An acidic beta-mannanase from Penicillium sp C6: gene cloning and over-expression in Pichia pastoris

作者：Cai, Hongying; Shi, Pengjun; Huang, Huoqing; Luo, Huiying; Bai, Yingguo; Yang, Peilong; Meng, Kun; Yao, Bin^*

来源：World Journal of Microbiology and Biotechnology, 2011, 27(12): 2813-2819.

DOI：10.1007/s11274-011-0759-6

• Penicillium sp C6
• beta-Mannanase
• Pichia pastoris
• Over-expression

Using degenerate polymerase chain reaction (PCR), thermal asymmetric interlaced (TAIL)-PCR, and reverse transcription (RT)-PCR techniques, a new beta-mannanase gene, denoted as man5C6, was obtained from Penicillium sp. C6. The gene has an open reading frame of 1,155 bp, and codes for a polypeptide (Man5C6) of 384 amino acids including a putative 26-residue signal peptide. The deduced amino acid sequence showed highest identity (59.2%) with an experimentally verified beta-mannanase from Podospora anserine belonging to glycoside hydrolase family 5. Man5C6 was successfully expressed in Pichia pastoris, and secreted up to 2.5 g in 1 l medium. Recombinant Man5C6 was easily purified to electrophoretic homogeneity by a sing-step chromatography. The purified recombinant enzyme exhibited optimal activity at pH 4.5, and remained > 55% of its maximum activity at pH 3.0-7.0. The temperature optimum was found to be 70A degrees C. The specific activity, and K (m) and V (max) values were 226.5 U mg(-1), 12.3 mg ml(-1) and 2,400.2 mu mol min(-1) mg(-1), respectively, for locust bean gum, and 78.7 U mg(-1), 0.2 mg ml(-1) and 894.6 mu mol min(-1) mg(-1), respectively, for konjac flour. These properties make Man5C6 a potential candidate for high-level production of beta-mannanase with low cost and simple processing technology.

• 出版日期2011-12
• 单位中国农业科学院