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

• 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
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-007-1287-z

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.