Immobilization of Soybean α-amylase on Gelatin and its Application as a Detergent Additive
The detailed kinetic study of the immobilized enzyme is usually important and pertinent for the better management of the enzyme preparation. Therefore, the suitable comparison of the various kinetic parameters of the immobilized α-amylase (on gelatin) with that of the soluble enzyme has been conducted. The purified α-amylase was immobilized on gelatin using glutaraldehyde as an organic hardener with an optimum immobilization of 82.5% followed by characterization with respect to pH, temperature, substrate concentration, effect of thiols and thiol inhibitors. The optimum pH shifted from 5.5 to 7.0 i.e., towards the basic side upon immobilization. The optimum temperature of gelatin immobilized α-amylase was 85°C as compared to soluble enzyme (75°C) showing an improvement in thermal stability. A decrease in Michaelis constant, Km from 2.427 to 1.870 mg mL-1 upon immobilization was observed. An increase in activity with all the thiols was observed. No marked inhibition with the thiol inhibitors (similar to the soluble enzyme) is an indication of the fact that there are no free thiol or sulfhydryl groups in soybean α-amylase which are necessary for catalysis. The reutilization capacity of the immobilized enzyme was up to seven cycles. The potential utility of the gelatin immobilized enzyme in removal of starch stain from cloths by various commercially available detergents and sodium lauryl sulphate was tested. The immobilized α-amylase could be considered as a potential candidate for use as a cleaning additive in detergents in order to facilitate the removal of starch stains due to which it may find potential application in laundry detergents.
January 19, 2011; Accepted: April 21, 2011;
Published: June 18, 2011
The immobilization of enzyme on insoluble supports has been a topic of active
research in enzyme technology and is essential for their application to individual
processes, for example in food technology, biotechnology, biomedicine and analytical
chemistry. Enzyme immobilization is preferred to enhance enzyme stability, multiple
and repetitive use and to remove the enzyme from the reaction mixture easily
(Hasirci et al., 2006; Pandya
et al., 2005; Altun and Cetinus, 2007). Since
amylases have many applications in the food and fermentation industries, it
is not surprising that several methods have been developed for the preparation
of immobilized α-amylase, each having its own advantages and disadvantages
specific to the methods. Some examples of different supports used for α-amylase
immobilization are: glass beads and glutaraldehyde fixation (De
Cordt et al., 1994), polymeric microspheres (Tumturk
et al., 2000), coconut fiber (Dey et al.,
2002), cellulose fibers from bagasse (Varavinit et
al., 2002), functionalized silica (Lim et al.,
2003), Cu2+-chelated poly (ethylene glycol dimethacrylate-n-vinylimidaole)
(Kara et al., 2005), poly(dimmer acid-co-alkyl
polyamine) particles (Hasirci et al., 2006),
UV-curable polymers (Kahraman et al., 2006),
sodium alginate, chitin, sephadex and dowex (El-Banna et
al., 2007), functionalized glass beads (Kahraman
et al., 2007), calcium alginate (Ertan et
al., 2007), adsorption on zirconia (Reshmi et
al., 2007), cellulose fiber (Egwim and Oloyede,
2008), cyclic carbonate (Turunc et al., 2009),
polyaniline (Pascoal et al., 2010), coconut coir
(Borgio, 2011). The several procedures for the immobilization
of an enzyme involve: (1) entrapment of the enzyme molecules in polymeric structures,
(2) physical adsorption of the enzyme onto a solid support, (3) covalent binding
to an activated support, (4) copolymerization of the enzyme molecules with the
polymers and (5) cross linking between the enzyme molecules. Among these immobilization
methods, covalent binding is usually advantageous as it: a) increases the thermal
and storage stability of the enzyme, b) prevents leakage and c) decreases diffusion
problems of the substrate and products in the reaction medium (Hasirci
et al., 2006). Recently, α-amylase has been covalently immobilized
onto poly (hydroxyethylmethacrylate) and poly (methyl methacrylate-acrylic acid)
microspheres and zirconium membranes (Arica et al.,
1995; Sun et al., 1999; Tien
and Chiang, 1999).
α-amylase (EC 184.108.40.206; 1,4-α-d-glucanohydrolase) is one among the
various starch hydrolyzing enzymes which is of considerable interest in terms
of industrial applicability. It is particularly employed in starch processing
industries for the hydrolysis of polysaccharides such as starch into simpler
sugar constituents (Ikram-ul-Haq et al., 2002).
It catalyzes the hydrolysis of glucosidic linkages as starch to produce smaller
oligosaccharides. This property of α-amylase is exploited for its practical
use in removal of starch stain from cloths as starch is a major polysaccharide
used as a food ingredient such as pasta, chocolate, baby food, sauce and gravy.
The removal of starch from surfaces is important as starch can be an attractant
for dust and dirt as well (Hmidet et al., 2009).
Usually, the enzymes used in detergents are unstable and are at the risk of
being attacked and inhibited by the detergent action. Immobilization may be
found as a better solution to this which not only protects the enzyme from inhibition
by various detergent components but also facilitates its reuse (Maliks
et al., 2000). Various literatures are available showing the washing
efficiency of α-amylases from various sources (Hmidet
et al., 2009; Rani et al., 2007; Dhingra
et al., 2006; Mitidieri et al., 2006).
In the present study, effects of various immobilization parameters in order
to obtain maximum yield of immobilized α-amylase was studied. The immobilized
enzyme was characterized with respect to pH, temperature, substrate concentration
and effect of thiols and thiol inhibitors. Besides, its application as a detergent
additive in order to remove starch stain from cloth was checked.
MATERIALS AND METHODS
Materials: Soybean seeds were procured from the local market. Sodium alginate, Maltose, Gelatin and 3,5-dinitrosalicylic acid (DNS) was purchased from Sigma Chemicals Co. (St. Loius, MO, USA). Soluble starch was from Qualigens Fine Chemicals, Mumbai. All other reagents were analytical grade chemicals either from BDH or E. Merck, India. The study was conducted during January 2010 to December 2010.
Isolation and purification of α-amylase: α-amylase was isolated
from soybean (Glycine max) seeds, procured from the local market and
was purified to electrophoretic homogeneity as described Prakash
and Jaiswal (2010).
Immobilization of α-amylase on gelatin: Gelatin solution of different concentrations was prepared by dissolving gelatin in distilled water by heating at 50°C and thereafter cooling to room temperature. One milliliter of the purified enzyme (0.44 mg protein) was added to 9.0 mL gelatin solution with thorough mixing. The suspension was casted on preassembled glass plates and when the suspension jellified, 10 mL of 10% glutaraldehyde solution was added and left for complete hardening. The gelatin was then cut into small beads (5x5 mm), washed thoroughly with distilled water to remove any enzyme attached to the surface of beads and stored in 25 mM sodium acetate buffer (pH 5.5) at 4°C.
α-amylase assay: α-amylase activity was estimated following
the method as described by Bernfeld (1955).
Protein estimation: Protein was estimated by the method of Lowry
et al. (1951) with Folin-ciocalteau reagent calibrated with crystalline
bovine serum albumin.
Steady-state kinetics and storage stability of immobilized enzyme: The
optimum pH for the immobilized α-amylase was determined by varying the
pH of assay buffer (Tris-acetate) from 4.0-9.0. The enzymatic activity was determined
by the method described for enzyme assay. The optimal temperature was studied
by varying the temperature of immobilized enzyme in assay buffer from 0-100°C.
Km was determined by Lineweaver and Burk (1934)
with variation in the substrate (starch) from 5 to 40 mg mL-1. In
order to test the reusability of the immobilized α-amylase, the activity
in the beads was assayed several times for the hydrolysis of soluble starch.
The activities of the first batch were taken to be 100%. After each amylase
activity assay of the beads, they were removed, washed thoroughly with distilled
water at 4°C. Then, the beads were reassayed for α-amylase activity
and the same process was repeated till the seventh use.
Effect of thiols and thiol inhibitors: The effect of different thiols and thiol inhibitors on enzyme activity was observed with the varying concentration of these reagents in the reaction mixture and then assaying the enzyme activity. The activity assayed in the absence of these reagents was taken as 100% and residual activity determined in their presence was calculated.
Application of immobilized α-amylase: Clean cotton cloth pieces of size 5x5 cm were stained with 0.2 mL aqueous starch solution. Stock solutions (2.0 g L-1) of commercially available detergents like Surf Excel, Ariel, Rin, Tide, Ghadi and sodium lauryl sulphate (a major component of detergent) were prepared in distilled water. Anthrone reagent (0.02%) was prepared in chilled concentrated sulphuric acid. The stained cloth pieces were subjected to wash treatments in separate flasks: (1) in distilled water only (2) in detergent only (3) in detergent and immobilized α-amylase. Each flask was incubated at 40°C for 20 min under continuous agitation (150 rpm). After incubation, the washing performance in each case was determined. The cloth piece from each flask was taken out and the volume of washout so left was made up to 25 mL with distilled water. A 1.0 mL of these washouts was mixed with 2 mL of anthrone reagent in tubes and then were placed in boiling water bath for 10 min and cooled to room temperature. The color obtained was determined spectrophotometrically at 630 nm. A standard curve was prepared by taking varying concentration of glucose in the range of 0-100 μg. The amount of glucose produced was extrapolated from the standard curve. The value of glucose was multiplied by 0.9 to convert glucose value to starch.
Statistical analysis: Data were analyzed using SPSS (version 16.0) package on a microcomputer. All values expressed were the means for three replicate experiments carried out with a fresh batch of purified enzyme.
RESULTS AND DISCUSSION
Immobilization of α-amylase on gelatin: In the present study, the enzyme was immobilized on gelatin matrix and covalently crosslinked by the organic hardener glutaraldehyde. The optimization of immobilization of α-amylase in gelatin was done by varying the concentration of gelatin from 5 to 30% (w/v). As it is evident from the Table 1, maximum immobilization was achieved at 20% gelatin concentration. The results revealed that at low gelatin concentration, unstable and fragile gelatin beads were obtained which led to poor immobilization which may be due to the larger pore size in the gel which probably caused leaching of the enzyme. At concentrations above 20%, the percent immobilization was low which may be the result of steric hindrance due to high concentration of gelatin.
The glutaraldehyde may be said to act as a hardening agent as it was not easy to handle the matrix (with the enzyme entrapped within) in its absence. Moreover, the glutaraldehyde concentration above 10% led to a decline in percent immobilization which again may be the result of the steric hindrance caused by the presence of the glutaraldehyde which may have affected the accessibility of the substrate to amylase (Table1). Thus, the optimum immobilization of 82.5% was observed with 20% gelatin and 10% glutaraldehyde concentration.
Steady-state kinetics: An enzyme in solution can have altered pH optima upon immobilization. Depending upon the surface and residual charges on the solid matrix and the nature of the enzyme bound, the pH value in the immediate vicinity of the enzyme molecule may change and thus cause a shift in the pH of the enzyme activity.
The effect of pH on the activity of gelatin immobilized amylase is shown in
Fig. 1. The pH optima of soluble and immobilized amylase were
5.5 (Prakash and Jaiswal, 2010) and 7.0, respectively,
i.e., a shift of 1.5 Units towards the basic side was resulted from immobilization
of α-amylase on gelatin. A similar trend of shift in pH of 2.4 U towards
the basic side (from 5.6 to 7.0) was observed by Tripathi
et al. (2007) from immobilization of α-amylase on both chitosan
|| Percent immobilization with varying gelatin concentration
|| Effect of pH on soluble and gelatin immobilized α-amylase
Similarly, El-Shora and Youssef (2008) also found a
shift of pH optimum from 7.5 to 8.0 on immobilization of glutamate dehydrogenase
on gelatin beads via cross-linking with glutaraldehyde. No change in optimum
pH was reported in case of commercial α-amylase (Bacillus sp.) immobilized
onto pHEMA microspheres and to a composite temperature sensitive membrane, respectively
(Tien and Chiang, 1999; Chen et
al., 1998). A shift towards acidic region has been observed when α-amylase
was immobilized on poly(methylacrylate-acrylic acid) microspheres (Aksoy
et al., 1998) and on zirconium dynamic membranes (Tien
and Chiang, 1999). The alteration in the pH optimum of the enzyme in solution
and upon immobilization to a solid matrix is largely dependent on the residual
surface charges on the solid matrix and the nature of the bound enzyme. The
effect of temperature on the activity of soluble and immobilized enzyme is shown
in Fig. 2. The soluble enzyme exhibited a temperature optimum
of 75°C (Prakash and Jaiswal, 2010) and this value
shifted to 85°C for the immobilized enzyme. This increase in the temperature
optima may be due to the conformational integrity of the enzyme structure upon
covalent binding to the support material (Tien and Chiang,
1999; Bayramoglu et al., 1992; Demircioglu
et al., 1995; Lee et al., 1993).
On comparison of the kinetic constants of soluble and immobilized amylase,
a decrease in Km [from 2.427 (soluble) to 1.87 (gelatin immobilized)]
was observed (data not shown). A similar decrease in Km was observed
upon immobilization of α-amylase onto cyclic carbonate (Turunc
et al., 2009). The Km value is known as the affinity
of the enzymes to substrates (Cosulich et al., 2000;
Park et al., 2005) and the lower value of Km
emphasize the higher affinity between enzymes and substrates. The present results
show that affinity of the enzyme to its substrate was increased by immobilization.
The reusability of the gelatin immobilized enzyme was upto seven cycles. Almost 90% activity was retained upto three cycles, but with subsequent runs, there was a decline in the activity of the immobilized enzyme. The activity loss upon reuse could be due to weakening in the strength of binding between the matrix and enzyme on repeated use and hence the enzyme might leach out from the matrix, therefore yielding a loss in activity. Besides, the frequent encountering of the substrate into the same active site might distort it which would dwindle the catalytic efficiency either partially or fully.
|| Effect of temperature on soluble and gelatin immobilized
|| Effect of thiols on gelatin immobilized α-amylase activity
Effect of thiols and thiol inhibitors: The immobilized α-amylase
showed an increase in activity by all the thiols (DTT, L-cysteine and ME) studied
(Fig. 3). The activation by DTT was much more pronounced showing
about 2.9 fold increase at 2.5 mM concentration. In comparison, L-cysteine and
ME showed only 2 fold and 1.84 fold increase in activity, respectively. This
observed activation of the soybean α-amylase may be due to the protection
of the-SH groups of the enzyme by the added thiol as the thiol reagents itself
is consumed by oxygen, leaving the-SH groups of the enzyme intact for the catalytic
functions. A similar activation was reported in case of watermelon urease (Prakash
and Upadhyay, 2003).
As observed from the effect of the thiol inhibitors; NEM, IAA and pHMB (Fig. 4), there was no marked inhibition maintaining more or less about 70% residual activity at 10 mM indicating that there are not free sulfhydryl groups available in soybean α-amylase to be directly involved in catalysis.
Application of the gelatin immobilized α-amylase in starch stain removal:
The ability of the gelatin immobilized α-amylase for starch stain removal
was assessed by washing starch stained cotton cloth pieces in detergents alone
and in presence of immobilized α-amylase. The starch content removed from
the cloth as washout was determined. The washing performance of the detergents
is shown in Table 2. The higher amount of starch content indicates
better washing. In distilled water, all gave almost similar washing.
|| Colorimetric determination of starch content in the washout
|| Effect of thiol inhibitors on gelatin immobilized α-amylase
The washing performance of the detergents supplemented with immobilized α-amylase
was better as compared to its performance with detergent only. This is based
on the property of amylases which are known to hydrolyze the starch and thus
removes the starch stain from clothes freely and rapidly. Thus, the use of immobilized
α-amylase in starch stain removal by detergents has increased their washing
efficiency without being affected by the components of detergents (like surfactants,
builders, co-builders, bleach, inhibitors, etc.
The results revealed that gelatin immobilized α-amylase formed irreversible and stable aldemine bonds that led the immobilized enzyme to acquire higher temperature optima, increased tolerance to higher pH range and could be reused for starch hydrolysis. All these criteria, could, therefore be successfully utilized in continuous production of maltose. It was found that immobilized α-amylase improved the washing efficiency of detergents and removed starch stains from clothes which are difficult to remove under normal washing conditions. Thus, the immobilized α-amylase could be considered as a potential candidate for use as a cleaning additive in detergents in order to facilitate the removal of starch stains. Due to this promising feature it may find potential application in laundry detergents.
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