Expression and characterization of 4-α-glucanotransferase genes from Manihot esculenta Crantz and Arabidopsis thaliana and their use for the production of cycloamyloses

4-α-Glucanotransferase or disproportionating enzyme (D-enzyme, DPE) catalyzes the α-1.4 glycosyl transfer between oligosaccharides. Type I D-enzyme (DPE1) can transfer maltosyl unit from one 1.4-α-d-glucan to an acceptor mono- or oligo-saccharide, which reflects the physiological role of DPE1 in plant starch metabolism. In this study, the genes encoding DPE1 from Arabidopsis thaliana (AtDPE1) and Manihot esculenta Crantz cultivar KU50 (MeDPE1) were cloned and expressed in Escherichia coli and purified to homogeneity. MeDPE1 encoded 585 amino acid residues, including a 56 residue signal peptide, while AtDPE1 encoded 576 amino acid residues with a 45 residue signal peptide. The molecular mass of both mature enzymes, estimated from deduced amino acid sequence, were the same at 59.4 kDa, with a pI of 5.13. The predicted structures of both enzymes showed the conserved 250's loop and three catalytic amino acid residues, characteristics of disproportionating enzymes in the GH77 glycoside hydrolase family. Biochemical characterization showed that both purified recombinant enzymes were homodimers in solution, with similar optimum pH and temperature for disproportionating activity at pH 6–8 and 37 °C. Using potato amylose as a substrate, AtDPE1 can produce cycloamyloses in the range 16–50 glucose residues, while products from the action of MeDPE1 on the same substrate were in a wider range of 16 to DP > 60. These recombinant enzymes are useful tools for elucidation of their functional roles in starch metabolism and for applications in the starch industry.     

Thermophilic α-glucan phosphorylase from Clostridium thermocellum: Cloning,characterization and enhanced thermostability

ORF Cthe0357 from the thermophilic bacterium Clostridium thermocellum ATCC 27405 that encodes a putative α-glucan phosphorylase (αGP) was cloned and expressed in Escherichia coli. The protein with a C-terminal His-tag was purified by Ni²⁺ affinity chromatography; the tag-free protein obtained from a cellulose-binding module–intein–αGP fusion protein was purified through affinity adsorption on amorphous cellulose followed by intein self-cleavage. Both purified enzymes had molecular weights of ca. 81,000 and similar specific activities. The optimal conditions were pH 6.0–6.5 and 60 °C for the synthesis direction and pH 7.0–7.5 and 80 °C for the degradation direction. This enzyme had broad substrate specificities for different chain length dextrins and soluble starch. The thermal inactivation of this enzyme strongly depended on temperature, protein concentration, and certain addictives that were shown previously to benefit the protein thermostability. The half lifetime of 0.05 mg αGP/mL at 50 °C was extended by 45-fold to 90 h through a combined addition of 0.1 mM Mg²⁺, 5 mM DTT, 1% NaCl, 0.1% Triton X-100, and 1 mg/mL BSA. The enzyme with prolonged stability would work as a building block for cell-free synthetic enzymatic pathway biotransformations, which can implement complicated biocatalysis through assembly of a number of enzymes and coenzymes.     

Purification and characterization of a novel thermostable α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp.

The purification and characterization of an extracellular α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp. was investigated in this report. The α-l-AFase was purified to homogeneity with a purification fold of 1030. The purified α-l-AFase had a specific activity of 20.6 U mg^?1. The molecular mass of the enzyme was estimated to be 52.9 kDa and 51.6 kDa by SDS–PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.0 and 70 °C, respectively. The enzyme was stable over a broad pH range of 4.0–10.0 and also exhibited excellent thermostability, i.e., the residual activities reached 75% after treatment at 60 °C for 1 h. The enzyme showed strict substrate specificity for the α-l-arabinofuranosyl linkage. The K_m and V_max values for p-nitrophenyl (pNP)-α-l-arabinofuranoside were calculated to be 1.43 mM and 68.3 μmol min^?1 mg^?1 protein, respectively. Furthermore, the gene encoding α-l-AFase was cloned and sequenced and found to contain a catalytic domain belonging to the glycoside hydrolase (GH) family 43 α-l-AFase. The deduced amino acid sequence of the gene showed the highest identity (67%) to the putative α-l-AFase from Neurospora crassa. This is the first report on the purification, characterization and gene sequence of an α-l-AFase from Chaetomium sp.     

Purification and biochemical characterization of a novel SDS and surfactant stable,raw starch digesting,and halophilic α-amylase from a moderately halophilic bacterium,Nesterenkonia sp. strain F

An extracellular halophilic α-amylase from Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography, with a 10.8-fold increase in specific activity. The molecular mass of the amylase was estimated to be 100 kDa and 106 kDa by SDS–PAGE and gel filtration chromatography, respectively. The enzyme showed maximal activity at pH 7.5 and 45 °C. The amylase was active in a wide range of salt concentrations (0–4 M) with its maximum activity at 0.5 M NaCl or 1 M KCl and was stable at the salts concentrations between 1 M and 4 M. Fe³⁺, Cu²⁺, Zn²⁺ and Al³⁺ strongly inhibited the enzyme, whereas Ca²⁺ stimulated the amylase activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. The enzyme showed remarkable stability towards 0.5% SDS and sarcosyl, and 2% each of Triton X-100, Tween 80 and Tween 20. K_m value of the amylase for soluble starch was 4.5 mg/ml. The amylase hydrolyzed 38% of raw wheat starch and 20% of corn starch in a period of 48 h. The major products of soluble starch hydrolysis were maltose, maltotriose and maltotetraose, indicating an α-amylase activity.     

A recombinant levansucrase from Bacillus licheniformis 8-37-0-1 catalyzes versatile transfructosylation reactions

This work disclosed the broad transglycosylation capability of the levansucrase from Bacillus licheniformis 8-37-0-1 for the first time. The levansucrase was firstly purified from the strain 8-37-0-1 and found to be a monomer of ∼51 kDa with KETQDYKKSY as the N-terminus. Then, the gene encoding the enzyme was cloned and it contained an ORF of 1449 nucleotides, encoding a 482 amino-acid protein with a predicted 29 amino-acid signal peptide. The deduced mature protein without the signal showed the same N-terminus to the purified enzyme. The mature enzyme was subsequently expressed in Escherichia coli. The recombinant enzyme showed similar biochemical properties to the native one. It produced maximal yield of 7.1 mg/mL levan (M_r 9.6 × 10⁶) from 0.8 M sucrose (pH 6.5) at 40 °C for 24 h in vitro. When using sucrose as the donor, the enzyme displayed a wide range of acceptor specificity and was able to transfer fructosyl to a series of sugar acceptors including hexose, pentose, β- or α-disaccharides, along with the difficult branched alcohols that have not been investigated before. Chemical structures of the resultant products were analyzed by MS and NMR spectra.     

Applications of a high maltose forming,thermo-stable α-amylase from an extremely alkalophilic Bacillus licheniformis strain AS08E in food and laundry detergent industries

An alkalophilic bacterial strain was isolated from the soil sample of Assam, North-East India. This strain was found capable of growing and producing α-amylase at extremely alkaline pH (12.5). By molecular characterization, this bacterium was identified as Bacillus licheniformis strain AS08E. Statistical optimization of media components resulted in 3-fold increase in the production of α-amylase from this bacterium. From this strain, a major extracellular α-amylase of ∼55 kDa was purified to homogeneity with a 14.5-fold increase in its specific activity. The N-terminal sequence of this enzyme showed extensive identity with α-amylases purified from thermostable bacteria. The purified enzyme showed optimum activity at pH 10.0 and 80 °C, and demonstrated stability toward various surfactants, organic solvents, and commercial laundry detergents. The spectroflurometric analysis suggests that the enzyme has a strong binding affinity toward soluble starch. TLC analysis of starch degradation product displays this α-amylase as a high maltose-forming enzyme. The future application of this enzyme in food and detergent industries is highly promising.     

α-Amylase from germinating soybean (Glycine max) seeds – Purification,characterization and sequential similarity of conserved and catalytic amino acid residues

Starch hydrolyzing amylase from germinated soybeans seeds (Glycine max) has been purified 400-fold to electrophoretic homogeneity with a final specific activity of 384 units/mg. SDS–PAGE of the final preparation revealed a single protein band of 100 kDa, whereas molecular mass was determined to be 84 kDa by MALDI–TOF and gel filtration on Superdex-200 (FPLC). The enzyme exhibited maximum activity at pH 5.5 and a pI value of 4.85. The energy of activation was determined to be 6.09 kcal/mol in the temperature range 25–85 °C. Apparent Michaelis constant (K_m(app)) for starch was 0.71 mg/mL and turnover number (k_cat) was 280 s^?1 in 50 mM sodium acetate buffer, pH 5.5. Thermal inactivation studies at 85 °C showed first-order kinetics with rate constant (k) equal to 0.0063 min^?1. Soybean α-amylase showed high specificity for its primary substrate starch. High similarity of soybean α-amylase with known amylases suggests that this α-amylase belongs to glycosyl hydrolase family 13. Cereal α-amylases have gained importance due to their compatibility for biotechnological applications. Wide availability and easy purification protocol make soybean as an attractive alternative for plant α-amylase. Soybean can be used as commercially viable source of α-amylase for various industrial applications.     

Production and characterization of a thermostable endo-type β-xylanase produced by a newly-isolated Streptomyces thermocarboxydus subspecies MW8 strain from Jeju Island

A xylanase-producing, Gram-positive, aerobic, and spore-forming bacterium was isolated from a soil sample collected from Jeju Island and was classified as a novel subspecies of Streptomyces thermocarboxydus on the basis of 16S rRNA gene sequence similarity, the results of DNA–DNA hybridization analysis, and phenotypic characteristics. The novel strain was named as S. thermocarboxydus subsp. MW8 (=KCTC29013 = DSM52054). This strain produced extracellular xylanase. Xylanase from the strain was purified to homogeneity and had an apparent molecular weight of 52 kDa. The NH₂-terminal sequence (Ala-Glu-Ile-Arg-Leu) was distinct from those of previously reported xylanases. The purified xylanase produced xylobiose as the end-product of birchwood xylan hydrolysis. The K_m and V_max values of the purified xylanase on birchwood xylan were 1.71 mg/ml and 357.14 U/mg, respectively. The optimum pH and temperature for the enzyme were found to be 7.0 and 50 °C, respectively, and the enzyme exhibited significant heat stability. In addition, the enzyme was active over broad pH ranges: 84% of the maximum activity at pH 5.0, 84–88% at pH 6.0, 88% at pH 8.0, and 75–81% (pH 9.0). These enzymatic properties may be very useful for use in bio-industrial applications.     

Cloning,expression and characterization of highly thermo- and pH-stable maltogenic amylase from a thermophilic bacterium Geobacillus caldoxylosilyticus TK4

Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes, belong to glycoside hydrolase family 13. A gene corresponding to MA in Geobacillus caldoxylosilyticus TK4 (GcaTK4MA) was cloned into pET28a(+) vector and expressed in Escherichia coli with 6xHis-tag at the N-terminus. Herein, we report on the biochemical properties of a new thermo- and pH-stable MA. GcaTK4MA has similar properties those of other MAases in terms of the primary structure, preference for CD over starch and having an extra domain at its N- and C-terminals. The recombinant protein was purified efficiently by using one-step nickel affinity chromatography. The purified enzyme exhibited optimal activity for β-CD hydrolysis at 50 °C and pH 7.0. When the enzyme was separately incubated at 4 °C and 50 °C in the buffer solutions (pH 3.0–9.0) up to 7 days, it was seen that the enzyme had the higher stability at 50 °C than 4 °C. The enzyme retained about 80% of its original activity when it was incubated at 50 °C for 7 days. The enzyme activity was significantly inhibited by SDS and EDTA at the final concentration of 1%. These results suggest that this is the first reported MA having an extremely pH- and thermal stabilities.     

Amylolytically-resistant tapioca starch modified by combined treatment of branching enzyme and maltogenic amylase

Tapioca starch was modified using branching enzyme (BE) isolated from Bacillus subtilis 168 and Bacillus stearothermophilus maltogenic amylase (BSMA), and their molecular fine structure and susceptibility to amylolytic enzymes were investigated. By BE treatment, the molecular weight decreased from 3.1 × 10⁸ to 1.7 × 10⁶, the number of shorter branch chains (DP 6–12) increased, the number of longer branch chains (DP >25) decreased, and amylose content decreased from 18.9% to 0.75%. This indicated that α–1,4 linkages of amylose and amylopectin were cleaved, and moiety of glycosyl residues were transferred to another amylose and amylopectin to produce branched glucan and BE-treated tapioca starch by forming α–1,6 branch linkages. The product was further modified with BSMA to produce highly-branched tapioca starch with 9.7% of extra branch points. When subject to digestion with human pancreatic α-amylase (HPA), porcine pancreatic α-amylase (PPA) and glucoamylase, highly-branched tapioca starch gave significantly lowered α-amylase susceptibility (7.5 times, 14.4 times and 3.9 times, respectively), compared to native tapioca starch.     

Purification and functional characterization of the first stilbene glucoside-specific β-glucosidase isolated from Lactobacillus kimchi

This study aimed to develop viable enzymes for bioconversion of resveratrol-glucoside into resveratrol. Out of 13 bacterial strains tested, Lactobacillus kimchi JB301 could completely convert polydatin into resveratrol. The purified enzyme had an optimum temperature of 30–40 °C and optimum pH of pH 5.0 against polydatin. This enzyme showed high substrate specificities towards different substrates in the following order: isorhaponticin >> polydatin >> mulberroside A > oxyresveratrol-3-O-glucoside. Additionally, it rarely hydrolyzed astringin and desoxyrhaponticin. Based on these catalytic specificities, we suggest this enzyme be named stilbene glucoside-specific β-glucosidase. Furthermore, polydatin extracts from Polygonum cuspidatum were successfully converted to resveratrol with a high yield (of over 99%). Stilbene glucoside-specific β-glucosidase is the first enzyme isolated from lactic acid bacteria capable of bio-converting various stilbene glucosides into stilbene.     

BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification,biochemical and molecular characterization

A novel fibrinolytic enzyme, subtilisin BSF1, from a newly isolated Bacillus subtilis A26 was purified, characterized and the gene was isolated and sequenced. The subtilisin BSF1 was purified to homogeneity by five-step procedure with a 4.97-fold increase in specific activity and 6.28% recovery. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and gel filtration. The purified enzyme exhibited high fibrinolytic activity on fibrin agar plates.Interestingly, the enzyme was highly active over a wide range of pH from 7.0 to 12.0, with an optimum at pH 9.0. The relative activities at pH 10.0 and 11.0 were 97.8% and 85.2% of that at pH 9.0. The optimum temperature for enzyme activity was 60 °C. The activity of subtilisin BSF1 was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The N-terminal amino acid sequence of the first 11 amino acids (aa) of the purified fibrinolytic enzyme was AQSVPYGISQI.The bsf1 gene encoding the subtilisin BSF1 was isolated and its DNA sequence was determined. The bsf1 gene consisted of 1146 bp encoding a pre-pro-protein of 381 amino acids organized into a signal peptide (29 aa), a pro-peptide (77 aa) and a mature domain (275 aa). The deduced amino acids sequence of the mature enzyme (BSF1) differs from those of nattokinase from B. subtilis natto and subtilisin DFE from Bacillus amyloliquefaciens DC-4 by 5 and 39 amino acids, respectively.     

The stability and thermodynamic parameters of a very thermostable and calcium-independent α-amylase from a newly isolated bacterium,Anoxybacillus beppuensis TSSC-1

Anoxybacillus beppuensis TSSC-1 (GenBank Number, EU710556), a thermophilic bacterium isolated from a hot spring reservoir, was found to optimally secrete a monomeric α-amylase at 55 °C and pH 7. The enzyme was purified to homogeneity by a single-step purification on phenyl sepharose 6FF, achieving a 58% yield, 10,000 U/mg specific activity and 19.5 fold purification. The molecular weight, K_m and V_max were 43 kD, 0.5 mg ml^?1 and 3571.42 μmol ml^?1 m^?1, respectively. The enzymatic catalysis of soluble starch was optimum at 80 °C and pH 7. The thermodynamic parameters, K_d, t_1/2, ΔH*, ΔS*, E and ΔG*, were consistent. The very compact structure of the enzyme and the transitional enzyme–substrate complex resisted denaturation at extreme temperatures and alkaline pH. The K_d and t_1/2 measurements were consistent with the high thermostability and pH tolerance observed. The structural stability of the enzyme was also reflected by the values of ΔH*, ΔS*, E and ΔG*. While the enzyme did not exhibit metal ion dependency, it was resistant to chemical denaturation. The broad thermo- and pH-tolerance of this enzyme suggests potential commercial opportunities.     

Expression and characterization of antimicrobial peptide CecropinAD in the methylotrophic yeast Pichia pastoris

Hybrid antibacterial peptide CecropinAD (CAD) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach to express the hybrid peptide CAD in the methylotrophic yeast Pichia pastoris, the cDNA sequence encoding CAD was obtained by recursive PCR (rPCR) and cloned into the vector pPICZα-A. The Sac I-linearized recombinant plasmid pPICZα-CAD was transformed into P. pastoris GS115 by electroporation. Expression of recombinant CAD was induced for 96 h with 1.0% methanol at 28 °C, pH 5.0. The recombinant CAD was purified by two steps of reversed-phase HPLC and 1.8 mg pure active CAD was obtained from 100 ml culture. Tricine-SDS-PAGE and mass spectrometry analyses demonstrated that the molecular weight of the purified CAD was 3.8 kDa. Analysis of circular dichroism (CD) revealed that CAD mainly has α-helixes in the presence of 10 mM phosphate buffer (pH 7.2), 50% TFE/water solution (pH 2.0), or 30 mM SDS (pH 10.8). FACScan analysis showed that the antibacterial mechanism of CAD is to act on the cell membrane to disrupt bacterial cell structure. Antimicrobial assays demonstrated that recombinant CAD has a broad spectrum of anti-microbial property against fungi, as well as Gram-positive and Gram-negative bacteria, but does not have hemolytic activity against human erythrocytes. Our results suggest that recombinant antimicrobial peptide CAD may serve as an attractive candidate for the development of therapeutic antimicrobial drugs.     

A novel thermostable GH5_7 β-mannanase from Bacillus pumilus GBSW19 and its application in manno-oligosaccharides (MOS) production

A novel thermostable mannanase from a newly isolated Bacillus pumilus GBSW19 has been identified, expressed, purified and characterized. The enzyme shows a structure comprising a 28 amino acid signal peptide, a glycoside hydrolase family 5 (GH5) catalytic domain and no carbohydrate-binding module. The recombinant mannanase has molecular weight of 45 kDa with an optimal pH around 6.5 and is stable in the range from pH 5–11. Meanwhile, the optimal temperature is around 65 °C, and it retains 50% relative activity at 60 °C for 12 h. In addition, the purified enzyme can be activated by several ions and organic solvents and is resistant to detergents. Bpman5 can efficiently convert locus bean gum to mainly M2, M3 and M5, and hydrolyze manno-oligosaccharides with a minimum DP of 3. Further exploration of the optimum condition using HPLC to prepare oligosaccharides from locust bean gum was obtained as 10 mg/ml locust bean gum incubated with 10 U/mg enzyme at 50 °C for 24 h. By using this enzyme, locust bean gum can be utilized to generate high value-added oligosaccharides with a DP of 2–6.     

A Ca-independent α-amylase that is active and stable at low pH from the Bacillus sp. KR-8104

Bacillus sp. KR-8104 was selected from a set of 18 bacteria strains isolated from soil samples and screened for production of amylase. The maximum productivity obtained at pH 5–6 and 60–65 h after cultivation in production medium. New extracellular Ca-independent α-amylase was highly purified using ion exchange and hydrophobic interaction chromatography, which showed a single band with an apparent molecular weight of 59 kDa by SDS-PAGE. This enzyme is active in a wide pH range with its maximum activity at low pH values (4.0–6.0) and has the 90% of its maximum activity at pH 3.5. The α-amylase is optimally active at 75–80 °C. The presence or absence of Ca²⁺ and EDTA did not affect enzyme activity and thermal stability.     

Purification,modification and inhibition mechanism of angiotensin I-converting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate

Angiotensin I-converting enzyme (ACE) inhibitory peptide from silkworm pupa (Bombyx mori) was purified, modified, as well as inhibition mechanism by using molecular docking analysis. Silkworm pupa protein was hydrolyzed by neutral protease and the obtained hydrolysate was subjected to various types of chromatography to acquire peptide isolate. Then the molecular mass and amino acid sequence of the peptide was determined by MALDI-TOF/TOF MS. Subsequently, thermal and digestive stability of the peptide were explored through a high temperature processing and a simulated gastrointestinal digestion. Finally, the peptide was modified to smaller peptides and investigated their potentiate activities. Results showed that the peptide from silkworm pupa was determined to be Gly-Asn-Pro-Trp-Met (603.7 Da) with IC₅₀ 21.70 μM. Stability testing showed that ACE inhibitory activities were not significantly changed at temperature from 40 to 80 °C as well as during in vitro gastrointestinal digestion. The inhibitory activity of four modified peptides were Trp-Trp > Gly-Asn-Pro-Trp-Trp > Asn-Pro-Trp-Trp > Pro-Trp-Trp, and the IC₅₀ of Trp-Trp was 10.76 μM Docking simulation revealed that the inhibitory activity was closely related to the spatial structure of peptide and zinc ions. The purified peptide and four modified peptides may be beneficial as functional food or drug for treating hypertension.     

Cloning and overexpression of raw starch digesting α-amylase gene from Bacillus subtilis strain AS01a in Escherichia coli and application of the purified recombinant α-amylase (AmyBS-I) in raw starch digestion and baking industry

Considering the economic and industrial relevance of α-amylases used in food and starch industries, a raw starch digesting α-amylase gene (amyBS-I) from Bacillus subtilis strain AS01a was cloned and expressed in Escherichia coli BL21 cells. The gene also includes its signal peptide sequence (SPS) for facilitating the efficient extracellular expression of recombinant α-amylase (AmyBS-I) in correctly folded (enzymatically active) form. The native AmyBS-I consists of 659 amino acids with a molecular mass and pI of 72,387 Da and 5.8, respectively. The extracellular secretion of AmyBS-I after response surface optimization of culture conditions was found to be 7-fold higher as compared to its production under non-optimized conditions. Purified AmyBS-I demonstrated optimum activity at 70 °C and pH 6.0. It shows K_m and V_max values toward soluble starch as 2.7 mg/ml and 454 U/ml, respectively. Further, it does not require Ca²⁺ ion for its α-amylase activity/thermo-stability, which is an added advantage for its use in the starch industry. The AmyBS-I also hydrolyzed a wide variety of raw starches and produced maltose and glucose as main hydrolyzed products. The bread dough supplemented with AmyBS-I showed better amelioration of the bread quality as compared to the bread supplemented with commercial α-amylase.     

Psychrophilic α-amylase from Aeromonas veronii NS07 isolated from farm soils

Among 120 isolates examined in this study, three isolates were selected for amylase production on starch agar plates following incubation at 10 °C. Identification by 16SrRNA on selected bacterium disclosed the highest similarity for protean regions of this gene as Aeromonas veronii NS07. A 63 kDa psychrophilic amylase enzyme from NS07 strain was purified by two-steps chromatography. The enzyme had the highest specific activity at pH 4 and was active at the range of temperatures from 0 to 50 °C, although the optimum temperature for enzyme activity was found at 10 °C. Analysis of the N-terminal amino acid sequencing disclosed 20 amino acids from purified amylase which had no similarity with other known α-amylases, indicating that the presented enzyme was novel. Amylase activity was enhanced in relation to optimum activity with the presence of sodium sulphate (161%), MnCl₂ (298%), CaCl₂ (175%), FeCl₂ (182%), MgCl₂ (237%), ZnCl₂ (169%), NiCl₂ (139%), NaCl (158%), each at 5 mM, while EDTA, phenylmethane sulphonylfluoride (PMSF) (3 mM), urea (8 M) and SDS (1%) inhibited the enzyme up to 5%, 2%, 80% and 18%, respectively. NS07 strain seems to be suitable as biocatalyst for practical use in liquefaction of starch at low temperatures, detergent and textile industries.     

Structural characterization of thermostable,solvent tolerant,cytosafe tannase from Bacillus subtilis PAB2

Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (K_m = 0.445 mM, V_max = 125.8 mM/mg/min and K_cat = 2.88 min^–1). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 °C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 °C temperature. It exhibited half-life (t_1/2) of 4.5 h at 60 °C. The enzyme comprised a typical secondary structure containing α-helix (9.3%), β-pleated sheet (33.6%) and β-turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 °C and showed Q₁₀, ΔG_d and ΔS_d values of 2.08, 99.37 KJ/mol and 252.38 J mol⁻¹ K⁻¹, respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study.