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Appl Biochem Biotechnol. 2013 Apr;169(8):2326-40. doi: 10.1007/s12010-013-0156-8. Epub 2013 Feb 28.

Cloning, expression, and characterization of β-mannanase from Bacillus subtilis MAFIC-S11 in Pichia pastoris.

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1
State Key Laboratory of Animal Nutrition, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, 100193, People's Republic of China.

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.

PMID:
23446982
DOI:
10.1007/s12010-013-0156-8
[Indexed for MEDLINE]
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