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Increasing the Thermostability of CRL1 by Protein Semi-rational Design

Author: HanYunZuo
Tutor: FengYan
School: Jilin University
Course: Biochemistry and Molecular Biology
Keywords: Fold a Candida lipase Thermal stability B factor
CLC: Q814
Type: Master's thesis
Year: 2011
Downloads: 121
Quote: 0
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Abstract

Fold Candida lipase (Candida rugosa lipase 1, CRL1) are widely used in oil, food, chemical, pharmaceutical and other fields to catalyze the hydrolysis of fats, ester synthesis, transesterification, chiral reactions, is currently the most widely used industrial applications one lipases. However, the nature of the native enzyme is often insufficient to meet the actual needs of the application, need to be further optimized using artificial means to improve its usefulness. For CRL1, its as a normal enzyme, poor thermal stability, optimum temperature is 40 ℃, 50 ℃ half-life in less than one hour, and at 80 ℃ rapidly inactivated. This deficiency limits its practical industrial application of a high temperature environment. This study is aimed at improving the thermal stability CRL1. Currently in protein engineering modification of the enzyme molecule methods are mainly directed evolution, rational design and semi-rational design. The first two methods have their advantages and disadvantages. Rational design of the success rate is high, but the idea of ??design must rely on protein structure - function relationships in-depth understanding. Although directed evolution to consider this issue can be avoided, but the need to establish an efficient and sensitive screening method, and the huge capacity of mutant libraries for screening. Semi-rational design approach combines the advantages of both methods. It is added on the basis of directed evolution of rational design elements, the mutation position is limited to one or a few points above, a smaller storage capacity can be obtained in good mutants. In the present study, we previously constructed in our laboratory from a successful recombinant plasmid pPicZαA-CRL1, making use of semi-rational design methods to improve the thermal stability CRL1. We select the B value as the basis for the point mutation. B is defined as the electrons from its equilibrium position of the statistical average, is reflected in the protein atom positions in the crystal structure parameters of uncertainty when these atoms represent electron density ambiguity parameter is converted into a protein amino acid residue The B value, it may represent the size of the rigid amino acid residues. As the central part of the enzyme activity greater flexibility. And, in a high temperature environment of the enzyme as a whole before the transition to the folded state, the enzyme active site changes resulting enzyme usually occurs first loss of vitality. We limit the mutations from the catalytic active center Ser209 10? Less than 60 amino acids. In the subsequent design of the point mutation, the B value of the size of the basic principle of the top three amino acid residues were analyzed. Glu126 and Leu302 predicted CRL1 may enhance the stability of the key mutation. The next step will use the saturation point mutation sites of these two methods were carried out mutation increased thermal stability screened mutants.

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