Cloning and optimized expression of a neutral endoglucanase gene (ncel5A) from Volvariella volvacea WX32 in Pichia pastoris

摘要

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 NCeBA, 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 NCeBA (rNCel5A) activity, numbered as CSNCel4-3, was chosen for optimizing expression conditions. In optimized conditions, the expressed rNCel5A activity was up to 4612 U/ml. SDS-PAGE and enzyme activity assays demonstrated that the rNCeBA, a glycosylated protein with an M.W. of about 42 kDa, was extracellularly expressed in P. pastoris. The rNCeBA showed the highest activity at pH 7.5 and 55°C and was stable at a broad pH range of 6.0-9.0 and at a temperature of 55°C or below.