Abstract
The β-mannanase gene (1,029 nucleotide) from Bacillus subtilis MAFIC-S11, encoding a polypeptide of 342 amino acids, was cloned and expressed in Pichia pastoris. To increase its expression, the β-mannanase gene was optimized for codon usage (mannS) and fused downstream to a sequence-encoding modified α-factor signal peptide. The expression level was improved by 2-fold. This recombinant enzyme (mannS) showed its highest activity of 24,600 U/mL after 144-h fermentation. The optimal temperature and pH of mannS were 50 °C and 6.0, respectively, and its specific activity was 3,706 U/mg. The kinetic parameters V max and K m were determined as 20,000 U/mg and 8 mg/mL, respectively, representing the highest ever expression level of β-mannanase reported in P. pastoris. In addition, the enzyme exhibited much higher binding activity to chitin, chitosan, Avicel, and mannan. The superior catalytic properties of mannS suggested great potential as an effective additive in animal feed industry.
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This work was supported by the National Natural Science Foundation of PR China (No. 31072061) and the Scientific and Technical Supporting Programs (2013BAD10B01 and 2011BAD26B02).
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Lv, J., Chen, Y., Pei, H. et al. Cloning, Expression, and Characterization of β-mannanase from Bacillus subtilis MAFIC-S11 in Pichia pastoris . Appl Biochem Biotechnol 169, 2326–2340 (2013). https://doi.org/10.1007/s12010-013-0156-8
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DOI: https://doi.org/10.1007/s12010-013-0156-8