time. However, little information is available to differentiate these isozymes other than their electrophoretic mobility. It has now been found that the two forms of glucoamylase from several different Aspergillus strains do differ in their rate of attack on starch and glycogen. The Michaelis constant of the least charged isoeyme on glycogen . . approaches infinity, whereas on starch it is several times greater than the more charged form. On small oligosaccharides and maltose the two forms show the same K, and V,,, values. In addition to kinetic differences, the two isozyme forms differ in their molecular weight.

In recent years, glucoamylase (a-1,4glucan glucohydrolase, EC 3.2.1.3) has assumed considerable industrial importance because of its ability to hydrolyze starch nearly quantitatively to glucose with little formation of reversion products as compared to acid hydrolysis of starch. Glucoamylase from strains of black Aspergillus was shown to be a mixture of two enzymes by Pazur and Ando (l), Smiley and Hensley (2), and Watanabe and Fukimbara (3). Pazur and Ando (1) only studied the glucoamylase from A. niger that was present in the largest Watanabe and Fukimbara (3) amount. studied both forms produced by a culture of A. awamori. Lineback et al. (4) conducted a thorough study of the two forms of glucoamylase from A. niger. They showed the two forms to be identical in many physicochemical properties. The only pronounced difference was in their electrophoretic mobility. This paper demonstrates that the two forms do differ in kinetic properties. The difference is most pronounced when glycogen is the substrate and least pronounced when smaller oligosaccharides are the substrate.

MATERIALS

AND

METHODS

Source of glucoamylase. Glucoamylase was prepared according to the method of Smiley et al. (5) from A. niger NRRL 337, A. niger NRRL 3122, and A. awamori NRRL 3112. Commercial samples of glucoamylase were obtained from Miles Chemical Co.2 and Wallerstein Laboratories. Both commercial samples were made with Aspergillus. Source of carbohydrate. Soluble starch according to Lintner was purchased from Pfanstiehl Chemical Co. Corn dextrin (10 D.E.) was kindly furnished by Corn Products International. Glycogen (rabbit liver) was a product of Pfanstiehl Chemical Co. Maltose was purchased from Difco Corp. Other starches used had been prepared previously at this laboratory. Preparation of isozymes. Concentrated crude culture filtrates or commercial enzyme water extracts were chromatographed on diethylaminoethyl (DEAE)-cellulose by a method adapted from one described by Todu and Akabori (6). Columns (25 x 590 mm) were filled to a depth of 350 mm with DEAE-cellulose that had been successively washed with 0.5 M HCl, distilled HZO, 0.5 M NaOH, and finally distilled water until the pH values of the washings were below 8.0. The cellulose was then equilibrated with 0.1 M sodium acetate at pH 4.2. Several volumes of the same buffer were *The mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.

1 This is a laboratory of the Northern Marketing and Nutrition Research Division, Agricultural Research Service, U. S. Department of Agriculture. 694

KINETICS

OF

GLUCOAMYLASE

r,m through the column after which an appropriate size sample of the crude glucoamylase was layered on top of the bed and washed in with a small amount of buffer. Elution was started with 0.1 M acetate at pH 4.2 and continued until the first broad peak was eluted. A gradient elution was then begun by adding dropwise 1.0 M acetate buffer at pH 4.2 to a 400-ml stirred reservoir of 0.1 M acetate buffer. The flow rate was 30 ml/hr. The first glucoamylase peak emerged shortly after starting the gradient, and was completely eluted when the gradient was about 0.35-0.4 M. The second peak then came off and was eluted over the gradient of 0.45-0.55 hr. The acetate gradient was essentially linear. Bcetat,e was determined by the method of Hutchens and Kass (7). The glucoamylase peaks were located by continuous ultraviolet (uv) light monitoring of the effluent stream with an Isco uv monitor. The tubes containing each uv-absorbing peak were pooled, dialyzed against, running tap water for 2-3 hr, and then concentrated in ~)UC?LO to 10 ml. Assay of glu.coamylase acfivity. The concentrated fractions were assayed according to the method described by Miles Chemical Co. (8). One glrlcoamylase unit is defined as the amount of enzyme necessary to form 1 g of glucose from 4 g of starch substrate in 1 hr at 60”. For ease in handling large numbers of samples, the volume of starch subst,rate was reduced to 5 ml and the resnlt,ing glucose was measured on a Technicon arltoanalyzer by reduction of alkaline ferricyanide reagent, The method is a modification of the one proposed by Hoffman (9) and adapted for use on the autoanalyzer. Protein. Prot,ein was determined by the method of Lowry et al. (10). Kinetic studies. Michaelis constants and I’,,,, valltes were determined on a Technicon autoalralyzer with starch, dextrin, glycogen, and malt,ose as substrates. The substrate was diluted to sllitable levels and placed in the sample changer of the instrument. The substrates were mixed with a cont,inuous stream of enzyme and incubated at the desired temperat,nre for approximately 6 min by plumping the reaction mixture through a glass holding-coil immersed in a water bath. After the holding time, solutions were pumped to the dialyzer and reducing sugars were determined by the standard reducing sugar met,hod employing the autoanalyzer. Sugars produced from starch and dextrin were determined by reduction of ferricyanide, and for dextrin, the reducing value of the unreact,ed substrate was subtracted from the total reducing value. No corrections nlere necessary for starch or glycogen substrate. Glucose produced from maltose was determined on the autoanalyzer by

695

ISOZYMES

the glucose oxidase method of Hill and Kessler (ll), except that Tris buffer was substituted for phosphate buffer. The data obtained were treated by either the Lineweaver-Burk or Hofstee method to determine K,” and V,,, values. Gel Jiltration. Bio-Gel P-100 (Bio-Rad Corp.) columns were used to determine the approximate molecular weight of each glucoamylase isozyme. Ten grams of the gel was hydrated with 400 ml of 0.005 ?d acetate buffer pH 3.9 in 1 M NaCl. The hydrated gel slurry was poured into a 1.2 X 200-cm column and equilibrated with several volumes of buffer. The elution volume of marker proteins of known molecular weight was determined. Gamma globulin was used to determine the void volume of the column. When t,he ratio of the elution volume of marker prot.eins to the void volume was plotted against the log of the molecular weight, a straight line resulted. The marker proteins consisted of bovine serum albumin, trypsin, a-chymotrypsin, ovalbumin, and cytochrome c. The molecular weights of the glucoamylase isozyme were estimated by t,heir elut,ion volume from the same column. This method is essentially the same as described by Whitaker (12). RESULTS

Chromatographic behavior. Representative results are plotted in Fig. 1. Two peaks of activity always occurred regardless of the Aspergillus strain used. The first peak eluted

0.6

la

11

0,2c----i’--+

0.2 0

1.0

NRRL 3112

0.4

---.-

---------,,------jo.,

F____J-----+---,~---,7-.. ... ..-

J Elution

t, -...

0

Volume -

FIG. 1. DEAE-cellulose chromatography of Aspergillus sp. culture filtrates: (A) NRRL 3112, (B) NRRL 3122; (C) cochromatography of 3112 and 3122; ---- acetate gradient. * Amount of enzyme required to form 1 g of glucose from 4 g of starch in 1 hr at 60”.

696

ET AL.

SMILEY

will be referred to as glucoamylase II and the second as glucoamylase I. This designation is consistent with the custom of referring to the isozyme with the greatest electrophoretic mobility as No. 1. As seen in Fig. 1A and B both A. awamori NRRL 3112 and A. niger NRRL 3122 elute similarly. To check on their similarity further, dialyzed culture filtrates of both organisms were combined and cochromatographed. Figure 1C reveals that the two peaks produced by each organism do chromatograph together on DEAE-cellulose. All Aspergillus strains used behaved similarly, including all sources of commercial glucoamylase tested. When the separate isozymes were isolated and tested against soluble starch, both gave nearly quantitative yields of n-glucose. The individual peaks were dialyzed and concentrated under vacuum and rechromatographed on DEAE-cellulose. Only one peak of activity appears in the expected position, which occurrence indicates that the isolation procedure does not seem to be responsible for the existence of the two forms of the enzyme. Watanabe and Fukimbara (3, 13), likewise, found two forms of glucoamylase produced by A. awamori. They concluded that there were two distinct forms of glucoamylase based on inhibition studies. They showed that the two forms of glucoamylase from their strain of A. awamori differed in their stability to acid. The two glucoamylases produced by NRRL 3112 were tested for acid stability by the method described by Watanabe and Fukimbara (3), but neither enzyme was labile to acid (Table I) TABLE ACID

TREATMENT

BY Aspergillus

awamori

Crude

a Incubated b Amount glucose from

PRODUCED

NRRL

3112

Glucoamylase unit/ml*

HI at pH 29 0 1 3 24

I

OF GLUCO.LMYL.~SE

filtrate

0.196 0.199 0.191 0.193

Glucoamylase

I Glucoamylase

0.064 0.066 0.062 0.065

0.057 0.056 0.054 0.058

at 30”. of enzyme required 4 g of starch substrate

II

to form 1 g of in 1 hr at 60”.

TABLE EFFECT

OF SUBSTRATE

BY THE Two

Substrate

(5 mg/ml)

Maltose Maltodecaose 16 D.E. dextrin Starch Glycogen

II ON GLUCOSE

GLUCOAMYL.~SES A. awamori

FORMATION

PRODUCED

BY

VI’

MI”

UI/UlI

0.42 1.72 1.99 2.55 2.42

0.37 1.51 1.85 0.74 0.37

1.14 1.18 1.08 3.4 6.54

a UI and VII = mg glucose at 60”. Enzyme concentration substrate.

produced/ml was 0.015

in 6 min units/mg

Kinetic studies with the two forms of glucoamylase. Judging from our studies and from the work of Pazur and Okada (14), Watanabe and Fukimbara (13), and Lineback et al. (4) the two forms of glucoamylase are not artifacts. A search was therefore made to see if any differences could be found in the reaction rates of the two isozymes on different substrates. Table II shows that the initial rates of glucose formation from smaller oligosaccharides and 16 D.E. dextrin by both isozymes are about the same. When either starch or glycogen is the substrate, the rate of glucose formation by the isozymes is quite different; glucoamylase II is much slower than glucoamylase I on these two substrates. Glycogen is an especially poor substrate for glucoamylase II, its rate being only about one-sixth that of glucoamylase I on this polymer. Glucoamylase II produces glucose from starch at about twice the rate it does from glycogen, but this rate is still only one-third that shown by glucoamylase I. Aside from maltose, glucoamylase I attacks all substrates at about the same rate. Maltose is known to be a rather poor substrate for glucoamylase (15). The rate of reaction of glucoamylase II depends on the degree of polymerization (DP) of the substrate. The degree of branching may also be a factor, but pullulanasetreated amylopectin was only slightly better as a substrate for glucoamylase II than untreated amylopectin. Also, 16 D.E. dextrin presumably is branched but is fully susceptible to amylolytic attack by glucoamylase II. The DP effect is shown more

KINETICS

OF

GLUCOAMYLASE

fully in Table III. Apparently maltopentaose is the smallest oligosaceharide to show near maximum reaction rates with both isoxymes. This finding confirms the conclusions of Hiromi (16) that the maximum rate of glucose formation by glucoamylase depends on a DP of about 4-5. How substrate concentration influences the rate of glucose formation with various TABLE INFLUENCK WSE

substrates was studied. Since the reaction rate of glucoamyIase II on glycogen is linear with concentration and the relative velocity (u/s) is constant (Table IV), glycogen fails to saturate the enzyme at any level used. The relative velocity of glucoamylase II on starch and 16 D.E. dextrin does decrease with increasing concentrations, and this indicates that the higher concentrations of these substrates tend to saturate the system. Glucoamylase I behaves similarly on all three substrates and did interact satisfactorily with all substrates tested. Data similar to that in Table IV were obtained for all the glucoamylase samples used in the study, and the results were graphed by either a Hofstee or LineweaverBurk plot (17). The resulting K, values are shown in Table V. On the one hand, glucoamylase I enzymes had similar K, values on all substrates tested; these values ranged from about 10 to 25 mg/ml. On the other hand, glucoamylase II enzymes do not attack glycogen sufficiently to get a K, value, and the K, on starch is considerably higher than the K, of glucoamylase I on this substrate. Further evidence that glucoamylase I differs from glucoamylase II is summarized in Table VI. Glucoamylase I is three to four times more active than glucoamylase II on starches from different sources. Gel filtration studies with Bio-Gel P-100 indicate that glucoamylase I from NRRL3112 has a molecular weight of about 71,600,

III

DF SUBSTRATE

ON VELOCITY

FORMATION BY ISOZYMr;S .ASE PROUUCED BY A.

OF GLU-

OF GLUCO.AMYLawamori Glucoamylase II (0.005 CU); mg glucose/ ml/6 min (VII)

ur,‘u11 -I-

-I DP-2c DP-3 DP-4 DP-5 DP-6 DP-7 DP-8 I)P-9 DP-10 16 11.E. dextrin Soluble starch Glycogen

0.479 0.853 1.62 1.78 1.88 1.97 1.73 1.84 1.72 1.99 2.55 2.42

a GU = glucoamylase Table I. b Glucose det;ermined peroxidase method. c DP-n = degree of oligosaccharides.

0.405 0.777 1.39 1.64 1.68 1.74 1.53 1.57 1.51 1.85 0.74 0.37 units/ml. by

the

1.18 1.10 1.17 1.09 1.12 1.13 1.13 1.17 1.14 1.08 3.4 6.54

See footnote glucose

polymerization

oxidaseof a-1,4-

TABLE SUBSTRATE

CONCENTRATION

AFFECTS ISOZYMES

RATE WITH

IV OF GLUCOSE

VARIOUS

Glycogen Substrate bx/mJ)

4 8 12 16 20 24 D Reaction autoanalyzer.

”

4s

2.05 3.65 4.90 5.95 6.65 7.00

0.513 0.456 0.408 0.372 0.333 0.292

conditions:

FORMATION

BY GLUCOAMYLASE

SUBSTRATES~

Starch Glucoal;lylase

G’ucoaImy’ase

697

ISOZYMES

”

0.30 0.50 0.70 0.90 1.10 0.015

G1ucoa;ly’ase v/s

0.038 0.042 0.044 0.045 0.046 units

”

2.50 4.15 5.45 6.55 7.00 7.60

of enzyme/O.2

16 D.E. Gluco;r;lylase

4s

0.625 0.520 0.455 0.410 0.350 0.317

u

1.00 1.20 1.80 2.15 2.50 2.95

ml of substrate.

dextrin Glucoa;;lylase

G*“coa~y’ase v/s

”

4s

0.250 0.150 0.150 0.134 0.125 0.123

1.9 3.20 4.40 5.10 5.75 6.20

0.475 0.400 0.367 0.319 0.288 0.259

Incubation

at 60” for

”

1.8 2.85 3.75 4.65 5.10 5.60 6 min

4s

0.450 0.357 0.312 0.291 0.255 0.216 on an

698

SMILEY TABLE

V

K,, VALUEX OFGLUCO.ZMYL.~SE ISOZYMES~SOLAT~D FROMSEVERALSOURCES ON GLYCOGEN,~T.~RCH, 16 D.E. DEXTRIN, .~ND MALTOSE

Substrate

I

Enzyme wurce

Km be/ml) Gluco1 Glucoamylase I amylw II

Glycogen

Starch

16

D.E. dextrin

Maltose

a (1) and

NRRL 3112 NRRL 3112 Wallerstein Miles Diazyme NRRL 3112 NRRL 3112 Wallerstein Miles NRRL 3112 NRRL 3112 Wallerstein Miles NRRL 3112 NRRL 3122 Wallerstein (2) refer

(l)a (2)

(1) (2)

(1) (2)

(1)

to two

separate

TABLE

-

14.9 13.7 25.5 25.5 14.7 10.9 19.2 18.5 18.9 21.3 21.3 21.7 16.7 mn 16.1 rnr, 16.9 mn

50 62.5 25.0 66.8 18.9 21.3 20.0 20.0 17.2 mM 17.2 mM 16.9 mM

preparations.

VI

INITI~LV~;LOCITY (TV) OF GLUCOAMYLASIZ ONDIFFERENTSTARCHSUBSTRATES

1~02~~~s

Mg glucose/ml/min Substrate (4 mg/ml)

Gluco-

Corn starch Waxy maize starch Potato starch Rice starch Q A. auramori

NRRL

amY?

Glucoamylase II

1.68 3.00

0.58 0.74

2.90 4.05

1.82 2.58

0.65 0.81

2.80 3.19

3112,

0.005

VI/U11

units/ml.

whereas glucoamylase II from the same source had a molecular weight of approximately 57,500. These values agree quite well with those determined by Lineback (18) who found form I to be 74,900 and II 54,300 by the ultracentrifuge, using the Archibald approach to sedimentation equilibrium. DISCUSSION

Lineback and co-workers (4) conducted a thorough study on chemical properties of glucoamylase isozymes. Aside from elec-

ET

AL.

trophoretic mobility, the two forms appear identical. Watanabe and Fukimbara (3) did notice a difference in acid stability between the two forms of glucoamylase from A. awamori. Although we failed to show any difference in acid stability, ample evidence is available to show that the two forms are not artifacts of the isolation procedure (1, 3, 4) and therefore presumably have some reason for existence. We established that the two forms do differ significantly in their action on large polymers, such as starch and glycogen. In fact, glycogen was such a poor substrate for glucoamylase II that the usual kinetic parameters of K, and I’,,,,, could not be determined when Hofstee plots of the data were used. Lineweaver-Burk plots for K, gave high values which are considered unreliable for the reasons discussedby Dow-d and Riggs (17). Starch was a better substrate for glucoamylaxe II, but it was still well below glucoamylase I in activity. Possibly the two isozymes require different conditions for optimum activity, but this seems unlikely because both had the same pH and temperature optimum. Neither isozyme was influenced in activity by Ca2+, Cl-, or other metallic ions. The two isozyme forms do not appear to have a monomer-dimer relationship since the molecular weights of about 72,000 vs. 5S,OOO are too close together. Furthermore, no evidence for association or dissociation from one form to the other was noticed in this study or in the one conducted by Lineback et al. (4), who obtained negative results using urea and urea plus P-mercaptoethanol as dissociating agents. The reason remains unclear for the presence of the two isozyme forms of glucoamylaseproduced by black Aspergillus. ACKNOWLEDGMENT We thank Mr. Martin Cadmus for invaluable assistance in development of the autoanalyzer procedure for kinetic studies. REFERENCES 1. P~ZUR, J. H., AND ANDO, T., J. Viol. Chem. 234, 1966 (1959). 2. SMILEY, K.L., AND HENSLEY, D.E.,Bacteriol. Proc., 12 (1964). 3. WATANABE, K., AND FUKIMBAR.4, T., J. Ferment. Technol. 43,690 (1965).

KINETICS

OF

GLUCOAMYLASE

4. LISEl3.\cIc, u. lt., l&SELL, I. ,I., AND I<.%MUSSEN, C., Arch. Biochenz. Biophys. 134, 539 (1969). 5. SYILEY, K. L., C~DMUS, M. C., HENSLEY, T). E., MD L.ZGODII, A. A., Appl. kficrobid. 12, 455 (1964). G. TODU, H., AND AKMORI, S., J. Biochem. 63, 102 (1963). i. HUTCHENS, J. O., AND K,\ss, B. M., J. Biol. Chem. 177, 571 (1949). 8. Miles Laboratories, Tech. Bull. 2-122 (1962). 9. HOFFM.YX, W. S., J. Biol. Chem. 120, 365 (1927). 10. LOWRY, I). H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951).

699

ISOZYMES

J. B., AND KESSLER, G., J. Lab. Clin. 11. HILL, Xed. 57, 970 (1961). 12. WHIT.~KER, J. It., And. Chem. 36,195O (1963). 13. WATANABE, K., ment. Technol.

.\ND FUI~IMBIK~, 45,226 (19F7).

14. Pazv~, J. H., AND OK.\I).Z, 241, 4146 (1960). 15. I~EESE, E. T., MAGUIRE, F. W., Can. J. Biochem. K., Biochem. 16. HIROMI, 40, 1 (1970).

D. R.,

personal

J.

J. Biol.

FerChem.

A. H., AND PARRISH, 46,25 (1968).

Biophys.

17. UOJVD, J. E., END RIGGS, 240, 863 (1965). 18. LINEBXK,

s.,

T.,

Res.

Commun.

1). S., J. Biol. communication.

Chem.