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A β-glucosidase gene (bsbgl1a) from Bacillus sp. CGMCC 1.16541 was expressed in Escherichia coli BL21 and subsequently characterized. The amino acid sequence shared 83.64% identity with β-glucosidase (WP_066390903.1) from Fictibacillus phosphorivorans. The recombinant β-glucosidase (BsBgl1A) had a molecular weight of 52.2 kDa and could hydrolyze cellobiose, cellotriose, cellotetrose, p-nitrophenyl-β-D-glucopyranoside (pNPG), and p-nitrophenyl-β-D-xylopyranoside (pNPX). Optimal activity for BsBgl1A was recorded at 45 °C with a pH between 5.6 and 7.6, and 100% of its activity was maintained after a 24 h incubation between pH 4 and 9. Kinetic characterization revealed an enzymatic turnover (Kcat) of 616 ± 2 s -1 (with cellobiose) and 3.5 ± 0.1 s -1 (with p-nitrophenyl-β-D-glucopyranoside). Interestingly, the recombinant enzyme showed cupric ion (Cu 2+ ), sodium dodecyl sulfate (SDS) and alcohol tolerance at 10 mM for Cu 2+ and 10% for both SDS and alcohol. Additionally, BsBgl1A had high tolerance for glucose (Ki = 2095 mM), which is an extremely desirable feature for industrial applications. Following the addition of BsBgl1A (0.05 mg/ml) to a commercial cellulase reaction system, glucose yields from sugarcane bagasse increased 100% after 1 day at 45 °C. This work identifies a Cu 2+ , SDS, alcohol, and glucose tolerant GH1 β-glucosidase with potential applications in the hydrolysis of cellulose for the bioenergy industry.

Chitosanase plays an important role in the production of chitooligosaccharides (CHOS), which possess various biological activities. Herein, a glycoside hydrolase (GH) family 46 chitosanase-encoding gene, csnB , was cloned from marine bacterium Bacillus sp. BY01 and heterologously expressed in Escherichia coli . The recombinant chitosanase, CsnB, was optimally active at 35 °C and pH 5.0. It was also revealed to be a cold-adapted enzyme, maintaining 39.5% and 40.4% of its maximum activity at 0 and 10 °C, respectively. Meanwhile, CsnB showed wide pH-stability within the range of pH 3.0 to 7.0. Then, an improved reaction condition was built to enhance its thermostability with a final glycerol volume concentration of 20%. Moreover, CsnB was determined to be an endo-type chitosanase, yielding chitosan disaccharides and trisaccharides as the main products. Overall, CsnB provides a new choice for enzymatic CHOS production.

The interaction between Ca 2+ and alkaline protease has been studied. Enzyme activity measurements demonstrated that Ca 2+ with a suitable concentration could increase the activity of alkaline protease. Particle size and zeta potential measurements showed that Ca 2+ bound to alkaline protease by electrostatic interaction. The combination of Ca 2+ and alkaline protease promoted the structure of alkaline protease to become more stable, which was favorable for the increase of enzyme activity. Fluorescence spectra showed that alkaline protease had two Ca 2+ binding sites and potent binding force to Ca 2+ . Thermodynamic parameters ΔH, ΔS and ΔG obtained by fluorescence spectra confirmed that the interaction between Ca 2+ and alkaline protease was also electrostatic interaction and the binding process could proceed spontaneously. The results of molecular docking were also consistent with the conclusions obtained from the zeta potential and fluorescence spectrum measurements. In addition, the washing performance test showed that the detergent added with Ca 2+ and alkaline protease had the best washing performance on protein-contaminated swatches JB02 and blood swatches., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A novel extracellular protease (SAPRH) was hyper-produced (9000 U/mL) from Bacillus safensis RH12, a newly isolated enzyme from a Tunisian offshore oil field. The enzyme was purified to homogeneity, using salt-precipitation, heat-treatment and FPLC anion-exchange chromatography. The purified enzyme was a monomer of molecular mass of ~28 kDa. The NH 2 -terminal 23 amino-acid sequence of SAPRH showed high homology with those of Bacillus-proteases. SAPRH displayed optimal activity at pH 9 and 60 °C. It was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), indicating that it belongs to the serine-proteases family. Moreover, SAPRH was extremely stable at a broad range of temperature and pH retaining 85% of its activity at 50 °C and 75% at pH 11. The enzyme exhibited excellent stability and compatibility with surfactants and commercial detergents, revealing 90% stability with SDS and 100% stability with Class commercial laundry detergent. One of the most distinctive properties is its catalytic efficiency, which is higher than that of Alcalase 2.5 L, typeDX (commercial enzyme) and SAPB from B. pumilus CBS. Interestingly, the results of the wash performance analysis demonstrated considerably good de-staining at 40 °C for 30 min with low supplementation (500 U/mL). Accordingly, such a protease could be considered as a good detergent-additive in detergent industry., (Copyright © 2018. Published by Elsevier B.V.)

A novel gene (ANK58566) encoding a cold-active α-amylase was cloned from marine bacterium Bacillus sp. dsh19-1 (CCTCC AB 2015426), and the protein was expressed in Escherichia coli. The gene had a length of 1302 bp and encoded an α-amylase of 433 amino acids with an estimated molecular mass of 50.1 kDa. The recombinant α-amylase (AmyD-1) showed maximum activity at 20 °C and pH 6.0, and retained about 35.7% of activity at 4 °C. The AmyD-1 activity was stimulated by Ca 2+ and Na + . However, the chelating agent, EDTA, inactivated the enzyme. Moreover, AmyD-1 displayed extreme salt tolerance, with the highest activity in the presence of 2.0 M NaCl and 60.5% of activity in 5.0 M NaCl. The K m , V max and k cat of AmyD-1 in 2.0 M NaCl were 2.8 mg ml -1 , 21.8 mg ml -1 min -1 and 933.5 s -1 , respectively, at 20 °C and pH 6.0 with soluble starch as substrate. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) revealed that the end products of starch hydrolysis by AmyD-1 were glucose, maltose, maltotriose, maltotetraose, and malt oligosaccharides. Thus, AmyD-1 is one of the very few α-amylases that can tolerate low temperatures and high salt concentrations, which makes it to be a potential candidate for research in basic and applied microbiology.

This study reports the ultrasound-assisted improvements in biocatalytic activity and production of protease from organic solvent-tolerant, alkaliphilic Bacillus circulans MTCC 7942. The strain withstands 10min ultrasound treatment (33kHz); this treatment promotes the growth of bacterium. The protease production was improved by 1.5-fold on multiple ultrasound treatment at 0, 6, 12h intervals. Although the substrate casein remains unaffected by ultrasound treatment (33kHz, 10min); the treatment had boosted the enzyme activity of purified protease. Also, the ultrasound treated purified protease showed enhancement in enzyme activity in presence of n-dodecane, n-octane, n-hexadecane, n-decane, xylene, butanol, toluene, pyridine and chloroform., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An extracellular alkaline stable protease BS1 from a new bacteria strain, Bacillus safensis S406, isolated from the Sfax solar saltern, was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-75 gel filtration, Mono-Q anion-exchange chromatography and ultrafiltration, with a 12.70-fold increase in specific activity and 20.29% recovery. The enzyme has a molecular weight of 29kDa and appeared as a single band on native-PAGE. The optimum pH and temperature values of its proteolytic activity were pH 11.0 and 60°C, respectively. BS1 was tested for the deproteinization of shrimp wastes to extract chitin. An enzyme-protein ratio of 10U/mg of proteins allows to eliminate 93% of protein linked to the chitin after 3h hydrolysis at 45°C. Being very active in alkaline conditions, the potential application of BS1 in laundry formulation was investigated. The enzyme showed high stability in the presence of non-ionic surfactants and some commercial liquid and solid detergents, suggesting its eventual use in detergent formulations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aim of the present work was to study the ability of a halophilic bacterial laccase to efficient delignification in extreme conditions. Here, a highly stable extracellular laccase showing ligninolytic activity from halophilic Aquisalibacillus elongatus is described. The laccase production was strongly influenced by NaCl and CuSO 4 and under optimal conditions reached 4.8UmL -1 . The monomeric enzyme of 75kDa was purified by a synthetic affinity column with 68.2% yield and 99.8-fold purification. The enzyme showed some valuable features viz. stability against a wide range of organic solvents, salts, metals, inhibitors, and surfactants and specificity to a wide spectrum of substrates diverse in structure and redox potential. It retained more than 50% of the original activity at 25-75°C and pH 5.0-10.0. Furthermore, the enzyme was found to be effective in the delignification of sugar beet pulp in an ionic liquid that makes it useful for industrial applications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A newly alkaline proteases producing strain was isolated from sea water. The strain was identified as Bacillus invictae on the basis of biochemical characteristics and 16S rRNA sequence analysis. The crude protease activity showed an optimal activity at approximately 60°C and in wide pH interval ranging from 9.0 to 11.0. At least six clear caseinolytic protease bands were observed in a zymogram. Phenylmethylsulfonyl fluoride (PMSF), a serine-protease inhibitor, was found to inhibit completely the protease activity. The crude alkaline proteases showed high stability toward solid and liquid detergents. Furthermore, wash performance analysis revealed that the crude enzyme could effectively remove blood stain when added to commercial detergent. In addition, the crude proteases were found to be effective in the deproteinization of shrimp shell waste. The percent of protein removal after 3h of hydrolysis at 50°C with an E/S ratio of 10U/mg of protein or after fermentation by the strain were about 76% and 82%, respectively. Thus, the results of the present study showed that the crude proteases of B. invectae could be effectively used in several industrial applications, as an eco-friendly agent., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Chiral lactic acid and its ester derivatives are crucial building blocks and platforms in the generation of high value-added drugs, fine chemicals and functional materials. Optically pure D-lactic acid and its ester derivatives cannot be directly generated from fermentation and are quite expensive. Herein, we identified, heterologously expressed and functionally characterized one Bacillus esterase BSE01701 from the deep sea of the Indian Ocean. Esterase BSE01701 could enzymatically resolve inexpensive racemic methyl lactate and generate chiral D-methyl lactate. The enantiomeric excess of desired chiral D-methyl lactate and the substrate conversion could reach over 99 % and 60 %, respectively, after process optimization. Notably, the addition of 60 % (v/v) organic co-solvent heptane could greatly improve both the enantiomeric excess of D-methyl lactate and the conversion. BSE01701 was a very promising marine microbial esterase in the generation of chiral chemicals in industry.

Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K 2 HPO 4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70°C, with K m and V max values of chitinase to be 5.6mg/mL and 0.87μmol/min, respectively. Ag + , Co 2+ , iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn 2+ , Cu 2+ , Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

An efficient method was introduced for soluble expression of recombinant laccase (rpCotA(SL-1)) from a newly isolated halo-thermotolerant Bacillus sp. SL-1 in modified Escherichia coli, trxB2/gor2 mutant (Origami™ B (DE3)). The yield of purified soluble laccase in Origami strain under micro-aerobic condition was ∼20mg/L of bacterial culture, showing significant improvement over the laccase produced in E.coli BL21 strain under aerobic condition. The specific activity of 13U/mg for purified laccase produced in micro-aerobic condition was higher than that of 1.07U/mg observed for the purified enzyme obtained in aerobic condition in Origami. The kinetic Km and kcat parameters for laccase-induced oxidation reactions were 46μM and 23s(-1) for ABTS (2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and 19.6μM and 24s(-1) for SGZ (syringaldazine) substrates, respectively. The rpCotA(SL-1) displayed thermostability at 70°C and tolerance to specified concentrations of NaCl, NaN3, EDTA and SDS as inhibitors. The enzyme was relatively stable in the presence of different concentration of organic solvents, however the residual activity was adversely affected as the dipole moment of the solvents increase. Here we successfully report the production of soluble and functional laccase in Origami at the expression level suitable for industrial application., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A glycoside hydrolase family 5 β-mannanase-encoding gene was cloned from Bacillus sp. HJ14 isolated from saline soil in Heijing town. Coding sequence of mature protein (without the predicted signal peptide from M1 to A30) was successfully expressed in Escherichia coli BL21 (DE3). Purified recombinant mannanase (rMan5HJ14) exhibited optimal activity at pH 6.5 and 65 °C. The enzyme showed good salt tolerance, retaining more than 56 % β-mannanase activity at 3.0-30.0 % (w/v) NaCl and more than 94 % of the initial activity after incubation with 3.0-30.0 % (w/v) NaCl at 37 °C for 60 min. Almost no mannanase activity was lost after incubation of rMan5HJ14 with trypsin, proteinase K, and Alcalase at 37 °C for 60 min. Surfactants and chelating agents, namely SDS, CTAB, Tween 80, Triton X-100, EDTA, and sodium tripolyphosphate, showed little or no effect (retaining >82.4 % activity) on enzymatic activity. Liquid detergents, namely Tupperware, Walch, Bluemoon, Tide, and OMO, also showed little or no effect (retaining >72.4 % activity) on enzymatic activity at 0.5-2.0 % (v/v). The enzyme further presents a high proportion (11.97 %) of acidic amino acid residues (D and E), which may affect the SDS and NaCl tolerance of the enzyme. Together, the mannanase may be an alternative for potential use in liquid detergent industry.

In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-L-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba(2+). This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.

Laccases are particularly promising enzymes for biotechnology and bioremediation purposes. They are among the most effective enzymes capable of catalyzing the degradation of phenolic compounds with poor water solubility. The technological utility of lacasses can be enhanced greatly by their use in ionic liquids rather than in conventional organic solvents or in their natural aqueous reaction media. In the current study, a laccase from Bacillus HR03 has been engineered through a semi rational method. By screening a library of 450 clones, Glu188Tyr and Glu188Phe showed a distinct improvement in thermal stability and ionic liquid tolerance. In comparison with the wild type, selected mutants exhibited higher kcat/Km against ABTS in the imidazolium based ionic liquids, (1-ethyl-3-methyl imidazolium chloride [EMIm][Cl], butyl-3-methyl imidazolium chloride [BMIm][Cl] and hexyl-3-methyl imidazolium chloride [HMIm][Cl]). Glu188Tyr had a catalytic efficiency, two times greater when compared to the wild type in [HMIm][Cl]. Far-UV circular dichroism (CD) exhibited no significant changes in the secondary structure of the mutants and wild type. Glu188Tyr revealed a more compact structure using Near-UV CD and fluorescence spectroscopy that could account for its high thermal stability. According to bioinformatic analysis, π-π and anion-π interactions played the dominant role in stabilizing both variants., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Lipases form one of the most important groups of biocatalysts used in biotechnology. We studied the lipase from the bacterium Cohnella sp. A01 due to the versatility of thermophilic lipases in industry. In this study lipase 3646 gene from the thermophilic bacterium Cohnella sp. A01 was expressed in Escherichia coli and the enzyme was purified by a two-steps anion exchange chromatography. The purified lipase appeared to have a molecular weight of approximately 29.5kDa on SDS-PAGE. The values of Km and Vmax, calculated by the Michaelis-Menten equation, were 1077μM and 61.94U/mg, respectively. The kinetic characterization of the purified enzyme exhibited maximum activity at 70°C and pH 8.5. Activities at 50, 55 and 60°C for 120min were measured 58%, 47% and 41%, respectively. The enzyme was also highly stable at the pH range of 8.5-10.0 for 180min. The effect of EDTA indicated that the enzyme is not a metalloenzyme. The stability of lipase 3646 in the presence of organic solvents, detergents, metal ions and inhibitors suggested that this lipase could be exploited in certain industries such as detergent and leather. Lipase 3646 was determined structurally to be 37.5% α-helix, 12.8% β-sheet, 22.7% β-turn and 27% random coil., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The recombinant Pichia pastoris harboring the endoxylanase gene (TSEV1xyl) of Bacillus halodurans TSEV1 yielded a high titer of extracellular xylanase (502±23 U ml(-1)) on induction with methanol. The purified recombinant xylanase (TSEV1xyl) displayed optimal activity at 80°C and pH 9.0. The glycosylated recombinant xylanase exhibited higher thermostability (T1/2 of 45 min at 80°C) than the native enzyme (T1/2 of 35 min at 80°C). The agroresidues subjected to pretreatment (soaking in alkali followed by microwave irradiation) liberated xylooligosaccharides (XOS) upon hydrolysis with the recombinant xylanase. The removal of unhydrolyzed agroresidues, xylanase and xylose from the hydrolysate by two-step ultrafiltration led to the purification of XOS as confirmed by TLC as well as HPLC analysis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

An extracellular chitinase (ChiA-65) was produced and purified from a newly isolated Bacillus licheniformis LHH100. Pure protein was obtained after heat treatment and ammonium sulphate precipitation followed by Sephacryl S-200 chromatography. Based on matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 65,195.13 Da. The sequence of the 27 N-terminal residues of the mature ChiA-65 showed high homology with family-18 chitinases. Optimal activity was achieved at pH 4 and 75 °C. Among the inhibitors and metals tested, p-chloromercuribenzoic acid, N-ethylmaleimide, Hg(2+), and Hg(+) completely inhibited enzyme activity. Chitinase activity was high on colloidal chitin, glycol chitin, glycol chitosane, chitotriose, and chitooligosaccharide. Chitinase activity towards synthetic substrates in the order of p-NP-(GlcNAc)n (n = 2-4) was p-NP-(GlcNAc)2 > p-NP-(GlcNAc)4 > p-NP-(GlcNAc)3. Our results suggest that ChiA-65 preferentially hydrolyzed the second glycosidic link from the non-reducing end of (GlcNAc)n. ChiA-65 obeyed Michaelis-Menten kinetics, the Km and kcat values being 0.385 mg, colloidal chitin/ml and 5000 s(-1), respectively. The chiA-65 gene encoding ChiA-65 was cloned in Escherichia coli and its sequence was determined. Above all, ChiA-65 exhibited remarkable biochemical properties suggesting that this enzyme is suitable for bioconversion of chitin waste., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The objective of the present study was the isolation, molecular cloning and biochemical characterization of a thermophilic organic solvent-resistant lipase from Bacillus sp. DR90. The lipase gene was expressed in Escherichia coli BL21(DE3) using pET-28a(+) vector. The purification of recombinant lipase was conducted by nickel affinity chromatography and its biochemical properties were determined. The lipase sequence with an ORF of 639 bp contains the conserved pentapeptide Ala-His-Ser-Met-Gly. His-tagged recombinant lipase had a specific activity of 1,126 U/mg with a molecular mass of 26.8 kDa. The cloned lipase was optimally active at pH 8.0 and 75 °C representing high stability in broad ranges of temperature and pH. High performance liquid chromatography was used to determine the major compounds released during the lipase-catalyzed reaction of p-nitrophenyl derivatives as well as the substrate specificity. The purified lipase showed high compatibility towards various organic solvents, surfactants and commercial solid/liquid detergents; therefore the recombinant DR90 lipase could be considered as a probable candidate for future applications, predominantly in detergent processing industries.

This work aimed at studying the recombinant expression of an alkali- and thermo-stable xylanase from Bacillus pumilus in Kluyveromyces lactis and its use in deinking of civic paper waste. Efficient expression with a 3-fold increase in the activity than the native organism was achieved. An inducer concentration of 2.5% and medium pH of 9.0 was the best for enzyme expression. Purified enzyme showed an optimum activity at temperatures 50 and 60°C and pH 9.0 and 10.0, respectively. At pH 12.0, enzyme retained 74% and 26% activity after 2 and 3h of incubation, respectively. After incubation at 50 and 60°C for 1h, the enzyme showed 100% retention of activity, and remained active for 4h at 60°C retaining 23% residual activity. Partially purified recombinant enzyme showed higher deinking efficiency (273%) of laser print waste paper than crude xylanase from Bacillus and commercial acidic enzyme. This xylanase with superior stability characteristics could be a suitable candidate in paper and pulp industries., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ~0.5-2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (K(M)) but to pH dependence of catalytic turnover: The k(cat) of B. clausii cotA was 1 s⁻¹ at pH 6 and 5 s⁻¹ at pH 8 in contrast to 6 s⁻¹ at pH 6 and 2 s⁻¹ at pH 8 for of B. subtilis cotA. Overall, k(cat)/K(M) was 10-fold higher for B. subtilis cotA at pH(opt). While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500-700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ~20 minutes half-life at 80°C, less than the ~50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH~8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization.

The present study deals with the characterization of halotolerant protease produced by Bacillus aquimaris VITP4 strain isolated from Kumta coast, Karnataka, India. The studies were performed at 40 °C and pH 8 in Tris buffer. Metal ions such as Mn(2+) and Ca(2+) increased the proteolytic activity of the enzyme by 34 and 30 %, respectively, at 10 mM concentration. Cu(2+) at 1 mM concentration was found to enhance the enzyme activity by 16 %, whereas inhibition was observed at higher concentration (>5 mM). Slight inhibition was observed even with lower (>1 mM) concentrations of Zn(2+), Hg(2+), Fe(3+), Ni(2+), and Co(2+).The activity of protease was completely inhibited by phenylmethylsulfonyl fluoride, indicating that the VITP4 protease is a serine protease. The presence of ethylenediaminetetraacetic acid and 1,10-phenanthroline (>5 mM) moderately inhibited the activity, suggesting that the enzyme is activated by metal ions. The protease was purified to homogeneity with a purification fold of 15.7 with ammonium sulfate precipitation and 46.65 with gel filtration chromatography using Sephadex G-100, resulting in a specific activity of 424 ± 2.6 U mg(-1). The VITP4 protease consists of a single polypeptide chain with a molecular mass of 34.7 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight. Among the different substrates used (casein, egg albumin, gelatin, and bovine serum albumin), the activity was higher with casein with V max, K m, and k cat values of 0.817 mg ml min(-1), 0.472 mg ml(-1), and 2.31 s(-1), respectively. Circular dichroism studies revealed that the VITP4 protease has a predominantly β-sheet structure (51.6 %) with a temperature for half denaturation of 85.8 °C in the presence of 1 mM CaCl2. Additionally, the VITP4 protease was found to retain more than 70 % activity in the presence of 10 mM concentration of different detergents (CTAB, urea, and sodium dodecyl sulfate) and surfactants (Triton X-100, Tween-20, and Tween-80), and the results of wash performance test with various commercial detergents confirmed that it can be used in detergent formulations.

A total of 18 protease producing bacterial strains were isolated from detergent effluent in South Korea using skim milk agar medium. A strain (BK-P21A) was selected and identified as Bacillus koreensis based on morphological, biochemical and molecular characterizations (16S rRNA gene sequence analysis). Optimized culture conditions for the production of protease were pH 8.5, 30 °C, sucrose (2%) and yeast extract (0.2%) during 36 h of incubation. Furthermore, the protease was partially purified by ammonium sulphate precipitation (80%) and again by Superdex 200 10/300 GL and Superdex 75 10/300 GL column chromatography, which resulted in 5.0 fold purification and a yield of 23%. The molecular mass of the protease was estimated to be 48 kDa by SDS-PAGE. The purified enzyme was further characterized and found to be most active at pH 9.0 and 60 °C. The activity of the purified protease was enhanced by CaCl₂ and CoCl₂, but inhibited by PMSF, which indicated it was a serine type protease. Moreover, the protease was moderately stable in surfactants and 81% stable in H₂O₂. Finally, the enzyme was more active and stable (94-126.5%) in various hydrophilic organic solvents. Considering the stability of protease towards the alkaline pH, high temperature and organic solvents (50%), the enzyme from B. koreensis can be used as an alternative biocatalyst for several industrial applications mainly for peptide synthesis in nonaqueous solvents., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In the present study, we report the optimisation of batch conditions for improved α-1,4-glucan-glucanohydrolase (GGH) secretion by a nitrous acid (NA)-treated Bacillus alcalophilus. The wild (isolate GCB-18) and NA-derivative (mutant GCBNA-4) were grown in a medium containing 10 g/L nutrient broth, 10 g/L starch, 5 g/L lactose, 2 g/L ammonium sulphate, 2 g/L CaCl2 and phosphate buffer (pH 7.6). Sodium dodecyl sulphate (SDS) was used as an enzyme inducer while batch fermentations were carried out at 40 °C. The mutant produced GGH in 40 h which was 15-fold higher than the wild in presence of SDS. Thermodynamic studies revealed that the mutant culture exhibited the capability for improved enzyme activity over a broad range of temperature (35-70 °C). The enzyme was purified by cation-exchange column chromatography with ~80 % recovery. The performance of fuzzy-logic system control was found to be highly promising for the improved substrate conversion rate. The correlation (1.045E + 0025) among variables demonstrated the model terms as highly significant indicating commercial utility of the culture used (P < 0.05).

A thermo-alkali-stable laccase gene from Bacillus licheniformis was cloned and expressed in Pichia pastoris. The recombinant laccase was secreted into the culture medium with a maximum activity of 227.9 U/L. The purified laccase is a monomeric glycoprotein, and its molecular weight was estimated to be 65 kDa on SDS-PAGE after deglycosylation. Optimal enzyme activity was observed at pH 6.2 and 70°C with syringaldazine as substrate. The recombinant laccase was highly stable in the pH range 7-9 after 10 days at 30°C. The enzyme displayed remarkable thermostability at 50-70°C, with a half-life of inactivation at 70°C of 6.9 h. It also exhibited high tolerance to NaCl and organic solvents like the native spore laccase. The purified laccase could rapidly decolorize reactive blue 19, reactive black 5 and indigo carmine in the presence of acetosyringone. More than 93% of the tested dyes were decolorized in 4 h at pH 9.0., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A novel extracellular serine protease (70 kDa by SDS-PAGE) was purified and characterized. This enzyme retained more than 93% of its initial activity after preincubation for 30 min at 37 °C in the presence of 25% (v/v) tested organic solvents and showed feather degradation activity. The purified enzyme was deactivated at various combinations of pH and temperature to examine the interactive effect of them on enzyme activity. The deactivation process was modeled as first-order kinetics and the deactivation rate constant (k(d)) was found to be minimum at pH 9 and 37 °C. The kinetic analysis of enzyme over a range of pH values indicated two pK values at 6.21 and at 10.92. The lower pK value was likely due to the catalytic histidine in the free enzyme and higher pK value likely reflected deprotonation of the proline moiety of the substrate but ionization of the active site serine is another possibility. Inhibition kinetic showed that enzyme is serine protease because enzyme was competitively inhibited by antipain and aprotinin as these compounds are known to be competitive inhibitors of serine protease. The organic solvent, thermal and pH tolerances of enzyme suggested that it may have potential for use as a biocatalyst in industry., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In a bioprospecting effort towards novel thermostable lipases, we assessed the lipolytic profile of 101 bacterial strains isolated from the volcanic area of Santorini, Aegean Sea, Greece. Screening of lipase activity was performed both in agar plates and liquid cultures using olive oil as carbon source. Significant differences were observed between the two screening methods with no clear correlation between them. While the percentage of lipase producing strains identified in agar plates was only 17%, lipolytic activity in liquid culture supernatants was detected for 74% of them. Nine strains exhibiting elevated extracellular lipase activities were selected for lipase production and biochemical characterization. The majority of lipase producers revealed high phylogenetic similarity with Geobacillus species and related genera, whilst one of them was identified as Aneurinibacillus sp. Lipase biosynthesis strongly depended on the carbon source that supplemented the culture medium. Olive oil induced lipase production in all strains, but maximum enzyme yields for some of the strains were also obtained with Tween-80, mineral oil, and glycerol. Partially purified lipases revealed optimal activity at 70-80°C and pH 8-9. Extensive thermal stability studies revealed marked thermostability for the majority of the lipases as well as a two-step thermal deactivation pattern.

Enzymes are highly complex systems with a substantial degree of structural variability in their folded state. In the presence of cosolvents, fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways; alternatively, certain conformations can be stabilized or destabilized. To understand the contribution of osmolytes to the stabilization of structural changes and enzymatic activity of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC), we monitored amylolytic activity, circular dichroism, and fluorescence as a function of osmolytes. In the presence of trimethylamine N-oxide (TMAO) and sorbitol, BACΔNC activity was retained significantly at elevated temperatures. As compared to the control, the secondary structures of this enzyme were essentially conserved upon the addition of these two kinds of osmolytes. Fluorescence results revealed that the temperature-induced conformational change of BACΔNC was prevented by TMAO and sorbitol. However, glycerol did not provide profound protection against thermal denaturation of the enzyme. Sorbitol was further found to counteract guanidine hydrochloride- and SDS-induced denaturation of BACΔNC. Thus, some well-known naturally occurring osmolytes make a dominant contribution to the stabilization of BACΔNC.

The characterization of a spore laccase from Bacillus vallismortis fmb-103, isolated from textile industry disposal sites, is described. The activity was 6.5 U/g of dry spore with ABTS as the substrate. The enzyme was quite stable at high temperature. It retained more than 90% of its initial activity after 10h at 70 °C. The enzyme demonstrated broad pH stability in both acidic and alkaline conditions. There was almost no activity loss at pH 3 over an extended period of time, and the relative activity remained at 82% and 38% at pH 7 and pH 9 after 10 days. NaN(3), SDS, L-cysterine, Dithiothreitol, EDTA and NaCl inhibit the enzyme activity. Triphenylmethane dyes, including malachite green, brilliant green and aniline blue were efficiently degraded by the enzyme after 24h in combination with a mediator with efficiencies of 76.84%, 96.56% and 81.17%, respectively. The reusability of spore laccase for decolorization dyes was also examined., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bacillus mycoides S122C was identified as carboxymethyl cellulase (CMcellulase)-producing bacteria from the Azorean Bacillus collection (Lab collection), which was isolated from local soil samples. The bacteria was identified by 16S rRNA sequence and designated as B. mycoides S122C. NCBI blast analysis showed that the B. mycoides S122C 16S rRNA sequence has high identity compared to other B. mycoides strains. CMcellulase was purified from the culture filtrates using anion-exchange chromatography. After mono-Q purification, the protein folds and recovery were 13.7 and 0.76 %, respectively. SDS-PAGE analysis showed that the molecular weight of the purified CMcellulase protein was estimated to be about 62 kDa and that it was composed of a single subunit. MALDI-MS/MS analysis yielded each four peptides of the purified protein; it has identity to other cellulases. The purified CMcellulase showed high activity with CMcellulose followed by β-glucan as a substrate. Optimum temperature and pH for the purified CMcellulase activity were found to be at 50 °C and pH 7.0, respectively. The purified CMcellulase was stable with about 60 % activity in broad pH ranges from 5 to 10 and temperature of 40 to 60 °C. However, purified CMcellulase was stable at about 70 % at 70 °C and also stable overall at 78 % for surfactants. CMcellulase activity was inhibited by ions such as HgCl(2), followed by CuSo(4), FeCl(2), and MnCl(2), while CoCl(2) activated CMcellulase activity. The purified CMcellulase activity was strongly inhibited by EDTA.

A xylanase gene (xyn10A) was cloned from Bacillus sp. SN5 and expressed in Escherichia coli. It encoded a 348-residue polypeptide of ~45 kDa. The deduced amino acid sequence had 68 % identity with the endo-1,4-beta-xylanase from Paenibacillus lactis 154 that belonged to family 10 of the glycoside hydrolases. Purified recombinant Xyn10A had maximum activity at 40 °C and pH 7.0, with the specific activity of 105 U/mg and a Km of 0.6 mg/ml for beechwood xylan. Xyn10A retained more than 80 % activity between 25 and 45 °C and 29 % activity at 5 °C. It exhibited the highest activity (134 %) in 0.5 M NaCl and still retained 90 % activity in 2.5 M NaCl. It retained about 87 % activity after incubation in 2 M NaCl for 24 h. The cold-active and halo-tolerant properties of Xyn10A make it promising for application in the food industry, especially in the processing of saline food and sea food.

A halotolerant isolate Bacillus sp. L1 producing extracellular cellulase was isolated from Yuncheng, China. Production of the enzyme started from mid-exponential phase of bacterial growth and reached a maximum level during the post-stationary phase. The cellulase was purified to homogeneity with molecular mass of 45 kDa. Substrate specificity test indicated that it was an endoglucanase for soluble cellulose. Optimal enzyme activity was found to be at 60 °C, pH 8.0, and 7.5 % NaCl. Furthermore, it was highly active and stable over broad ranges of temperature (30-80 °C), pH (7.0-9.0), and NaCl concentration (2.5-15 %), thus showing its excellent thermostable, alkali-stable, and halotolerant nature. The cellulase activity was greatly inhibited by ethylenediaminetetraacetic acid, indicating that it was a metalloenzyme. Significant inhibition by phenylmethylsulfonyl fluoride and phenylarsine oxide revealed that serine and cysteine residues were essential for the enzyme catalysis. Moreover, the cellulase was highly active in the presence of surfactants, and it showed high stability in the presence of water-insoluble organic solvents with log P (ow)at least 0.88. Results from this study indicate that the purified cellulase from isolate L1 may have considerable potential for industrial application owing to its useful properties.

An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-β-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins.

A new bacterial strain exhibiting laccase activity was isolated from forest soil and was identified as Bacillus licheniformis LS04. The spore laccase of B. licheniformis LS04 demonstrated a broad pH range for catalyzing substrates. It was quite stable at high temperature and alkaline pH. There was no loss of laccase activity after 10 days incubation at pH 9.0, and about 16% of the initial activity was detected after 10h at 80°C. In addition, the spore laccase was tolerant towards 1M of NaCl and 30% of organic solvents. Reactive black 5, reactive blue 19 and indigo carmine were decolorized by the spore laccase in the absence of mediator. Meanwhile, the decolorization process was efficiently promoted when acetosyringone was present, with more than 80% of color removal in 1h at pH 6.6 or 9.0. The unusual properties indicated a high potential in industrial applications for this novel spore laccase., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

An extracellular haloalkaline, thermoactive, solvent stable, SDS-induced serine protease was purified and characterized from an alkali-thermo tolerant strain Bacillus sp. SM2014 isolated from reverse osmosis reject. The enzyme was purified to homogeneity with recovery of 54.4% and purity fold of 64. The purified enzyme was composed of single polypeptide of molecular mass about 71 kDa. The enzyme showed optimum activity at alkaline pH 10 and temperature 60°C. The km and Vmax for the enzyme was 0.57 mg/ml and 445.23 U/ml respectively. The enzyme showed novel catalytic ability at high pH (10), temperature (60°C) and salinity (3M). Moreover, the stability of enzyme in organic solvents (50% v/v) of logP ≥ 2 signified the prospective of this enzyme for peptide synthesis. The compatibility of the enzyme with surfactants and various detergent matrices together with wash performance test confirmed its potential applicability in laundry industry., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40 degrees C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu2, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.

Significant progress has been made in isolating novel alkaline β-mannanases, however, there is a paucity of information concerning the structural basis for alkaline tolerance displayed by these β-mannanases. We report the catalytic domain structure of an industrially important β-mannanase from the alkaliphilic Bacillus sp. N16-5 (BSP165 MAN) at a resolution of 1.6 Å. This enzyme, classified into subfamily 8 in glycosyl hydrolase family 5 (GH5), has a pH optimum of enzymatic activity at pH 9.5 and folds into a classic (β/α)(8)-barrel. In order to gain insight into molecular features for alkaline adaptation, we compared BSP165 MAN with previously reported GH5 β-mannanases. It was revealed that BSP165 MAN and other subfamily 8 β-mannanases have significantly increased hydrophobic and Arg residues content and decreased polar residues, comparing to β-mannanases of subfamily 7 or 10 in GH5 which display optimum activities at lower pH. Further, extensive structural comparisons show alkaline β-mannanases possess a set of distinctive features. Position and length of some helices, strands and loops of the TIM barrel structures are changed, which contributes, to a certain degree, to the distinctly different shaped (β/α)(8)-barrels, thus affecting the catalytic environment of these enzymes. The number of negatively charged residues is increased on the molecular surface, and fewer polar residues are exposed to the solvent. Two amino acid substitutions in the vicinity of the acid/base catalyst were proposed to be possibly responsible for the variation in pH optimum of these homologous enzymes in subfamily 8 of GH5, identified by sequence homology analysis and pK(a) calculations of the active site residues. Mutational analysis has proved that Gln91 and Glu226 are important for BSP165 MAN to function at high pH. These findings are proposed to be possible factors implicated in the alkaline adaptation of GH5 β-mannanases and will help to further understanding of alkaline adaptation mechanism.

Bacillus sp. ADR secretes an extracellular laccase in nutrient broth, and this enzyme was purified up to 56-fold using acetone precipitation and DEAE-cellulose anion exchange chromatography. The molecular weight of purified laccase was estimated to be 66 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified laccase oxidized 2,6-dimethoxy phenol, o-tolidine, hydroquinone, L-DOPA and guaiacol. The optimum pH for oxidation of o-tolidine, 2,6-dimethoxy phenol and guaiacol were 3.0, 4.0 and 5.0, respectively. The purified laccase contained 2.7 mol/mol of copper. The laccase was stable up to 40 °C and within the pH range of 7.0-9.0. Well-known inhibitors of multicopper oxidases such as, sodium azide, L-cysteine and dithiothreitol showed significant inhibition of laccase activity. The purified enzyme decolorized structurally different azo dyes with variable decolorization rates and efficiencies of 68-90%. This study is useful for understanding the precise use of Bacillus sp. ADR in the decolorization of textile dyes containing industrial wastewater., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Ionic liquids are recognized as green solvents for carbohydrates dissolution. However, only a limited number of studies have been carried out to investigate their effect on carbohydrate hydrolyzing enzymes. We have investigated the influence of two water miscible ionic liquids on the activity, stability and structure of two related α-amylases from Bacillus amyloliquefaciens and Bacillus lichiniformis. Upon changes in ionic liquids concentrations, both enzymes activity and stability were reduced. Associated thermodynamic and conformational changes were observed using differential scanning calorimetry and fluorescence techniques. Thermal denaturation was accompanied by aggregation in both aqueous buffer and [BMIm][Cl] but [HMIm][Cl] significantly suppressed aggregation., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The gene gdh encoding an organic solvent-tolerant and alkaline-resistant NAD(P)-dependent glucose 1-dehydrogenase (LsGDH) was cloned from Lysinibacillus sphaericus G10 and expressed in Escherichia coli. The recombinant LsGDH exhibited maximum activity at pH 9.5 and 50 °C. LsGDH displayed high stability at a wide pH ranging from 6.5 to 10.0 and was stable after incubation at 30 °C for 1 week in 25 mM sodium phosphate buffer (pH 6.5) in the absence or presence of NaCl. The activity of LsGDH was enhanced by Li+, Na+, K+, NH4+, Mg2+, and EDTA at pH 8.0. LsGDH exhibited high tolerance to 60% DMSO, 30% acetone, 30% methanol, 30% ethanol, 10% n-propanol, 30% isopropanol, 60% n-hexanol and 30% n-hexane. The relationship between stability and chain length of the alcohols fit a Gaussian distribution model (R2≥0.94), and demonstrated lowest enzyme stability in C4-alcohol. The results suggested that LsGDH was potentially useful for coenzyme regeneration in organic solvents or under alkaline conditions., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

An alkaline active xylanase, XynBYG, was purified from an alkaliphilic Bacillus pumilus BYG, which was newly isolated from paper mill effluent. It had an optimum pH of 8.0-9.0, and showed good stability after incubated at pH 9.0 for 120 min. The optimum temperature for the activity was 50°C, and the enzyme retained below 55% of its original activity for 30 min at 55°C. The gene coding for XynBYG consists of 687 bp and encodes 229 amino acids. Similarity analysis indicated that XynBYG belong to family 11 glycosyl hydrolases. Site-directed mutagenesis was performed to replace five sites (Tyr/Ser) to Arg/Glu and the results demonstrated that the optimum temperature of the mutant Y7 (S39R-T146E) increased 5°C and the half-life of inactivation (T1/2) at 60 and 65°C was 1 h and 25 min, respectively. Thus, it provides a potential xylanase that can meet the harsh conditions in the industrial applications.

This paper describes the purification and characterization of a novel acidophile alpha-amylase from newly isolated Bacillus sp. Ferdowsicous. The enzyme displayed a molecular weight of 53 kDa and it was stable over a range of pH from 3.5 to 7 with an optimum around 4.5. The optimum temperature for activity was found to be around 70 degrees C and the enzyme remained active to more than 75% up to 75 degrees C for 45 min. The enzyme activity was decreased by Zn(2+)and EDTA but inhibited by Hg(2+), whereas the activity was increased by approximately 15% by Ba(2+) and Fe(2+). Na(+), Mg(2+), K(+), Ca(2+), PMSF, Triton X-100 and beta-mercaptoethanol had any considerable effect on its activity. The enzyme activity on the amylose as substrate was 1.98 times greater than amylopectin. Partial N-terminal sequencing demonstrated no significant similarity with other known alpha-amylases, indicating that the presented enzyme was new. Considering its promising properties, this enzyme can find potential applications in the food industry as well as in laundry detergents., (Copyright 2010 Elsevier B.V. All rights reserved.)

An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 degrees C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H(2)O(2), 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P(1) position, in descending order. The k(cat), K(m) and k(cat)/K(m) values for Z-Val-Lys-Met-MCA were 16.8+/-0.14 min(-1), 5.1+/-0.28 microM, and 3.3+/-0.28 microM(-1) min(-1) respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS.

In this study, the objective was to investigate an exponential feeding strategy for fed-batch production of thermostable alpha-amylase (E.C. 3.2.1.1.) from the Bacillus caldolyticus (DSM405). The parameters for establishing compositions of feed media and feeding rate were obtained by statistical analysis of batch and continuous shake flask experiments. These parameters were casitone to starch ratio of 2.67g(casitone)g(starch)(-1), maintenance coefficient 0.174g(casitone)g(DW)(-1)h(-1), cell yield 0.62g(DW)g(casitone)(-1) and mu(opt)=0.2h(-1). The exponentially fed fermentation resulted in yield of 120Uml(-1) alpha-amylase that was thermostable up to 105 degrees C. Results of the exponentially fed fermentation have been discussed in the light of a feed-back controlled fed-batch fermentation reported earlier by the authors. A comparison of the temperature and pH effects on amylase produced by B. caldolyticus and on several other commercially available amylases has also been presented.

A novel hot spring thermophile, Anoxybacillus gonensis A4 (A. gonensis A4) was investigated in terms of capability of tributyrin degradation and characterization of its thermostable esterase activity by the hydrolysis of p-nitrophenyl butyrate (PNPB). It was observed that A. gonensis A4 has an esterase with a molecular weight of 62 kDa. The extracellular crude preparation was characterized in terms of substrate specificity, pH and temperature optima and stability, kinetic parameters and inhibition/activation behaviour towards some chemicals and metal ions. Tributyrin agar assay showed that A. gonensis A4 secreted an esterase and V(max) and K(m) values of its activity were found to be 800 U/L and 176.5 microM, respectively in the presence of PNPB substrate. The optimum temperature and pH, for A. gonensis A4 esterase was 60-80 degrees C and 5.5, respectively. Although the enzyme activity was not significantly changed by incubating crude extract solution at 30-70 degrees C for 1 h, the enzyme activity was fully lost at 80 degrees C for same incubation period. The pH-stability profile showed that original crude esterase activity increased nearly 2-fold at pH 6.0. The effect of some chemicals on crude esterase activity indicated that A. gonensis A4 produce an esterase having serine residue in active site and -SH groups were essential for its activity.

The introduction of disulfide bonds has been used as a strategy to enhance the stability of Bacillus circulans xylanase. The transition temperature of the S100C/N148C (DS1), V98C/A152C (DS2), and A1GC/G187,C188 (cXl) in comparison to the wild type was increased by 5.0, 4.1 and 3.8 degrees C, respectively. Interestingly, a combination of two disulfide bonds of DS1 and cXl (cDS1, circular disulfide 1) led to a 12 degrees C increase in the transition temperature. Importantly, an increase in the melting point and DeltaDeltaG values of the cDS1 mutant was cooperative. These results suggest that the mechanism of stabilization by disulfide bonds under irreversible denaturation condition is achieved through: (1) a change in the rate-limiting step on the denaturation pathway; (2) destabilizing the unfolded state without affecting the relative rate constants on the denaturation pathway (like cXl mutant); and (3) or combination of the two (cDS1 mutant).

Chemical cross-linking of a mesophilic alpha-amylase from Bacillus amyloliquefaciens (BAA) was carried out. Intra-molecular cross-links between lysine residues upon treatment of the enzyme with ethylene glycol bis(succinic acid N-hydroxy succinimide ester) resulted in enhancement of thermostability as indicated by irreversible thermoinactivation experiments. Enhancement of thermostability coincided with a dramatic protection against aggregation, combined with a decrease in surface hydrophobicity. Deamidation, another important mechanism of irreversible thermoinactivation, was also diminished upon modification. While no significant changes in the kinetic parameters are evident, rigidification of the protein structure is suggested by circular dichroism (CD) and fluorescence studies.

Bacillus licheniformis MB-2, isolated from a hot spring water in Manado, Indonesia, secreted a unique chitosanase. Media consisted of 0.24% chitosan, 0.25% casiton, 1% MgSO4, 1.4% K2HPO4, 0.02% CaCl2 x 2H2O, 0.002% FeSO4 x 7H2O (w/v) was used for enzyme production. Purification of the enzyme through the hydrophobic interaction chromatography system (butyl Sepharose 4 FF) resulted in two major active fractions; the F2 fraction was shown as a single band at both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis with apparent molecular mass of 75 kDa. The enzyme worked best at 70 degrees C and pH between 6.0 and 7.0. When incubated at 70, 80, and 90 degrees , the t(1/2) values were 26.56, 18.44, and 16.74 min, respectively with the k constant being at 0.026, 0.037, and 0.04/min. When heated at 90 degrees C, the enzyme retained its activity up to 8 h in the presence of 1 mM MnCl2. The enzyme's activity was unaffected by the presence of 1 M NaCl and 6 M urea but was decreased by 2 M of guanidine hydrochloride. Albeit the enzyme did not degrade colloidal and glycol chitin, it hydrolyzed glycol chitosan up to 0.8% and colloidal chitosan up to 11%. The 85% deacetylated (DDA) soluble chitosan was the most susceptible to this enzyme, followed by 90% and 100% DDA chitosan. The K(m app) values of the 85, 90, and 100% DDA soluble chitosans were found as 0.23, 0.24, and 0.58 mg/mL, whereas the Vmax values were 843, 668, and 261 U/mg, respectively. The hydrolysis products of F2 chitosanase at 24 h incubation (70 degrees C) were pentasaccharide (GlcN)5 and hexasaccharide (GlcN)6. The preliminary test showed inhibitory effect of chitooligosaccharides resulted from enzymatic degradation toward Pseudomonas aeruginosa, Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus, Escherichia coli, and Staphylococcus aureus.

A gene (apr 46) encoding a protease was cloned from Bacillus licheniformis RSP-09-37. It had an ORF of 1725 bp, encoding a pre-protein of 575 amino acids (63.2 kDa), which was functionally expressed and processed in E. coli JM 109. The mature protein, Apr 46, consists of 500 amino acids with a calculated molecular mass of 55 kDa. This protease shows 29-50% homology to known serine proteases and conserved domains. N-terminal sequencing suggests that Apr 46 protease is identical to a B. licheniformis RSP-09-37 protease, which is further supported by a similar stability in acetonitrile.

We demonstrated for the first time that alkylhydroxybenzenes (the d1 microbial autoregulatory factors involved in stress responses of cells) are capable of stabilizing enzymes in aqueous media and increasing their catalytic activity. The stabilizing effect of a chemical analogue of alkylhydroxybenzenes, C7-AHB, was established in in vitro studies with enzymes of microbial origin: a protease produced by Bacillus licheniformis, cellulase produced by Trichoderma viride, and alpha-amylase produced by Bacillus subtilis. This effect manifested itself in considerable extension of the temperature and pH ranges of the enzymatic activity. The modulation of the catalytic activities of the stabilized enzymes depended on the C7-AHB concentration and on the time of preincubation of the complexes obtained. We demonstrated that not only enzymes but also their polymeric substrates formed complexes with C7-AHB, and this also significantly influenced the efficiency of hydrolytic reactions. We also conducted comparative studies on the efficiency of hydrolytic reactions in systems in which the structure of enzymes and/or substrates was modified with C7-AHB.