The Experts below are selected from a list of 345 Experts worldwide ranked by ideXlab platform
Rajni Hattikaul - One of the best experts on this subject based on the ideXlab platform.
-
crystal structure of an alkaline serine protease from Nesterenkonia sp defines a novel family of secreted bacterial proteases
Proteins, 2008Co-Authors: Na Yang, Jie Nan, Erik Brostromer, Rajni HattikaulAbstract:Crystal structure of an alkaline serine protease from Nesterenkonia sp. defines a novel family of secreted bacterial proteases Na Yang, Jie Nan, Erik Brostromer, Rajni Hatti-Kaul, and Xiao-Dong Su* 1 Shenzhen Graduate School of Peking University, Shenzhen 518055, China 2 National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, China 3 Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, S-221 00 Lund, Sweden
-
Nesterenkonia aethiopica sp nov an alkaliphilic moderate halophile isolated from an ethiopian soda lake
International Journal of Systematic and Evolutionary Microbiology, 2006Co-Authors: Osvaldo Delgado, Jorge Quillaguaman, Shahrzad Bakhtiar, Bo Mattiasson, Amare Gessesse, Rajni HattikaulAbstract:T Strain DSM 17733(T), isolated from the shore of Lake Abjata in Ethiopia, is a heterotrophic, alkaliphilic, moderately halophilic, Gram-positive, strictly aerobic, non-motile, non-endospore-forming bacterium. The organism grows optimally at 30-37 degrees C, pH 9 and 3 % (w/v) NaCl. Analysis of the cell wall showed the presence of murein of the type L-Lys-Gly-L-Glu, variation A4 alpha. The G + C content of the genomic DNA was 69(.)0 mol%. Sequence analysis of 16S rRNA gene sequence of strain DSM 17733(T) placed the isolate in the genus Nesterenkonia. DNA-DNA hybridization of DSM 17733(T) with those organisms with the closest phylogenetic affiliation, i.e. Nesterenkonia halobia, Nesterenkonia facusekhoensis and Nesterenkonia xinjiangensis, gave relatedness values of 48.5 %, 63(.)7 % (repetition, 57(.)2 %) and 35(.)7 % (repetition, 29(.)3 %), respectively. On the basis of both phenotypic and phylogenetic criteria and the low levels of DNA-DNA relatedness with the phylogenetically closest species N. xinjiangensis and N. halobia, it is proposed that the isolate be classified in a novel species, Nesterenkonia aethiopica sp. nov. The type strain is DSM 17733(T) (=CCUG 48939(T)). (Less)
-
substrate specificity of alkaline protease from alkaliphilic feather degrading Nesterenkonia sp al20
Enzyme and Microbial Technology, 2005Co-Authors: Shahrzad Bakhtiar, Rui Jose Estiveira, Rajni HattikaulAbstract:The substrate specificity of an alkaline protease, produced by Nesterenkonia sp. AL20 grown on chicken feather as the nutrient source, was assessed using oxidized insulin B-chain and derivatized peptide substrates. The initial cleavage of the. insulin chain was determined to be at Tyr(16) -Leu(17) and Tyr(26) -Thr(27), followed by Gln(4)-His(5), Phe(25)-Tyr(26) and Leu(15)-Tyr(16) bonds. An additional cleavage site at Ser(9)-His(10) was found during hydrolysis for a long time. Among the peptide substrates, the enzyme exhibited activity mainly with tetrapeptide substrates with hydrophobic residues located at P1 site in the order Tyr > Phe > Len. Decreasing the size of the peptide resulted in a drastic reduction of activity, suggesting the enzyme to possess relatively narrow substrate specificity in comparison to several other serine proteases. AL20 protease showed good activity towards casein and hemoglobin as substrates, low activity with keratin azure, and poor activity with elastin-orcein. (c) 2005 Elsevier Inc. All rights reserved. (Less)
-
stability characteristics of a calcium independent alkaline protease from Nesterenkonia sp
Enzyme and Microbial Technology, 2003Co-Authors: Shahrzad Bakhtiar, Bo Mattiasson, Amare Gessesse, Maria M Andersson, Rajni HattikaulAbstract:Thermodynamic stability of an alkaline protease from a new alkaliphilic Nesterenkonia sp. AL-20, was investigated and compared with that of Subtilisin Carlsberg. The amount of calcium bound to the AL-20 protease was determined to be only about 0.14 mol/mol of protease. Differential scanning calorimetry scan of the enzyme at increasing temperature showed the denaturation of the enzyme to be a two-state process with melting temperature, Tm of about 74 °C at pH 10.0, which was unaltered upon addition of calcium as well as after treatment with chelating agents. The thermodynamic parameters were nearly the same over a pH range of 7.0–10.0. Tm was reduced to 69.7 °C at pH 6.0 and 72 °C at pH 11.0. The secondary structure of the protease was unaffected during storage at 50 °C, even in the presence of 1% SDS as observed by circular dichroism. The protease activity was extremely stable in the presence of hydrogen peroxide and various sequestering agents used in detergents.
-
crystallization and preliminary x ray analysis of an alkaline serine protease from Nesterenkonia sp
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Shahrzad Bakhtiar, Jitka Vevodova, Rajni HattikaulAbstract:A novel calcium-independent serine protease from an alkaliphilic bacterium, Nesterenkonia sp. AL20, has been purified and crystallized at 296 K using sodium formate as the main precipitant. This enzyme is optimally active at pH 10, exhibits high stability towards autolytic digestion and its stability is not affected by the presence of EDTA or detergents. The triangular prism-shaped crystals diffracted X-rays to beyond 1.5 A at a synchrotron beamline, with space group R3 and unit-cell parameters a = b = 92.26, c = 137.88 A. A complete data set has been collected to 1.39 A resolution. The asymmetric unit is estimated and confirmed by self-rotation function calculation to contain two molecules, giving a crystal volume per protein mass (VM) of 2.68 A3 Da-1 and a solvent content of 54%. (Less)
Mohammad Ali Amoozegar - One of the best experts on this subject based on the ideXlab platform.
-
Purification and characterization of a halophilic α-amylase with increased activity in the presence of organic solvents from the moderately halophilic Nesterenkonia sp. strain F
Extremophiles, 2012Co-Authors: Mohammad Shafiei, Abed-ali Ziaee, Mohammad Ali AmoozegarAbstract:An extracellular halophilic α-amylase was purified from Nesterenkonia sp. strain F using 80 % ethanol precipitation and Q-Sepharose anion exchange chromatography. The enzyme showed a single band with an apparent molecular weight of 110 kDa by SDS-PAGE. The amylase exhibited maximal activity at pH 7–7.5, being relatively stable at pH 6.5–7.5. Optimal temperature for the amylase activity and stability was 45 °C. The purified enzyme was highly active in the broad range of NaCl concentrations (0–4 M) with optimal activity at 0.25 M NaCl. The amylase was highly stable in the presence of 3–4 M NaCl. Amylase activity was not influenced by Ca^2+, Rb^+, Li^+, Cs^+, Mg^2+ and Hg^2+, whereas Fe^3+, Cu^2+, Zn^2+ and Al^3+ strongly inhibited the enzyme activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. K _m value of the amylase for soluble starch was 6.6 mg/ml. Amylolytic activity of the enzyme was enhanced not only by 20 % of water-immiscible organic solvents but also by acetone, ethanol and chloroform. Higher concentration (50 %) of the water-miscible organic solvents had no significant effect on the amylase activity. To the best of our knowledge, this is the first report on increased activity of a microbial α-amylase in the presence of organic solvents.
-
draft genome sequence of Nesterenkonia sp strain f isolated from aran bidgol salt lake in iran
Journal of Bacteriology, 2011Co-Authors: Sajjad Sarikhan, Reza Azarbaijani, Laleh Parsa Yeganeh, Abolhassan Shahzadeh Fazeli, Mohammad Ali Amoozegar, Ghasem Hosseini SalekdehAbstract:ABSTRACT The draft genome of the aerobic, Gram-positive, halophilic chemoorganotroph Nesterenkonia sp. strain F consists of a 2,812,133-bp chromosome. This study is the first to report the shotgun-sequenced draft genome of a member of the genus Nesterenkonia.
-
Purification and characterization of an organic-solvent-tolerant halophilic α-amylase from the moderately halophilic Nesterenkonia sp. strain F
Journal of Industrial Microbiology & Biotechnology, 2011Co-Authors: Mohammad Shafiei, Abed-ali Ziaee, Mohammad Ali AmoozegarAbstract:A halophilic α-amylase produced by Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange, and Sephacryl S-200 gel filtration chromatography. The purified amylase exhibited specific activity of 357 unit/mg protein that corresponds to twofold purification. The molecular mass of the amylase was determined to be 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The optimal pH and temperature for enzyme activity were 6.5 and 45°C, respectively. The amylase was active over a wide range of salt concentrations (0–4 M) with maximum activity at 0.75–1 M NaCl. The α-amylase activity was stimulated by Ca^2+ and inhibited by ethylenediamine tetraacetic acid (EDTA), suggesting that this enzyme is a metalloenzyme. The purified enzyme showed remarkable stability towards surfactants (Tween 20, Tween 80, and Triton X-100), and its activity was increased by β-mercaptoethanol. The halophilic α-amylase was stable in the presence of various organic solvents such as benzene, chloroform, toluene, and cyclohexane. These properties indicate wide potential applications of this α-amylase in starch-processing industries.
-
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
Process Biochemistry, 2010Co-Authors: Mohammad Shafiei, Abed-ali Ziaee, Mohammad Ali AmoozegarAbstract:Abstract 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. Fe 3+ , Cu 2+ , Zn 2+ and Al 3+ strongly inhibited the enzyme, whereas Ca 2+ 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.
-
evaluation of hexavalent chromium reduction by chromate resistant moderately halophile Nesterenkonia sp strain mf2
Process Biochemistry, 2007Co-Authors: Mohammad Ali Amoozegar, Ali Ghasemi, Mohammad Reza Razavi, Saied Reza NaddafAbstract:Abstract A Gram-positive moderately halophilic chromate reducing bacterial strain was isolated from effluents of tanneries, and identified as Nesterenkonia sp. strain MF2 by phenotypic characterization and 16S rRNA analysis. The strain could tolerate up to 600 mM of chromate and completely reduced 0.2 mM highly toxic and soluble Cr(VI) (as CrO42−) into almost non-toxic and insoluble Cr(III) in 24 h under aerobic condition. The maximum chromate removal was exhibited in 1.5 M NaCl at 35 °C and pH 8.0. Initial Cr(VI) concentration until 0.4 mM did not have a significant effect on Cr(VI) reduction. The isolate was capable of chromate reduction in the presence of various concentrations of salts. The chromate reduction corresponded with growth of bacteria and reached a maximum level at the end of exponential phase.
Shahrzad Bakhtiar - One of the best experts on this subject based on the ideXlab platform.
-
Nesterenkonia aethiopica sp nov an alkaliphilic moderate halophile isolated from an ethiopian soda lake
International Journal of Systematic and Evolutionary Microbiology, 2006Co-Authors: Osvaldo Delgado, Jorge Quillaguaman, Shahrzad Bakhtiar, Bo Mattiasson, Amare Gessesse, Rajni HattikaulAbstract:T Strain DSM 17733(T), isolated from the shore of Lake Abjata in Ethiopia, is a heterotrophic, alkaliphilic, moderately halophilic, Gram-positive, strictly aerobic, non-motile, non-endospore-forming bacterium. The organism grows optimally at 30-37 degrees C, pH 9 and 3 % (w/v) NaCl. Analysis of the cell wall showed the presence of murein of the type L-Lys-Gly-L-Glu, variation A4 alpha. The G + C content of the genomic DNA was 69(.)0 mol%. Sequence analysis of 16S rRNA gene sequence of strain DSM 17733(T) placed the isolate in the genus Nesterenkonia. DNA-DNA hybridization of DSM 17733(T) with those organisms with the closest phylogenetic affiliation, i.e. Nesterenkonia halobia, Nesterenkonia facusekhoensis and Nesterenkonia xinjiangensis, gave relatedness values of 48.5 %, 63(.)7 % (repetition, 57(.)2 %) and 35(.)7 % (repetition, 29(.)3 %), respectively. On the basis of both phenotypic and phylogenetic criteria and the low levels of DNA-DNA relatedness with the phylogenetically closest species N. xinjiangensis and N. halobia, it is proposed that the isolate be classified in a novel species, Nesterenkonia aethiopica sp. nov. The type strain is DSM 17733(T) (=CCUG 48939(T)). (Less)
-
substrate specificity of alkaline protease from alkaliphilic feather degrading Nesterenkonia sp al20
Enzyme and Microbial Technology, 2005Co-Authors: Shahrzad Bakhtiar, Rui Jose Estiveira, Rajni HattikaulAbstract:The substrate specificity of an alkaline protease, produced by Nesterenkonia sp. AL20 grown on chicken feather as the nutrient source, was assessed using oxidized insulin B-chain and derivatized peptide substrates. The initial cleavage of the. insulin chain was determined to be at Tyr(16) -Leu(17) and Tyr(26) -Thr(27), followed by Gln(4)-His(5), Phe(25)-Tyr(26) and Leu(15)-Tyr(16) bonds. An additional cleavage site at Ser(9)-His(10) was found during hydrolysis for a long time. Among the peptide substrates, the enzyme exhibited activity mainly with tetrapeptide substrates with hydrophobic residues located at P1 site in the order Tyr > Phe > Len. Decreasing the size of the peptide resulted in a drastic reduction of activity, suggesting the enzyme to possess relatively narrow substrate specificity in comparison to several other serine proteases. AL20 protease showed good activity towards casein and hemoglobin as substrates, low activity with keratin azure, and poor activity with elastin-orcein. (c) 2005 Elsevier Inc. All rights reserved. (Less)
-
a thermostable alkaline protease from a new alkaliphilic Nesterenkonia sp
2004Co-Authors: Shahrzad BakhtiarAbstract:This thesis concerns a novel alkaline protease produced by an extremophilic microbial specie, designated as AL20, isolated from a feather sample collected at the shore of the alkaline soda lake Abjata in the Ethiopian Rift Valley. The isolate is a heterotrophic, alkaliphilic, halotolerant gram-positive, strictly aerobic, non-motile, non-spore forming bacterium. The organism grew at 37°C, pH 10, and 1M NaCl. Comparison of the 16S rDNA sequence showed that the organism was phylogenetically closely related to Nesterenkonia species, although differences in G+C content and low DNA-DNA hybridisation demonstrated that the organism represents a new species, which was named Nesterenkonia abyssinica. The alkaline protease was produced by the organism in alkaline medium (pH 10), using feather as the only sole source of carbon and nitrogen. The enzyme has a molecular mass of 22 876±5 Da and isoelectric point of 4.2. The enzyme was optimally active at 70 °C and over a broad pH range (7-11). The amount of calcium bound to the AL20 protease was determined to be only about 0.14 mol/mol of protease. The thermal unfolding of the enzyme was consistent with the classical two-state model in which the enzyme unfolded at about 74 °C and pH 10. The midpoint of thermal unfolding (Tm) was unaltered upon addition of calcium as well as after treatment with chelating agents. The thermodynamic parameters were nearly the same over a pH range of 7-10. The secondary structure of the AL20 enzyme, apparently a mixture of a-helix and b-sheet, remained intact after incubation for 24 h at 50 oC, and in the presence of 1 % SDS as observed by circular dichroism. The enzyme exhibited unusual stability in the presence of hydrogen peroxide and various sequestering agents used in detergents. The enzyme was found to be highly active against larger peptide substrates containing aromatic or hydrophobic residues e.g. Phe, Tyr, Leu at the P1 site and neutral amino acid e.g Pro, Val at the P2 site. With the oxidized insulin B-chain as substrate, the bonds initially cleaved by the enzyme were Tyr16-Leu17 and Tyr26-Thr27 followed by Gln4-His5, Phe25-Tyr26 and Leu15-Tyr16 bonds. An additional cleavage site at Ser9-His10 was found during hydrolysis for long time. The enzyme hydrolysed casein and hemoglobin efficiently. It also showed keratin hydrolysing activity and was able to hydrolyse elastin-orcein to a detectable level. Finally, the enzyme was subjected to crystallization, X-ray analysis of the triangular prism-shaped crystals diffracted beyond 1.5 A. A complete data set to 1.39 A resolution was collected. (Less)
-
stability characteristics of a calcium independent alkaline protease from Nesterenkonia sp
Enzyme and Microbial Technology, 2003Co-Authors: Shahrzad Bakhtiar, Bo Mattiasson, Amare Gessesse, Maria M Andersson, Rajni HattikaulAbstract:Thermodynamic stability of an alkaline protease from a new alkaliphilic Nesterenkonia sp. AL-20, was investigated and compared with that of Subtilisin Carlsberg. The amount of calcium bound to the AL-20 protease was determined to be only about 0.14 mol/mol of protease. Differential scanning calorimetry scan of the enzyme at increasing temperature showed the denaturation of the enzyme to be a two-state process with melting temperature, Tm of about 74 °C at pH 10.0, which was unaltered upon addition of calcium as well as after treatment with chelating agents. The thermodynamic parameters were nearly the same over a pH range of 7.0–10.0. Tm was reduced to 69.7 °C at pH 6.0 and 72 °C at pH 11.0. The secondary structure of the protease was unaffected during storage at 50 °C, even in the presence of 1% SDS as observed by circular dichroism. The protease activity was extremely stable in the presence of hydrogen peroxide and various sequestering agents used in detergents.
-
crystallization and preliminary x ray analysis of an alkaline serine protease from Nesterenkonia sp
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Shahrzad Bakhtiar, Jitka Vevodova, Rajni HattikaulAbstract:A novel calcium-independent serine protease from an alkaliphilic bacterium, Nesterenkonia sp. AL20, has been purified and crystallized at 296 K using sodium formate as the main precipitant. This enzyme is optimally active at pH 10, exhibits high stability towards autolytic digestion and its stability is not affected by the presence of EDTA or detergents. The triangular prism-shaped crystals diffracted X-rays to beyond 1.5 A at a synchrotron beamline, with space group R3 and unit-cell parameters a = b = 92.26, c = 137.88 A. A complete data set has been collected to 1.39 A resolution. The asymmetric unit is estimated and confirmed by self-rotation function calculation to contain two molecules, giving a crystal volume per protein mass (VM) of 2.68 A3 Da-1 and a solvent content of 54%. (Less)
Peter Hirsch - One of the best experts on this subject based on the ideXlab platform.
-
International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1145–1150 DOI: 10.1099/ijs.0.02118-0 Nesterenkonia lacusekhoensis sp. nov., isolated from hypersaline Ekho Lake, East Antarctica, and emended description of the genus
2015Co-Authors: Dsmz Deutsche, B J Tindall, Norbert Weiss, Sammlung Von, Peter HirschAbstract:e-mail: phirsch!ifam.uni-kiel.de An aerobic and heterotrophic isolate, designated IFAM EL-30T, was obtained from hypersaline Ekho Lake (Vestfold Hills, East Antarctica). The isolate consisted of Gram-positive cocci or short rods which occasionally exhibited branching. The organism was moderately halotolerant, required thiamin.HCl and was stimulated by biotin and nicotinic acid. It grew well with glucose, acetate, pyruvate, succinate, malate or glutamate, and hydrolysed DNA but not gelatin, starch or Tween 80. Nitrate was aerobically reduced to nitrite. Chemical analysis revealed diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and an unidentified glycolipid as the major polar lipids. The cellular fatty acids were predominantly of the anteiso and iso methyl-branched types, and the major menaquinones were MK-7 and MK-8. The peptidoglycan type was A4a, L-Lys-L-Glu. The DNA base ratio was 66<1 mol% GMC. Comparisons of 16S rRNA gene sequences showed that the unidentified organism was phylogenetically closely related to Nesterenkonia halobia, although a sequence divergence value of S3 % demonstrated that the organism represents a different species. On the basis of phenotypic and genotypic evidence, it is proposed that the unknown bacterium be designated as a new species of the genus Nesterenkonia, namely Nesterenkonia lacusekhoensis sp. nov., the type strain being IFAM EL-30T (flDSM 12544T flCIP 107030T). An emended description of the genus Nesterenkonia is given
-
Nesterenkonia lacusekhoensis sp nov isolated from hypersaline ekho lake east antarctica and emended description of the genus Nesterenkonia
International Journal of Systematic and Evolutionary Microbiology, 2002Co-Authors: Matthew D Collins, Paul A Lawson, Matthias Labrenz, B J Tindall, Norbert Weiss, Peter HirschAbstract:An aerobic and heterotrophic isolate, designated IFAM EL-30T, was obtained from hypersaline Ekho Lake (Vestfold Hills, East Antarctica). The isolate consisted of Gram-positive cocci or short rods which occasionally exhibited branching. The organism was moderately halotolerant, required thiamin.HCI and was stimulated by biotin and nicotinic acid. It grew well with glucose, acetate, pyruvate, succinate, malate or glutamate, and hydrolysed DNA but not gelatin, starch or Tween 80. Nitrate was aerobically reduced to nitrite. Chemical analysis revealed diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and an unidentified glycolipid as the major polar lipids. The cellular fatty acids were predominantly of the anteiso and iso methyl-branched types, and the major menaquinone6 were MK-7 and MK-8. The peptidoglycan type was A4alpha, L-Lys-L-Glu. The DNA base ratio was 66.1 mol% G+C. Comparisons of 16S rRNA gene sequences showed that the unidentified organism was phylogenetically closely related to Nesterenkonia halobia, although a sequence divergence value of > 3% demonstrated that the organism represents a different species. On the basis of phenotypic and genotypic evidence, it is proposed that the unknown bacterium be designated as a new species of the genus Nesterenkonia, namely Nesterenkonia lacusekhoensis sp. nov., the type strain being IFAM EL-30T (= DSM 12544T = CIP 107030T). An emended description of the genus Nesterenkonia is given.
Mohammad Shafiei - One of the best experts on this subject based on the ideXlab platform.
-
a novel ph and thermo tolerant halophilic alpha amylase from moderate halophile Nesterenkonia sp strain f gene analysis molecular cloning heterologous expression and biochemical characterization
Archives of Microbiology, 2021Co-Authors: Nastaran Solat, Mohammad ShafieiAbstract:A novel pH and thermo-tolerate halophilic alpha-amylase from moderately halophilic bacterium, Nesterenkonia sp.strain F was cloned and expressed in Escherichia coli. 16S rRNA sequence of the strain shared 99.46% similarities with closely related type species. Also, the genome sequence shared ANI values below 92% and dDDH values below 52% with the closely related type species. Consequently, it is proposed that strain F represents a novel species. The AmyF gene was 1390 bp long and encodes an alpha-amylase of 463 amino acid residues with pI of 4.62. The deduced AmyF shared very low sequence similarity (< 24%) with functionally characterized recombinant halophilic alpha-amylases. The recombinant alpha-amylase was successfully purified from Ni–NTA columns with a molecular mass of about 52 KDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active over a wide range of temperature (25–75 °C) and pH (4–9) with optimum activity at 45 °C and 7.5, respectively. Also, although it was active over a various concentrations of NaCl and KCl (0–4 M), increasing activity of the enzyme was observed with increasing concentration of these salts. Low concentrations of Ca2+ ion had no activating effect, but high concentrations of the ion (40–200 mM) enhanced activity of AmyF. The enzyme activity was increased by increasing concentrations of Mg2+, Zn2+, Hg2+ and Fe3+. However, it was inhibited only at very high concentrations of these metal ions. Cu2+ did not decrease the amylase activity and the highest activity was observed at 100 mM of the ion. These properties indicate wide potential applications of this recombinant enzyme in starch processing industries. This is the first isolation, cloning and characterization of a gene encoding alpha-amylase from Nesternkonia genus.
-
Purification and characterization of a halophilic α-amylase with increased activity in the presence of organic solvents from the moderately halophilic Nesterenkonia sp. strain F
Extremophiles, 2012Co-Authors: Mohammad Shafiei, Abed-ali Ziaee, Mohammad Ali AmoozegarAbstract:An extracellular halophilic α-amylase was purified from Nesterenkonia sp. strain F using 80 % ethanol precipitation and Q-Sepharose anion exchange chromatography. The enzyme showed a single band with an apparent molecular weight of 110 kDa by SDS-PAGE. The amylase exhibited maximal activity at pH 7–7.5, being relatively stable at pH 6.5–7.5. Optimal temperature for the amylase activity and stability was 45 °C. The purified enzyme was highly active in the broad range of NaCl concentrations (0–4 M) with optimal activity at 0.25 M NaCl. The amylase was highly stable in the presence of 3–4 M NaCl. Amylase activity was not influenced by Ca^2+, Rb^+, Li^+, Cs^+, Mg^2+ and Hg^2+, whereas Fe^3+, Cu^2+, Zn^2+ and Al^3+ strongly inhibited the enzyme activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. K _m value of the amylase for soluble starch was 6.6 mg/ml. Amylolytic activity of the enzyme was enhanced not only by 20 % of water-immiscible organic solvents but also by acetone, ethanol and chloroform. Higher concentration (50 %) of the water-miscible organic solvents had no significant effect on the amylase activity. To the best of our knowledge, this is the first report on increased activity of a microbial α-amylase in the presence of organic solvents.
-
Purification and characterization of an organic-solvent-tolerant halophilic α-amylase from the moderately halophilic Nesterenkonia sp. strain F
Journal of Industrial Microbiology & Biotechnology, 2011Co-Authors: Mohammad Shafiei, Abed-ali Ziaee, Mohammad Ali AmoozegarAbstract:A halophilic α-amylase produced by Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange, and Sephacryl S-200 gel filtration chromatography. The purified amylase exhibited specific activity of 357 unit/mg protein that corresponds to twofold purification. The molecular mass of the amylase was determined to be 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The optimal pH and temperature for enzyme activity were 6.5 and 45°C, respectively. The amylase was active over a wide range of salt concentrations (0–4 M) with maximum activity at 0.75–1 M NaCl. The α-amylase activity was stimulated by Ca^2+ and inhibited by ethylenediamine tetraacetic acid (EDTA), suggesting that this enzyme is a metalloenzyme. The purified enzyme showed remarkable stability towards surfactants (Tween 20, Tween 80, and Triton X-100), and its activity was increased by β-mercaptoethanol. The halophilic α-amylase was stable in the presence of various organic solvents such as benzene, chloroform, toluene, and cyclohexane. These properties indicate wide potential applications of this α-amylase in starch-processing industries.
-
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
Process Biochemistry, 2010Co-Authors: Mohammad Shafiei, Abed-ali Ziaee, Mohammad Ali AmoozegarAbstract:Abstract 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. Fe 3+ , Cu 2+ , Zn 2+ and Al 3+ strongly inhibited the enzyme, whereas Ca 2+ 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.
