Hongling Shi

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

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 4612 U/ml. SDS-PAGE and enzyme activity assays demonstrated that the rNCel5A, a glycosylated protein with an M.W. of about 42 kDa, was extracellularly expressed in P. pastoris. The rNCel5A 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.

Determination of amino acids and dipeptides is correlated significantly with optimum temperatures of microbial lipases

Amino acids and dipeptides that are correlated significantly with lipase optimum temperatures were searched for in 34 microbial lipase sequences by a stepwise regression method. The positive dipeptides were found to be IR, KS, NY, SA, ST and YR, whereas negative ones were DK, DY, IS, KA, WS, YS and QI. The calculated optimum temperatures from an optimal regression equation of dipeptides fitted the corresponding experimental optimum temperatures of lipases very well, and the maximal absolute difference was only 3.43°C. The spatial positions of the related dipeptides were searched for in two known crystal structures of a thermophilic and mesophilic lipase, respectively. Most of the positive dipeptides were sited in the α-helices, while the negative ones were located mainly in the β-strands or coils and about half of them existed in the N- or C-terminii of the lipases. The results obtained will be very useful in lipase engineering for enhancing lipase thermostability.