immobilisation of amylase by various techniques -...
TRANSCRIPT
Indian Journal of Fihrc & Tcxtilc Rcscarch Vol. �l). MardI �OO). PI'. 7)-X I
Immobilisation of amylase by various techniques S R Shukla" & Lalil Jajpura
Dcpartmcnt of rihrcs and Tcxtiic Proccssing Tcchnology. Institutc of Chcmical Tcchnology. Univcrsity of Mumhai. Matunga. Mumbai 40() O I l)
/?ecein'd 2(j Septell/ber 20W: 1I('('epted 23 April 2()().J
Thc enzymc u-amylasc was subjccted to immobilisation by entrapment and covalent binding mcthods. Calcium alginatc gcl clltrapmcnt tcchniquc was uscd for thc cnzyme cntrapmcnl. Nylon 6 hcads and kniltcd fahric wcrc thc supports choscn for thc covalcnt bonding tcchniquc using glutaraldchydc with and without chitosan. Thc charaL·tcristics of thc immobiliscd cnzyme havc hccn discusscd. Covaicnt bonding gavc bcltcr stability and rcusability of thc immohiliscd cnzymc than thc calcium alginatc bead cntrapmenl.
Keywords: u-Amylasc. Calcium alginatc. Enzymc immobilisation. Glutaraldchyde. Nylon (,
fPC Code: Inl. CI.7 f)06B I WOO
1 Introduction The textile wet processing industry is one of the
major contributors of pollution to the global
environment. particularly through effluent generation
of diverse characteristics. The cotton fabric pre
treatment undergoes desizing to remove added starch.
scouring to remove naturally present impurities and
bleaching to make the fabric white. The conventional
chemicals used for these purposes contribute
substantially to the chemical and biological load of
the eftluent. It is. therefore. desirable to replace them
by the ecofriendly alternatives and in this respect. the
enzymes play a key role.
In recent years, the interest in the use of enzymes
in textile processing has increased many folds. The
mostly used enzymes 1·1 are et-amylase for desizing of
starch-sized cotton, catalase as a peroxide neutraliser
after bleaching. hemicellulase and pectinase for
relting of hast fibres. lipase for scouring. and cellulase
and protease for denim washing. biopolishing. etc.
Although the enzymcs are ccofriendly in nature, the
enzyme treatments are sometimes much costlier
compared to the conventional chemical treatments.
After completion of a desircd reaction, the enzymc is
discarded as is the case with any other chemicals used
for treatments. The activity of an enzyme, howcver, is
"To whom all tht: cOITt:spondcncc should bc addrcsscd. Phonc: 2-11-15616: Fax: + <)1-22-2-11-1561-1: E-mail: sanjccl'[email protected]()m
scarcely diminished during reactions carried out under
optimum conditions. Draining away the enzyme IS thus a wasteful practice.
Reusing the enzyme several times is a possible
remedy to ovcrcome this problem. The increase in the
concentration of product, byproduct and other
impurities in the same treatment bath. however. cause
a drastic decrease in the enzyme activity.
An immohilised enzyme may simply be rccovcred
at the time of drainage of the reaction waste products
and be reused in fresh treatment bath in a batch-wise
manner. In a continuous flow rcactor containing
immohilised enzyme. the substrate may be fed
continuously, the product bei ng collected
simultaneously. Enzyme may thus be recovered and
reused. greatly improving the economy of the
operation. Enzyme can be immobilised by various
techniques, such as adsorption, entrapment.
microencapsulation and covalent bonding-l·s.
Before weaving a cotton fahric. the starch sill' is
appl ied to the warp threads to strengthen them to
sustain mechanical adversities. Removal of this starch
through desizing operation after weaving is essential
to impart absorbency for further wet processing. The
enzyme a-amylase catalyses the hydrolysis of 1.4 linked D-glucose units of starch size to produce maltose and larger oligosaccharides. which are
I bl . <)·11 � so u e In water .
Commonly used cntrapment mcdia for enzyme
immobilisation are polyacrylamides, calcium alginate
76 INDIAN J. FIBRE TEXT. RES., MARCH 200S
and gelatine. In all the protocols, enzymes are well mixed with monomers/polymers and cross-linking agents in a solution. The solution is then exposed to polymerisation promoters to start the process of gel formation and moulded into the desired shape and size. Spherical beads, whose size can be controlled conveniently, are generally prepared and then hardened to enhance structural integrityI2-14.
Enzymes have been covalently bound to insoluble cellulose derivatives by various methods. The enzyme must, of course, be linked at some distance from the active site of the enzyme. Such immobilised enzymes retain their activities, although their immobility may reduce the reaction rate. The anionic or cationic nature of the carrier may alter the pH optimum for the reaction. The binding of the enzyme to carrier may result in steric hindrance and impose restrictions on its specificity. This is most apparent where the substrate is a large molecule, such as protein, rather than a small molecule like a peptide IS.
Nylon is a particularly convenient immobilisation matrix; it is inexpensive, inert, non-toxic, readily available and can be obtained in a number of forms. The earliest practical procedure involved partial acid hydrolysis of the nylon surface to generate more number of amino and carboxyl end groups, which could be coupled to proteins with glutaraldehyde. The enzyme can be immobilised onto nylon support with glutaraldehyde crosslinking using chitosan as a spacerl6-
19. It is an unbranched 13 ( 1 � 4)-linked polymer of 2-acetamido-2-deoxY-D-glucose (N-acetyl-D-glucoseamine), and chitosan is a collective name given to a group of N-deacetylated chitin derivatives20.21.
In the present work, alginate salt was used for entrapment of the enzyme a-amylase, and nylon in the form of beads and knitted fabric was used for its immobilisation by coupling with glutaraldehyde in absence and presence of chitosan as spacer molecule. The optimisation and stability studies for the entrapped or immobilised amylase towards pH and temperature were carried out. The activity was measured by estimating the amount of reducing groups formed on reaction with starch using DNSA reagent22.
2 Materials and Methods 2.1 Materials
Aquazyme Ultra 250 L (Novo Nordisk), an aamylase, supplied by Zytex Pvt. Ltd, Mumbai, was used for immobilisation.
Chitin, used for preparation of chitosan, was supplied by S.D. Fine Chemicals Ltd. Soluble starch,
supplied by Loba Chemie, was used as the substrate for reaction with the enzyme.
2.2 Methods 2.2.1 Preparation of 3, 5-Dinitro Salicylic Acid (DNSA) Reagent
The DNSA reagent was prepared by dissolving 1 0 g DNSA, 1 0 g NaOH, 200 g rochelle salt, 0.5 g sodium metabisulphite and 2 g phenol in 1 litre distilled water.
2.2.2 Determination of Enzyme Activity by DNSA Method
After enzymatic reaction with starch, the glucos� formed was measured, for which 2 m1 of the incubated solution and 2m! DNSA reagent were taken in a test tube, stoppered with rubber plug, kept for 10 min in a water bath at boil and then diluted to 24 ml with distilled water. The -N02 group of DNSA was reduced to -NH2 group by glucose, which was formed by the enzymatic reaction with starch. The absorbance of the red colour formed was measured at 540 nm on UV -Visible spectrophotometer by Techcomp 8500. The quantity of glucose formed due to enzyme reaction was then estimated by comparing this absorbance value with the known absorbance on the already prepared standard glucose curve.
2.2.3 Enzyme Entrapment by Calcium Alginate Gel
The solutions of sodium alginate (40 gIL) and the enzyme Aquazyme Ultra 250 L (0.5mllL) were prepared in distilled water and mixed together in equal volumes. The mixture was extruded drop wise through a 25 ml pipette into a beaker containing 1 00 mM calcium chloride solution from a height of 7.5 cm (Fig. 1 ).
Spherical calcium alginate gel beads were formed having entrapped enzyme. These were left to harden
• • •
Mixture of Enzyme and
Sodium A lginate Solution
Calcium Chloride Solution
• • Calcium Alginate
• • &..���-- Beads with Entrapped
• ••• • Enzyme
Fig. I - Preparation of enzyme entrapped in calcium alginate bead
SHUKLA & JAJPURA: IMMOBILISATION OF AMYLASE BY VARIOUS TECHNIQUES 77
in calcium chloride solution for 3 h, washed twice with the same solution and stored in a refrigerator. These were subsequently used for the starch hydrolysis reaction.
2.2.41mmobilisation of Amylase Enzyme on Nylon 6 Support
Nylon 6 knitted fabric and beads of 2 mrn diameter were used as an enzyme support for immobilization through covalent bonding.
2.2.4. 1 Partial Hydrolysis of Nylon
Pieces measuring - 10mm xl 0 mm were cut from the knitted nylon fabric . For hydrolysis of the amide bonds, the nylon beads and fabric pieces were placed in a flask fitted with a magnetic stirrer and then
. incubated in 2.9 M hydrochloric acid for 2 h at room temperature (2S°C). The reaction was terminated by thorough washing with water and then with 0. 1 M sodium phosphate buffer of pH 8.0.
2.2.4.2 Activation of Nylon Beads and Knitted Fabric
The amino groups of nylon support were activated by placing the samples in 2.S% glutaraldehyde solution in 0. 1 M sodium phosphate buffer of pH 8.0 with material-to-liquor ratio 1 : 10. The reaction was carried out at 2SoC for IS min. Unreacted glutaraldehyde was removed by washing with water and then with buffer of pH 8.0.
2.2.4.3 Synthesis of Chit os an
Chitosan was prepared by refluxing 2S g chitin powder with ISO g of SO% NaOH solution in 1 litre three-necked flask fitted with a water condenser and a mechanical stirrer. The mixture was refluxed for 4 h at 120°C and the completion of reaction was detected by complete solubility of the product in dilute acetic acid. The reaction mixture was cooled to room temperature and the product was filtered and washed with water till it was neutral. Final washing was given with ethanol to get crude chitosan.
Further purification was done by dissolving chitosan in 400 ml of 2% HCl. The mixture was well stirred and filtered. Filtrate was made slightly alkaline by gradual addition of sodium hydroxide solution with constant stirring until a precipitate was obtained. The precipitate was isolated by filtration, washed and dried at 40°C. It was further dried in a desiccator and then powdered. The powder was passed through 80 mesh sieve.
2.2.4.4 Bonding of Spacer Molecule
The chitosan spacer molecules were attached to the nylon beads and fabric by treatment with O.S % (w/v)
chitosan dissolved in 0. 1 M sodium phosphate buffer for 3 h at 2SoC using material-to-Iiquor ratio of 1 : 10. This was followed by thorough washing with water and then with buffer of pH 8.0. 2.2.4.5 lmmobilisation after Activation of Nylon
The chitosan-treated nylon supports were reactivated by 2.S % glutaraldehyde using the procedure mentioned in section 2.2.4.2. These samples were then incubated in S.O % enzyme solution at 4°C for 24 h with material-to-Iiquor ratio of 1 : IS . The enzyme in 0. 1 M sodium phosphate pH 8.0 buffer should be as concentrated as conveniently possible (ideally 1 .0 mg/ml or more) but with prolonged time (24 h or more), even the solution as dilute as 0. 1 mg/ml may give satisfactory levels of binding . Thereafter, the supports were thoroughly washed with water followed by pH 8.0 buffer solution and then with 1M sodium chloride solution in 0. 1 M sodium phosphate buffer. These nylon supports, immobilised with enzyme, were stored at pH 8.0 and temperature SoC in a refrigerator.
2.2.5 Determination of Activity of Immobilised Enzyme 2.2.5.1 Activity Determination of Entrapped Enzyme
Five ml of reaction mixture containing 0:8 ml of 2 % starch solution along with different arnounts of CaCh buffered at pH 4.2 was added to a test tube containing accurately weighted (- O.S g) entrapped enzyme beads. After an incubation period of 30 min at SOOC, the reaction mixture was collected and the aliquot was estimated for the reducing sugar produced by DNSA method to determine the activity of the immobilised enzyme.
The effect of calcium chloride on the activity of calcium alginate entrapped enzyme was studied by taking out 4.2 ml of different concentrations of calcium chloride in S rnl of reaction mixture. The beads were stored in varying concentrations of calcium chloride for 2, 1 2 and 24 h and the stability of entrapped enzyme was measured by the method discussed earlier.
2.2.5.2 Activity Detennination of Covalently Immobilised Enzyme on Nylon
The activity of the enzyme immobilised on nylon support was determined by adding S ml of reaction mixture containing 0.8 ml of 2 % starch solution and 4.2 ml of different buffer solutions in test tubes containing accurately weighted (- 0.3 g) immobilised enzyme nylon beads and fabric. After an incubation period of 30 min at SOOC, the reaction mixture was collected and the aliquots were estimated for the
78 INDIAN 1. FIBRE TEXT. RES., MARCH 2005
reducing sugar produced by DNSA method. The effect of time on reaction of immobilised enzyme and of starch concentration was also studied.
2.2.6 Determination of Optimum pH of Immobilised Enzyme
In the experimental work, acetate, phosphate and glycine-sodium hydroxide buffer solutions were used respectively for pH ranges 3.5 - 5.5, 6.0 - 8.0 and 9.0 -1 0.0. Optimum pH for the activity of immobilised enzyme was determined by adding 5 ml of reaction mixture containing 0.8 ml of the starch solution and 4.2 ml of the appropriate buffer in a test tube containing the immobilised enzyme. After an incubation period of 30 min at 50oe, the reaction mixture was collected and the aliquots were estimated for the reducing sugars by DNSA method.
2.2.7 Determination of pH Stability of Immobilised Enzyme
The immobilised enzyme samples were incubated at 250e for 24 h in 5 ml of 0.1 M buffer solutions at the appropriate pH. Residual activity in the support was then determined as described in section 2.2.5.2.
2.2.8 Determination of Optimum Temperature of Immobilised Enzyme
Activity of the enzyme immobilised onto the nylon support was estimated at various temperatures ranging from 300e to 900e at lOoe interval. The assay was carried out as described in section 2.2.5.2.
2.2.9 Determination ofThermostability of Immobilised Enzyme
The temperature stability of the immobilised enzyme was determined at sOe and over a temperature range of 30 - 900e at lOoe interval. Enzyme immobilised support was incubated in S ml of pH 8 buffer solution at the appropriate temperature for 2 h. After the incubation period was over, the support was removed and the residual activity of the immobilised enzyme was determined as described earlier in section 2.2.5.2.
2.2.10 Recycling EffICiency of Immobilised Enzyme
Immobilised enzyme was added to S ml of mixture containing 0.8 ml starch solution (2%) and 4.2 ml of pH 8.0 phosphate buffer for 30 min at sooe. Reaction mixture was then decanted and reducing sugars estimated using DNSA reagent. After washing the once used immobilised enzyme support with distilled water and pH 8.0 buffer solution, it was reused in fresh batches of substrate under similar conditions.
3 Results and Discussion Good linear correlation between the glucose
concentration and the absorbance was observed at
Amax of S40 nm. Correlation coefficient and standard error were found to be 0.9987 and 0.0196 respectively. Activity of the enzyme was thus gauged indirectly by measuring the change in absorbance of DNSA solution.
The amylase enzyme was entrapped in calcium alginate gel. It was observed that with the increase in time and starch concentration, the absorbance increased. After the completion of reaction, the calcium alginate beads got swollen and after prolonged reaction time the beads got cracked, making them unsuitable for any further reaction involving reuse of the enzyme entrapped in these beads.
Since the alginate beads were found to be stable in calcium chloride solution, the reaction with starch was carried out in it. Fig. 2 shows the effect of concentration of calcium chloride solution on the activity of enzyme entrapped in the freshly prepared beads. When the reaction was carried out in 0.25% calcium chloride solution, the activity of entrapped enzyme was found to be maximum, which decreased with the further increase in calcium chloride concentration in the reaction mixture. This may be due to increased hardening of beads, which reduces the accessibility of starch molecules to the entrapped enzyme and lor reduces the leaching out of the enzyme molecules to react with starch. The absence of calcium chloride in the reaction mixture gave less activity of the enzyme.
The activity of entrapped enzyme was then checked by keeping the beads in calcium chloride solution of different concentrations for 24 h (Fig. 3).
2.0 .-----------------�
1.6
1.2
0.6
0.4
.0 L-__ � __ � ___ � __ � __ � o 0.5 1.0 1.5 2.0 2.5
CaCl, cone. (%)
Fig. 2 - Effect of CaCl2 concentration on entrapped enzyme reaction
SHUKLA & JAJPURA: IMMOBILISA TION OF AMYLASE BY VARIOUS TECHNIQUES 79
It may be observed that the maximum activity of the entrapped enzyme was for 2 h storage in 0.25% calcium chloride solution and it decreased with prolonged duration. This may be attributed to the leaching out of some of the enzyme from the beads since they got swollen and became more open.
Fig. 4 indicates that for the first use, the maximum activity was observed for the beads reacted with starch in water alone. Here, the beads were used as such without washing from the stock stored in calcium chloride solution for a few days. As compared to the beads reacted with starch in presence of different concentrations of calcium chloride, the ones in virtual absence of calcium chloride (a very little calcium chloride comes from the beads used without washing) allows most of the enzyme to leach out and react. In second use, therefore, it may be observed that very little activity, the lowest among all, was available for reaction. In all other cases, with the increasing amount of calcium chloride in solution, the beads hardened to more extent, thereby causing hindrance either to leaching out of the enzyme or to the accessibility of starch molecules towards enzyme. Thus, in 2% calcium chloride solution, lowest activity was observed for first use and it showed least decrease among all the cases, keeping the enzyme well entrapped and inaccessible till the beads cracked at the fifth reuse.
a-amylase was immobilised by covalent bonding on nylon beads and fabric through glutaraldehyde activation. Chitosan was used as a spacer. The results
1.0 r-----------------�
0.8
Q) 0.6 () c: l\I -e 0 I/) �
0.4 <{ � B B El � 6 A t-
0.2
O 0 0.5 1.0 1.5 2.0 2.5
CaCI2 cone. (%)
Fig. 3- Effect of CaCI2 storage time on entrapped enzyme stability [ --e- after 2h, -e- after 1 2 h, and -A- after 24 h]
on pH optimisation of the enzyme reaction are shown in Fig. 5. The maximum activity of the immobilised enzyme was observed at pH 6.0 and by using chitosan, it was found to be little higher. The enzyme immobilised on nylon fabric by using chitosan (nylon fabric-GA-Chi-GA-Enz) when reacted with starch in pH 6.0 buffer showed activity about 1 8% more than that observed without using chitosan (nylon fabricGA-Enz), whereas in the case of beads, nylon beadGA-Chi-GA-Enz gave activity more by about 70% as
1.4 �-----------------...
1.2
1.0
Q) (,) 0.8 c::: ro .c ... 0 I/) 0.6 .c « 0 .4
0.2
O�-L---�----� ___ -L� 2 3 4
Reuse Cycle
Fig. 4 -Reusability of entrapped enzyme at different CaCI2 concentrations [ __ 0 %, -e 0.25 %, � 0.50 %, ...... 1.00 %, -.-1 .50 %, and-- 2.00 %]
0.9 0.8 0.7 0.6
f! c: 0.5 l\I -e 0 0.4 ., � <{ 0.3 0.2 0.1 G-
a 3 4
� 5 6
pH
e
7 B 9 10
Fig. 5 - Effect of pH on nylon-immobilised enzyme activity [ --- Nylon fabric-GA-Chi-GA-Enz,-e- Nylon fabric-GA-Enz, -Nylon bead-GA-Chi-GA-Enz, and -e- Nylon bead -GA-Enz]
80 INDIAN J. FIBRE TEXT. RES., MARCH 2005
compared to nylon bead-GA-Enz. The fabric support already has more open structure and available surface area as compared to the beads, causing higher level of bonding with the enzyme. The effect of enhancement of enzyme activity on fabric by using chitosan was, therefore, less pronounced as compared to that observed for the beads.
The maximum stability of the enzyme immobilised on nylon support was observed in the buffer solution of pH 6-7 (Fig. 6). Towards acidic pH, the enzyme stability decreased, although not as severely as in the case of free enzyme. The fabric support also showed significant decrease in the stability, since the enzyme is more accessible than in the case of beads.
The results on effect of temperature (Fig. 7) indicate that the maximum activity of the immobilised enzyme was observed at 70°C and with the increase in temperature it decreased, although slightly as compared to that of the free enzyme.
Fig. 8 shows the comparative stability of the free enzyme stored in distilled water and calcium chloride solution and that of the enzyme immobilised on nylon supports. The maximum stability was observed for enzyme immobilised on nylon beads and minimum for free enzyme stored without calcium chloride. The order of temperature stability was found to be nylon beads-GA-Chi-GA-Enz > nylon fabric-GA-Chi-GAEnz > free enzyme with calcium chloride > free enzyme without calcium chloride.
1 20 �----------------...,
1 00
� 80
.� � 60 0> <= 'c '(ij � 40
20
o L-__ �� ___ � ____ � ___ � 2 4 6 8 1 0
pH
Fig. 6 - Effect of pH on nylon-immobilised enzyme stability [ -+- Nylon fabric-GA-Chi-GA-Enz, -- Nylon bead-GA-ChiGA-Enz, and -A- Free enzyme)
120�----------------�
1 00
� 80 .� >
� 60
40 �---�---�----�----� 20 40 60
Temperature tel 80 1 00
Fig. 7- Effect of temperature on nylon-immobilised enzyme activity [ __ Nylon fabric-GA-Chi-GA-Enz, -+- Nylon bead
GA-Chi-GA-Enz, and -&- Free enzyme)
� � .2: U «
100 ���------------------------, 90 80 70 60 50 40 30 20 1 0
o �----�------�----�------���� o 20 40 60
Temperature tc) 80 100
Fig. 8 - Effect of temperature on free and immobilised enzyme stability [ -A- Free enzyme stored in distilled water, -k- Free
enzyme stored in 0.25 % CaCI2 solution, ___ Nylon fabric-GA-Chi-GA-Enz, and -- Nylon bead-GA-Chi-GA-Enz ]
1 .6 ,.----------------------------,
8 c: ..
1 .2
� 0.8 �
0.4
o 2 4 6 8 1 0 1 2 1 4 16 Reuse Cycle
Fig. 9 - Reusability of nylon-immobilised enzyme [ __ Nylon fabric-GA-Chi-GA-Enz. and --Nylon bead-GA-Chi-GA-Enz]
SHUKLA & JAJPURA: IMMOBILISATION OF AMYLASE BY VARIOUS TECHNIQUES 81
The immobilised enzyme was taken in starch reaction mixture and then reused number of times after washing it first with distilled water and then with pH 8.0 buffer solution. Fig. 9 shows the number of such reaction cycles and the residual activity of the enzyme. After 8- 10 times reuse, the immobilised enzyme retained around 50% of its original activity and even after 1 5lh reuse, 32% residual activity was retained by the enzyme immobilised on nylon fabric.
Thus, it is clear that different ways are possible to practically immobilise enzymes and such enzymes are quite capable of being reused for a number of times in a given reaction. The immobilisation technique may proved to be an efficient way of conserving the precious enzymes. However, from application point of view, the reuse of enzymes immobilised on solid supports causes certain difficulties. It will be very much useful for reactions conducted in fluid medium. Thus, in the case of textile processing applications, the use of catalase in residual peroxide bath and in effluent treatment should be quite promising.
4 Conclusions
The stability of calcium alginate beads improved when calcium chloride was used in the enzyme reaction as well as during storage. The cracking of beads occurred on reuse, the enzyme leached out and thus the reusability was limited. On the other hand, the covalently immobilised enzyme on nylon support was much stable and could be reused number of times. For the same weight, the nylon fabric gave more yield than nylon beads. With chitosan as a spacer during covalent binding with glutaraldehyde, the yield increased. The enzyme immobilised on nylon could be reused up to 8-10 times with 50% residual activity.
Once the retained activity of immobilised enzyme which decreases after every reuse is understood, it is
possible to add the required amount of fresh free Or immobilised enzyme to carry out the reaction in a particular reused cycle.
References 1 Mitra A, Saylee P & Rathi C L, Chern Weekly, 12 ( 1995)
155. 2 Shukla S R, Sharma U & Kulkarni K, Colourage, 42 (2000)
19. 3 Annis P A & Etters J N, Am Dyest Rep, 87 ( 1998) 18. 4 Messing R A, Immobilised Enzyme for Industrial Research
(Academic Press, New York), 1975, 9 1. 5 Tanyolac D, Yuruksoy B I & Ozdural A R, Biochem Eng i, 2
( 1998) 179. 6 Handa T, Hirose A, Akino T, Watanabe K & Tsuchiya H,
Biotechnol Bioeng, 25 ( 1983) 2957. 7 Ohtsuka Y, Kawaguchi H & Yamamoto T, i Appl Polym Sci,
29 ( 1984) 3295. 8 Das G & Prabhu K A, Enzyme Microb Technol, 12 ( 1990)
625. 9 Ajgaonkar D, Talukdar M & Wadekar V, Sizing-Materials,
Methods, Machines (Textile Trade Press Publication, Ahemdabad), 1982, 57.
10 Chapattwala M & Gandhi R, Colourage, 40 ( 1993) 15. 11 Weil J H, General Biochemistry, 6th edn (New Age
International Limited, New Delhi), 1 996, 167. 12 Bernath F R & Vieth W R, Immobilised Enzyme in Food and
Microbial Processes (Plenum Press, New York), 1974, 176. 13 Watanabe H, Matsuyama T & Yamamoto H, Biochem Eng i,
8 (2001) 171. 14 Hulst A C, Tramper J, Riet K V & Westerbeek M M,
Biotechnol Bioeng, 27 ( 1985) 870. 15 Silman J H & Katchalski E, Ann Rev Biochem, 35 (1966)
873. 16 Ishrove F H, Williams R J H, Niven G W & Andrews A T,
Enzyme Microbial Technol, 28 (2001) 225. 17 Sundaram P V & Hornby W E, Febs Lett, 10 ( 1970) 325. 18 Inman D J & Hornby W E, Biochim i, 129 ( 1972) 255. 19 Barker S A, Somers J & Epton R, Carbohyd Res, 14 ( 1970)
786. 20 Taylor R F, Protein Immobilisation; Fundamentals and
Application (Marcel Dekker Inc., New York), 1991, 90. 2 1 Stanley W L, Watters G G, Kelly S H & Olson A C,
Biotechnol Bioeng, 20 ( 1978) 135. 22 Miller G L, Anal Chem, 3 1 ( 1959) 426.