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ABSTRACT: A moderately halophilic protease producer, Bacillus sp. strain isolated from sea water is described. The protease is purified to homogeneity by ammonium sulphate precipitation and CM cellulose chromatography. The serine protease has a molecular mass of 29kDa. Enzymatic characterization of protease revealed K(m) 2.22mgmL(-1), V(max) 1111.11UmL(-1), pH optimum 9.0, t(1/2) 190min at 60°C and salt optima 1% (w/v) NaCl. The protease is remarkably stable in hydrophilic and hydrophobic solvents at high concentrations. The purified preparation is unstable at room temperature. Ca(2+) ions are required for preventing this loss of activity. Interestingly, the activity and stability are modulated differentially. Whereas, divalent cation Ca(2+) are involved in maintaining stability in solution at room temperature by preventing unfolding, monovalent Na(+) and K(+) ions participate in regulating the activity and assist in refolding of the enzyme. Application of the protease is shown in efficient removal of blood stain.
Bioresource technology 11/2012; · 4.25 Impact Factor
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Journal of Basic Microbiology 06/2012; · 1.27 Impact Factor
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ABSTRACT: Xylanase from Scytalidium thermophilum was immobilized on Eudragit L-100, a pH sensitive copolymer of methacrylic acid and methyl methacrylate. The enzyme was non-covalently
immobilized and the system expressed 70% xylanase activity. The immobilized preparation had broader optimum temperature of
activity between 55 and 65°C as compared to 65°C in case of free enzyme and broader optimum pH between 6.0 and 7.0 as compared
to 6.5 in case of free enzyme. Immobilization increased the t1/2 of enzyme at 60°C from 15 to 30min with a stabilization factor of 2. The Km and Vmax values for the immobilized and free xylanase were 0.5% xylan and 0.89μmol/ml/min and 0.35% xylan and 1.01μmol/ml/min respectively. An Arrhenius plot showed an increased value of activation energy for immobilized xylanase (227kcal/mol)
as compared to free xylanase (210kcal/mol) confirming the higher temperature stability of the free enzyme. Enzymatic saccharification
of xylan was also improved by xylanase immobilization.
World Journal of Microbiology and Biotechnology 04/2012; 21(6):1123-1128. · 1.53 Impact Factor
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ABSTRACT: Maltooligosaccharides especially maltotriose and maltotetraose producing amylases are highly desirable for application in bread making and other food industries. A maltotriose and maltotetraose producing amylase from moderately halophilic Marinobacter sp. EMB8 is described. Under optimized culture conditions, 48.0 IU/mL amylase was obtained. The enzyme was purified to homogeneity by ultrafiltration, DEAE cellulose and Sephadex G-75 column chromatography with 52% yield and 76-fold purification. It was a monomeric protein of 72 kDa. The amylase had many novel features viz. stability up to 20% NaCl, 80 °C temperature, pH 6.0-11.0 and in wide range of organic solvents at high concentrations. The enzyme efficiently hydrolyzed starch into maltooligosaccharides rich in maltotriose and maltotetraose. These novel properties make the Marinobacter sp. amylase a potentially useful enzyme.
Bioresource technology 12/2011; 116:247-51. · 4.25 Impact Factor
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ABSTRACT: Abstract Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are ubiquitous biocatalysts known to catalyze the hydrolysis of water insoluble triglycerides in aqueous medium and carry out the reverse reaction (synthesis) under organic solvent rich medium. Microbial lipases have received a great deal of attention in the field of food technology, pharmaceutical sciences, chemical and detergent industries due to their stability, selectivity, mild operation conditions and broad substrate specificity. Despite these advantages, low activity and stability displayed in organic medium has restricted their commercial application in organic synthesis. Researchers have explored alternative ways to modify the enzymes making them suitable for use in non-conventional media. In this context, harvesting lipases from “Solvent Tolerant Microbes” has recently become an attractive approach. These microbes are able to grow in the presence of high concentrations of organic solvents, generally known to have detrimental effect on microorganisms. Such microbes survive through novel adaptation mechanisms and secretion of solvent stable enzymes having efficient functionality in solvent-rich media. These enzymes could be useful for bioconversion in non-conventional media. In the current review, this approach is described with an emphasis on characteristics, applications and genetic aspect of lipases from the genus Pseudomonas.
09/2011; 29(5):161-171.
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ABSTRACT: A novel protease from the halophilic bacterium Geomicrobium sp. EMB2 (MTCC 10310) is described. The activity of the protease was modulated by salt, and it exhibited remarkable stability in organic solvents, at alkaline pH, and in other denaturing conditions. The structural changes under various denaturing conditions were analyzed by measurements of intrinsic fluorescence and circular dichroism spectroscopy. Circular dichroism showed that the secondary structure of the protease was predominantly α-helical but unfolded in salt-free medium. The structure is regained by inclusion of NaCl in the range of 2-5%. The presence of NaCl exerted a protective effect against thermal, organic solvent, and guanidine hydrochloride denaturation by preventing unfolding.
Biochemistry (Moscow) 06/2011; 76(6):686-93. · 1.06 Impact Factor
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ABSTRACT: The high activity biocatalyst for use in organic media is termed Protein Coated Micro Crystals (PCMC). These PCMC are formed by the immobilization of protein by adsorption onto solid support i.e. K2SO4. In present study, the PCMC formation was attempted using Pseudomonas aeruginosa lipase and termed PAL PCMC. These novel particles consists of water soluble micron sized crystalline particles coated with the protein. A further study for their thermal stability in organic solvent and their structure analysis by transmission electron microscope (TEM) was done. The PAL PCMC shows very good long-term stability at room temperature and 90% of their initial activity was retained after 12 hrs at 45oC and 60.5 % after 8 days at room temperature when stored in 1-Propano (l/1wt%H2O).
Springer. 01/2011;
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ABSTRACT: Large amount of seed cake is generated as by-product during biodiesel production from Jatropha seeds. Presence of toxic phorbol esters restricts its utilization as livestock feed. Safe disposal or meaningful utilization of this major by-product necessitates the degradation of these phorbol esters. The present study describes the complete degradation of phorbol esters by Pseudomonas aeruginosa PseA strain during solid state fermentation (SSF) of deoiled Jatropha curcas seed cake. Phorbol esters were completely degraded in nine days under the optimized SSF conditions viz. deoiled cake 5.0 g; moistened with 5.0 ml distilled water; inoculum 1.5 ml of overnight grown P. aeruginosa; incubation at temperature 30 °C, pH 7.0 and RH 65%. SSF of deoiled cake seems a potentially viable approach towards the complete degradation of the toxic phorbol esters.
Bioresource technology 01/2011; 102(7):4815-9. · 4.25 Impact Factor
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ABSTRACT: 1 tgcaagtcga gcggatgaag agagcttgct ctctgattca gcggcggacg ggtgagtaat
61 gcctaggaat ctgcctggta gtgggggaca acgtttcgaa aggaacgcta ataccgcata
121 cgtcctacgg gagaaagcag gggaccttcg ggccttgcgc tatcagatga gcctaggtcg
181 gattagctag ttggtgaggt aatggctcac caaggcgacg atccgtaact ggtctgagag
241 gatgatcagt cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg
301 gaatattgga caatgggcga aagcctgatc cagccatgcc gcgtgtgtga agaaggtctt
361 cggattgtaa agcactttaa gttgggagga agggttgtag attaatactc tgcaattttg
421 acgttaccga cagaataagc accggctaac tctgtgccag cagccgcggt aatacagagg
481 ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcgc gtaggtggtt tgttaagttg
541 gatgtgaaag ccccgggctc aacctgggaa ctgcattcaa aactgacaag ctagagtatg
601 gtagagggtg gtggaatttc ctgtgtagcg gtgaaatgcg tagatatagg aaggaacacc
661 agtggcgaag gcgaccacct ggactgatac tgacactgag gtgcgaaagc gtggggagca
721 aacaggatta gataccctgg tagtccacgc cgtaaacgat gtcaactagc cgttgggagc
781 cttgagctct tagtggcgca gctaacgcat taagttgacc gcctggggag tacggccgca
841 aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat
901 tcgaagcaac gcgaagaacc ttaccaggcc ttgacatcca atgaactttc cagagatgga
961 ttggtgcctt cgggaacatt gagacaggtg ctgcatggct gtcgtcagct cgtgtcgtga
1021 gatgttgggt taagtcccgt aacgagcgca acccttgtcc ttagttacca gcacgttatg
1081 gtgggcactc taaggagact gccggtgaca aaccggagga aggtggggat gacgtcaagt
1141 catcatggcc cttacggcct gggctacaca cgtgctacaa tggtcggtac agagggttgc
1201 caagccgcga ggtggagcta atcccataaa accgatcgta gtccggatcg cagtctgcaa
1261 ctcgactgcg tgaagtcgga atcgctagta atcgcgaatc agaatgtcgc ggtgaatacg
1321 ttcccgggcc ttgtacacac cgcccgtcac accatgggag tgggttgcac cagaagtagc
1381 tagtctaa
GenBank. 01/2011; JF508446.1.
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ABSTRACT: Jatropha curcas is a major biodiesel crop. Large amount of deoiled cake is generated as by-product during biodiesel production from its seeds. Deoiled J. curcas seed cake was assessed as substrate for the production of xylanase from thermophilic fungus Scytalidium thermophilum by solid-state fermentation. The seed cake was efficiently utilized by S. thermophilum for its growth during which it produced good amount of heat stable extracellular xylanase. The solid-state fermentation conditions were optimized for maximum xylanase production. Under the optimized conditions viz. deoiled seed cake supplemented with 1% oat-spelt xylan, adjusted to pH 9.0, moisture content 1:3 w/v, inoculated with 1×10(6) spores per 5 g cake and incubated at 45 °C, 1455 U xylanase/g deoiled seed cake was obtained. The xylanase was useful in biobleaching of paper pulp. Solid-state fermentation of deoiled cake appears a potentially viable approach for its effective utilization.
Bioresource technology 01/2011; 102(2):1722-6. · 4.25 Impact Factor
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ABSTRACT: The toxicity of two commonly used nanoparticles, silver and zinc oxide on mesophilic and halophilic bacterial cells has been investigated. Enterobacter sp., Marinobacter sp., Bacillus subtilis, halophilic bacterium sp. EMB4, were taken as model systems. The nanotoxicity was more pronounced on Gram negative bacteria. ZnO nanoparticles reduced the growth of Enterobacter sp. by 50%, while 80% reduction was observed in halophilic Marinobacter sp. In case of halophiles, this may be attributed to higher content of negatively charged cardiolipins on their cell surface. Interestingly, bulk ZnO exerted minimal reduction in growth. Ag nanoparticles were similarly cytotoxic. Nanotoxicity towards Gram positive cells was significantly less, possibly due to presence of thicker peptidoglycan layer. The bacterium nanoparticle interactions were probed by electron microscopy and energy dispersive X-ray analysis. The results indicated electrostatic interactions between nanoparticles and cell surface as the primary step towards nanotoxicity, followed by cell morphological changes, increase in membrane permeability and their accumulation in the cytoplasm.
Bioresource technology 01/2011; 102(2):1516-20. · 4.25 Impact Factor
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ABSTRACT: Thirty-eight haloalkaliphilic bacterial strains were isolated from Sambhar Salt Lake, India and screened for their ability to secrete haloalkaliphilic proteases. Among them, a moderately halophilic, mesophilic and alkaliphilic potent strain Geomicrobium sp. EMB2 produced an extracellular protease, which was remarkably stable in organic solvents, salt, surfactants, detergents and alkaline pH. Statistically based experimental designs were applied to study the interactions and optimization of medium constituents for efficient protease production by Geomicrobium sp. EMB2. An overall 20-fold increase in protease production was achieved in the optimized medium (721 U/ml) as compared with the unoptimized medium (37 U/ml). The high production level coupled with novel properties makes it a prospective industrial enzyme. The Geomicrobium sp. EMB2 isolate is deposited in Microbial Type Culture Collection, Chandigarh, India with accession number MTCC 10310.
New Biotechnology 10/2010; 28(2):136-45. · 2.76 Impact Factor
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ABSTRACT: A moderately haloalkaliphilic bacterium, Geomicrobium sp. EMB2, was isolated from the Sambhar Salt Lake located in the western part of India. It secreted an alkaline protease, which was stable and active in the presence of a wide range of organic solvents. The protease was purified by hydrophobic interaction chromatography on Phenyl Sepharose 6 Fast Flow matrix, and a 22.6-fold purification with 51.2% recovery was attained. The apparent molecular mass was estimated to be 38 kDa. The enzyme was stable in the pH range 6.0-12.0, the optimum being 10.0. The Km and Vmax towards casein were found to be 0.10 mM and 526 U/min, respectively. The protease was most active at 50 degrees C. It appeared to be serine type, owing to its sensitivity to phenylmethylsulphonyl fluoride (PMSF). It withstood a range of detergents and surfactants, and exhibited remarkable stability in the presence of solvents having a log P >2. The presence of NaCl or osmolytes exerted a protective effect and further enhanced the stability of the enzyme. These properties make this novel protease potentially useful for catalysis in non-aqueous medium.
Environmental Technology 09/2010; 31(10):1061-72. · 1.41 Impact Factor
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ABSTRACT: Solvent-tolerant bacteria have emerged as a new class of micro-organisms able to grow at high concentrations of toxic solvents. Such bacteria and their solvent-stable enzymes are perceived to be useful for biotransformations in nonaqueous media. In the present study, the solvent-responsive features of a lipase-producing, solvent-tolerant strain Pseudomonas aeruginosa PseA have been investigated to understand the cellular mechanisms followed under solvent-rich conditions.
The solvents, cyclohexane and tetradecane with differing log P-values (3.2 and 7.6 respectively), have been used as model systems. Effect of solvents on (i) the cell morphology and structure (ii) surface hydrophobicity and (iii) permeability of cell membrane have been examined using transmission electron microscopy, atomic force microscopy and other biochemical techniques. The results show that (i) less hydrophobic (low log P-value) solvent cyclohexane alters the cell membrane integrity and (ii) cells adapt to organic solvents by changing morphology, size, permeability and surface hydrophobicity. However, no such changes were observed in the cells grown in tetradecane.
It may be concluded that P. aeruginosa PseA responds differently to solvents of different hydrophobicities. Bacterial cell membrane is more permeable to less hydrophobic solvents that eventually accumulate in the cytoplasm, while highly hydrophobic solvents have lesser tendency to access the membrane.
To the best of our knowledge, these are first time observations that show that way of bacterial solvent adaptability depends on nature of solvent. Difference in cellular responses towards solvents of varying log P-values (hydrophobicity) might prove useful to search for a suitable solvent for carrying out whole-cell biocatalysis.
Letters in Applied Microbiology 10/2009; 49(3):372-7. · 1.62 Impact Factor
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ABSTRACT: Solvent-tolerant microbes are a newly emerging class that possesses the unique ability to thrive in the presence of organic solvents. Their enzymes adapted to mediate cellular and metabolic processes in a solvent-rich environment and are logically stable in the presence of organic solvents. Enzyme catalysis in non-aqueous/low-water media is finding increasing applications for the synthesis of industrially important products, namely peptides, esters, and other trans-esterification products. Solvent stability, however, remains a prerequisite for employing enzymes in non-aqueous systems. Enzymes, in general, get inactivated or give very low rates of reaction in non-aqueous media. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media by immobilization, surface modifications, mutagenesis, and protein engineering. Enzymes from solvent-tolerant microbes appear to be the choicest source for studying solvent-stable enzymes because of their unique ability to survive in the presence of a range of organic solvents. These bacteria circumvent the solvent's toxic effects by virtue of various adaptations, e.g. at the level of the cytoplasmic membrane, by degradation and transformation of solvents, and by active excretion of solvents. The recent screening of these exotic microbes has generated some naturally solvent-stable proteases, lipases, cholesterol oxidase, cholesterol esterase, cyclodextrin glucanotransferase, and other important enzymes. The unique properties of these novel biocatalysts have great potential for applications in non-aqueous enzymology for a range of industrial processes.
Critical Reviews in Biotechnology 02/2009; 29(1):44-54. · 6.47 Impact Factor
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ABSTRACT: Solvent tolerant Pseudomonas aeruginosa strain PseA has been studied for lipase activity. This strain has earlier been reported to be secreting alkaline and solvent stable protease. It produced an extra cellular lipase with suitable properties for detergent applications viz. (i) alkaline in nature, (ii) stability and compatibility towards bleach oxidants, surfactants and detergent formulations and (iii) resistant to proteolysis. Since the culture supernatant contains both protease and lipase which are together required in detergent formulations, enzymes from P. aeruginosa seem ideal for use as detergent additive. P. aeruginosa lipase exhibited remarkable stability in wide range of organic solvents at 25% (v/v) concentration. This property can be useful for solvent bioremediation and biotransformations in non-aqueous media. Media optimization for cost effective production of lipase was carried out by response surface methodology which led to 5.58-fold increase in lipase production (4580 IU/ml) over un-optimized media.
Bioresource Technology 08/2008; 99(11):4796-802. · 4.98 Impact Factor
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ABSTRACT: Deoiled Jatropha seed cake was assessed for its suitability as substrate for enzyme production by solid-state fermentation (SSF). Solvent tolerant Pseudomonas aeruginosa PseA strain previously reported by us was used for fermentation. The seed cake supported good bacterial growth and enzyme production (protease, 1818 U/g of substrate and lipase, 625 U/g of substrate) as evident by its chemical composition. Maximum protease and lipase production was observed at 50% substrate moisture, a growth period of 72 and 120 h, and a substrate pH of 6.0 and 7.0, respectively. Enrichment with maltose as carbon source increased protease and lipase production by 6.3- and 1.6-fold, respectively. Nitrogen supplementation with peptone for protease and NaNO(3) for lipase production also enhanced the enzyme yield reaching 11,376 U protease activity and 1084 U lipase activity per gram of Jatropha seed cake. These results demonstrated viable approach for utilization of this huge biomass by solid-state fermentation for the production of industrial enzymes. This offers significant benefit due to low cost and abundant availability of cake during biodiesel production.
Bioresource Technology 05/2008; 99(6):1729-35. · 4.98 Impact Factor
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ABSTRACT: An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8-13, optimally at 9-11. It was stable with 0-4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 degrees C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25-80 degrees C (optimum at 50 degrees C), the purified enzyme had temperature optimum at 37 degrees C, which shifted to 80 degrees C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K(cat)). The enzyme was also calcium dependent and with 2 mM Ca(+2), the activity reached to maximum at 50 degrees C. The crude enzyme was highly thermostable (37-90 degrees C); however, the purified enzyme lost its stability above 50 degrees C and its half life was enhanced by 30 and sevenfold at 60 degrees C with 1 M NaCl and 50 mM Ca(+2), respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.
Journal of Industrial Microbiology and Biotechnology 03/2008; 35(2):121-31. · 2.73 Impact Factor
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ABSTRACT: We have previously isolated a solvent-stable protease from a novel solvent-tolerant strain of Pseudomonas aeruginosa (PseA). Here we report cloning and characterization of the gene coding for this solvent-tolerant protease. A homology search of the N-terminal amino acid sequence of the purified PseA protease revealed an exact match to a P. aeruginosa PST-01 protease gene, lasB. The c-DNA sequence of the PST-01 protease was used to design primers for the amplification of a 1,494-bp open reading frame encoding a 53.6-kDa, 498-amino-acid PseA LasB polypeptide. The deduced PseA LasB protein contained a 23-residue signal peptide (2.6 kDa) followed by a propeptide of 174 residues and a 33-kDa mature product of 301 residues. A phylogenetic analysis placed PseA lasB closest to the known zinc metalloproteases from P. aeruginosa. This gene was also found to contain a conserved HEXXH zinc-binding motif, characteristic of all zinc metallopeptidases. The 3D structure analysis of PseA protease revealed the presence of 7 alpha-helices (36% of the sequence). The molecule was found to have two disulfide bonds (between Cys-227 and Cys-255 and between Cys-467 and Cys-494) and had a number of hydrophobic clusters at the protein surface. These hydrophobic patches (21% of the sequence) and disulfide bonds may possibly be responsible for the solvent-stable nature of the enzyme.
Journal of Molecular Microbiology and Biotechnology 09/2007; 15(4):234-43. · 1.95 Impact Factor
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ABSTRACT: Three phase partitioning (TPP), a technique used in protein purification has been evaluated, for extraction of oil from three different plant sources viz: mango kernel, soybean and rice bran. The process consists of simultaneous addition of t-butanol (1:1,v/v) and ammonium sulphate (w/v) to a crude preparation/slurry. Under optimized condition, the protein appears as an interfacial precipitate between upper t-butanol containing oil and lower aqueous phase. Pretreatment of the slurries with a commercial enzyme preparation of proteases, Protizyme, followed by three phase partitioning resulted in 98%, 86% and 79% (w/w) oil yields in case of soybean, rice bran and mango kernel, respectively. The efficiency of the present technique is comparable to solvent extraction with an added advantage of being less time consuming and using t-butanol which is a safer solvent as compared to n-hexane used in conventional oil extraction process.
Bioresource Technology 03/2007; 98(3):696-9. · 4.98 Impact Factor
