Improvement Of Thermostability Of Rhizopus Chinensis Lipase By Rational Design And The Function Of Its Prosequence

Lipase is one of the most widely used enzymes. As an important source of microbiallipases, a variety of Rhizopus lipases have been made into commercial enzymes and used infood, pharmaceutical, chemical, environmental and other industries because of the highcatalytic efficiency. Rhizopus chinensis lipase is a kind of lipase with independent intellectualproperty rights in our laboratory. In the previous studies, we had got some achievements in therespect of stability and heterogenous high-level expression and expanded its range ofindustrial application.In order to improve the thermostability of R. chinensis lipase and get acknowledge of therelationship between structure and function, on one hand, we improved thermostability ofR.chinensis lipase by rational design; on the other hand, the function of prosequence was alsostudied by means of studying the effects of prosequence on R. chinensis lipase’s folding andsecretion in Pichia pastoris. The main results include:(1) Obtained a mutant lipase with improved thermostability. According to homologymodeling and sequence comparing, D190V mutation was introduced into wide-type r27RCLby site-directed mutagenesis, The mutant lipase D190V and wild-type lipase r27RCL wereexpressed in P. pastoris and the enzymatic properties were characterized. The results revealedthat the optimum temperature of D190V was5°C higher than that of the wild-type, thehalf-life(T1/2) of D190V at65°C exceeded that of r27RCL by1-fold, and the Kmdecreased by23.3%. According to the analysis of structures, the reason of improving thermostability for thevariant by only an amino acid substitution D190V was probably due to the improved stabilityof the α-helix located and the strengthened hydrogen bonding force in the protein structure.(2) The function of the prosequence for in vitro folding of lipase was studied, and therefolding difference of integral prosequence lipase(proRCLCNQ)、processed prosequencelipase(r27RCLC) and mature sequence lipase(mRCLC) at28°C and various concentrationsof GSH and GSSG. The results showed that denatured lipases could refold to activeenzymes spontaneously with no added GSH/GSSG, and propeptide enhanced this processobviously. When GSH/GSSG increased from0to2:1(GSSG:2mmol L-1), the data ofrefolding for6h showed that the recovery of proRCLCNQ、r27RCLC and mRCLCincreased from13.6%,23.7%,8.7%to19.7%,74.5%,40.9%respectively. Theimprovement degree(r27RCLC mRCLC proRCLCNQ) revealed that the processedpropeptide largely contributed to the spontaneous recovery at various concentrations ofGSH and GSSG, while the integral propeptide didn’t motivated the recovery at highconcentration of GSH. The region of residues1-67of the propeptide is not essential forthe recovery of lipase, while the region of residues68-94promoted the recovery of lipaseobviously.(3) The effects of prosequence on the expression and secretion of lipase was studied,and the difference of various N-terminal deleted prosequence lipase gene(proRCLC)expressed in P. pastoris was also compared. The results suggested that the region ofresidues1-16of the propeptide is not essential for expression and secretion of the lipase, Deleting it resulted in high-level expression and secretion of lipase. lipase could notexpressed successfully when deleting the whole region of residues1-67.Deleting1-35and1-47affected cells growth, decreased expression and secretion, and resulted in the increaseof extracellular impurity proteins apparently。...