Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600

Based on the original thermostable alpha-amylase gene from Bacillus licheniformis , two amino acids were site-directed mutagenised by polymerase chain reaction to obtain a new gene. This gene, with Leu134→Arg and Ser320→Ala, was substituted for acid-resistant capability previously. To favor purifica...

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Published in:Applied microbiology and biotechnology 2008-02, Vol.78 (1), p.85-94
Main Authors: Liu, Yi-han, Lu, Fu-ping, Li, Yu, Yin, Xiang-bin, Wang, Yi, Gao, Chen
Format: Article
Language:English
Subjects:
alpha-Amylases - chemistry
alpha-Amylases - genetics
alpha-Amylases - isolation & purification
alpha-Amylases - metabolism
Amino Acid Sequence
Amino Acid Substitution - genetics
Amino acids
Ammonium
Ammonium Sulfate - metabolism
Animals
Anion exchange
Bacillus licheniformis
Bacillus subtilis
Bacillus subtilis - enzymology
Bacillus subtilis - genetics
Bacillus subtilis - metabolism
Biological and medical sciences
Biotechnologically Relevant Enzymes and Proteins
Biotechnology
Chemical Fractionation
Chromatography, Gel
Chromatography, Ion Exchange
Deoxyribonucleic acid
DNA
Electrophoresis, Polyacrylamide Gel
Enzyme Activators - pharmacology
Enzyme Stability
Enzymes
Fractionation
Fundamental and applied biological sciences. Psychology
Gene expression
Genes
Genetic engineering
Genetic technics
Genetic testing
Hydrogen-Ion Concentration
Kinetics
Life Sciences
Metals - pharmacology
Methods. Procedures. Technologies
Microbial Genetics and Genomics
Microbiology
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Peptides
Plasmids
Polymerase chain reaction
Proteins
Sequence Alignment
Site specific mutagenesis
Studies
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container_issue 1
container_start_page 85
container_title Applied microbiology and biotechnology
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creator Liu, Yi-han
Lu, Fu-ping
Li, Yu
Yin, Xiang-bin
Wang, Yi
Gao, Chen
description Based on the original thermostable alpha-amylase gene from Bacillus licheniformis , two amino acids were site-directed mutagenised by polymerase chain reaction to obtain a new gene. This gene, with Leu134→Arg and Ser320→Ala, was substituted for acid-resistant capability previously. To favor purification of the product, high-level expression and secretion of mature, authentic and stable recombinant mutagenised alpha-amylase were achieved with protease-deficient strain Bacillus subtilis WB600 as the host. The recombinant mutagenised alpha-amylase with the activity of 4,700 U/mL was then purified by ammonium sulphate fractionation, anion exchange and gel filtration, consecutively. By multi-step purification, the specific activity of the recombinant protein was up to 916.7 U/mg with a 187.1-fold purification. The mutagenised protein was found to be more acid resistant than the native protein. The optimum pH and stable range of pH with the mutagenised protein was 4.5 and 4.0 to 6.5, respectively, compared with pH 6.5 and 5.5 to 7.0 as the favorite pH and pH stability range of the native protein.
doi_str_mv 10.1007/s00253-007-1287-z
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This gene, with Leu134→Arg and Ser320→Ala, was substituted for acid-resistant capability previously. To favor purification of the product, high-level expression and secretion of mature, authentic and stable recombinant mutagenised alpha-amylase were achieved with protease-deficient strain Bacillus subtilis WB600 as the host. The recombinant mutagenised alpha-amylase with the activity of 4,700 U/mL was then purified by ammonium sulphate fractionation, anion exchange and gel filtration, consecutively. By multi-step purification, the specific activity of the recombinant protein was up to 916.7 U/mg with a 187.1-fold purification. The mutagenised protein was found to be more acid resistant than the native protein. The optimum pH and stable range of pH with the mutagenised protein was 4.5 and 4.0 to 6.5, respectively, compared with pH 6.5 and 5.5 to 7.0 as the favorite pH and pH stability range of the native protein.</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-007-1287-z</identifier><identifier>PMID: 18157528</identifier><identifier>CODEN: AMBIDG</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>alpha-Amylases - chemistry ; alpha-Amylases - genetics ; alpha-Amylases - isolation &amp; purification ; alpha-Amylases - metabolism ; Amino Acid Sequence ; Amino Acid Substitution - genetics ; Amino acids ; Ammonium ; Ammonium Sulfate - metabolism ; Animals ; Anion exchange ; Bacillus licheniformis ; Bacillus subtilis ; Bacillus subtilis - enzymology ; Bacillus subtilis - genetics ; Bacillus subtilis - metabolism ; Biological and medical sciences ; Biotechnologically Relevant Enzymes and Proteins ; Biotechnology ; Chemical Fractionation ; Chromatography, Gel ; Chromatography, Ion Exchange ; Deoxyribonucleic acid ; DNA ; Electrophoresis, Polyacrylamide Gel ; Enzyme Activators - pharmacology ; Enzyme Stability ; Enzymes ; Fractionation ; Fundamental and applied biological sciences. 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subjects alpha-Amylases - chemistry
alpha-Amylases - genetics
alpha-Amylases - isolation & purification
alpha-Amylases - metabolism
Amino Acid Sequence
Amino Acid Substitution - genetics
Amino acids
Ammonium
Ammonium Sulfate - metabolism
Animals
Anion exchange
Bacillus licheniformis
Bacillus subtilis
Bacillus subtilis - enzymology
Bacillus subtilis - genetics
Bacillus subtilis - metabolism
Biological and medical sciences
Biotechnologically Relevant Enzymes and Proteins
Biotechnology
Chemical Fractionation
Chromatography, Gel
Chromatography, Ion Exchange
Deoxyribonucleic acid
DNA
Electrophoresis, Polyacrylamide Gel
Enzyme Activators - pharmacology
Enzyme Stability
Enzymes
Fractionation
Fundamental and applied biological sciences. Psychology
Gene expression
Genes
Genetic engineering
Genetic technics
Genetic testing
Hydrogen-Ion Concentration
Kinetics
Life Sciences
Metals - pharmacology
Methods. Procedures. Technologies
Microbial Genetics and Genomics
Microbiology
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Peptides
Plasmids
Polymerase chain reaction
Proteins
Sequence Alignment
Site specific mutagenesis
Studies
title Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600
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