Immobilization of B. amyloliquefaciens α-amylase and ...

Romanian Biotechnological Letters Vol. 16, No. 6, 2011 Copyright © 2011 University of Bucharest Printed in Romania. All rights reserved

ORIGINAL PAPER

6690

Immobilization of B. amyloliquefaciens α-amylase and comparison of some of its enzymatic properties with the free form

Received for publication, May 10, 2011

Accepted, May 25, 2011

ELİF DEMİRKAN1*, SERHAN DİNCBAS1, NİHAN SEVİNC1 and FİGEN ERTAN2 1Science and Art Faculty, Departmen of Biology, Uludag University, Bursa, Turkey 2Science Faculty, Departmen of Biology, Trakya University, Edirne, Turkey Corresponding author: Dr. Elif Demirkan Tel: +90 224 294 17 94, Fax: +90 224 294 18 99 E-mail: [email protected]

Abstract

The enzyme α-Amylase from Bacillus amyloliquefaciens was entrapped by drop-wise addition of an aqueous mixture of sodium alginate in the solution of a Ca2+ salt. Effects of immobilization conditions were investigated. Optimum alginate and CaCl2 concentrations were found to be 2% and 5% (w/v), respectively. The immobilization yield was 89%. Amylase activity increased with increasing enzyme loading on the beads. The best size and amount of beads for achieving enzyme activity were found to be 3 mm and 0.4 g, respectively. When coated with silicate, amylase-entrapped beads retained the highest catalytic activity. The highest operational stability was six reuses with 51% residual activity. Some properties of immobilized enzyme were determined, and compared with those of free enzyme. Product profile of the various raw starches has been determined by thin layer chromatography.

Key words: α-amylase, Bacillus amyloliquefaciens, calcium alginate, immobilization, optimization, thin layer chromatography Introduction

The enzyme α-amylase (EC 3.2.1.1; α-1,4-D-glucan glucanohydrolase) hydrolyzes α-1,4-glucosidic bonds in starch in an endo fashion and forms low molecular weight products. These enzymes are of great significance in present day biotechnology and find applications in areas including food, baking, brewing, fermentation, detergent applications, textile desizing, paper industries and analysis in medicinal and clinical chemistry [1]. Microbial amylases are available commercially, and they have almost completely replaced chemical hydrolysis of starch in the starch processing industry [2]. Amylolytic enzymes (including α-amylase, β-amylase, glucoamylase, α-glucosidase, and pullulanase) account for US$ 225 million of the US$ 2 billion annual worldwide sales of industrial enzymes. α-Amylases seem to be the most versatile enzymes in the industrial enzyme sector and account for approximately 25 % of the enzyme market [3, 4]. B. subtilis, B. stearothermophilus, B. licheniformis and B. amyloliquefaciens are known to be good producers of α-amylase, and these have been widely used for commercial production of the enzyme for various applications [5]. The use of enzymes in a soluble or free form must be considered as wasteful because the enzyme generally cannot be recovered at the end of the reaction. A new and valuable area of enzyme technology is that concerned with the immobilization of enzymes on insoluble polymers, such as membranes and particles that act as supports or carriers for the enzyme activity [6]. Generally, enzymes are immobilized by physical adsorption, ionic binding, covalent binding, cross-linking and entrapment methods [7]. Entrapment method consists of enclosing the enzyme in an aqueous solution inside a semipermeable membrane capsule. In this immobilization technique, the particle structure allows contact between the substrate and enzyme to be achieved, and it is possible to immobilize several enzymes at the same time [8].

ELİF DEMİRKAN, SERHAN DİNCBAS, NİHAN SEVİNC and FİGEN ERTAN

Romanian Biotechnological Letters, Vol. 16, No. 6, 2011 6691

Calcium alginate has been explored for enzyme and cell immobilization by entrapment and it is widely used most commonly in the form of spherical beads because bead preparation is a safe, easy, and rapid process [9]. Immobilized enzymes are preferred over their native counterparts because of their potential for repetitive use. In addition, the reaction product is not contaminated with the enzyme, and the immobilized enzyme has a longer half-life and predictable decay rate [10].

Immobilization of an enzyme allows for easy separation of the enzyme from the products and for recycling of the enzyme. Immobilized enzymes are used in food technology, biotechnology, biomedicine and analytical chemistry [11, 12].

In this study, immobilization conditions were investigated for B. amyloliquefaciens α- amylase as a model enzyme. The effects of alginate concentration, calcium concentration, silica concentration, loading of enzyme onto the alginate, bead size, and bead amount have all been determined. The reusability of the beads was tested, and the free and immobilized enzyme activities for starch hydrolysis were examined. Product profile of starches has been determined by thin layer chromatography. Some properties of immobilized enzyme were determined and compared with those of free enzyme. Materials and Methods Bacterial Strain and Culture Conditions

A new strain of B.amyloliquefaciens was obtained from a Biochemicals plant (ORBA Biochemistry, Istanbul). Basal medium for liquied culture consists of (%): starch, 1; peptone, 0.5; corn steep liquor, 0.5; (NH4)2SO4, 0.8; MgSO4.7H2O, 0.2; CaCI2.2H2O, 0.05; K2HPO4, 1.4; KH2PO4, 0.6, and the pH was adjusted to 7.0 before autoclaving [13]. Cultivation was carried out at 37 oC on a rotary shaker operated at 150 rpm for 64 h. Cells were removed by centrifugation at 6000 rpm for 10 min. The supernatant was used as the source of the enzyme Enzyme Activity

α-Amylase activity was assayed by the starch-iodine method [14]. One enzyme unit was defined as the amount of enzyme that hydrolyzed 1 mg of starch (0.1%) in 10 min at 37 oC and pH 5.9 (unit/mL). Enzyme Immobilization

Calcium alginate beads were prepared with an equal volume of crude enzyme solution and sodium alginate solution to form a 2% (w/v) final concentration of sodium alginate in the mixture. The mixture of enzyme and alginate was taken into a syringe (0.8 mm diameter), and beads were formed by dropping the solution into CaCl2 (5% w/v) solution with gentle stirring at 4 oC for 1 h. The formed beads were recovered by filtration and were thoroughly washed with distilled water to remove excess CaCl2 and free α-amylase. The beads were dried using filter paper (Whatman no. 1) followed by exposure to the open air for 1 h before use [15, 16]. The filtered CaCl2 solution and the washings were collected for protein concentration determination. Protein content of free enzyme and the collected washings was determined by using the Lowry method [17].

The activity yield was defined as the yield of enzyme that was immobilized in alginate beads and was expressed as

where: aimm is the specific activity of immobilized enzyme (U mg protein-1) and afree is the specific activity of the free enzyme (U mg protein-1) [18].


6692 Romanian Biotechnological Letters, Vol. 16, No. 6, 2011

Optimization of Critical Parameters for Immobilization The properties of the beads were studied by using beads of varying permeability and

rigidity. The effect of sodium alginate concentration on the bead permeability was studied by using various amounts of sodium alginate (1-5% w/v). The formation of beads was induced by 5% (w/v) CaCl2 solution. The effect of CaCl2 concentration on the rigidity of the beads in presence of the 2% (w/v) sodium alginate was studied by using different concentrations of CaCl2 solution (2-6% w/v). In addition, the affects of bead size and bead amount on the activity were examined by using differently sized syringe needles (0.45, 0.8, 2, and 3 mm diameter) and by varying the bead amounts (0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50 g beads). The bead sizes were measured by a caliper. Different enzyme loading in the beads was investigated by adding varying amounts of enzyme (1000, 2000, 3000, 4000, 5732 units) to aqueous sodium alginate 2% (w/v) solution.To prevent the enzyme from leaking out of the gel beads, the beads were coated with silica (0.5, 1.0, 2.0% w/v). Reusability of the Immobilized α-amylase

To test the stability of the amylase entrapped in the Ca-alginate beads, the beads were used several times for the hydrolysis reaction. Each run lasted for 10 min, after which the beads were separated, washed with distilled water and stored at 4 oC until the next use. The reaction medium was then replaced with fresh medium. The activity of beads in the first run was defined as 100%. Starch Hydrolysis Using the Free and the Immobilized α-amylase

The activities of the free and the immobilized enzymes were tested in the presence of various starch sources such as a soluble starch (potato) and commercial starches (potato, corn, rice, wheat). Each the free and the immobilized enzyme were mixed with buffered solution containing 1% of the various starches. The reaction mixture was incubated at 37 oC for 10 min, and α-amylase activity was assayed by the starch-iodine method. Soluble potato was purchased from Merck. Commercial raw starches were obtained from the local markets. Properties of the Free and the Immobilized α-amylase

The influence of temperature and pH on the activities of both the free and the immobilized amylases was determined by carrying out the reaction at varying temperatures ranging from 37 to 80 oC and at varying pH ranging from 4.0 to 8.0.

To study the effect of metal ions on enzyme activities, the free and the immobilized amylases were incubated with metal salts at a concentration of 5 mM. Relative activities were expressed as a percentage of untreated control that is taken as 100%. Hydrolysis Products of Starches

The profile of products formed from various starches hydrolyzed by the immobilized and the free α-amylase was determined at 5 h of reaction time. Products of hydrolysis of starches with α-amylases were visualized on silica gel plates (0.5 mm thick and 20 cm x 20 cm size) by thin layer chromatograhy (TLC). Results and Discussion

Alginate as an immobilization medium has been extensively studied mainly because of its ease of formulation, mild gelation conditions, biocompatibility, acceptability as a food additive [19, 20]. It has been reported that alginate and CaCl2 concentrations used in enzyme and cell entrapment range from 2-4% (w/v) and 2-5% (w/v), respectively [21, 22]. Therefore, to determine the optimum alginate and CaCl2 concentration for enzyme immobilization, various amounts of sodium alginate (1-4%, w/v) and CaCl2 (2-6%, w/v) were used. Maximum amylase activity was obtained with an alginate concentration of 2% (w/v) and CaCl2 concentration of 5% (w/v) (Fig. 1), and as a result of these, the immobilization efficiency of



α-amylase was approximately 89%. At high alginate concentrations, the beads were more permeable than those formed at low alginate concentrations.

Figure. 1. Effect of alginate and CaCI2 concentration on immobilization. In different concentratrions of Sodium alginate and CaCl2 on the bead permeability and the bead rigidity were studied. Sodium alginate concentration was between 1-5% w/v and, CaCl2 concentration was between 2-6% w/v. Each treatment was performed in triplicate.

The more permeable beads showed activity losses due to a rapid leakage of the enzyme from the beads. It has been reported that capsules formed from sodium alginate solutions lower than 0.5% (w/v) had gelled poorly, produced sticky surfaces and were fragile and difficult to handle [21]. A 4% (w/v) solution of sodium alginate provided the highest immobilization yield (75%) of B. circulans α-amylase [11]. Amylase activity increased in the high concentration of alginate (3% w/v) [23]. The highest immobilization yield (60.3%) was obtained with a sodium alginate solution of 2% (w/v) [24]. In physical adsorption, the binding forces between the enzyme and the matrix are weak in comparison with covalent or ionic binding [25].The size of the beads in which amylase is entrapped may be one of the most important parameters of amylase immobilization. In this study, alginate beads were generated reproducibly by changing the size of a needle (0.45, 0.8, 2, and 3 mm diameter) through which a mixture of amylase and alginate was dropped into a CaCl2 solution. The measured bead sizes were approximately 2, 3, 3.5, and 4 mm. As the bead size increased, activity of amylase entrapped in the beads decreased. Entrapped enzyme in beads of 3 mm diameter had the maximum activity (Fig. 2). Others have also reported that the activity of immobilized enzymes had decreased with increasing bead size due to mass transfer resistances [26, 27]. With α-amylase immobilized in 5 mm beads, the level of starch hydrolysis was only 32% [15]. The effect of bead amount on the enzymatic activity was investigated, and maximum activity was realized with 0.4 g of beads (Fig. 2).



Figure. 2. Effects of bead size and bead amount on enzyme activity. Beads containing crude enzyme were prepared in the presence of sodium alginate (2% w/v) in CaCl2 (5% w/v) solution. Each treatment was performed in triplicate.

To investigate the effects of loading of α-amylase to alginate, different concentrations

of immobilized α-amylase (1000, 2000, 3000, 4000, 5732 units) were tested. The result showed that loading efficiency was affected by increasing the loading amount (Fig. 3). The maximum activity was found at a loading enzyme concentration of 5732 U/ml, and this result may depend on the increased enzyme concentration. These results are similar to those reported for α-amylase produced by Aspergillus sclerotiorum [16], and Bacillus subtilis [21]. Alginate gels have a wide pore size distribution due to the open lattice structure of the matrix. It has been known that the net pores of Ca-alginate gel beads are so large that enzymes can sometimes leak out from the gel [28]. To overcome the leakage of entrapped enzyme, coating of the alginate gel surface with chitosan or silicate has been attempted [29-31]. Hence, α-amylase was immobilized in silica-alginate composites. Different concentrations of silica were added to alginate solution before gelation. The maximum enzyme activity was in the presence of 0.5% silica. When the beads with a 0.5, 1, and 2% (w/v) silica coating level were treated with amylase, the residual activities were 90, 86, 73%, respectively, It has also been reported that the presence of silica gel at concentrations up to 1% (w/v) in the sodium alginate solution do not significantly affect the extent of starch hydrolysis. Further increasing of the silica gel concentration to 4% (w/v) resulted in a 40% decrease in the yield of reducing sugars [21]. Other literature reported the decrease in the specific activity after immobilization of α-amylase using mesoporous silica that retained 80% of the specific activity of free enzyme [1].



0

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Figure. 3. Effect of amount of loading enzyme on enzyme activity. Beads containing crude enzyme were prepared in the presence of sodium alginate (2% w/v) in CaCl2 (5% w/v) solution. Each treatment was performed in triplicate.

The operational stability of the immobilized enzyme is one of the most important

factors affecting the utilization of an immobilized enzyme system. Immobilized beads prepared from 2% (w/v) alginate and 5% (w/v) CaCl2 were suitable for up to 7 repeated uses, and 38% of the initial activity was retained. The highest operational stability was 6 reuses with 51% residual activity (Fig. 4). In this case, the activity decreased and could have been due to enzyme denaturation and physical loss of enzyme from the carriers because beads were overused.

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Figure. 4. Reusability of immobilized α-amylase enzyme. The repeated use of the amylase entrapped in the Ca-alginate beads was tested as several times. Each treatment was performed in triplicate.

In contrast, some researchers have reported different results. Capsules prepared from

2% (w/v) sodium alginate and 5% (w/v) CaCl2 were suitable for up to 20 repeated uses, losing only 30% of their initial efficiency [21]. B.circulans α-amylase immobilized by entrapment in calcium alginate beads retained 83% of the initial activity after 7 cycles [11]. The immobilized α-amylase on a cation exchange resin and glass beads retained 70 and 73.4% from the initial activity after up to 6 cycles [24].



Starches from soluble potato and commercial potato, rice, wheat and corn were hydrolyzed with the free and the immobilized α-amylase. Figure 5 compares the hydrolysis efficiency of the free and the immobilized enzyme on soluble and commercial (com.) starches. The immobilized enzyme was more efficient in the hydrolysis of starches than the free enzyme. Starch hydrolysis by the immobilized amylase was ranked as com.potato > soluble potato> com.corn > com.wheat > com.rice. For the free enzyme, the rank order was soluble potato > com.corn > com.potato > com.wheat. No hydrolysis via the free enzyme in the presence of com.rice was determined, but com.rice was hydrolyzed by the immobilized enzyme with a ratio of 50%. The degree of hydrolysis for the immobilized enzyme was 20-50% more than with the free enzyme. It has been reported that 90% hydrolysis was obtained for starches from potato, rice and wheat, whereas soluble starch and corn starch showed 69% and 64% hydrolysis, respectively [15]. On the other hand, α-amylase of B. amyloliquefaciens was effective on potato, corn, wheat and rice starches. The free enzyme exhibited 85% hydrolysis of raw potato starch [32].

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Figure. 5. Hydrolysis efficiency of the free and the immobilized enzyme on soluble andcommercial (com.) starches. The abilities of hydrolysis of the free and immobilized α-amylase enzyme in the presence of soluble potato and commercial potato, corn, wheat, rice of 1% w/v were investigated. Enzyme and starch solustions were incubated at 37 oC for 10 min. Each treatment was performed in triplicate.

It has been reported that the maximum hydrolysis of potato starch by immobilized α-

amylase (as compared to hydrolysis of rice and corn starch and to the lower rates of hydrolysis) was attributed to the interference of the gel matrix of capsules in the diffusion of starch molecules to the enzymatically active core [21]. Enzymes that are capable of digesting raw potato starch are economically attractive because they can increase the range of starch sources useful in direct hydrolysis [2]. In this study, the immobilized α-amylase enzyme was found to be highly effective on commercial potato starch. The mechanism of adsorption on starch granules of enzymes is still unclear, but some researchers perhaps explain it through a C-terminal binding domain [33, 34]. It has been reported that the number and size of granules, and the starch structure were significant for enzyme attack [35].

To characterize the free and the immobilized enzyme, the effects of temperature and pH were studied. The optimum temperature and pH were found to be 55 oC and 6.4 for the



free enzymes, whereas the optima for the immobilized enzyme was 60 oC and 6.8, respectively (Fig. 6, Fig. 7). The immobilized amylase activity was higher than the free enzyme at these temperatures and pH conditions. The immobilized enzyme activities were higher than the free one over a range of different pH conditions. The free enzyme is rapidly inactivated at an alkaline pH, whereas the immobilized enzyme retained about 51% activity at pH 8.0. This showed that the immobilization matrix might be able to protect the enzyme against denaturation at high temperature and pH.

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Figure. 6. Effect of temperature on the free and the immobilized α-amylase activity. The optimum temperature of the free and the immobilized amylase was determined by using different temperatures ranging from 37 to 80 oC. Each treatment was performed in triplicate.

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Figure. 7. Effect of pH on enzyme activity of the free and the immobilized α-amylase. The optimum pH of the free and the immobilized amylase was determined by using different pH ranging from 4.0 to 8.0. Each treatment was performed in triplicate.

On the other hand, the immobilized α-amylase studies in the literature are mostly low

temperature applications (e.g. below 50 oC) [36, 37]. The effect of the metal salts on the



activity of the free and the immobilized amylase was investigated (Table 1). Among the various metal ions studied, apparent activity of the immobilized enzyme was increased in the presence of Ba2+, whereas the activation by metal ions (Ca2+, Mg2+ and Ba2+) became more pronounced with the free enzyme. The metal Cu2+ caused inhibition on the activity of the free enzyme, but the immobilized enzyme showed a little activity. It has been reported that immobilization protected the enzyme from the inhibition by some metal ions (Co2+, Cu2+ and Fe3+) [38]. Table 1. Effect of metal salts on residual activity (%) of the free and the immobilized α-amylase. Values are shown as means of triplicates. Free α-amylase immobilized α-amylase None 100 100 CaCI2 115 84 MgSO4 145 84 MnCI2 98 72 BaCI2 110 125 CuCI2 0.0 16 LiSO4 98 76

To determine hydrolysis products from various starches by the immobilized and the free α- amylase after 5 h of reaction, product distibutions were analysed by thin layer chromatography (TLC). With the free amylase, the end product profiles of starch hydrolysis was determined as the formation of glucose, maltose, and maltotriose (G1-G3) after 5 h reaction (Fig. 8). TLC analysis of starch hydrolysis products by the immobilized amylase after 5 h showed the formation of glucose, maltose, maltotriose, and maltotetrose, maltopentose and maltohexose (G1-G6) as seen in Fig. 9.

Figure. 8. Thin layer chromatographic analysis of the main products from the hdyrolysis of starches by the free enzyme at 5 h. 1-Standarts (Glucose and Maltose), 2-Soluble Potato, 3-Com. Potato, 4-Com. Corn, 5-Com. Wheat, 6-Com. Rice. Glucose (G1), Maltose (G2) and Maltotriose (G3).



Figure. 9. Thin layer chromatographic analysis of the main products from the hdyrolysis of starches by the immobilized enzyme at 5 h. 1-Standarts (Glucose and Maltose), 2-Soluble Potato, 3-Com. Potato, 4-Com. Corn, 5-Com. Wheat, 6-Com. Rice. Glucose (G1), Maltose (G2), Maltotriose (G3), Maltotetrose (G4), Maltopentose (G5) and Maltohexose (G6).

The main hydrolysis product of various starches by both amylases were maltose as major product. The immobilized enzyme showed broader product distribution. The products formed by the free enzyme and the immobilized enzyme were obtained as G2>G3>G1 and G2>G3>G5>G6>G4>G1, respectively, after 5 h. Conclusions

B.amyloliquefaciens α-amylase was succesfully immobilized in calcium alginate

beads. The optimum conditions selected for effective α-amylase encapsulation were 2% (w/v) sodium alginate, 5% (w/v) CaCl2 and loading enzyme concentration 5732 U/ml. Optimum bead size and bead amount were 3 mm and 0.4 g, respectively, for maximum α-amylase activity. The formation of alginate/silica capsules was proposed in this work. When coated with silicate, amylase-entrapped beads retained the highest catalytic activity. Immobilized α-amylase was also used in the hydrolysis of various starchy substrates and compared with free enzyme. Soluble and commercial potato starches were hydrolyzed at a greater extent than the other starches tested. The enzyme entrapped in alginate beads could be reused and retained 51% activity at the end of six cycles. Compared to the free enzyme, the immobilized enzymes exhibited much more activity in the high temperature and pH range. The free enzyme is rapidly inactivated at an alkaline pH, whereas the immobilized enzyme retained about 51% activity at pH 8.0. A widely product distibutions were obtained with the immobilized enzyme. TLC results showed that immobilization of α-amylase is very useful for starch hdyolysis and economically produce malto-oligosaccharides. All these criteria can be successfully utilized in practical application and immobilized α-amylase can be used more successfully than free α-amylase in the starch hyrdolysis process



Acknowledgment

This research was funded by Uludag University Commission of Scientific Research Projects, Grant F-2008/24, which is highly appreciated.

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Immobilization of B. amyloliquefaciens α-amylase and ...

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