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

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Abstract

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.

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Acknowledgments

The authors would like to thank Dr. Sui-Lam Wong for the supply of plasmid pWB980. Dr. Sui-Lam Wong is a senior medical scholar from University of Calgary. This work was supported by Key Program of Tianjin Science and Technology Development Plan (06YFGPSH03500).

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Authors and Affiliations

  1. Tianjin Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, 300457, Tianjin, People’s Republic of China

    Yi-han Liu, Fu-ping Lu, Yu Li, Xiang-bin Yin & Yi Wang

  2. College of Life Science, Nankai University, 300071, Tianjin, People’s Republic of China

    Chen Gao

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  1. Yi-han Liu
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  2. Fu-ping Lu
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  3. Yu Li
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  4. Xiang-bin Yin
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Correspondence to Fu-ping Lu.

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Liu, Yh., Lu, Fp., Li, Y. et al. Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600. Appl Microbiol Biotechnol 78, 85–94 (2008). https://doi.org/10.1007/s00253-007-1287-z

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  • DOI: https://doi.org/10.1007/s00253-007-1287-z

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