Glucoamylase 0 from a Protease-Negative, Glycosidase- Negative Aspergillus awamori var. kawachi Mutant

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1983, p /83/395-8$2./ Copyright 1983, American Society for Microbiology Vol. 45, No. 3 Production and Characteristics of Raw Starch-Digesting Glucoamylase from a Protease-Negative, Glycosidase- Negative Aspergillus awamori var. kawachi Mutant PERFECTO Q. FLOR AND SHINSAKU HAYASHIDA Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Kyushu University 46, Fukuoka 812, Japan Received 24 August 1982/Accepted 23 November 1982 Production of a raw starch-digesting glucoamylase (GA ) by proteasenegative, glycosidase-negative mutant strain HF-15 of Aspergillus awamori var. kawachi was undertaken under submerged culture conditions. The purified GA was electrophoretically homogeneous and similar to the parent glucoamylase I (GA I) in the hydrolysis curves toward gelatinized potato starch, raw starch, and glycogen and in its thermostability and ph stability, but it was different in molecular weight and carbohydrate content (25, and 24.3% for GA, 9, and ca. 7% for GA I, respectively). The chitin-bound GA hydrolyzed raw starch but the chitin-bound GA I failed to digest raw starch because chitin was adsorbed at the raw starch affinity site of the GA I molecule. The removal of the raw starch affinity site of GA with subtilisin led to the formation of a modified GA (molecular weight, 17,), which hydrolyzed glycogen 1%, similar to GA and GA I, and was adsorbed onto chitin and fungal cell wall but not onto raw starch, Avicel, or chitosan. The modified GA I (molecular weight, 83,) derived by treatment with subtilisin hydrolyzed glycogen up to only 8% and failed to be adsorbed onto any of the above polysaccharides. The N-bromosuccinimideoxidized GA lost its activity toward gelatinized and raw starches, but the abilities to be adsorbed onto raw starch and chitin were preserved. It was thus suggested that both the raw starch affinity site essential for raw starch digestion and the chitin-binding site specific for the binding with chitin in the cell wall could be different from the active site, located in the three respective positions in the GA O molecule. Aspergillus awamori var. kawachi (parent strain) produced raw starch-digesting glucoamylase I (GA I; molecular weight, 9,) selectively in submerged culture under the conditions of culture A (5). GA I was converted in vitro into the different types of glucoamylases such as modified GA I (GA I'; molecular weight, 83,) and glucoamylase II (molecular weight, 57,) by stepwise degradations with proteases and glycosidases (9, 11, 21). The enzymatic capacity for hydrolysis and adsorption to raw corn starch was lost, but the ability to hydrolyze gelatinized starch and glycogen was retained in those modified glucoamylases. The conversion of GA I into raw starch-indigesting GA I' and glucoamylase II also occurred during the submerged production of the enzyme and was correlated with the presence of proteases and glycosidases in the culture filtrate (5, 9, 11, 21). We also report the presence of a raw starch affinity site different from the active site in the GA I molecule, and this site was found to be responsible for the adsorption of the enzyme onto chitin and raw 95 starches (8). This raw starch affinity site was found to be specifically cleaved by subtilisin, leading to the formation of a chitin- and raw starch-inadsorbable GA I' (8, 9). The binding of GA I with chitin also led to the formation of GA 1-chitin complex which had no raw starch digesting ability because chitin was adsorbed at the raw starch affinity site of the enzyme (8). However, the complex retained its ability to hydrolyze gelatinized starch and glycogen, similar to the unbound enzyme. Recently, we reported that both the a-amylase and glucoamylase of the protease-negative glycosidase-negative mutant HF-15 of A. awamori var. kawachi were intensively adsorbed onto chitin without the aid of a cross-linking agent (glutaraldehyde) and that the resulting mutant amylase-chitin complex digested raw corn starch to glucose, similar to the unbound amylase (7). This study reports the production, purification, and properties of the raw starch-digesting glucoamylase (GA ) produced by a mutant

2 96 FLOR AND HAYASHIDA strain HF-15 of A. awamori var. kawachi and presents evidence for the presence of a chitinbinding site in the glucoamylase different from the active and raw starch affinity sites. MATERIALS AND METHODS Organism. The organism used was a mutant, strain HF-15, selected from the parent strain of A. awamori var. kawachi. This mutant produces selectively a high amount of raw starch-digesting glucoamylase under any cultural condition as previously reported (6). Stock cultures were maintained in potato dextrose agar and kept at 4 C. Seed culture. The mutant HF-15 was grown in potato dextrose agar slants for 7 days at 3 C and then inoculated into a previously prepared sterile synthetic medium A (5), slightly modified by using tap water. The seed culture was incubated at 3 C with continuous reciprocal shaking for 48 h before transfer to the main culture. Primary submerged culture. The primary culture was prepared by using the slightly modified medium A. The main culture medium (12 liters) was prepared in a 2-liter fermentor (Marubishi-Rikasochi Laboratory), sterilized at 12 C for 2 min, cooled to 32 C, and inoculated with the previously prepared 48-h seed culture at a concentration of 5% (vol/vol). The culture was aerated continuously at the rate of 12 liters/min and agitated at 2 to 25 rpm. The fermentation temperature was maintained at 32 C. The culture filtrate was collected after the fermentation period for the purification of the mutant raw starch-digesting glucoamylase. The glucoamylase was designated GA O because it was different in several properties from GA I. Assay of proteases, glycosidases, glucoamylase, and raw starch adsorption and digestion. These procedures were described in a previous paper (6). Preparation of GA I and GA I'. Purified GA I and GA I' from A. awamori var. kawachi were prepared according to the methods reported previously (5, 9, 11, 21) Ġeneral analytical procedures. The total carbohydrate content of the enzyme was determined by the phenol-sulfuric acid method (2), using mannose as a standard. Neutral sugar components were identified by paper chromatography with Whatman no. 1 filter paper and developed by ascending technique in a solvent system of n-butanol:pyridine:water (6:4:3 [vol/vol]) at room temperature. Sugars were detected by the dipping method with silver nitrate-sodium hydroxide reagent (18). Neutral sugars were confirmed by gas-liquid chromatography by the method of Sweeley et al. (17), using a Hitachi gas chromatograph (model 163) with a flame ionization detector and stainless steel column (2 m by 3 mm) containing l1o silicone SE-52 on 8 to 1 mesh chromosorb WAW at 125 to 25 C, programmed at the rate of 2 C/min. The N-acetylglucosamine content of the enzyme was assayed according to the method of Johnson (12). Polyacrylamide gel disc electrophoresis was carried out on 7.5% polyacrylamide gel with a Tris buffer system, ph 8.3 (1). The gel was stained with.5% Coomassie brilliant blue R-25. Destaining was done by using a solution containing 75 ml of acetic acid, 5 ml of methanol, and 875 ml of deionized water. APPL. ENVIRON. MICROBIOL. The molecular weight of the purified enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2). Protein bands were detected by staining the gel with Coomassie brilliant blue R-25 solution (Coomassie brilliant blue, 1.25 g; 5% methanol-water solution, 454 ml; acetic acid, 46 ml). The destaining solution was the same as that used in polyacrylamide gel electrophoresis. The protein content was determined by the method of Lowry et al. (13), using bovine serum albumin as standard. Purification of GA. Based on the procedures above, the mutant HF-15 was cultivated for 68 to 72 h for enzyme production, and the resulting culture filtrate was collected for enzyme purification. Unless otherwise specified, all operations were carried out at 4C. (i) Step 1. Ammonium sulfate filtrate (4, ml) was added to a portion of the culture filtrate to 6% (wt/vol) concentration and kept overnight. The resulting precipitate was collected by filtration with Celite and then dissolved in a small volume of pure water. After the removal of Celite by centrifugation, the supernatant was dialyzed against running tap water for 2 days. (ii) Step 2. After dialysis, the enzyme solution was adjusted to ph 2. with 1 N sulfuric acid and left to stand for 48 h. The precipitate was removed by centrifugation at 12, rpm for 1 min, and the supernatant was adjusted to ph 9. with ammonia water and allowed to incubate for 18 h. The resulting precipitate was again discarded, and the ph of the supernatant was adjusted to ph 5.. The above treatments completely inactivated all the contaminating a- amylase, protease, and glycosidase but not glucoamylase in the culture filtrate. (iii) Step 3. Solid ammonium sulfate was added to the supernatant to give a 3o (wt/vol) concentration. After 24 h, the precipitate was discarded and an additional amount of ammonium sulfate was added to give a 5% (wt/vol) concentration. After allowing the solution to stand overnight, the precipitate was collected, dissolved in a minimal volume of water, and dialyzed for 2 days with deionized water. The desired amount of Rivanol was added to the dialyzed sample, and the resulting precipitate was dissolved in.5 M sodium acetate buffer, ph 5.. The Rivanol was removed by acid clay, and the supernatant was further fractionated with solid ammonium sulfate at a concentration of 3 to 5% (wt/vol). The precipitate formed after standing overnight was dissolved in a minimal volume of pure water, dialyzed for 2 days with deionized water, and concentrated. (iv) Step 4. The concentrated enzyme was mixed with a small volume of.5 M phosphate buffer, ph 5.5, and applied on a DEAE-Sephadex A-5 column (2. by 9 cm) previously equilibrated with.5 M phosphate buffer, ph 5.5. The enzyme was eluted with 5 ml of a linear gradient from.5 to 1 M phosphate buffer, ph 5.5. The eluate was collected in a volume of 5 ml per tube at a rate of 2 ml/h. Only one main peak with glucoamylase activity was observed in fractions 78 through 119. Fractions 85 to 118 were combined, desalted by filtration through a Sephadex G-5 column, and concentrated. (v) Step 5. The concentrated enzyme was again mixed with.5 M phosphate buffer, ph 5.5, and then

3 VOL. 45, 1983 applied for the second time on DEAE-Sephadex A-5, following the same procedure as that in step 4. Figure 1 shows the good correspondence between protein peak and glucoamylase activity. Fractions 9 to 11 were combined, desalted in Sephadex G-5, and lyophilized. The lyophilized sample was designated as purified mutant glucoamylase (GA ) and kept in an evacuated dessicator at 4 C. The recovery and specific activity of GA during the purification procedure are summarized in Table 1. Polysaccharide substrates. Purified chitin from crab shells and chitosan were purchased from Sigma Chemical Co. Alkali-treated fungal cell wall was prepared from the mycelium of mutant HF-15, following a previously reported method (8). Raw corn starch (Japanese pharmacopoeia) was purchased from Wako Pure Chemical Industries Ltd., and Avicel (microcrystalline cellulose) was supplied by Asahi Kasei Co. Chemical modification of GA by N-bromosuccinimide. N-Bromosuccinimide was purchased from Wako Pure Chemical Industries Ltd. The oxidation of tryptophan residues was monitored by using a Hitachi spectrophotometer (model 124) fitted with a Hitachi recorder (model 56). RESULTS Enzyme production. During the cultivation of mutant HF-15 for 4 days under the submerged culture conditions in the 2-liter fermentor, a portion of culture filtrate was examined for enzyme activities and ph. The maximum amount of glucoamylase was observed after 72 h, whereas a-amylase was highest after 84 h (Fig. 2). A small peak of acid protease (1.9 U) was observed after 6 h and then gradually decreased. N-Acetyl-o-D-glucosaminidase appeared after 24 h, with a peak at 36 h. The mutant failed to produce a-mannosidase and neutral and alkaline proteases throughout the co Q A. AWAMORI MUTANT RAW STARCH-DIGESTING AMYLASE 97 culture period. The ph of the culture filtrate decreased rapidly from 7. to 3. after 36 h and then increased gradually up to 6.7 at % h. No addition of CaCO3 or adjustment of ph was undertaken throughout the culture period. The parent strain produced 4.2 U of a-mannosidase after 1 h and 18.2 U of acid protease after 6 h under the same culture conditions. Homogeneity of purified GA. The lyophilized sample showed a single protein band on polyacrylamide gel electrophoresis. Properties of mutant glucoamylase. (i) Molecular weight. Logarithmic plots of reference proteins versus their relative mobilities are shown in Fig. 3. The molecular weight of GA was estimated to be 25, by sodium dodecyl sulfate electrophoresis. (ih) Carbohydrate composition. The total carbohydrate content of GA was estimated to be 24.3% as mannose. The neutral sugar component was only mannose as confirmed by paper and gas-liquid chromatography. (iii) N-Acetylglucosamme content. The amino sugar content was identified by paper chromatography. When the acid hydrolysate of the enzyme was chromatographed in ethyl acetate:pyridine:water (1:4:3), sprayed with acetylacetone-1-butanol-koh solution and ethanol (16), and heated at 15 C, a single red spot corresponding to the Rf value of standard N- acetylglucosamine was observed after spraying with a solution of N,N-dimethyl-p-aminobenzaldehyde (1 g) in a mixture of ethanol (3 ml), concentrated hydrochloric acid (3 ml), and N- butanol (3 ml) followed by heating at 15 C. The N-acetylglucosamine content of the enzyme was estimated to be 2.1% according to the 5 C C- i OF S i Y a E Fraction number ( 5ml ) FIG. 1. Step 2 DEAE-Sephadex A-5 chromatography of GA. Symbols:, absorbance at 28 nm;, glucoamylase activity. Experimental details are described in the text.

4 98 FLOR AND HAYASHIDA TABLE 1. Purification of the raw starch-digesting GA of A. awamori var. kawachi mutant HF-15 Vol Total Total Sp act Recovery Purification step (Ml) activity protein (U/Mg of R (U) (mg) protein) Crude filtrate 4, 155,2 5, %o (NH4)2SO4 fractionation 2 6, 1, After acid and alkaline treatment 25 27, to 5% (NH4)2SO4 2 22, fractionation and Rivanol treatment First DEAE-Sephadex A , Second DEAE-Sephadex A , method of Johnson (12). (iv) Thermostablity and ph stability. Portions of enzyme solutions prepared in.1 N phosphate buffer, ph 5.5, were kept for 15 min at various temperatures. Both GA and GA I were stable at 6 C, but the latter was less stable above 65 C (Fig. 4). To determine the ph stability, the enzyme was dissolved in the following buffer systems: Clark-Lubs (ph 1. to 2.); Sorensen citrate (ph 2.5 to 5.); Sorensen phos- phate (ph 5.5 to 7.5); Clark borate (ph 8. to 1). The mixture was kept at 3 C for 2 h. The residual activity- was measured under the standard assay conditions (6). GA was slightly more stable at lower and higher ph as compared with GA I (Fig. 4). (v) Hydrolysis curves for various substrates. The hydrolytic curves exhibited by GA for various amylaceous substrates follow the same pattern as those for GA I. This shows that both enzymes could hydrolyze raw corn starch and glycogen to glucose up to 1% and gelatinized potato starch up to 9% (5, 9, 11, 21) x 1o1 ;s _ 2 _ APPL. ENVIRON. MICROBIOL TIME (DAYS) FIG. 2. Time courses of enzyme production of GA under submerged culture conditions. Symbols:, glucoamylase activity; l, a-amylase; U, acid protease; A, N-acetyl-,3-D-glucosaminidase; A, a-mannosidase; *, ph. Experimental details are described in the text Mobility FIG. 3. Estimation of molecular weight of GA O by sodium dodecyl sulfate electrophoresis. Molecular weights of standard proteins: 1, chymotrypsinogen (2.5 x 1w); 2, ovalbumin (4.5 x 14); 3, bovine serum albumin (6.7 X 14); A. awamori var. kawachi (parent) GA 1 (9. x 14); 5, gamma globulin (16 x 14); 6, GA O (25 x 14); 7, apoferritin (48 x 14). Experimental details are described in the text.

5 VOL. 45, 1983 A. AWAMORI MUTANT RAW STARCH-DIGESTING AMYLASE 99 1_ 8.5. Go Temperature ( OC ) ph 1 FIG. 4. Thermal stability (A) and ph stability (B) of GA and GA I. Symbols:, GA ;, GA I. Experimental details are described in the text. Enzyme activities were measured after allowing the reaction mixtures to stand at ph 5.5 at various temperatures for 15 min to determine thermostability and to stand at 3C for 2 h at various ph values to determine ph stability. The residual activities were expressed as a percentage of the activity in the untreated control. Evidence for a chitin binding site on mutant GA. (i) Digestion of raw corn starch by the chitinbound glucoamylases. Three grams of highly purified chitin was first allowed to swell by the addition of 3 ml of.1 M citrate buffer, ph 3.6, and then saturated with 25 ml of previously prepared enzyme solution (45 U/ml in.2 M citrate buffer, ph 3.8) and allowed to stand for 2 h at 4 C. The chitin-glucoamylase complex was washed several times with distilled water until no activity was observed in the washings. GA and GA I were tightly bound onto chitin (Table 2) and were not removed by the washing treatment. As shown in Fig. 5, the chitin-bound GA O digested raw corn starch, whereas the chitinbound GA I failed to exhibit any raw starch digestibility. (ii) Effect of masking the raw starch affnity site of GA before adsorption onto chitin. The raw starch affinity site of GA was masked with raw corn starch before adsorption onto chitin by the following procedure. Raw corn starch (35 g) was saturated with GA solution (3 mg of enzyme dissolved in 15 ml of.2 M citrate buffer, ph 3.8), which was mixed occasionally, kept at 4 C for 2 h, and then centrifuged to collect the raw starch-ga complex. One gram of chitin (suspended in 15 ml of.2 M citrate buffer, ph 3.8) was added and mixed occasionally at 4 C. After 6 h, the mixture was centrifuged to remove the raw starch-ga -chitin complex. To the resulting complex, 15 ml of.2 M citrate buffer, ph 7.8, was added, and the suspension was mixed occasionally at 4 C. After 24 h, the GA -chitin complex was separated from the raw starch by filtration with cloth gauze. The use of higher ph (neutral to alkaline) caused the desorption of the enzyme from the starch granules without affecting the adsorption of the enzyme onto chitin. As previously reported (3, 8), the adsorption of the enzyme onto raw corn starch was ph dependent but was ph independent for adsorption onto chitin. After washing the complex several times with pure water, its raw starch digestibility was compared with the unmasked GA -chitin complex and unbound 1 o?5o -J TIME (DAYS) FIG. 5. Digestion of raw corn starch by chitinbound GA I and GA. Symbols:----- chitin-bound enzyme; ~, unbound enzyme; 4, GA I; A, GA. The reaction mixture containing.3 g of raw corn starch, 48 ml of.2 M citrate buffer, ph 3.8, 24 U of unbound or chitin-bound enzyme, with ml of toluene was incubated statically at 3C. At suitable intervals, the reducing sugar formed was determined by the micro-bertrand method and the degree of hydrolysis was calculated.

6 91 FLOR AND HAYASHIDA enzyme. The raw starch digestion rate of the masked GA -chitin complex increased by almost twofold as compared with the unmasked GA -chitin complex with the same level of enzyme activity (Fig. 6). (iii) Modification of mutant GA with subtilisin. GA was modified with subtilisin (Nagarse Sangyo Co.; 1 x 14 PUN/g [PUN, optical density equivalent to one gamma L-tyrosine released in 1 min at 3 C and ph 7.5]) similar to the modification of GA I as previously reported (3, 9). Crystalline subtilisin was added to 1 ml of a.1 M phosphate buffer, ph 6., containing 1 mg of GA. The reaction mixture was incubated at 3 C for 24 h, after which the ph was adjusted to 2.6 and the mixture was left to stand for 18 h at 4 C. The subtilisin was inactivated and precipitated by this treatment. The precipitate was removed by centrifugation, the ph was adjusted to 5.5, and the precipitate was applied to a DEAE-Sephadex A-5 column (2. by 9 cm) previously equilibrated with.5 M phosphate buffer. The enzyme was eluted with a linear gradient from.5 M to 1 M phosphate buffer, ph 5.5. The eluate was collected in a volume of 5 ml per tube at the rate of 2 ml/h. The chromatographic pattern of the subtilisindigested GA is shown in Fig. 7. The subtilisindigested GA yielded four fractions: A, B, C, and D. Fractions B and D were both inactive peptides, whereas fraction C was active and thus -J 1 Oṙ TIME (DAYS) FIG. 6. Effect of masking on the raw corn starch digestion of chitin-bound GA O. Symbols: O, unbound GA ; A, masked chitin-bound GA ;, unmasked chitin-bound GA. The reaction mixture was similar to that described in the legend to Fig. 5. Experimental details are described in the text. I I I 1. APPL. ENVIRON. MICROBIOL. 5 1 FRACTION NO. 5 X. 252 FIG. 7. DEAE-Sephadex A-5 chromatography of the subtilisin-digested GA. Absorbance at 49 nm was determined after adding the phenol-sulfuric acid reagent. Experimental details are described in the text. designated as GA O'. Fraction A was undigested GA. The active fraction C (GA ') was collected, desalted, and further purified by DEAE-Sephadex A-5 column chromatography. The molecular weight of the purified GA O' was estimated to be 17, by sodium dodecyl sulfate electrophoresis, with a total carbohydrate content of 15.2% with mannose as standard. Furthermore, the hydrolysis curves for gelatinized starch and glycogen of GA O' were different from those of GA I', but they were similar to GA and GA I as previously reported (3, 5, 8). GA O' failed to be adsorbed onto raw corn starch because the raw starch affinity site was removed by subtilisin, but it retained its adsorbability onto chitin in contrast with GA I', which lost its ability to adsorb onto both raw corn starch and chitin. This indicated that GA contained a separate binding site for chitin. Both of the subtilisin-modified glucoamylases, however, failed to digest raw corn starch. The adsorption of different types of glucoamylases onto various polysaccharides is shown in Table 2. GA O' was adsorbed onto chitin and fungal cell wall but not onto Avicel, chitosan, and raw corn starch, whereas GA I' failed to be adsorbed onto any of the above polysaccharides. Both GA and GA I were adsorbed onto chitin, fungal cell wall, and raw corn starch but not onto Avicel and chitosan. Effect of N-bromosuccinimide on GA. When 1 mg of GA was treated with various quantities of N-bromosuccinimide (.2 M acetate buffer, ph 4.), no significant effect on the enzymatic activity of the enzyme was observed with up to 5,uM (reaction mixture was 4 ml containing 5,uM N-bromosuccinimide and 1 mg of GA incubated at 27 C for 3 min) (Fig. 8). Further oxidation gradually inactivated the enzyme. However, the N-bromosuccinimide-oxidized GA (4 ml of 5 pmol of N-bromosuccini- a ta

7 VOL. 45, 1983 A. AWAMORI MUTANT RAW STARCH-DIGESTING AMYLASE 911 TABLE 2. Adsorption of glucoamylases onto polysaccharidesa Adsorption rate (U/g) Substrate GA GA GA GA O' I I' Chitin Alkali-treated fungal cell wall Raw corn starch Avicel Chitosan a Various amounts of enzyme were separately dissolved in 5 ml of.2 M citrate buffer, ph 3.6, and then applied onto various amounts of polysaccharides. The mixtures were left to stand for 3 min at 4 C. The enzyme activities were determined before and after adsorptions, and the adsorption rate was calculated. mide in.2 M acetate buffer, ph 4., per mg of GA ) retained completely its adsorbability onto raw corn starch and chitin, even after losing 98% of its catalytic activity (Table 3). DISCUSSION In previous reports, we have shown that the protease-negative, glycosidase-negative mutant strain ofa. awamori var. kawachi, HF-15, overproduced raw starch-digesting glucoamylase under any conditions of submerged or solid cultures (6). Recently, we further reported the use of the above glucoamylase in the saccharification of raw ground corn for high-concentration ethanol fermentation (1). The purified mutant glucoamylase was electrophoretically homogeneous with a molecular weight of 25,, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a total carbohydrate content of 24.3%, in contrast with GA I, which has a molecular weight of 9, and a carbohydrate content of ca. 7% (11, 21). The carbohydrate content of the enzyme was composed mainly of mannose and N-acetylglucosamine, similar to GA I (11). GA and GA I exhibited similar thermostability, ph stability, and hydrolysis curves toward gelatinized potato starch, glycogen, and raw starches. However, the chitin-bound GA I retained its ability to hydrolyze gelatinized potato starch and glycogen but lost its ability to digest raw starches because the chitin was adsorbed at the raw starch affinity site (8). The specific removal of the raw starch affinity site of GA I with alkaline protease (subtilisin) led to the formation of an inactive peptide containing the raw starch affinity site, and GA I', with lower hydrolytic properties toward gelatinized potato starch and glycogen and without adsorbability onto raw corn w O,af N-BROMOSUCCINIMIDE ( IMOLE) FIG. 8. Effect of N-bromosuccinimide concentration on the enzymatic activity of GA. The reaction mixtures containing 1 mg of enzyme in 4 ml of N- bromosuccinimide solutions (.2 M acetate buffer, ph 4.) at various concentrations were incubated for 3 min at 27C. The residual activity was determined by following the standard assay procedure. starch and chitin (8, 9). The purified glycopeptide containing the raw starch affinity site of GA I was adsorbable onto both raw corn starch and chitin (8). On the contrary, the chitin-bound GA hydrolyzed not only gelatinized potato starch and glycogen but also raw corn starch, in contrast with the parent chitin-bound GA I. Also, the masking of the raw starch affinity site of GA with raw corn starch before contact with chitin caused an almost twofold increase in the raw TABLE 3. Adsorption of N-bromosuccinimideoxidized GA onto raw corn starch and chitina Absorbance (at 28 nm) Prepn GA N-Bromosuccinimide- (control) oxidized GA Before adsorption After adsorption.82.8 onto chitin After adsorption.1.98 onto raw corn starch a Pure raw corn starch (1.5 g) and chitin (5 mg) were washed several times with pure water, and then 4 ml of N-bromosuccinimide-oxidized GA (4 ml of 5,uM N-bromosuccinimide in.2 M acetate buffer, ph 4., containing 1 mg of GA, incubated for 3 min at 27 C before use) was mixed into each substrate; the mixture was then left to stand for 3 min at 4 C. After centrifugation the absorbance of the supernatant fluid was determined with a Hitachi spectrophotometer (model 124).

8 912 FLOR AND HAYASHIDA starch digestion rate of the prepared chitinbound enzyme. Similar to GA I, the removal of the raw starch affinity site of GA with subtilisin resulted in the loss of raw starch adsorbability and digestibility. However, the GA O' which was formed after the treatment with subtilisin showed a molecular weight of 17, and a carbohydrate content of 15.2%, which were substantially higher than those of GA I' (molecular weight, 83,; carbohydrate content, 4.6%) reported previously (9, 11). In addition, GA O' still possessed the ability to hydrolyze gelatinized potato starch and glycogen with the same hydrolysis curves as those of GA and GA I (11). Furthermore, it is very interesting to note that GA O' was adsorbed onto chitin and alkali-treated cell wall but not onto raw corn starch, chitosan, and Avicel (microcrystalline cellulose) in contrast with GA I', which failed to be adsorbed onto any of the above substrates. The chitin-bound GA O' hydrolyzed gelatinized starch and glycogen to glucose to a large extent, similar to the unbound enzyme. This indicated that the active site of GA was not affected by the adsorption of the enzyme onto chitin or the removal of the raw starch affinity site by subtilisin. The oxidation of GA by N-bromosuccinimide resulted in the loss of catalytic activity, indicating that tryptophan was involved in the active site of the enzyme similar to chymotrypsin (19), Rhizopus niveus glucoamylase (14), bacterial saccharifying and liquefying oa-amylase, and Taka-amylase A (4, 15). However, the loss of catalytic activity of the N-bromosuccinimide-oxidized GA did not prevent the adsorption of the enzyme onto raw corn starch and chitin. The above facts concerning GA indicate that (i) the raw starch affinity site of the enzyme is essential in the digestion of raw starches; (ii) the raw starch affinity site is different from the active site of the enzyme and is located in a separate position; and (iii) GA possessed a separate extra binding site specific to chitin in addition to the raw starch affinity and active sites, whereas GA I contained only the raw starch affinity site and active site. The formation of multiple types of raw starchdigesting glucoamylase and the structural relationship between GA and GA I will be reported later. LITERATURE CITED 1. Davis, B. J Disc electrophoresis-ii. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121: APPL. ENVIRON. MICROBIOL. 2. Dubois, M., K. A. Glles, J. K. Hamilton, P. A. Rebers, and F. Smith Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: Flor, P. Q., and S. 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