An acidic β-mannanase from Penicillium sp. C6: gene cloning and over-expression in Pichia pastoris
Autor: | Peilong Yang, Huiying Luo, Yao Bin, Yingguo Bai, Pengjun Shi, Kun Meng, Huoqing Huang, Hongying Cai |
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Rok vydání: | 2011 |
Předmět: |
chemistry.chemical_classification
biology Physiology Glycoside hydrolase family 5 General Medicine Molecular cloning biology.organism_classification Applied Microbiology and Biotechnology Molecular biology Amino acid law.invention Pichia pastoris chemistry.chemical_compound Open reading frame chemistry Biochemistry law Recombinant DNA Locust bean gum Peptide sequence Biotechnology |
Zdroj: | World Journal of Microbiology and Biotechnology. 27:2813-2819 |
ISSN: | 1573-0972 0959-3993 |
Popis: | 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 Km and Vmax 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. |
Databáze: | OpenAIRE |
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