(623m) Improving Thermostability of Cis-Epoxysuccinate Hydrolase from Rhodococcus Opacus By Semi-Rational Designs and Directed Evolution

Cis-epoxysuccinate hydrolases (CESH), belonging to soluble epoxide hydrolases (sEHs, EC 3.3.2.10), catalyze the reaction of hydrolyzing the oxirane ring of cis-epoxysuccinate (CES), adding one water molecule onto it and synthesizing L-(+)-tartaric acid. The first report about biologically preparing L-(+)-tartaric acid was carried out by Miura and his colleagues in 1977, and the similar enzymes with the same function were found in various kinds of bacterial afterwards. The gene sequence (GenBank DQ471957) of cis-epoxysuccinate hydrolases from Rhodococcus opacus was deposited in 2007 and the catalytic properties of it were also studied thoroughly. Though has been industrially utilized, the low thermal resistance of CESH has caused extra production costs in continuously maintaining low temperatures and repeatedly preparing new biocatalysts. Low temperatures lead to low reaction rate, as well as low solubility of substrate and product, thus reduce the overall efficiency and increase the consumption of water and electric power. Consequently, improving the thermal resistance of CESH will have a positive influence to the biologically industrial production of L-(+)-tartaric acid. Because of the fact that there is still no exact information about the 3D structure and catalytic mechanism of CESH, we employed vitro protein engineering strategies including semi-rational designs and directed evolution to stabilize the CESH from Rhodococcus opacus. After screening 10,000 colonies from directed evolution, we obtained mutant Q122R whose half-life time at 50°C (t_1/2,50°C) increased by 1.75-fold and the T₅₀¹⁵ value (temperature under which the original activity of enzyme would decrease 50% in 15min) of it increased by 5.5°C. As was manifested in our work, the directed evolution was effective, but the screening of the mutant library was random. Thus, we turned to sequence-based semi-rational redesign and computational enzyme redesign. We compared the sequence of CESH with four thermostable enzymes from haloacid dehalogenase-like hydrolases (HAD) superfamily (GenBank cl21460), and predicted seven mutants. After analyzing the thermal resistance of those proposed mutants, we obtained F26V and I83R whose t_{1/2, 50°C} increased by 2.47- and 0.21-fold and the T₅₀¹⁵ value of them increased by 4.5 and 0.5°C individually. After constructing the homology model of CESH and carrying out simulated mutagenesis, we proposed ten mutants within which the mutants D8K and S90R turned out to be positive substitutions. The t_{1/2, 50°C} of mutants D8K and S90R ascended by 1.98- and 0.35-fold and the T₅₀¹⁵ value of them increased by 5.1 and 1.0°C respectively. To maximize the effects of the positive substitutions, we introduced site-saturated mutagenesis into amino acid residues Asp8, Phe26, Ile83, Ser90, and Gln122. After screening over 2,000 colonies from the site-saturated mutant library, we isolated mutant F26W whose t_{1/2, 50°C} increased by 3.38-fold and the T₅₀¹⁵ value of it increased by 7.2°C. At last, an integrated mutant whose substitutions included D8K, F26W, I83R, S90R, and Q122R was obtained, the optimum reaction temperature of it extended from 50°C to 60°C, the t_{1/2, 50°C} increased by 33.49-fold and the T₅₀¹⁵ value increased by 20.0°C. As far as we know, the final mutation obtained in this study is the most thermostable CESH.