ELSEVIER 0960-8524(95)00132-8

Bioresome TMnology55(1996)55-61 8 1996 Ekxier Science Limited

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SAGOSTARCHASABIOMASSSOURCE:RAWSAGO STARCHHYDROLYSISBYCOMMERCIALENZYMES

W. J. Wang,” A. D. Powellb & C. G. Oates” *

a Department of Biochemistry and bDeparment of Botany, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511

(Received 24 August 1995; accepted 2 September 1995)

Abstract Raw sago starch and sago starch pretreated by heating at 60°C for 2 hours in sodium acetate buffer (pH 3.5) were hydrolysed using commercial glucoamyhzse-AMG (EC 3.2.1*3), a-amylases-BAN, Fungamyl and Terma- my1 (EC 3-2-l -I), debranching amylase-Promozyme (EC 3*2*1*41), and their mixtures in sodium acetate buffer; pH 5.0 at 35°C. Raw sago starch was a poor substrate for enzyme action compared to corn and tapioca starches tested under the same conditions, although pretreating the starch increased the extent and rate of hydrolysis. A strong synergism between gluco- amylase and a-amylase on the hydrolysis of both untreated and pretreated sago starch was observed. The hydrolysis products were characterized by high-per- formance size-exclusion chromatography (HPSEC). The total carbohydrate concentration of hydroljsed sago starch decreased but the amylose and amylopectin ratios in the residues remained the same. Copyright 0 1996 Elsevier Science Ltd.

Key words: Amylase, glucoamylase, sago starch, raw starch hydrolysis, starch oligosaccharide profiles.

INTRODUCTION

It is estimated that about 60 million tonnes of sago starch, extracted from sago palms, are produced per annum in south-east Asia. In view of its abundance, sago may be utilized for the production of ferment- able sugars. Utilization of sago as part of a sustainable agrotechnological system will provide a source of starch and lead to conservation of marshy areas of land.

Starch, in its native form, exists in relatively inert granular structures which are composed of macro- molecules arranged in a polycrystalline state. These granules are insoluble in water and resistant to many chemicals and enzymes. Disruption of the granular structure by heating in water (gelatinization), enhan- ces its chemical reactivity towards hydrolytic enzymes. There has been much interest recently in enzymes capable of digesting raw (native) starch *Author to whom correspondence should be addressed.

granules. This would bring about a reduction in the costs associated with the high temperatures required for gelatinization (Ueda, 1981; Bergmann et al., 1988; Fogarty & Kelly, 1990).

The use of sago starch for bioconversion is limited by high paste viscosity and resistance of the raw granule to enzyme digestion (Sakano et aZ., 1986; Takao et al., 1986). No systematic investigations of sago starch degradation by commercial amylases have been reported. One report outlines the action of an amylase of unspecified activity extracted from Penicillium brunneum, which exhibited poor hydro- lytic activity towards untreated sago starch (Haska & Ohta, 1991).

The present investigation explored the potential for utilizing sago starch in a manner that requires minimum energy input. A number of systems, which include enzyme mixtures, have been investigated to improve the hydrolysis of other poorly hydrolysable starch granules (Fujii et aZ., 1988; Hayashida et aZ., 1988). The conclusions from these studies are based upon the liberation of reducing groups rather than the solubilization of granular material. Such proce- dures give no indication of the source of liberated reducing groups. These could either be derived from the granule or further breakdown of solubilized oligosaccharides. We analysed the data in this work using both criteria.

Several enzymes and enzyme mixtures were used in order to achieve a better understanding of the process and mechanism of raw sago-starch hydroly- sis. Attempts were also made to improve the starch hydrolysis by a combined acid and moderate heat treatment. The morphological changes in starch granules resulting from this action were observed by SEM.

METHODS

Starch Sago starch (Metro&on sp.) from Sarawak, Malay- sia, was supplied by a commercial producer, Wah Chang International Group of Companies (Singa- pore). It was specially processed for our laboratory

55

56 W J. Wang, A. D. Powell, C. G. Oates

use. Corn starch was obtained from Sigma Chemical 120. Deionized water was used as a mobile phase. Co., St Louis, MO, USA. Tapioca starch was also Apparent molecular weights were determined using obtained from Wah Chang International Group of oligosaccharide and dextran molecular-weight stan- Companies. dards.

Starch pretreatment was carried by a modified Haska method (Haska & Ohta, 1991). One gram starch and 5 ml O-1 M sodium acetate buffer solu- tion, pH 3.5, were incubated in an oven at 60°C for 2 h.

Enzymes Isoamylase, derived from a strain of Pseudomonas amyloderamosa, was purchased from Hayahibara Biochemical Laboratories, Inc., 2-3 Shimoishii- 1-chome, Okayama, 700 Japan. All other enzymes were commercial preparations, kindly donated by Novo Nordisk, Regional Office, Kuala Lumpur, Malaysia (Table 1).

Raw and degraded starch residue samples were prepared by a modified Jackson method (Jackson et al, 1988). Fifteen millilitres distilled water was added to 30 mg starch granules and the suspension was gelatinized by placing in a boiling water bath for 10 min. The suspension was cooled, dispersed fur- ther by sonication (30 set) and the final solution passed through a 8.0 ,um SC Millipore filter prior to HPSEC analysis.

Analytical methods The degree of hydrolysis was determined using two different methods:

Hydrolysis of raw starch

Liberation of reducing groups: D.H.(%)

A reaction mixture containing 2.0% starch granules, 0.1 M acetic acid-sodium acetate buffer pH 5-O and 40 ppm Ca*+ was incubated at 35°C with constant shaking. Hydrolysis was initiated by addition of 1% (weight of enzyme/weight of starch) of the enzyme. Aliquots were removed periodically and centrifuged at 2500 rpm for 10 min (Jouan BR.A 3.11). The supernatant was mixed with an equal volume of O-4 mM HgC12 and incubated in a water bath at 90°C for 20 min to inactivate the enzyme(s) (Govindasamy et al., 1992). This solution was then used for reducing sugar determination or hydrolysis products analysis by HPSEC. The precipitate (degraded starch resi- due) was washed with water, filtered through Whatman No. 1 filter paper and dried at room tem- perature.

(Reducing sugar produced by enzyme hydrolysis) = (Reducing sugar produced by acid hydrolysis)

x 100

Reducing sugar was determined by the method of Dygert et al. (1965) using glucose as the standard. Acid hydrolysis was carried out by treating starch granules with 1.0 M HCl at 100°C for 2 h.

Solubilization of granular material: solubilized material was determined by HPSEC, previously calibrated with suitable oligosaccharide standards (Govindasamy et al., 1992).

HPSEC The analysis procedure was performed following the method of Govindasamy et al. (1992). A Waters Associates (Milford, MA, USA) series liquid chromatography system, with a model 510 pump, WISP model 712 autosampler, a model 410 differ- ential refractometer detector and three Ultrahydrogel columns, was used. Columns, main- tained at 4O”C, were connected in the order: Ultrahydrogel linear followed by two Ultrahydrogel

Scanning electron microscopy Dried starch granules were sprinkled on to double- backed adhesive tape attached to a circular specimen stub and coated with gold using Balzers SCD 004 sputter coater. The samples were viewed and photographed using a Philips SEM 515 scanning electron microscope on AGFAPAN-APX 100 films.

RESULTS AND DISCUSSION

Untreated or treated sago starch incubated under the test conditions remained unchanged in the

Table 1. A list of Novo Nordisk enzymes used in this work

Enzyme (commercial name)

Origin Activity”

Glucoamylase (AMG) Bacterial a-amylase (BAN) Fungal cl-amylase (Fungamyl) Thermostable cc-amylase(Termamy1) Debranching amylase (Promozyme)

Aspergilus niger Bacillus subtilis Aspergdlus oryzae Bacillus lichenifonnis Bacillus acidopullulyticus

300 U/ml 480 U/g 800 U/g 120 u/g 200 u/g

a All activities were defined by Novo Nordisk.

Enzymic hydrolysis of saga starch 57

absence of enzyme. To fully appreciate the mechan- isms of hydrolysis, liberation of reducing-end groups, indicating chain scission, and solubilization of granu- lar material are compared.

Hydrolysis of untreated starch Single enzymes were not effective in producing soluble products from raw sago-starch granules: the highest being AMG, producing 11.8% soluble products after 40 h (Fig. 1). For all enzymes, a low rate of solubilization was noted with no evidence of a completed reaction, indicating that further reac- tion could have been achieved with longer incubation times.

Inclusion of AMG in enzyme mixtures in all cases resulted in glucose as the sole product. Low-molecu- lar-weight oligosaccharides accumulated using Termamyl alone, which is in agreement with Govin- dasamy et al. (1992). Reaction with Termamyl and BAN resulted in a product mixture with more lower- molecular-weight compounds than expected (Table 2). The unique product composition and amounts of

solubilized material formed by each of the amylases (Fig. 1; Table 2) could reflect their subsite variability and ultimate adsorption on to the granule. Funga- my1 preferentially accumulated glucose rather than maltose (Fig. 2). Such high levels of glucose accumu- lation by Fungamyl could not have resulted from extraneous glucoamylase activity but may have reflected abnormality of adsorption or positioning of the substrate with the enzyme. This enzyme is very poorly adsorbed on to maize starch (Sandstedt & Ueda, 1969).

Termamyl and BAN both exhibited a strong syn- ergistic effect with glucoamylase in the digestion of raw sago starch. At 40 h, Termamyl in the presence of AMG produced 35% solubilization of the raw starch, while a BAN+AMG mixture brought about 29% solubilization (Fig. 1). The synergistic effect between a-amylase and glucoamylase has been explained as resulting from the action of a-amylase at the granule surface, which supplies new non- reducing groups for AMG. The AMG acting on these groups strips the molecules from the surface,

F T A T+P A+P A+F A+0 A+T A+T+P

Enzyme(s) Fig. 1. Hydrolysis of untreated sago starch after 40 h at 35°C pH 5.0. Enzymes used, A: AMG, B: BAN, F: Fungamyl,

T: Termamyl and P: Promozyme.

Table 2. Percentage composition of oligosaccharides separated by HPSEC after enzymatic hydrolysis of non-treated and treated sago starch for 40 and 32 h, respectively

Oligosaccharide

Gl G2 G3 G4 G.5 G6 G7 7<DP<70

Non-treated BAN Termamyl Term + Prom Treated Termamyl

11.5 17.3 35.1 0 17.0 0 4.6 14.5 3.5 17.7 21.8 0 43.8 : 1.7 11.4 5.1 34.8 24.5 0 34.9 0 0

1.7 13.7 18.5 0 56.4 0 1.6 8.2

WJ. Wang A. D. PoweU, C. G. Oates

2

d

I 1 .

20 4

RETENTION TIME ( min)

Fig. 2. HPSEC profile of the soluble products of sago starch hydrolysed by Fungamyl after 40 h. (A) Fungamyl

hydrolysis products; (B) standard glucose.

thereby allowing access for the cr-amylase to the next layer (Fujii et aZ., 1988).

Fungamyl did not exhibit significant synergism with AMG; the solubilization resulting from their combined action being approximately 19%. This contrasts with a number of reports (Ueda, 1980; Abe et af., 1988) describing fungal amylase stimulation of glucoamylase digestion of raw starch. Absence of a synergistic effect may reflect the inability of this amylase to adsorb to the granule surface: as previ- ously suggested, the pattern of attack by this enzyme on raw sago starch may be abnormal. The hydrolysis capacity of fungal amylases is known to correlate with their ability to adsorb on the granule surface (Fogarty & Kelly, 1990). In addition, both bacterial enzymes are known to digest the granule through a multiple-attack mechanism, where the substrate is repeatedly attacked following adsorption before desorption occurs. This is not the case for Fungamyl, which is known to digest starch through a non-repet- itive attack process (Reilly, 1985). The synergistic mechanism previously described would not be as effective under such conditions.

Preferential accumulation of low-molecular-weight oligosaccharides due to or-amylase hydrolysis has been suggested as indicating a highly branched structure of the granule components (Atkins & Ken- nedy, 1985). Therefore the influence of a-1,6 bonds on the restriction of amylase activity was investi- gated by the addition of debranching enzymes in

combination with amylases. Solubilization or release of reducing sugar were little changed when Promo- zyme or isoamylase were combined with Termamyl (Fig. 1).

Promozyme or isoamylase alone could not attack the sago-starch granules (D.H. <O*l%), suggesting that the debranching enzymes either cannot adsorb on to, or hydrolyse, the starch granule. Although Promozyme in combination with Termamyl did not significantly increase the degree of hydrolysis, oligo- saccharide profiles showed that more low-molecular-weight oligosaccharides were present, evidently formed from oligosaccharides of greater than G7 (Table 2); this may reflect the removal of branch points on early products of ol-amylase action. The hydrolysis products were the same when iso- amylase was substituted for Promozyme. Variation in the oligosaccharide profile, with no apparent effect upon the residual granular structure, suggests that Promozyme and isoamylase are acting on the released, soluble oligosaccharides rather than the granule material per se.

This observation was further substantiated by studies with high enzyme concentrations; whilst raised AMG and Termamyl (3% each) concentra- tions resulted in a corresponding increase in soluble material, higher levels of Promozyme (3%) elicited no further improvement in hydrolysis compared to the original concentration (Fig. 3).

The optimum pH for the mixtures AMG+Term and AMG+Term+Prom was pH 45 (Fig. 4). This pH is near to the optimum pH for AMG action, which suggests that glucoamylase plays a major role in raw starch degradation in the glucoamylase and cr-amylase mixtures.

A typical, non-hydrolysed sago-starch HPSEC profile was used as a reference in this study (Fig. 5). The relative peak area for each component was 72% for amylopectin and 28% for amylose. The small

50

E .‘A 40

s

g 30

Z

0 0 10 20 30 40

Time (h) Fig. 3. Effect of enzyme concentration in enzyme mix- tures on the hydrolysis of sago starch: (0) AMG + 3% Termamyl; (A) 3% AMG + Termamyl; (0) 3% (AMG + Termamyl) and (0) AMG + Termamyl + 3% Promo-

we.

Enzymic hya!ro&sis of sago starch 59

01 I I I 8 1 I 3.5 4.0 4.5 5.0 5.5 6.0 6.5

PH Pig. 4. Effect of pH on hydrolysis of sago starch with enzyme mixtures. Sago starch (2%) was hydrolysed in O-1 M sodium acetate buffer solution for 20 h. Enzyme mix- tures: (0) AMG + Termamyl; (0) AMG + Termamyl +

Promozyme.

h II Am

I\

I

L, I II ui_

I I

20 1

RETENTION TIME (mid Fig. 5. HPSEC profiles of non-hydrolysed sago-starch

granules. (A) Treated starch; (B) non-treated starch.

peak eluting at the end of the main amylopectin peak may be an amylopectin with a low degree of branching.

Complete hydrolysis of starch is known to proceed through a number of well defined stages (Atkins & Kennedy, 1985; Govindasamy et aL, 1992). High and intermediate molecular weight components are formed in the early stages and are used as sub- strates. These fractions are insoluble. We attempted to establish whether intermediate components were present in addition to the small amounts of soluble material. Enzyme hydrolysis (40 h) with single or enzyme mixtures did not change the retention times c . . or tne peaks and their ratio (Fig. 6j. Such compara- tive rates of degradation of amylose and amylopectin by HPSEC suggest that both macromolecular com- ponents are equally susceptible to attack. Contrary to expectations, amylose and amylopectin fractions, whilst decreasing in overall concentration, did not form intermediate products; apparent molecular weights of these fractions remained the same throughout the hydrolysis process (Figs 5 and 6). This suggested that neither amylose nor amylopectin was more resistant to the enzyme(s) and that, possibly, the macromolecules were digested in a non-random fashion, while the material facing away from the front of enzyme attack remained unhy- 1_.~1~.~~1 ululys~u.

Hydrolysis of pretreated starch Pretreatment of the starch did not increase the degree of hydrolysis or solubility of the control sample (D.H.<O-1%). In addition, the granule morphology remained intact (Fig. 7).

E

I 20

b

EENTION llM(min) Fig. 6. HPSEC profiles of enzyme-degraded treated (A) and non-treated (B) sago-starch residues, after 32 and 40 h hydrolysis, respectively. Enzymes used: (a) AMG and

(b) AMG + Termamyl.

60 W J. Wang, A. D. Powell, C. G. Oates

Fig. 7. Scanning electron micrographs of (A) native sago-starch granules and (B) pretreated sago-starch granules. denotes 10 PM.

Greater amounts of G5 products were produced by Termamyl hydrolysis of pretreated starch than by hydrolysis of untreated granules (Table 2). These profiles are similar to those formed when gelatinized starch is hydrolysed by Termamyl (Govindasamy et al., 1992).

Synergistic effects, similar to those with non- treated starch, were evident. Solubilization of sago starch with an AMG and Termamyl mixture was 2.5fold greater than the sum of the corresponding values of AMG and Termamyl alone (Table 3).

Pretreatment greatly increased the extent and rate of hydrolysis of starch granules by single enzymes and enzyme mixtures (Fig. 8; Table 4). The hydroly- sis, measured by reducing sugar analysis, for the AMG+Termamyl mixture was increased from

Table 3. Synergism between AMG and Termamyl in the hydrolysis of sago starch measured after 32 h by the

solubilization method

Solubilization (%)

Enzyme(s) AMG Termamyl AMG + Termamyl

Treated starch 15.0 165 81.6 Non-treated starch 9-O 4.3 30.0

approximately 30% to 81% (32 h). However, the

Bar

major macromolecular components were apparently unaffected by the pretreatment (Figs 5 and 6). This step also increased the degree of hydrolysis of corn and tapioca starches, which are known to be easily hydrolysed even without a pretreatment (Fig. 8; Table 4).

0 0 5 10 15 20 25 30 35

Time (hl Fig. 8. Hydrolysis of untreated (closed symbols) and treated (open symbols) starches with AMG and Termamyl mixture. Starches: (u, 0) sago; (A, A;) tapioca; (0, 0) corn.

Table 4. Initial hydrolysis rate (V,) and degree of hydrolysis (D.H.) at 32 h of treated and non-treated sago, tapioca and corn starches

Starch Enzyme(s) Treated

D.H. (%)

Non-treated

V. D.H. (%)

Sag0

Tapioca

Corn

AMG 0.02 Termamyl 0.02 AMG + Termamyl 0.11 AMG + Termamyl + Promozyme 0.11 AMG 0.47 Termamyl 0.16 AMG + Termamyl 0.83 AMG 0.23 Termamyl 0.12 AMG f Termarnyl 058

14.5

8Z.t 79.0 73.0 29.0

lOO*O 56.0 18.0 00.0

0.01 9.0 <o-o1 2.4

0.03 29.8 0.03 30.5 0.05 36.9 0.02 12.3 0.14 84.9 O-06 38.0 0.02 12.6 0.18 91.1

Enzymic hydrolysis of saga starch 61

It is possible that the poor hydrolysis of untreated sago starch was caused by the presence of substances inhibitory to the actions of the amylases, associated with or adhering to the starch granules, and that the pretreatment inactivated such compounds. Washing the starch granules thoroughly before enzymatic digestion did not increase the degree of hydrolysis with a AMG+Termamyl mixture (21.6% before wash and 21.4% after wash, at 20 h). Thus external water-soluble factors did not contribute to the resist- ance of sago starch towards enzyme action or lead to enzyme degradation.

CONCLUSION

Raw sago starch was resistant to the actions of glu- coamylase, a-amylases, debranching amylase, and mixtures of these. A significant synergism was observed for glucoamylase and cc-amylase. Deb- ranching amylases did not show any apparent effect on the degree of hydrolysis. Pretreating of the starch did not physically change the starch fractions or impose any observable effects on the granule morphology, but subsequent hydrolysis was greatly increased. Despite the improvement in the degree of hydrolysis by the pretreatment it was still slow com- pared to the hydrolysis of corn and tapioca starches. Macromolecules within native sago starch are not as susceptible to hydrolysis as are those in gelatinized starch. Enzyme actions would therefore seem to pro- ceed along restricted paths. Whilst this holds true for non-treated and pretreated starches, precise micromodes of action may be different.

ACKNOWLEDGEMENTS

The authors thank Miss Tan Suat Hoon for her advice on SEM work. The financial support of the National University of Singapore and the ASEAN- Australia Biotechnology project is much appreciated.

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