Molecular cloning, overexpression and

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of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens. Dhanya ... Key words: Bacillus amyloliquefaciens; α-amylase; cloning; overexpression; raw starch digestion ...... A Laboratory Manual, 2nd Edn. Cold Spring Harbor, Cold.
Biologia 65/3: 392—398, 2010 Section Cellular and Molecular Biology DOI: 10.2478/s11756-010-0042-6

Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens Dhanya Gangadharan1, Priya Ramachandran2, Gunasekaran Paramasamy2, Ashok Pandey1 & K. Madhavan Nampoothiri1* Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR, Trivandrum – 695 019, Kerala, India; e-mail: [email protected]; m.nampoothiri@rediffmail.com 2 Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai – 625 021, India 1

Abstract: Raw starch is the most abundant source of glucose in the world. Therefore, finding enzymes capable of digesting raw starch would find high industrial demand. The α-amylase gene of Bacillus amyloliquefaciens ATCC 23842 was amplified, cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme was purified to apparent homogeneity using ion exchange and gel filtration chromatography. The raw-starch digestibility of the purified enzyme was characterized by studying the hydrolysis and adsorption rate on a variety of raw starches (potato, cassava, corn, wheat and rice). The raw-starch digestion was further confirmed by scanning electron microscopy studies, which revealed an effective rate of hydrolysis. The kinetic studies revealed a relatively low Km of 2.76 mg/mL, exhibiting high affinity towards the soluble starch as the most preferred substrate and the inhibition kinetic studies revealed a high Ki value (350 mM). Key words: Bacillus amyloliquefaciens; α-amylase; cloning; overexpression; raw starch digestion Abbreviations: CBM, carbohydrate-binding module; IPTG, isopropyl β-D-thiogalactopyranoside; LB, Luria Bertani; SBD, starch-binding module; SEM, scanning electron microscopy.

Introduction Amylases constitute a class of industrial enzymes representing approximately 30% of the world enzyme production (Van der Maarel et al. 2002). α-Amylases (EC 3.2.1.1) hydrolyse α-1,4 bonds in starch components and related carbohydrates and bypass α-1,6 linkages in amylopectin and glycogen. Microbial amylases are preferred for their stability over plant and animal enzymes thus increasing their spectrum of industrial applications. Moreover, microbial enzymes in general have the advantages of cost effectiveness, consistency, less-time and easy-process optimization. The amylolytic enzymes find a wide spectrum of applications in food industry, such as production of glucose syrups, crystalline glucose, high fructose corn syrups, maltose syrups, reduction of viscosity of sugar syrups, reduction of haze formation in juices, solubilisation and saccharification of starch for alcohol fermentation in brewing industries, retardation of staling in baking industry, etc. Other applications include their use as additive to remove starchbased dirts in detergent industry, for the reduction of viscosity of starch for appropriate coating of paper in paper industry and also for warp sizing of textile fibres. In pharmaceutical industry they are used as a digestive aid (Sivaramakrishnan et al. 2006). Starch represents one of the most abundant storage

polysaccharides in nature and the most popular ingredient in food. Generally amylases are not effective on raw-starch granules because granules are very resistant to amylolytic digestion (Hyun & Zekius 1985). Extracellular hydrolytic enzymes from microbes that catalyses the degradation of starch granules or plant cell walls typically possess a modular architecture, and very often contain non-catalytic ancillary domains referred to as carbohydrate-binding modules (CBMs). CBMs with affinity for starch are commonly referred to as starchbinding domains (SBDs). Currently, nine CBM families, 20, 21, 25, 26, 34, 41, 45, 48, and 53, have been reported to contain SBDs (Christiansen et al. 2009). The adsorption of amylolytic enzymes to raw starch and their hydrolysis is correlated with the presence of the SBD (Mitsuiki et al. 2005). SBD is a functional domain usually composed of about one hundred amino acid residues, which adopt an open-sided, distorted βbarrel structure consisting of several β-strand segments (Penninga et al. 1996; Sorimachi et al. 1997; Mikami et al. 1999). SBD can bind to granular starch increasing the concentration of substrate at the enzyme active site and also can disrupt the structure of starch surface thereby enhancing the amylolytic rate (Sorimachi et al. 1997; Southall et al. 1999). SBDs are present in about 10% of amylases and their related enzymes. This domain is usually localized at the C-terminal end

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α-Amylase gene cloning for raw-starch digestion

of enzymes (Svensson et al. 1989). A few exceptions are the Rhizopus oryzae glucoamylase (Ashikari et al. 1986; Takahashi et al. 1985), the Thermoactinomyces vulgaris “α-amylase” (Abe et al. 2004) and the Thermotoga maritima pullulanase (Bibel et al. 1998), which present their SBDs at the N-terminus. Production of α-amylase byB. amyloliquefaciens ATCC 23842 through solid state and submerged fermentations has been reported previously by our group (Gangadharan et al. 2006, 2008). Biochemical characterization of the enzyme purified from the parent strain had proven its efficacy of raw-starch hydrolysis (Gangadharan et al. 2009). However, the present work describes the amplification of the α-amylase gene of the parent Bacillus strain and its cloning and overexpression in Escherichia coli. The ability to digest raw starch of the recombinant enzyme and its kinetic properties were studied. Material and methods Bacterial strains, chemicals and reagents The genomic DNA of Bacillus amyloliquefaciens ATCC 23842, Campbell strain T, CCTM La 2872, CECT, 483, LMG 12331, Logan B0173, Logan B0256, NCIMB 10785, NRRL B-14394, strain t, T ATCC23842 was isolated using the standard protocol described by Sambrook et al. (1989). Escherichia coli strains, DH5α T1 (Invitrogen, CA, USA) and BL21 (DE3) (Novagen, Inc., CA, USA) were used for transformation and overexpression studies, respectively. The strains were grown on Luria Bertani (LB) agar (Himedia, Mumbai, India) slants at 37 ◦C for 24 h and subcultured every two weeks. The expression vector, pET20b (Invitrogen, CA, USA) was used for cloning the amylase gene. The QIAprep Miniprep kit and QIAquick gel extraction kits were obtained from Qiagen (Germany). Restriction enzymes, T4 DNA ligase and DNA polymerase were procured from New England Biolabs and MBI Fermentas (both USA). The primers were procured from Microsynth (Switzerland). All other molecular biology grade chemicals used in the present study were procured from Sigma, Merck (USA) or Hi-media (India). Gene amplification and cloning All DNA manipulations were performed using standard protocols as described by Sambrook et al. (1989). The gene specific primers Ba sig F: 5’-CCGCATATGGTCGACATGATTCAAAAACGAAAG-3’ (which contained SalI site) and Ba Xho R: 5’-GCCCTCGAGTTATTTCTGAACATAAATGGAGAC-3’ (having XhoI site) for the amplification of the α-amylase gene from its genomic DNA by PCR were designed based on the nucleotide sequence of B. amyloliquefaciens (GenBank accession No.: J04152) available from GenBank (Benson et al. 2010). The PCR (Eppendorf, India) amplification was conducted in a 100 µL volume containing 2 mM MgCl2 , 200 mM of dNTP, 1 µL of the genomic DNA extracted from B. amyloliquefaciens, 1.0 mM each of the species-specific primers, 2.5 U of Taq DNA polymerase and the supplied buffer. The amplification program consisted of one cycle at 94 ◦C for 5 min, then 30 cycles at 94 ◦C for 40 s, 55 ◦C for 40 s, and 72 ◦C for 2 min and, finally, one cycle at 72 ◦C for 10 min. The amplified PCR product was eluted from

393 the gel and purified. The amplicon and the pET20b vector were digested with SalI/XhoI enzymes and then ligated as per the manufacturer’s instructions. The ligated mixture was transformed into E. coli BL21 (DE3). The recombinant plasmid carrying the α-amylase gene was designated as pET 20b::amy and used for sequence verification of the α-amylase gene. The α-amylase gene sequence obtained was deposited with GenBank (Benson et al. 2010). The protein sequence was deduced and analysed for the presence of catalytic triad and substrate-binding as well as metal-binding sites. Overexpression of recombinant amylase One % v/v of overnight grown E. coli recombinant cells were grown in LB broth containing 50 µg/mL of ampicillin at 37 ◦C until the OD reaches 0.6–0.7 at 600 nm. The expression of α-amylase was achieved by the addition of isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, and incubated for 3 h at 37 ◦C at 200 rpm. The culture was centrifuged at 5,009×g at 4 ◦C and the pellet was re-suspended in 2 mL of lysis buffer (PBS pH 7.5 with 1 mM PMSF and lysozyme 1 mg/mL) and kept in ice for 30 min. The lysozyme-treated cell suspension was then sonicated (Sonics, USA, Amplitude 47% and 30 s ON and OFF cycle for 2 min). The cell debris was centrifuged at 11,270×g for 20 min at 4 ◦C. The overexpression of αamylase in the soluble and pellet fractions was checked on a 12% SDS PAGE. Enzyme assays α-Amylase activity was determined by the method of Okolo et al. (1995). The reaction mixture contains 1.25 mL of 1% soluble starch (Hi-media, India), 0.5 mL 0.1 M acetate buffer (pH 5.0), and 0.25 mL of crude enzyme extract. After 10 min of incubation at 50 ◦C, the liberated reducing sugars (glucose equivalents) were estimated by the dinitrosalicylic acid method (Miller 1959). The colour developed was read at 575 nm using a Shimadzu UV-160A spectrophotometer. Glucose was used as the standard. The blank contained 0.75 mL of 0.1 M acetate buffer (pH 5.0) and 1.25 mL 1% starch solution. One unit (1 U) of the α-amylase activity is defined as the amount of enzyme releasing one µmol of glucose equivalent per min under the assay conditions. Total soluble proteins in the sample were estimated with crystalline serum albumin (Sigma) as per the standard protocol (Lowry et al. 1959). Purification and characterization of the raw-starch digestibility of the overexpressed α-amylase Although a C-terminal His-tag was present in pET20b, conventional methods were used for purification. The reason was that presence of the C-terminal His-tag had resulted in a considerable reduction of certain enzyme activities by other groups (unpublished data). To avoid this, a stop codon was introduced preceding the His-tag and conventional methods were used for purification. Gel filtration and ion exchange chromatography The soluble fraction of the overexpressed protein (25 mg) was loaded onto Q-Sepharose Fast Flow (Pharmacia) chromatographic column (1.6 cm × 20 cm) pre-equilibrated with 0.5 M Tris HCl buffer pH 9.0 at 4 ◦C and atmospheric pressure. Stepwise elution was performed (0.1–1.0 M NaCl in 50 mM Tris HCl buffer, pH 9.0) with flow rate of 1.5 mL/min. Two mL fractions were collected and the elution profile was determined by checking the absorbance at 280 nm. The αamylase active fractions were dialysed against 20 mM acetate buffer pH 5.0 and were loaded onto Sephadex G200

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gel filtration column pre-equilibrated with 50 mM sodium acetate buffer pH 5.0. The protein was eluted with 20 mM sodium acetate buffer (pH 5.0) at a flow rate of 0.5 mL/min. The fractions having the α-amylase activity were collected and 12% SDS-PAGE were performed to check the homogeneity of the purified enzyme and determine its molecular weight. The proteins were stained with Coomasie brilliant blue R-250. Raw-starch hydrolysis and adsorption rate The efficiency of the enzyme to digest various substrates (raw starches such as potato, cassava, corn, wheat and rice) was studied by incubating 100 U of the purified enzyme with 1% (w/v) of each substrate for 3 h. The amount of reducing sugars released in each case was estimated. The adsorption rate on different starches was determined. A protein concentration of approximately 200 µg/mL in 50 mM sodium acetate buffer (pH 6.0) was mixed with 100 mg of starch to a final volume of 1 mL. The resulting samples were mixed well at 4 ◦C for 1 h and then centrifuged to sediment the insoluble substrate. The residual protein in the supernatants was measured. The adsorption rate (AR) was defined by the following equation: AR (%) = [(B − A)/B] × 100, where A indicates the residual protein after adsorption and B represents the protein concentration in the original enzyme solution. Scanning electron microscopy (SEM) for raw-starch hydrolysis Raw starches (1% w/v) were hydrolysed by incubating with 100 U of the purified enzyme for 12 h. For electron microscopic studies of raw-starch degradation, the starchy pellets were washed twice with pure ethanol and centrifuged at 12,000×g for 10 min. These pellets were washed twice with t-butyl alcohol, centrifuged again, and lyophilized. The dried starch granules were mounted and examined by SEM (JEOL JSM 5600LV, 115 Japan) (Lo et al. 2002). Kinetic studies To understand the basic kinetics of the enzyme, various concentrations of soluble starch (Hi-media, India) (0.2– 2.0%, w/v) were used. The kinetic constants Km and Vmax were determined by Lineweaver-Burk method (Lineweaver & Burk 1934) which was analyzed by Hyper 32 software (Department of Biochemistry, University of Liverpool, Liverpool, UK). The end production inhibition of the purified enzyme was studied in the presence of maltose (200–800 mmol) at various substrate (soluble starch) concentrations (1–2.5 mL/min). The Dixon plot was used to evaluate the inhibitor constant (Ki ) of the purified α-amylase.

Results Cloning and over expression of the α-amylase of B. amyloliquefaciens in E. coli BL21 (DE3) The amplification of the α-amylase gene of B. amyloliquefaciens by PCR resulted in a 1.5 kb DNA fragment. The α-amylase gene was sequenced and the data deposited with GenBank (Benson et al. 2010) with the accession No. GU591658. The sequence analysis revealed 100% identity with B. amyloliquefaciens strain IH αamylase gene (Takkinen et al. 1983). The coding sequence of the α-amylase gene consisted of 1,542 bp, with an ATG initiation codon and a TAA stop codon. The protein sequence was deduced and the BLAST

resulted in highest similarity to the α-amylase of B. amyloliquefaciens (SwissProt: P00692), which encoded a protein precursor of 514 amino acids comprising a putative 29–residue long signal peptide. The conserved domain search revealed that the residues 36–405 exhibited high similarity towards the catalytic domains of the α-amylases from B. amyloliquefaciens (Takkinen et al. 1983), Bacillus stearothermophilus (Dauter et al. 1999), B. licheniformis (Yuuki et al. 1985) and Aeromonas hydrophila (Gobius & Pemberton 1988). The pair-wise amino acid sequence alignment with the α-amylase from B. amyloliquefaciens revealed the presence of four Ca2+ -binding sites: Asp231, Gly331, Asp438 and Asp461 among the 11 Ca2+ -binding sites reported by Takkinen et al. (1983). PCR product was cloned into pET20b vector and transformed into E. coli BL21 (DE3) cells for overexpression. Induction with 1 mM IPTG followed by cell disruption resulted in the accumulation of majority of the recombinant protein in the soluble fraction. The overexpression of the α-amylase gene in E. coli resulted in 1,850 U/mL of activity after 3 h induction with 1 mM IPTG, which was 1.7 fold higher compared to the maximum activity obtained with the parent Bacillus culture after 42 h of fermentation (Gangadharan et al. 2009). The molecular weight of the overexpressed protein was determined and found to be 58 kDa, which is well in accordance with the molecular weight of the purified α-amylase of the parent Bacillus strain (Gangadharan et al. 2009). Purification and characterization of the raw-starch digestion ability of the overexpressed α-amylase Ion exchange and gel filtration chromatography. The soluble fraction of the overexpressed protein was loaded onto the Q Sepharose column. The α-amylase active fractions were found between 28–45 eluted between 0.2– 0.4 M NaCl in 50 mM Tris HCl buffer which were dialysed against 50 mM acetate buffer pH 5.0 and subjected to gel filtration chromatography. The active fractions from the Sephadex G200 column were pooled and the homogeneity of the purified enzyme was checked by running SDS-PAGE. The apparent molecular weight of the purified enzyme was found to be 58 kDa. The purification steps had resulted in 43% recovery of the protein and the specific activity of the purified enzyme had increased by 1.74 fold (Table 1). Raw-starch hydrolysis and adsorption rate. The raw-starch hydrolysis and adsorption rate were found to be highest for potato starch followed by cassava, corn, wheat and rice. The hydrolytic activity of the purified enzyme on potato, cassava, corn, wheat and rice were 95, 73, 65, 55 and 45% respectively. The adsorption of the α-amylase on the raw starches was compared with soluble starch. Maximum adsorption was exhibited by soluble starch and the adsorption rate among raw starches followed the same pattern as for their hydrolytic activity (Fig. 1). SEM studies for raw-starch degradation. The rawstarch degradation was also analysed by SEM. The

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α-Amylase gene cloning for raw-starch digestion

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Table 1. Summary of purification steps of B. amyloliquefaciens α-amylase overexpressed in E. coli BL21 (DE3). Purification steps Soluble fraction (overexpressed protein) Q-Sepharose column Sephadex G200 column

Total protein (mg) Total activity (U/mL) Specific activity Yield (%) Purification fold 25 14.5 6.2

1850 1241 796

74 85.5 128.3

100 67.08 43.02

1.0 1.73 4.03

Fig. 1. Adsorption rate of the purified α-amylase of B. amyloliquefaciens overexpressed in E. coli on soluble and raw starches at 4 ◦C for 1 h.

non-hydrolysed starch granules were characterized by smooth surface (Fig. 2a, c, e, g, i) and the hydrolysed starches revealed deep holes and the magnitude of the holes could be directly correlated to the rate of hydrolysis (Fig. 2b, d, f, h, j). Kinetic studies. The enzymes showed MichealisMenten kinetics while hydrolyzing starch. The Km (2.76 mg/mL) and Vmax (4.76 mg/mLmin) values were derived from the Lineweaver-Burk plot (Fig. 3), which was highly comparable with the parent enzyme. The TLC of the products of hydrolysis of soluble starch revealed the formation of matooligosaccharides as reaction products (Gangadharan et al. 2009). An enzyme tolerant to reaction end-product would be of high value for industrial applications. Thus an inhibition kinetic study of the α-amylase was performed in the presence of maltose to study the end-product inhibition levels. The Ki 350 mM of the purified α-amylase with maltose was evaluated from Dixon plot as quite high (Fig. 4). Discussion Cloning the genes is mostly done for the hyperproduction of proteins with high specific activity that facilitates their easier purification. In the present study the raw-starch-digesting α-amylase gene of B. amyloliquefaciens has been successfully cloned and overexpressed in E. coli cells. Raw starch represents the most abundant storage polysaccharide in nature. There was a previous report on cloning and sequencing of raw-starch-digesting amylase from a Cytophaga sp. (Jeang et al. 2002), which showed high amino acid identity with the α-amylases from three Bacillus sp. (Southgate et al. 1993). Similarly, Demirkan et al. (2005) reported the cloning and expression of a B. amyloliquefaciens α-amylase gene, which was capable of raw-starch digestion. The complete nucleotide

Fig. 2. Scanning electron microscopic studies of raw-starch hydrolysis by the purified α-amylase of B. amyloliquefaciens overexpressed in E. coli. (a), (c), (e), (g) and (i) non-hydrolysed potato, cassava, corn, wheat and rice starch, respectively; (b), (d), (f), (h) and (j) – hydrolysed (by 100 U purified α-amylase for 12 h) potato, cassava, corn, wheat and rice starch, respectively.

sequence of the α-amylase gene from B. amyloliquefaciens, including the 5’- and 3’-flanking regions, was 1,542 nucleotides long and coded for 514 amino acid residues, giving a molecular mass of 58,427 daltons (Takkinen et al. 1983). Borgia & Campbell (1978) have reported that α-amylases of five strains of B. amyloliquefaciens were identical in pri-

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Fig. 3. Kinetic studies of the purified α-amylase of B. amyloliquefaciens overexpressed in E. coli (Lineweaver-Burk plot).

Fig. 4. Dixon plot for the inhibition kinetic study of purified αamylase for maltose. [S1 ] – 1.0 mg/mL (), [S2 ] – 1.25 mg/mL (•), [S3 ] – 2.5 mg/mL ( ) of soluble starch concentration.

mary structure or exhibited only conservative modifications. In the present study, the induction of the recombinant BL21 E. coli (DE3) cells resulted in 1.7 fold overexpression in the soluble fraction after cell disruption in comparison to the parent strain. Previously, the molecular weight of the α-amylase from B. amyloliquefaciens has been estimated to be between 40–60 kDa as determined by SDS-PAGE, sedimentation properties, gel filtration, amino acid composition analysis and cyanogen bromide fragment analysis (Detera & Friedberg 1979; Chung & Friedberg 1980; Palva et al. 1981). In the present case the overexpressed enzyme was purified and the molecular weight was determined to be 58 kDa. Hamilton et al. (1998) reported a higher molecular weight (63 kDa) for the Bacillus sp. IMD435 αamylase capable of raw-starch digestion, but without raw-starch-adsorbing ability. The behaviour of the overexpressed enzyme at different pH and temperature was well in accordance with

the parent enzyme (Gangadharan et al. 2009). Ca2+ ions were found to play an important role in maintaining the correct conformation, thermostability and activity of α-amylases (Janecek 1997). Liu et al. (2010) investigated the role of Ca2+ -binding residues Asp231, Asp233 and Asp438 of B. amyloliquefaciens α-amylase on the enzyme properties by site-directed mutagenesis, and they observed no significant changes in Asp231 and Asp438 mutations, while Asp233 mutation resulted in a dramatic reduction of catalytic efficiency and thermostability at 60 ◦C. The present study also showed an increase in thermostability at 60 ◦C in the presence of Ca2+ ions (data not shown). The study on raw-starch hydrolysis revealed high hydrolyzing rate in the case of tuber starches in comparison with cereal starches. It is well-known that there is high starch content (60%) in corn and wheat, while potato and cassava contains only 20–30% starch content. Corn and wheat starch are also characterized by low moisture and higher lipid content than potato and cassava starch, which could be factors contributing towards the easy accessibility and higher rate for hydrolysis in the case of potato and cassava starch. The amylose content of various starches were also studied and found that tuber starches, such as potato and cassava has lower amylose content than cereal starches. Studies have also shown that high amylose starches were highly resistant to enzymatic hydrolysis (MacGregor et al. 2002). Yetti et al. (2007) reported that the use of combined treatment with acid and heating below gelatinization temperature of sago starch improved the accessibility to enzyme attack by enhancing its hydrolysability due to formation of pores on the starch granule’s surface. The α-amylase of Bacillus sp. IMD434 displayed hydrolysis up to 32, 28 and 18% of raw corn, rice and wheat starch, respectively, after 24 h, while potato starch exhibited only 10% hydrolysis but no adsorption to the starches were examined (Kelly et al. 1995). High cost for starch-based products may be attributed to the high energy input required for the gelatinization of starch. Thus, the raw-starch-hydrolyzing enzymes have gained much importance for effective utilization of natural resources since they also overcome viscosity problems (Jeang et al. 2002). The experiments in the present study proved a strong correlation between the adsorption and hydrolysis rates. Hamilton et al. (1998) have classified bacterial α-amylases into 2 groups: (i) raw-starch-hydrolyzing and adsorbing α-amylases; and (ii) raw-starch-hydrolyzing but nonadsorbing α-amylases. Hayashida et al. (1990) reported the hydrolysis of raw starch by the α-amylase of Bacillus subtilis 65 although it showed no adsorption onto raw starch. Similar reports of amylolytic enzymes that which are capable of adsorbing or hydrolyzing raw starch have been reported (Itkor et al. 1989; Iefuji et al. 1996). Most of them possess the SBD at their Cterminal region (Svensson et al. 1989). The raw-starch hydrolysis was also analysed by SEM studies and the efficient hydrolysis was proven by the formation of deep holes. Similar studies with

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α-Amylase

gene cloning for raw-starch digestion

raw corn starch treated for 3 h with the α-amylase of Bacillus sp. strain TS-23 showed initial granular pitting and treatment for 12 h resulted in very large deep holes, while the surfaces of the non-hydrolysed starch granules were smooth (Lo et al. 2002). Vidilaseris et al (2009) reported that the raw-starch-degrading α-amylase from marine Bacillus sp. ALSHL3 produced large and deep holes on the surface of rice starch granules compared to starch from maize, whereas no holes were observed on the surface of cassava starch granules. The SEM studies on raw-starch digestion by glucoamylase from Cladosporium gossypiicola revealed the presence of small pits or pinholes on the peripheral region of the corn starch granules in the early stage of hydrolysis and these penetrated deep into the granule forming deep holes (Quigley et al. 1998). The efficiency of an enzyme towards a substrate can be explained by its kinetic properties. Low values of Km indicate high affinity of the enzyme for the substrate (van der Maarel et al. 2002). The previous kinetic studies of the α-amylase of B. amyloliquefaciens cloned into E. coli indicated a Km of 1.92 mg/mL and Vmax of 351 U/mLmin (Demirkan et al. 2005). The application of an enzyme for prolonged period demands tolerance to the reaction-end products. In the present study, a high value of Ki (350 mM) indicates the high tolerance of our α-amylase to reaction-end products and thus increases its industrial demand. The α-amylase of an ascomycete Fusicoccum sp. BCC 4124 was found to be tolerant up to 125 mM maltose (Champreda et al. 2007). Among the various substrates for α-amylase, raw starch is the dominant form that exists in nature. Therefore α-amylase capable of raw-starch hydrolysis would be of high industrial demand. Cloning and overexpression of the raw-starch-digesting α-amylase and its subsequent purification had increased the specific activity of the enzyme by 1.78 fold when compared with the parent enzyme and facilitated its comprehensive characterization. Acknowledgements The study was financially supported by Department of Biotechnology (DBT), New Delhi.

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Received October 19, 2009 Accepted February 25, 2010