Tan_2014_Appl.Biochem.Biotechnol_173_1752

Cysteine mutants of a cold-active lipase (PcLipI) from Penicillium cyclopium were designed by the software Disulfide by Design Ver. 1.20 in an effort to improve enzyme thermostability by addition of a disulfide bridge. Those mutants predicted by molecular dynamics simulation to have better thermostability than the wild type were first expressed in Escherichia coli BL21(DE3) and then, for further investigation, in Pichia pastoris GS115. By replacing Val(248) and Thr(251) with cysteines to create a disulfide bridge, the recombinant lipases reE-PcLip(V248C-T251C) (expressed in E. coli) and reP-PcLip(V248C-T251C) (expressed in P. pastoris) were obtained. Both had enhanced thermostability with half-lives at 35 degrees C about 4.5- and 12.8-fold longer than that of the parent PcLipI expressed in E. coli and P. pastoris, respectively. The temperature optima of reE-PcLip(V248C-T251C) and reP-PcLip(V248C-T251C) were 35 and 30 degrees C, which were each 5 degrees C higher than those of the parent PcLipI expressed in E. coli and P. pastoris. The K ms of reE-PcLip(V248C-T251C) and reP-PcLip(V248C-T251C) toward tributyrin were 53.2 and 39.5 mM, while their V maxs were 1,460 and 3,800 U/mg, respectively. PcLip(V248C-T251C) had better thermostability and catalytic efficiency than the other mutants and the parent PcLipI.