Molecular Engineering Approaches For Improving Thermostability Of Aspartate Aminotransferase From E.coli

Enzyme has remarkable properties of high reaction rates, great reaction specificity and mild reaction conditions, so it is widely used in the field of industry and pharmaceutics. However, its instability and scarce source limit its usage, so it should be improved for meeting the needs of the application. Now protein engineering is one of attractive field for improving the enzyme properties in the biotechnology.Aspartate aminotransferase is a ubiquitous pyridoxal 5'-phosphate-dependent enzyme. And the enzyme is necessary for the survival of all cells with the major roles in amino acid metabolism. In industry, it is useful in the production of L-phenylalanine, which is important raw materials of industry and medicine. And in the industrial technology if the temperature of the reaction increases, it would be beneficial to increase the solubility of substrate-phenylpyruvic acid and improve the productivity of L-phenylalanine with accelerating the decomposition of oxaloacetic acid. Unfortunately the instability of aspartate aminotransferase in higher temperature leads to the bottleneck of the technology of L-phenylalanine.The structural comparision of E.coli and thermophilic aspartate aminotransferases had three distinct differences. Based on the three structural distinctions, three approaches were developed for improving thermostability of E.coli aspartate aminotransferase, respectively. Five thermophilic aspartate aminotransferases were acquired by these methods.(1) Engineering aspartate aminotransferase by rational design.In the research of the rational design, on the basis of the residues composition in the loop on surface of aspartate aminotransferas and comparation between E. coil aspartate aminotransferase and thermophilic aminotransferase, hydrophobic residues L233 and F350 had been converted to N233 and K350 respectively. The half-life time of the Enz N233 by the mutagenesis of L233N was prolonged to 180 minutes at 55℃and that of the Enz K350 by the mutagenesis of F350K was reduced to 30 minutes, while that of the wild-type enzyme was 140 minutes. The research developed a simple method of thermostability improvement by the substitution of the hydrophobic residues to hydrophilic ones.(2) Engineering aspartate aminotransferases by semi-rational design For more thermostable aspartate aminotransferase, the research was followed on the interaction between the two subunits. By the analysis of the relationship between the structure and the thermostability, six selected residues in the subunit interface were replaced randomly with all 20 amino acids in a single experiment for the mutant libriary of the aspartate aminotransferase.4 more thermal stable aspartate aminotransferase (C1, C2, C3 and C4) were screened from 4000 mutant strains out of the libriary. Their half-lives of 4 enzyme variants were more than 840 minutes, while one of enzymes kept 87.9% of activity of wild-type aspartate aminotransferase at 37℃. This method firstly combined rational design and directed evolution to improve the thermal stability of the enzyme by enhancing the interaction of the two subunits.(3) Engineering aspartate aminotransferases by directed evolutionBased on feature of low homology of the aspartate aminotransferase family genes (aspC and tyrB), the mutant library was constructed with the fragment of restriction endonuclease by homologous recombination and nonhomologous recombination. The sites of restriction endonuclease were selected carefully to locate in homologous area of aspC and tyrB gene and had the base pair interval with more than 20 base pairs. These efforts led to the high 66% frequency of recombination of the library. By the control of the annealing temperature and cycles in the PCR, the non-homologous recombination was introduced to increase the mutation rate of the library. The result from DNA sequence proved the recombination genes including the exchange of the fragment of aspC and tyrB and deletion of the gene from the non-homologous recombination. These mutagenesises increased the mutation rate of the library and diversity of the mutant library. As a result, these works were helpful to explore the construction of mutant library and improve the function of aspartate aminotransferase.This paper firstly improved the thermostability of aspartate aminotransferase by enzyme engineering and studied on the enzyme-engineering technical platform.(1) A semi-rational design was firstly developed on the basis of the combination of structural analysis of the interaction between the two subunits with site-directed random mutagenesis to improve the thermal stability. By this method,4 more themostable aspartate aminotransferase were acquired effectively. All their half-lives extended 5.6 times than initial one at 55℃and that of the highest thermal-stability of the enzyme (C5) extended 16 times than initial one at 60℃. By the structural comparisons of four enzyme variants either hydrogenous network (such as C2, C4 and C5) or hydrophobic interaction (such as C2, C4 and C5) was formed to enhance the combination of the dimer. In addition, the changes of the distances between residues (such as W140,N297*,R292* and R386) in the active center of enzyme led to the variation of interaction between these residues and substrate, which maybe the main reason of binding affinities between the enzyme and substrate.(2) Firstly based on the structural feature in the loop on enzymatic surface, a simple rational design method was developed to evolve aspartate aminotransferase. This method can design the mutational residues to evolve the enzyme by comparing the differences among the enzyme with different thermostability. As a result, by the analysis of the structure of enzyme variant-Enz N233, the enhancement of hydrophile property was the main reason for thermostability improvement of the variant, while the enhancement of the interaction between the mutagenesis residue N233 and its adjacent residue E320 may also improve the enzyme stability. A counterexample of less thermal stable enzyme variant-Enz K350 was obtained by site-directed method. The structural analysis of the variant showed if there were residues with the same charge near the mutational residues, it might lead to less thermal stability of variant because the mutational residues excluded to the interior of enzyme by these adjacent'residues with the same charge.(3) The method of library construction for directed evolution of aspartate aminotransferase was developed firstly on the combination of homologous recombination and nonhomologous recombination. This method overcame the limitation of low mutation rate in the homologous recombination in low homologous genes and could keep the high frequency of recombination with the fragments of Restriction Endonuclease by the homologous recombination. The control of mutation rate of the mutant library by the condition of PCR (such as annealing temperature and PCR cycles) can be master of the probability of effective mutation. The high frequency of recombination can reduce the work of library screening.