Behaviour of Endomycopsis fibuligera glucoamylase towards raw starch

Behaviour of Endomycopsis fibuligera glucoamylase towards raw starch S e i n o s u k e U e d a a n d Badal C h a n d r a Saha Department o f Food Science and Technology, Faculty o f Agriculture, Kyushu University, Fukuoka 812, Japan

(Received 10 August 1982 ; revised 28 September 1982) An extracellular glucoamylase [exo-l,4-Ot-D-glucosidase, 1,4-Ot-D-glucan glucohydrolase, EC 3.2.1.3] of Endomycopsis fibuligera has been purified and some of its properties studied. It had a very high debranching activity (0.63). The enzyme was completely adsorbed onto raw starch at all the pH values tested (pH 2. O- 7. 6). Amylase in hibitor from Streptomyces sp. did not prevent the adsorption o f glucoamylase onto raw starch although the enzyme did not digest raw starch in the presence of amylase inhibitor. Sodium borate (0.1 M) eluted only 35% of the adsorbed enzyme from raw starch. The optimum pH for raw starch digestion was 4.5 whereas that of boiled soluble starch hydrolysis was 5.5. Waxy starches were more easily digested than non-waxy starches, and root starches were slowly digested by this enzyme. Keywords: Starches; glucoamylase, EC 3.2.1.3 ; raw starch; Endornyeopsisfibuligera

Introduction

Materials and methods

In recent years, there has been considerable interest in glucoamylase [exo-l,4-a-D-glucosidase, 1,4-a-D-glucan glucohydrolase, EC 3.2.1.3] because of its industrial importance. A number of enzymes can hydrolyse boiled soluble starch into sugars but not all of them are able to degrade raw starch. Glucoamylase can digest raw starch I and is used for the alcoholic fermentation of raw starch without cooking.2,3 Although fungal glucoamylase, which often exists in multiple forms, has been studied extensively,4-s only a few reports are available on yeast glucoamylase 9-11 Moreover, very little is known about raw starch adsorption and digestion by yeast glucoamylase. Recently, we have reported on raw starch adsorption, elution and digestion behaviour of glucoamylase I of black Aspergillus 12, 13 and also Rhizopus niveus glucoamylase.14 We found that the black Aspergillus glucoamylase system is different from that ofRhizopus niveus with respect to raw starch adsorption, elution and digestion. The degradation of raw starch by amylase may be related to its ability to adsorb to the starch granules, and glucoamylase, having a high debranching activity, is generally strong in raw starch adsorption and digestion, is In order to elucidate in more detail the raw starch adsorption, elution and digestion mechanism of glucoamylase, it is necessary to study the process with glucoamylase from various sources. Although glucoamylase from Endomycopsis fibuligera (Saccharomycopsis fibuligera IFO 0111) has been studied 9'1°'16 nothing has yet been reported about the mechanism of its raw starch adsorption, elution and digestion. The present work deals with the raw starch adsorption, elution and digestion characteristics of the glucoamylase from this strain.

Batch cultures of Endomycopsis fibuligera (Saccharomycopsis fibuligera IFO 0111) were grown in 500 ml conical flasks containing 100 ml of liquid medium (1% soluble starch and 0.5% yeast extract), pH 6.0, on a reciprocal shaker with a frequency o f ~ 1 5 0 cycles/min at 25°C for 4 days. The culture filtrate was treated with ammonium sulphate (0.8 saturation), kept overnight and then centrifuged. The precipitate obtained was dissolved in 0.05 M acetate buffer, pH 5.0, and then dialysed against the same buffer for 48 h. The dialysed solution was concentrated by membrane ultrafiltration (Amicon PM 10) and applied to a column of DEAE-Sephadex A-25 equilibrated with 0.05 M acetate buffer, pH 5.0. The column was first eluted with the same buffer, in which case one active protein peak was obtained. It was then eluted stepwise by increasing the concentration of NaC1 (0.05,0.1,0.2 and 0.5 M) in the same buffer; in all these cases no active enzyme peak was obtained. The active enzyme peak was then subjected to preparative isoelectric focusing according to the method of Vesterberg and Svensson 17 with a carrier ampholyte of pH 3.0-10.0. In this case also, only one active protein peak (pI 6.5) was obtained. This fraction was further purified by gel filtration on Sephadex G-100 using 0.05 M acetate buffer, pH 5.0. All the procedures were performed at 4°C, unless stated otherwise. The enzyme thus obtained displayed homogeneity on 7.5% polyacrylamide gel electrophoresis performed according to the method of Davis 18 and was used throughout this investigation. Glucoamylase activity was determined as follows. The reaction mixture containing 5 ml of 1% boiled soluble starch solution, 1 ml 0.2 M acetate buffer, pH 4.8, 1 ml deionized water and I ml of suitably diluted enzyme solution in a final volume of 8 ml was incubated at 40°C. After a 10 min reaction, 1 ml of the reaction mixture was withdrawn and

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Enzyme Microb. Technol., 1983, Vol. 5, May

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Endomycopsis fibuligera glucoamylase: S. Ueda and B. C. Saha

the amount of reducing sugar formed was measured by the micro-Bertrand method.19 One unit of glucoamylase activity is defined as the amount of enzyme which liberates 1 mg glucose in 1 ml of the reaction mixture under the above conditions. Debranching activity of glucoamylase was measured according to the method reported previously.6 The procedures we followed for raw starch adsorption, elution and digestion have been described in a previous report.14 Protein was determined by measuring the absorbance at 280 nm using bovine serum albumin as standard or by the method of Lowry et al. 20 Total carbohydrate content was measured by the phenol-sulphuric acid method. 21 Molecular weight was estimated by gel filtration on Sephadex G-100 according to the method of Andrews.22

amylase onto raw starch was investigated. Glucoamylase (1.71 units m1-1) and amylase inhibitor (16 ~g m1-1 with respect to sugar content of the inhibitor) solution (5 ml) at pH 4.5 was left for 20 min at 30°C and in this case the enzyme was almost completely inactivated. Then, the adsorption of the enzyme-inhibitor complex onto raw starch was carried out. Here, instead of assaying amylase activity of the supernatant, the absorbance at 280 nm was measured. A similar procedure was also followed for the inhibitor-untreated enzyme solution. It was found that the adsorption of the enzyme-inhibitor complex onto raw starch occurred almost completely like the untreated enzyme. So, the active site and the raw starch adsorption site in the enzyme molecule seem to be different. The same finding was obtained in the case of glucoamylase I of black Aspergillus 13 and Rhizopus niveus glucoamylases)4

Results and discussion

Elution o f adsorbed e n z y m e from raw starch

Purity and properties o f glucoamylase Table i summarizes the purification of glucoamylase ofEndomycopsis fibuligera. The glucoamylase thus purified

In the previous paper, 13 we reported that >90% of the adsorbed glucoamylase I of black Aspergillus could be easily eluted from raw starch by applying boric acid (0.05 M)--borax (0.1 M) buffer, pH 8.2, or simply 0.02 N sodium borate. It was assumed that the hydroxy groups of the starch molecule are involved in the adsorption of glucoamylase I of black Aspergillus and that when borate buffer or borate solution under mild alkaline conditions is applied the borate forms a complex with the hydroxy groups of starch and thus the enzyme molecule becomes detached from starch. But in the case ofRhizopus niveus glucoamylase, no significant elution could be achieved under similar conditions. One possible reason for the failure of borate to elute Rhizopus niveus glucoamylases from raw starch may be that the adsorption was less dependent upon pH and the enzymes were well adsorbed, even at pH 8.0. We tried to elute the adsorbed glucoamylase of Endornycopsisfibuligera from raw starch by borate buffer or sodium borate solution under the same conditions as reported. But in this case, as with Rhizopus niveus glucoamylase, no significant elution could be made. Only ~35% of the adsorbed enzyme was eluted by a much higher concentration of sodium borate (0.1 M) and the percentage elution was not increased, even after prolonging the time of elution from 30 min to 2 h. Although glucoamylase I of black Aspergillus can be eluted by sodium molybdate (0.5 M) optimally at pH 6.0 (unpublished data), no elution was done by molybdate under the same conditions in this case. The adsorbed enzyme was also not eluted at all at an acidic pH of 2.0 (glycine-HC1 buffer, 0.1 M). Thus, raw starch elution behaviour of glucoamylase of Endomycopsis fibuligera is different from that of glucoamylase I of black Aspergillus but somewhat similar to that of Rhizopus niveus glucoamylase. As the adsorption of glucoamylase onto raw starch is thought to be a physical phenomenon and it is not at all dependent upon pH in the case of Endomycopsis fibuligera enzyme, elution of the adsorbed enzyme may not be easily achieved by a change of pH.

had about 125-fold higher purity than the crude enzyme. It is interesting that the glucoamylase system contained only one form, in contrast to the glucoamylase systems of Aspergillus or Rhizopus sp. The enzyme had molecular weight ~ 4 0 000, carbohydrate content 9.5%, K m value towards soluble starch of 20 mg m1-1 and hydrolysis limit of soluble starch 87%. The debranching activity of the glucoamylase was found to be 0.63, which indicates that the enzyme has a very strong debranching activity.

Adsorption onto raw starch The glucoamylase (1.71 units m1-1) was found to be completely adsorbed onto raw starch within the pH range (2.0-7.6) tested. It is interesting that the adsorption of the enzyme was unaffected by pH (pH 2.0-7.6) under the conditions tested. The adsorption of black Aspergillus glucoamylase I was highly dependent upon pH: adsorption was optimal at pH 3.4 and it had a very sharp adsorption maximum.13 The adsorptions ofRhizopus niveus glucoamylases were not so dependent upon pH and the optimum adsorption of these enzymes occurred over a broad range of pH.14 The effect of time of adsorption of glucoamylase onto raw starch was tested and it was found that complete adsorption of the enzyme occurred within 15 min. The effect of temperature of adsorption was not so prominent for this enzyme. Adsorption decreased by ~6% only with the increase of temperature from 4 to 40°C, although low temperature generally favoured adsorption. 23 The effect of amylase inhibitor from Streptomyces sp. (partially purified 24) on the adsorption of the ghicoTable 1 Purification of Endomycopsis fibuligera glucoamylase

Total

Specific activity (units mgprotein)

Step

(mg)

Total activity (units)

Crude enzyme

110 819

4295

0.039

4 180 568 226 128

2675 2114 1042 631

0.64 3.72 4.61 4.93

protein

(culture filtrate) (NH4)2SO4 precipitation DEAE-Sephadex A-25 Isoelectric focusing Sephadex G-100

R a w starch digestion The optimum pH of boiled soluble starch and raw starch hydrolysis by the glucoamylase were found to be 5.5 and 4.5, respectively. The optimum pH of raw starch digestion by black Aspergillus glucoamylase I was 3.4 and it was different from that (4.5) of soluble starch hydrolysis)2 The optimum pH of raw starch and soluble starch hydrolysis were also different in the case ofRhizopus niveus glucoamylases. 14 From these three cases, it may be concluded that

Enzyme Microb. Technol., 1983, Vol. 5, May

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Papers Table 2 Digestion of different kinds of raw starch by the glucoamylase (1.71 units)

2 3

Starch (50 mg)

% Hydrolysis in 24 h

4

Wheat Rice Corn Cassava Sweet potato Potato Waxy rice Waxy corn

38.0 41.5 35.0 34.9 20.8 2.9 95.1 80.5

5 6

the optimum pH for raw starch digestion and soluble starch hydrolysis by a glucoamylase are different. The enzyme could not digest raw starch in the presence of amylase inhibitor. Table 2 shows a comparative study of the digestion of different kinds of raw starch by the enzyme. Waxy starches were more easily digested than cereal starches. Cassava starch, although it is a root starch, was easily digested, like corn starch. From the results obtained it is clear that the Endomycopsis fibuligera glucoamylase is similar to Rhizopus niveus glucoamylase but quite different from glucoamylase I of black Aspergillus with respect to raw starch adsorption, elution and digestion.

References 1

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Ueda,S. TrendsBiochem. ScL 1981, 6, 89-90

E n z y m e Microb. Technol., 1983, Vol. 5, M a y

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Ueda,S. and Koba, Y.J. Ferment. Technol. 1980, 58, 237-242 Ueda, S., Zenin, C. T., Monteiro, D. A. and Park, Y. K. Biotechnol. Bioeng. 1981, 23,291-299 Pazur,J. H., Knull, H. R. and Cepure, A. Carbohydr. Res. 197l, 20, 83-96 Ueda, S., Ohba, R. and Kano, S. Stdrke 1974, 26,374-378 Lineback, D. R., Russell, I. J. and Rasmussen, C. Arch. Biochem. Biophys. 1969, 134,539-553 Ueda, S. and Kano, S. Stdrke 1975, 27, 123-128 Saha,B. C., Mitsue, T. and Ueda, S. Stdrke 1979, 31,307-314 Hattori, Y. Agric. Biol. Chem. 1961, 25,737-743 Fukui, T. and Nikuni, Z. Agric. Biol. Chem. 1969, 33, 884-891 Sukhumavasi,J., Kato, K. and Harada, T. J. Ferment. Technol. 1975, 53,559--565 Medda, S., Saha, B. C. and Ueda, S.J. Fac. Agric., Kyushu Univ. 1982, 26, 139-149 Medda, S., Saha, B. C. and Ueda, S.Z Ferment. Technol. 1982, 60, 261-264 Saha, B. C. and Ueda, S.J. Ferment. Technol. in press Ueda, S. and Saha, B. C. Utilization Res. 1981, 1, 9-17 Hattori, Y. and Takeuchi, I. Agric. Biol. Chem. 1961, 25, 895-901; 1962, 26,316--322 Vesterberg,M. and Svensson, H. Aeta Chem Scand. 1966, 20, 820-834 Davis,B. J. Ann. NYAcad. ScL 1964, 121,404-427 Klein, G. Handbuch der Pflanzenanalyse H, Specielle Analyse. Wien 1932, vol. 1, p. 786 Lowry,O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. J. Biol. Chem. 1951, 193,265-275 Dubios, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. Anal. Chem. 1956, 28,350-356 Andrews,P. Biochem. J. 1965, 96,595-606 Walker,G. J. and Hope, P. M. Biochem. J. 1963, 86,452-462 Nakano, H., Tajiri, T., Koba, Y. and Ueda, S. Agric. Biol. Chem. 1981, 45, 1053-1060