Increasing The Thermostability Of CRL1 By Protein Semi-rational Design

? Candida rugosa lipase 1 (CRL1)is one of the most efficient catalyst in hydrolizations, stereoselective transformations and polyester synthesis in the industries of dairy foods, chemicals, pharmaceutical. . As a mesophilic lipase, CRL1 has contradiction in excellent catalysis activity and low thermostability which limit it practical application.Enzymes are subjected to three approaches to optimize the themostability in protein engineering: directed evolution, rational design, semi-rational design. The approaches of directed evolution and rational design both have advantage and disadvantage. The rational design usually has high efficiency but demands detailed understanding the structure information, catalytic mechanism and the structure-function relationship while the directed evolution needs high-though put screening in large mutant library. The semi-rational design can avoid the disadvantage of these two approaches by adding the rational element to directed evolution in order to limit the library to specified sites.In our study, we used the semi-rational design approach to increase the thermostability. This study was based on crystal structure analysis of CRL1 (wild type) and the atomic displacement parameters with the name of B-factor obtained from X-ray data. The B-factor was a parameter reflecting the smearing of atomic electron densities from their equilibrium positions as a result of thermal motion and positional disorder, corresponding to the parameter of flexibility. The residues with high B-factor were identified as suitable target sites along with the position and function of the target sites. The residues near the catalytic triad always change its native conformation before the entirety protein. So we focus on the residues within 10 ? from the catalytic triad Ser209.The B-factor and rational design method were used to predict the mutant sites and Leu302 and Glu126 were chose as the key amino acids to enhance the stability of CRL1.In the next step, the two sites will be mutated using saturation mutagenesis, respectively.