Immobilisation of amylases on krill chitin

FoodChemistry8 (1982)239-246

IMMOBILISATION OF AMYLASES ON KRILL CHITIN

JOZEF SYNOWIECKI,ZDZIS/LAWSIKORSKI• MARIAN NACZK

Technical University Politechnika Gdahska, Institute of Organic and Food Chemistry and Technology, Gdahsk, Poland (Received: 11 February, 1981)

ABSTRACT

Krill chitin was used as a support for diastase. The enzyme was immobil&ed by simple adsorption or in fhe presence of O"l °//oglutaraldehyde. Alternatively, the enzyme was supported on chitin previously activated with glutaraldehyde. The best results were achieved by the first procedure using chitin fractions of 22-52 mesh, obtained by demineralisation of raw dried krill offal with 2 2 ~ HCI (1.'10)for 2h at room temperature and deproteinisation with 28 % KOH solution for 2 h at 95 ° C. Supporting diastase without any treatment with glutaraldehyde did not reduce the enzyme activity. The optimum pH for binding of the diastase on chitin preparations was 6"2 in the presence of glutaraldehyde or 6"7 without the crosslinking agent. Immobilisation shifts the optimum pH for the activity of diastase by 0"5 units towards the acid side.

INTRODUCTION

Several investigators have reported on the utilisation of chitin, isolated from shells of edible shellfish, as support for papain (Finley et al., 1977), lactase, c~chymotrypsin, acid phosphatase (Stanley et al., 1975) and glucose isomerase (Stanley et al., 1976). For fixation of the enzymes on the support usually glutaraidehyde is used, in concentrations ranging from 0.1 to 2 ~o in the reaction mixture. In most cases supporting of the enzymes on crab chitin results in a negative shift of the optimum pH value for the enzyme activity by 0-7 to 1 pH units. However, binding of ~-chymotrypsin is accompanied by a 1.2-unit alkaline shift, which is thought to be caused by the low diffusion rate of the carboxylic groups released during hydrolysis of the protein (Stanley et al., 1975). Recently there is a growing interest in commercial exploitation of the vast resources of Antarctic krill. Processing of krill to various edible products yields 239 Food Chemistry 0308-8146/82/0008-0239/$02.75 © Applied Science Publishers Ltd, England, 1982 Printed in Great Britain

240

JOZEF SYNOWIECKI, ZDZISLAW SIKORSKI, MARIAN NACZK

from 8 to 10 % of offal (Sikorski et al., 1979), which can be a good source of highquality chitin and chitosan (Brzeski et al., 1980). The use of krill chitin in biochemical reactors could be one of several factors influencing the overall economy of krill fisheries. Chitin obtained from krill offals, contrary to that isolated from fresh water crayfish, was found to have a highly porous surface and therefore could be suitable to bind enzymes strongly by noncovalent bonds (Popowicz, pers. comm.).

MATERIALS AND METHODS

Raw dried krill offal was demineralised by treating with 22 % HC1 (1:10) for 2 h at room temperature and deproteinised with KOH solution of different concentrations during 2 h at 95 °C. The raw chitin was further purified by extraction with methanol (1:5), air dried at 80°C, and separated into fractions of different mesh sizes. For comparative purposes also, Fluka chitin and Kyowa chitosan were used. Characteristics of the supports are given in Table 1. Riedel diastase used in the experiments had maximum activity at 50-52 °C and pH 5.7. The results of thermal inactivation and of inhibitory action of Cu 2 + salts indicate that the enzyme preparation did not exhibit other amylolytic activity except that of fl-amylase. The same can also be concluded when the optimum pH and temperature of the enzyme are compared with those of published data. The enzyme was supported by adsorption on 50 mg of the chitin preparation during 1 h at 25 °C with intermittent mixing, followed by 20 h of standing at 4 °C, and by washing of the free diastase with 800 cm 3 of distilled water. Covalent binding of the enzyme was accomplished in the presence of 0.1% and 0.3 % glutaraldehyde TABLE 1 THE CHARACTERISTICS OF SUPPORTS USED IN THE EXPERIMENTS a (FRACTION

Support

Concentration

Chemical characteristics

of K O H solution

%

(%)

Ash

Nitrogen b (%)

0"5 3'0 10.0 28"0

0-08 0'06 0.04 0"05

6'39 6.27 6-17 6"23

Commercial chitin (Fluka)

3.62

7'73

Chitosan

0.30

--

Krill chitin

22-52 MESH)

. Activity of bound diastase Treatment of (rate of reaction available rag~rain) N i l 2 groups (#M/g chitin) 211 190 232 408

1.62 1.64 1.90 2" 18 0-19

--

1.36

a Mean values of results obtained in 4 - 6 experiments. The coefficient of variation was _<5 %. b For demineralised dry chitin.

IMMOB1LISATION OF AMYLASES ON KRILL CHITIN

241

in the mixture, all other parameters being equal. Alternatively, the diastase was supported on chitin previously activated with glutaraldehyde (50 mg of chitin in 5cm 3 2 % glutaraldehyde and 0.5cm 3 concentrated acetic acid for 24h at room temperature). The quantity of the enzyme bound to the support was assayed by determining the amount of starch hydrolysed by the diastase supported on 50 mg of chitin, measured according to Heinkel (1956); however, the quantity of the reagents was five times greater than in the original procedure. The quantity of hydrolysed starch (S) was calculated using the formula:

S -A°-A Ao

100%

where A o and A is the extinction of the starch solution prior to and after hydrolysis, respectively. For comparative purposes the activity of the immobilised enzyme was expressed as the degree of hydrolysis of starch or as the rate of reaction.

RESULTS AND DISCUSSION

The binding of the enzymes The most active preparations were obtained by simple adsorption of the enzyme on krill chitin deproteinised by 28 % KOH solution (Table 1). Glutaraldehyde brings about a decrease in activity of the bound diastase, both when used for pretreating of the support or for crosslinking of the enzyme (Table 2). The activity of diastase fixed with glutaraldehyde depends upon the concentration and time of action of the crosslinking agent and on the concentration of the enzyme (Table 3). About 39 to 46 % of the initial amount of diastase can be supported on chitin by simple adsorption without any significant loss in activity (Table 4). The yield of bound diastase depends upon the quality of the support, i.e. the molecular weight of TABLE 2 COMPARISON OF THE ACTIVITYOF DIASTASEIMMOBILISEDON CHITIN BY DIFFERENT TREATMENTSa

Treatment

The relatit, e actit, ity o f immobilised diastase

%)

Adsorption

100

Adsorption and binding by glutaraldehyde

56"9

Adsorption after pretreatment of chitin with glutaraldehyde

53'8

° Mean values of results obtained in six experiments. The coefficient of variation was < 5 %.

242

JOZEF SYNOWIECKI, ZDZIS4LAW S1KORSKI, MARIAN NACZK TABLE 3

I N F L U E N C E OF TI-IE C O N C E N T R A T I O N OF DIASTASE A N D G L U T A R A L D E H Y D E IN T H E R E A C T I O N M I X T U R E O N T H E ACTIVITY OF T H E B O U N D E N Z Y M E

Concentration of diastase in the reaction mixture ( % of the weight of chitin) 20 30 40

Concentration of glutaraldehyde in the reaction mixture

(%)

Act&ityofbound d&stase (%) 0 0.1 0.3

39 31 20

56 34 21

73 41 31

Mean values of results obtained in six experiments. The coefficient of variation was < 5 ~ .

the polymer as well as the content of minerals and residual proteins, which in turn depend on parameters of demineralisation and deproteinisation of the krill offals. The increase in the activity of amylase adsorbed on chitin preparations, obtained by demineralisation of the offal with 22 ~ HC1 and deproteinisation with more concentrated KOH solutions, suggests that for effective binding of the enzyme the availability of deacetylated amino groups of chitin is necessary. The best results were obtained on chitin fractions of 22-52 mesh (Fig. 1). The optimum range of pH for binding of the enzyme on chitin preparations is 6.2 in the presence of glutaraldehyde or 6.7 without the crosslinking agent (Fig. 2). The pH value, 6.2, is close to that of the isoelectric point, reported for different preparations of diastase. It is possible that isoelectric aggregation is partially responsible for the increased binding of the enzymes on chitin at this pH. However at pH6.7 ionic attraction phenomena seem to be additionally responsible for binding of the enzyme, At the optimum pH value most efficient immobilisation of diastase was accomplished at very low ionic strength (Fig. 3). At optimum pH and ionic strength the efficiency of binding or activity of the preparations decreased linearly with increasing temperature of the mixture (Fig. 4). After 1 h at 25 °C and a TABLE 4 D I S T R I B U T I O N OF DIASTASE BETWEEN T H E S U P P O R T A N D S O L U T I O N

The quantity of enzyme ( r a g ) a 1

2

3

4

Initial in the mixture

lmmobilised on 50 mg of chitin without treatment with glutaraldehyde

Residual in the solution

~ (2 + 3)

5 10 15 20

2.3 4.1 6"0 7.9

3'3 5.3 8.9 12"8

5.6 9-4 14.9 20-7

° Mean values of results obtained in six experiments. The coefficient of variation was _<8 %.

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pH

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ois o:s

Ionic strength

o.~s

i

Fig. 3. Activity of diastase immobilised on chitin at different ionic strengths (optimum pH enzyme binding = 6.2 or 6.7, T = 25 °C). Immobilisation by adsorption (O--O), adsorption in the presence of 0,1 % glutaraldehyde ( A - - ~ ) .

llal

40

Ternperoture (°C) Fig. 4. Activity of diastase immobilised on chitin at different temperatures (optimum pH for enzyme binding = 6.2 or 6.7, ionic strength = 0-05). Immobi|isation by adsorption ( ( ~ O ) , adsorption in the presence of 0.1% glutaraldehyde (A A).

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IMMOBILISAT1ON OF AMYLASES ON KRILL CHITIN

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pH of about 7 the activity of the enzyme supported by adsorption was 60 ~ and that fixed with glutaraldehyde was 36 9/0.After an additional holding time of 20 h at 4 °C the activity increased only by about 4 ~.

Properties of the immobilised enzyme Immobilisation on chitin brings about a shift of the optimum pH for the activity of diastase towards the acid side by about 0.5 units (Fig. 5). A similar effect of binding on chitin has been reported in the case of glucose isomerase (Stanley et al., 1976) and acid phosphatase (Stanley et al., 1975). There is no change in the optimum temperature for the activity of the enzyme, although the bound diastase is slightly more heat resistant (Fig. 6). The activity of diastase supported on krill chitin was three to five times greater than that supported on samples of Fluka chitin and Kyowa chitosan of the same mesh size supplied by the producers. Further treatment of the Fluka preparation with HC! and NaOH, identical to that applied for preparing the krill chitin, did not improve the result of enzyme binding.

ACKNOWLEDGEMENTS The authors acknowledge with thanks the financial support obtained under the research grant PR-Kryl administered by the Sea Fisheries Institute in Gdynia. The authors also express their thanks to Kyowa Oil and Fat Co. Ltd for supplying free samples of chitosan.

REFERENCES BRZESKI, M., MIECZKOWSKA, M., STOLZ, H., WOJTASZ, A., SOWA, K. & NEUGEBAUER, W. (1980). Technologia otrzymywania chityny i chitosanu. Paper presented at the Seminar on Chitin and Chitosan. Sea Fisheries Institute, Gdynia, January. FINLEY, J. W., STANLEY, W. L. & WATTERS, G. G. 0977). Removal .of chill haze from beer with papain immobilized on chitin. Biotechnol. Bioeng., 19, 1895. HEINKEL, K. (1956). Uber die Vergleichbarkeit erschiedener-Bestimmungsmethoden der Blutdiastasen. Klinische IVochenschrift, 34, 155. SIKORSKI, Z. E., BYKOWSKI, P. & KNYSZEWSKI, J. (1980). The utilization of krill for food. In: Food Process Engineering, Vol. 1." Food Processing Systems. (Linho, P. et al. (Eds)), Applied Science Publishers, London, pp. 845-55. STANLEY, W. L., WATTERS,G. G., CHAN, B. ~,~MERCER, J. M. (1975). Lactase and other enzymes bound to chitin with glutaraldehyde. Biotechnol. Bioeng., 17, 315. STANLEY, W. L., WATTERS,G. G., KELLY, S. H., CHAN, B. G., GARIBALDI,J. A. & SCHADE,J. E. (1976). Immobilization of glucose isomerase on chitin with glutaraldehyde and by simple adsorption. Biotechnol. Bioeng., 18, 439.