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

• Published in: World journal of microbiology & biotechnology Vol. 27; no. 12; pp. 2813 - 2819
• Main Authors: Cai, Hongying, Shi, Pengjun, Huang, Huoqing, Luo, Huiying, Bai, Yingguo, Yang, Peilong, Meng, Kun, Yao, Bin
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.12.2011; Springer
• Subjects: Applied Microbiology; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Environmental Engineering/Biotechnology; Fundamental and applied biological sciences. Psychology; Life Sciences; Microbiology; Original Paper; Over-expression; β-Mannanase; sp. C6; Ascomycota; Enzyme; Penicillium; Gene overexpression; Pichia pastoris; Fungi; Glycosylases; β-Mannosidase; Glycosidases; Penicillium sp. C6; Hydrolases; Fungi Imperfecti; Molecular cloning
• ISSN: 0959-3993; 1573-0972
• DOI: 10.1007/s11274-011-0759-6

Using degenerate polymerase chain reaction (PCR), thermal asymmetric interlaced (TAIL)-PCR, and reverse transcription (RT)-PCR techniques, a new β-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 β-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 70°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 μmol min −1 mg −1 , respectively, for locust bean gum, and 78.7 U mg −1 , 0.2 mg ml −1 and 894.6 μmol min −1 mg −1 , respectively, for konjac flour. These properties make Man5C6 a potential candidate for high-level production of β-mannanase with low cost and simple processing technology.