Abstract
A gene encoding β-1,3-1,4-glucanase was cloned by polymerase chain reaction (PCR) from Bacillus subtilis MA139. Sequencing result showed 97% homology to the corresponding gene from Bacillus licheniformis. The open reading frame (ORF) of the gene contained 690 bp coding for a 226 amino-acid matured protein with the estimated molecular weight of 24.44 kDa. The β-1,3-1,4-glucanase gene was subcloned into an expression vector of pET28a and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a nickel–nitrilotriacetic acid (Ni–NTA) column. The purified β-1,3-1,4-glucanase demonstrated 24.05 and 12.52 U ml-1 activities for the substrates of barley β-glucan and lichenan, respectively, and the specific activities were 728.79 and 379.1 U mg-1 for them, respectively. The optimal temperature and pH of the purified enzyme were 40°C and 6.4, respectively. When barley β-glucan was used as the substrate, K m was 5.34 mg ml-1, and K cat showed 7,206.71 S-1, thus the ratio of K cat and K m was 1,349.67 ml s-1 mg-1. The activity of β-1,3-1,4-glucanase was affected by a range of metal ions or ethylenediaminetetraacetic acid (EDTA).
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Acknowledgements
We thank Professor Sekman Wong of National University of Singapore for critically reading the manuscript and suggestions.
This work was supported by the National High Technology Research and Development Program of China (2007AA100601) and New Century Excellent Talent in University NCET-07-0807.
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Qiao, J., Dong, B., Li, Y. et al. Cloning of a β-1,3-1,4-Glucanase Gene from Bacillus subtilis MA139 and its Functional Expression in Escherichia coli . Appl Biochem Biotechnol 152, 334–342 (2009). https://doi.org/10.1007/s12010-008-8193-4
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DOI: https://doi.org/10.1007/s12010-008-8193-4