Cloning and optimized expression of a neutral endoglucanase gene (ncel5A) from Volvariella volvacea WX32 in Pichia pastoris(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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A cDNA fragment encoding a mature neutral endoglucanase with 366 amino acids was cloned from Volvariella volvacea WX32. Online analysis of amino acid sequence homology showed that the endoglucanase, designated as NCel5A, belongs to glycoside hydrolase family 5. The recombinant plasmid, pPIC9K-ncel5A, was transformed into Pichia pastoris GS115 by electroporation. Screening of multiple copies of the gene ncel5A in transformants was performed on YPD plates containing geneticin G418. One transformant expressing the highest recombinant NCel5A (rNCel5A) activity, numbered as GSNCel4-3, was chosen for optimizing expression conditions. In optimized conditions, the expressed rNCel5A activity was up to 4612U/ml. SDS-PAGE and enzyme activity assays demonstrated that the rNCel5A, a glycosylated protein with an M.W. of about 42kDa, was extracellularly expressed in P. pastoris. The rNCel5A showed the highest activity at pH 7.5 and 55℃ and was stable at a broad pH range of 6.0-9.0 and at a temperature of 55℃ or below.
著者
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Li Jianfang
School Of Food Science And Technology Jiangnan University
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Tang Cunduo
School of Biotechnology, Jiangnan University
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Shi Hongling
School of Medicine and Pharmaceutics, Jiangnan University
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Wu Minchen
School of Medicine and Pharmaceutics, Jiangnan University
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Shi Hongling
School Of Medicine And Pharmaceutics Jiangnan University
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Tang Cunduo
School Of Biotechnology Jiangnan University
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Wu Minchen
School Of Medicine And Pharmaceutics Jiangnan University
関連論文
- Cloning and optimized expression of a neutral endoglucanase gene (ncel5A) from Volvariella volvacea WX32 in Pichia pastoris(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
- Cloning and optimized expression of a neutral endoglucanase gene (ncel5A) from Volvariella volvacea WX32 in Pichia pastoris