Soluble and immobilized enzyme technology in bioconversion of barley starch

Soluble and immobilized enzyme technology in bioconversion of barley starch* Yu-Yen L i n k o , Arja Lindroos and P. Linko Department o f Chemistry, Helsinki University o f Technology, SF-02150 Espoo 15, Finland

(Received 25 January 1979; revised 26 March 1979) Homogeneous and heterogeneous biocatalysis were both investigated as tools f o r barley starch syrup production. Barley starch was first liquefied by soluble heat-stable Bacillus sp. ~t-amylase EC 3.2.1.1 (1,4-Ot-D-glucan glucanohydrolase) Termamyl 60 L at 95°C, pH 6.5, to obtain slurries o f varying DEvalues up to ~ 3 7. Alternatively, it was extruded with a Creusot-Loire BC 45 twin-screw extruder at 25% moisture, 150°C, f o r denaturation. A f t e r cooling and adjusting the pH to 4.5 or grinding, respectively, the pretreated starch was saccharified either by soluble or by immobilized AspergiUus niger glucoamylase EC 3.2.1.3 (1,4-t~-D-glucan glucohydrolase) at 60°C, pH 4.5, to obtain glucose syrup o f up to DE 96. The course o f hydrolysis was followed by automated Biogel P-2 chromatographic analysis. Glucoamylase was immobilized either on a p h e n o l - f o r m a l d e h y d e resin Duolite S 761 or on silanized Spherosil porous silica beads. Barley glucose syrup obtained was further continuously converted to high fructose syrup by a packed bed reactor o f Actinoplanes missouriensis whole cell glucose isomerase (EC 5.3.1.5) M a x a z y m e entrapped within or-cellulose beads. We could conclude that barley starch may be used as an alternative raw material f o r biocatalytic starch syrup production.

Introduction

Materials and methods

Barley, one of the major cereal grain crops in Finland, has largely_been utilized for the production of malt and animal feed. Dry substance of barley consists of ~63-65% starch (~22% amylose), 10% hemicellulose and gums, 11-13% protein, 2-3% lipids, 2% ethanol-soluble sugars, 6% indigestible fibre, and 3% ash. During the past ten years, partial replacement of malt by unmalted barley or other cereal grain and commercial enzyme preparations has been extensively investigated. For this purpose, barley 'syrup' is produced from grits or flour, employing c~-amylase, glucoamylase and proteolytic enzymes. The inclusion of/3-glucanase has also been suggested to avoid difficulties during subsequent processing due to undegraded barley ~-glucans. However, unlike potato, corn, wheat and rice, to our knowledge barley has not been utilized by the starch industry for syrup manufacture, largely because of processing difficulties caused by the high hemicellulose content and, consequently, the poor yields. Yet, with the recent development of modern technology for barley starch production, barley appears to provide an interesting alternative as a substrate for the enzymatic production of various modified starches and syrups for the food industry. We have studied both homogeneous and heterogeneous biocatalysis as tools for upgrading barley starch for various food applications.

Materials

* Presented at the Fifth International Congress of Food Science and Technology [IUFoST], Kyoto, Japan, September 1978.

0141 --0229/79/040273--06 $02.00 © 1979 |PC Business Press

Barley starch was obtained from H/imeen Peruna Oy, Jokioinen, and wheat starch from Raision Tehtaat Oy, Raisio, Finland. Aspergillus niger glucoamylase (EC 3.2.1.3) 150 L, activity 7500 pmol min- t ml-1, and Bacillus sp. c~-amylase (EC 3.2.1.1) Termamyl 60 L, activity 60 KNU/g (Novo units), were a gift from Novo Industri A/S, Copenhagen, Denmark. Actinoplanes missouriensis whole cell glucose isomerase (EC 5.3.1.3) Maxazyme GI, activity 1000/lmol min-I g - t , was a gift from Gist-Brocades N.V., Holland. Phenol-formaldehyde resin Duolite S 761 (0.250.76 mm) was obtained from Dia-Prosim, Vitry, France, and different types of porous silica beads, Spherosil XOA 400, XOB 075 and XOC 005 (each both 40-100/~m and 100-200 tzm) from Rhone- Poulenc S.A., Paris, France. or-Cellulose (DP 880) was obtained from Rauma-Repola Oy, Finland.

HTST-extrusion

cooking

A portion of barley starch was extruded with a CreusotLoire BC 45 twin-screw extruder, at 150°C, feed speed 175 g (d.s.)min-1 and revolution 150 min-i, after conditioning to 25% moisture. After cooling and air-drying overnight the extruded starch was hammer-milled (Retsch KG, Type SK 1, FRG) through 0.75 mm sieve.

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Papers Enzyme activity assays a-Amylase activity was determined by the dyed substrate method as described by Linko et al. l Glucoamylase activity was determined as follows. To 2 ml of glucoamylase solution in 0.02 M-sodium acetate buffer, pH 4.8, or to a suitable quantity of the immobilized enzyme in 2 ml of the same buffer, 2 ml of 2% Lintner's soluble starch (Thomas Morson & Sons Ltd, London, England) was added in the same buffer. Magnetic stirring was used for the immobilized enzyme. After a 30 min reaction at 45°C, the mixture was boiled for 3 min to inactivate the enzyme and subsequently cooled in cold water. The glucose formed was determined by the glucose oxidase peroxidase method according to Barton. 2 Glucose isomerase activity was determined as described by Linko et al. 3

Dextrose equivalent (DE) value Dextrose equivalent value was determined as described by Miles Laboratories, Inc. 4

Gel chromatography Quantitative gel chromatography of carbohydrates was performed according to a slightly modified method of John et al. s Biogel P-2 polyacrylamide gel, minus 400 mesh (Bio-Rad Laboratories, Richmond, California) was used in a 1.5 x 180 cm water-jacketed column at 65°C. A 1 ml sample (6.6 mg d.s.) was applied to the top of the column, and deaerated deionized water was used as eluant at a flow rate of 33.5 ml h -1 . The eluate was monitored spectrophotometrically at 618 nm for carbohydrate content by an automatic analyser with anthrone-sulphuric acid as reagent (1 g of anthrone, Fluka, in 11 H2SO4, 95-97% Merck, p.a., and 275 ml H20 with 1 ml of 30% Brij-35 as wetting agent).

Immobilization o f glucoamylase Phenol-formaldehyde resin Duolite S-761 was wetsieved, and only the particle size range of 0.25--0.76 mm was used. The resin was pretreated with 2% sodium hydroxyde (3 ml/g resin) for 1 h, washed with water, and subsequently treated with 1% hydrochloric acid (2.5 ml/g resin) for 30 rain. The resin was then washed with distillea water until the eluate reached pH 4. A suitable quantity of Novo glucoamylase 150 L was added, and the adsorption was allowed to take place overnight at 23°C with shaking. The preparation was crosslinked with 2.5% glutaraldehyde for 3 h at 23°C and thoroughly washed with distilled water. Spherosil was first silanized by refluxing with 10% 7-aminopropylmethoxysilane in xylene for 20 h, followed by acetone washing. Acetone was removed in a vacuum desiccator, and the product was finally dried overnight at 110°C. Dried silanized Spherosil was activated with 2.5% glutaraldehyde in 0.1 M-phosphate buffer, pH 7.0, for 30 min under vacuum, followed by shaking tot 1 h at 230C at atmospheric pressure. Novo glucoamylase 150 L was added immediately after washing, and the adsorption was first allowed to take place for 30 min at 23°C in a vacuum desiccator, followed by 1.5 h shaking at 4°C at atmospheric pressure.

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Immobilization o f whole cell glucose isomerase Glucose isomerase-active Actinoplanes missouriensis cells were entrapped within a-cellulose beads formed by extruding into water a suspension of cells in an a-cellulose solution in a melt of N-ethylpyridinium chloride and dimethylformamide, as described by Linko e t al. 3'6 The beads were further treated with 2.5% glutaraldehyde in 0.1 M-phosphate buffer, pH 7.0, at 23°C for 1 h for crosslinking, and washed with water.

Enzymatic hydrolysis o f barley starch A 33.3% (d.s.) slurry of barley starch was first liquefied by Termamyl 60 L a-amylase in a stirred tank stainless steel batch reactor at 95°C, pH 6.5 until a desired DE-value was reached, or 25% moisture starch was treated with an HTST extruder as described above. After cooling, 0.15 ml (1125/lmol rain -1) of glucoamylase per 100 g (d.s.), or the equivalent quantity of immobilized enzyme was added either to the hydrolysate after adjusting the pH to 4.5, or to a 10% (d.s.) slurry of extruded milled starch. The saccharification took place at temperatures varying from 30 to 70°C and at pH values of 4.0 to 5.0. In most cases, 60°C and pH 4.5 were employed.

Continuous isomerization o f barley starch syrup Barley starch glucose syrup was heat treated to inactivate glucoamylase and filtered. To the clear syrup of DE 95 • (38% sohds, 35% w/w glucose), 0.003 M-Mg2 + and 0.0003 MCo 2+ were added, the solution was deaerated and used as a substrate for isomerization in a packed bed column reactor (2.3 x 1.5 cm, total activity 162/1mol rain - l , flow rate 2 m l h - l ' 60°C).

Results and discussion

Hydrolysis o f barley starch with soluble a-amylase and glucoamylase The course of hydrolysis of 33.3% (d.s.) barley starch slurry at 95°C, pH 6.5, in a batch reactor is shown in Figure 1 in comparison to the hydrolysis of wheat starch under the same conditions. During the first 5 h with Termamyl 60 L a-amylase a slightly faster rate of hydrolysis was obtained with wheat starch. After 24 h, a DE-value of 34.6 was

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(33.3% d.s.) w i t h T e r m a m y l 60 L c~-amylase at 9 5 ° C , pH 6.5

B i o c o n v e r s i o n o f b a r l e y starch: Yu- Yen L i n k o et al.

obtained with wheat, and a somewhat higher value of 37.5 with barley starch. Consequently, various DE-values up to ~ 3 7 may be obtained with Termamyl liquefaction of barley starch at 95°C. The rate of hydrolysis is comparable to that obtained with wheat starch under similar conditions. Hydrolysates with DE-values of 12 and 28 were adjusted to pH 4.5 and saccharified with Novo glucoamylase 15 L at 60°C. Typical results are shown in Figure 2. A somewhat faster conversion was obtained with the DE 12 hydrolysate as substrate as compared with the DE 28 hydrolysate. After about 24 h a DE-value o f ' 9 6 was reached with both hydrolysates. No significant differences could be obtained with wheat starch under similar conditions. Typical corresponding results from automated Biogel P-2 chromatographic analysis are shown in Figure 3, indicating the progress of conversion. When barley starch was first liquefied to DE 28, and subsequently saccharified with glucoamylase to DE 49, a decrease in the glucose oligomers higher than G3, together with a considerable increase in glucose, maltose and maltotriose is clearly seen. At DE 95, only glucose together with small quantities of G 2 and G 3 could be detected on the chromatogram. The glucose syrup obtained could be easily fdtered through Whatman no. 1 paper to obtain a clear, slightly yellowish liquid. Subsequent treatment with cation and anion exchange resins and with active carbon gave a colourless syrup of pleasant flavour.

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Starch denaturation and liquefaction has been shown to take place without hydrolysis under conditions of high pressure and shear such as are encountered during HTSTextrusion cooking. 7'8 When 10% (d.s.) untreated barley starch slurry was directly hydrolysed with glucoamylase (0.3 ml/100 g d.s.) at 60°C, pH 4.5,DE ~ 8 9 was reached in 72 h, as shown in Figure 4. A DE of only ~65 was obtained with a 33.3% (d.s.) slurry under the same conditions. Similarly, Smith and Lineback 9 reported 66.3 and 86.5% hydrolysis of raw wheat and corn starch, respectively, with glucoamylase after 64 h at 37°C.

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Time (h) Figure 2 Liquefaction of 33.3% (d.s.) barley starch slurry with Termamyl 60 L e-amylase (o) at 95°C, pH 6.5, and subsequent saccharification with Novo glucoamylase 150 L at 60°C, pH 4.5. =, Initial DE 12; e, initial DE 28

No soluble carbohydrates could be detected in untreated barley starch by gel chromatography under the experimental conditions. When 25% moisture starch was pretreated for partial denaturation under pressure and shear at 150°C with a twin-screw extruder, a 10% (d.s.) suspension of the milled product was very rapidly saccharified with glucoamylase without prior liquefaction with a-amylase, as shown by Figures 4 and 5. ADE-value of ~ 9 8 was reached within 10 h. Gel chromatography of the extruded starch with DE 2 indicated the formation of high molecular degradation products during extrusion, but no detectable quantities of small molecular sugar (Figure 5).

Immobilization of glucoamylase Since Wilson and Lilly1° coupled glucoamylase on cellulose, glucoamylase has been immobilized on various supports

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such as bEAE-Sephadex, II ion exchange resin, 12 cellulose triacetate fibre, 13 acrylic gel, 14 gelatin, Is porous silica, 16 ceramics,17 cellulose beads, 18 and others, by a variety of techniques such as adsorption, ionic and covalent binding, chelation and entrapment. Furthermore, successful continuous saccharification of liquefied corn starch has been demonstrated by a number of pilot operations, one of which has been described in detail. 17 A number of immobilization techniques were investigated during this work. The best results were obtained with adsorption on phenol-formaldehyde resin Duolite S 761, and with covalent binding on silanized Spherosil porous silica beads. After thorough washing with distilled water, no leakage of enzyme could be detected with either carrier. The effect of the enzyme quantity on the activity of the immobilized enzyme preparation is shown in Figure 6. Relatively higher activities were obtained with Spherosil

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XOC 005 than with Duolite S 761. The activity of glucoamylase immobilized on different Spherosil types is shown in Table 1. The most porous Spherosil XOA 400 ( 4 0 100/am, pore volume 91 ml/100 g, surface area 500 m2/g) had the smallest activity. Weetall and Havevala 19 also reported that the immobilized enzyme activity varies greatly with the surface area and pore diameter of porous glass. The highest activity was obtained with Spherosil XOB 075 of 100-200/am pore volume and a pore diameter of 26 nm.

Saccharification o f liquefied starch with immobilized glucoamylase The results of saccharification of DE 28 barley starch hydrolysates using a 33.3% (d.s.) slurry both with Duolite S 761- and Spherosil XOC 005-immobilized glucoamylase in a stirred tank batch reactor at 60°C, pH 4.5 are shown in Figure 7. The results obtained with the Duolite immobilized enzyme were comparable to those obtained with the soluble enzyme. When the enzyme was covalently bound to Spherosil, a very rapid hydrolysis took place, and a DEvalue o f ~ 9 6 was reached in 10h. The very efficient saccharification with this enzyme preparation is further illustrated by the gel chromatography analysis shown in Figure 8. An initial DE-value of 12 was too low for efficient saccharification with the Duolite immobilized glucoamylase, as shown in Figure 9, while with the Spherosil XOC 005 enzyme a DE-value of 93 was obtained in 10h. A slow increase in maltose formation could be detected on the chromatograms (Figure 7).

lsomerization o f barley starch syrup The suitability of DE 95 glucose syrup obtained by saccharification of barley starch was examined using a packed bed column reactor employing a-cellulose beadentrapped whole cell glucose isomerase as a biocatalyst. The glucose syrup used as substrate contained 38% solids and

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Bioconversion o f barley starch: Yu-Yen Linko et aL Table 1 Enzyme activities obtained with Novo glucoamylase 150 L immobilized on various silanized Spherosil porous silica beads

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35% (w/w) glucose. Glucose isomerase is known to be very sensitive to Ca 2+ inhibition. 2° Consequently, if the oramylase liquefaction step requires Ca z+ as an activator, an ion exchange treatment for removal of calcium ions prior to isomerization is necessary. However, since neither the Termamyl 60 L a-amylase nor the Novo glucoamylase 150 L required an activator, the glucose syrup obtained could be used for isomerization after Filtration, without further purification. This would also be the case with syrups produced by direct saccharification of extruded starch. As shown in Figure 10, the column reactor exhibited good stability and a nearly constant 41% conversion was obtained during continuous isomerization for 10 days.

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tion of continuous saccharification process conditions with immobilized glucoamylase in respect to the possible reversion and transfer reactions at the high enzyme/substrate ratios encountered requires further investigation.

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Acknowledgement Financial support from the Ministry of Trade and Industry (Finland) and the Academy of Finland is gratefully acknowledged.

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References 1

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Figure 10 Continuous isomerization of DE 95 (38% solids, 35%, w/w, glucose) barley glucose syrup with cellulose bead-immobilized whole cell glucose isomerase (60°C, pH 7.5, 0.003 M-Mg 2+,

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Conclusion The results obtained suggest that barley starch may be an interesting alternative for biocatalytical glucose syrup production. The syrup obtained may be readily isomerized to high fructose syrup by existing technology. The optimiza-

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Linko, Y.-Y., Saariuen, P. and Linko, M. Biotcchnol. Bioeng. 1975, 17,153 Barton, R. R.Anal. Biochem. 1966, 16,258 Linko, Y.-Y., Viskari, R., Pohjola, L. and Linko, P. J. Solid Phase Biochem. 1978, 2 (3), 203 MilesLaboratories Inc. TechnicalBulletin, 1963, no. 1-174, p. 20 John, M.,Tr6nel,G. and Dellweg, H.J. Chromatogr. 1969,42, 476 Linko, Y.-Y., Pohjola, L. and Linko, P. Process Biochem. 1977, 12 (6), 14 Suzuki, S. in Proceedings SOS/70, Third International Congress o f Food Science and Technology Inst. Food Technol., Chicago, 1970, p. 484 Olkku, J. and Linko, P. in Food Quality and Nutrition Research Priorities for Thermal Processing (Downey, W. K., ed.) Applied Science Publishers, London, 1977, p. 375 Smith, J. S. and Lineback, D. R. Stiirke 1976,28,245 Wilson, R. J. H. and Lilly, M. D. Biotechnol. Bioeng. 1969, 11, 349 Solomon, B. and Levin, Y. Biotechnol. Bioeng. 1974, 16, 1161 Miyamoto, K., f:ujii, T., Tamaoki, N., Okazaki, M. and Miura, Y. J. Ferment. Technol. 1973,51,566 Como, C., Galli, G., Morisi, F., Bettontc, M. and Stopponi, A. Stiirke 1972, 24,420 Kr~imer,D. M., Lehmann, K., Plaisier, H., Reisner, W. and Spr6ssler, B. G. J. Poh'm. Sci. 1974,47, 89 Hupkes, J. V. Stiirke 1978, 30, 24 Lee, D. D., Lee, Y. Y., Reilly, P. J., Collins, E. V. and Tsao, G. T. Biotechnol. Bioeng. 1970, 18,253 Weetall, H. H., Vann, W. P., Pitcher Jr., W. H., Lee, D. D., Lee, Y. Y. and Tsao, G. T. in Immobilized Enzyme Technology: Research and Applications (Weetall, H. H. and Suzuki, S., eds) Plenum Press, New York, 1975, p. 269 Chert,L. F. and Tsao, G. T. Biotechnol. Bioeng. 1977, 19, 1463 Weetall, H. H. and Havevala, N. B. BiotechnoL Bioeng. Svmp. 1972, 3,241 Poutanen, K., Linko, Y.-Y. and Linko, P. Milch wissensehaft 33,435