The Molecular Modification For Enhancing The Thermostability Of Cadida Rugosa Lipase 1

Candida rugosa lipase 1(CRL1) has catalytic ability of hydrolysis, transesterification,and esterification activities with the highest expression levels compared with the other seven isoenzymes. Nowadays, it is widely used in many fields of biomedical(Chiral Separation), food processing and biodiesel because of its unique catalytic properties(such as stereo-selectivity). But as a mesophilic enzyme, CRL1 has poor thermal stability. This disadvantage has seriously hampered its further industrial applications. In this context, the thermal stability sites were predicted to enhance its catalytic performance through comparative modeling using three kinds of computer-assisted servers. Futhermore, the properties of resultant site-directed mutated CRL1 were experimrntally determined.Additionally, the 3-D structure of site-directed mutated CRL1 was preliminary investigated through comparative modeling to elucidate the structural basis of thermal stability. The main work and results were summarized as follows:(1) The eukaryotic vector pPICZ?-N6 × His-CRL1 harboring the optimized gene of CRL1 and six histidine on the N-terminal was constructed, and subsequently transformed into Pichia pastoris GS115 to obtain the recombinant strains GS115/pPICZ?-N6 ×His-CRL1. The secreted target protein CRL1 was purified using nickle column after induced by methanol. Furthermore, the properties of purified CRL1 were characterized.The optimal substrate is p-Nitrophenol butyrate(C4). The optimal temperature of purified CRL1 is 40 °C and the pH is 8.0. Its half-life is 4 min at 50°C.(2) The structural information of CRL1 molecules was obtained using comparative protein computer-assisted modeling servers including Fold X, Rosetta ddG of mutation and I-Mutant 2.0. Temperature mutation sites were predicted to enhance the thermal stability of CRL1. Ultimately, thirteen mutation sites were selected after excluded all functional sites that potential affect the enzyme's catalytic properties by analyzing the active sites of CRL1.(3) Compared to original CRL1, the Tm values of thirteen mutants were changed to some degree. Among them, the thermal stability of the mutant D457 F has been enhanced significantly whose Tm value has improved 9.4°C. Subsequently, the relevant enzymaticproperties of D457 F were investigated. The optimal temperature of mutant D457 F increased by 10°C, and its half-life at 50°C extended 6.5 times when compared with the original CRL1. More interesting, the specific activity has little differences between the mutant D457 F and original CRL1, which indicating that the replacement of residues at457 site has fewer effect on the enzyme's activity.(4) As we all know, the most stable conformation in the protein' secondary structure is ?-helix. Through homology modeling in SWISS-MODEL, we found that the mutant's structure of the ?-helix where 457 sites at has extended, while the introduction of a hydrophobic residue strengthen the internal hydrophobic interaction of this helix. In addition, we suspected that the bulky side chain of the aromatic amino acid(Phe) just filled the gap formed by the small side of Asp which increased the structural rigidity and thereby improved the protein's thermal stability.