Enhancing Tolerance Of Cis-Epoxysuccinate Hydrolase To Temperature By Directed Evolution And Semi-Rational Redesign

As a typical soluble epoxide hydrolases (sEHs, EC 3.3.2.10), cis-epoxysuccinate hydrolase (CESH) is capable of regio-and enantio-selectively opening the oxirane ring of cis-epoxysuccinate (CES), adding one water molecule to it and obtaining an optically active vicinal diol, L-(+)-tartaric acid.L-(+)-tartaric acid is deemed as a widely used organic compound in the manufacture of pharmaceutical products, nutritional supplements and cosmetics. Preparing L-(+)-tartaric acid using cis-epoxysuccinate hydrolases was first reported in 1977, and similar enzymes with same catalytic functions have also been found in several kinds of bacteria like Pseudomonas, Nocardia, Corynebacterium and Rhodococcus.Since CESH is efficient in catalyzing cis-epoxysuccinate into L-(+)-tartaric acid, it has been successfully applied in the industrial field. However, the low thermostability limits its use in large-scale applications. As has been substantiated, using protein engineering strategies in vitro could stabilize frangible enzymes, directed evolution and methods based on semi-rational redesign were utilized to improve the thermal resistance of CESH from Rhodococcusopacus. The specificity activity of wild-type CESH is 76.5 U/mg, its half-life time (t1/2,50℃) is 8.5 min, and the T5015 (temperature at which the activity of enzyme decreases by 50% in 15 min) is 44.0℃.The CESH variant 1X-1(Q122R) was harvested from directed evolution; its specificity activity is 75.8 U/mg, and the T12,50℃ and T5015 increased to 31.6 min, and 49.5℃. Directed evolution was employed on modifying mutant 1X-1, but no mutant manifested higher thermostability than 1X-1. To furtherlly enhance the thermostability of CESH, we turned to semi-rational design.Mutants F26V and I83R predicted by multiple sequence alignments were introduced into mutant 1X-1 individually. The specificity of mutant 2X-2 (Q122R, F26V) was 76.1 U/mg, the t1/2,50℃ was162.1 min, and the T5015 was 54.6℃; the specificity of mutant 2X-4 (Q122R, I83R) was 74.3 U/mg, the t1/2,50℃ was 44.0 min, and the T5015 was 50.4℃. Subsequently, mutants 2X-2 and 2X-4 were introduced to mutant 1X-1, generating mutant 3X (Q122R, F26V, I83R). Its specificity activity was 75.5 U/mg, and the t1/2, 50℃ and t5015 increased to170.8 min and 55.4℃ respectively. Computationally simulated mutation was used in constructing mutants 4X-4 (Q122R, F26V, I83R, D8K) and 4X-6 (Q122R, F26V, I83R, S90R). The specificity activity of mutant 4X-1 was 75.8 U/mg, the t1/2,50℃ was 225.2 min, and the T5015 was 61.6℃. The specificity activity of mutant 4X-6 was 75.2 U/mg,t1/2,50℃ was 180.5 min, and the T5015 was 56.5℃. Mutants 4X-1 and 4X-6 were recombined, generating mutant 5X (Q122R, F26V, I83R, D8K, S90R), whose specificity activity was 74.3 U/mg, the t1/2,50℃ increased to 237.1 min, and the Tso15 increased to 62.4℃. Site-saturated mutagenesis was employed on amino acid residues Asp8, Phe26, Ile83, Ser90, and Gln122 to maximize the thermostability of mutant 5X. Mutant 5X-1(Q122R, F26W, I83R, D8K, S90R) was isolated from saturated mutations; itst1/2,50℃ increased to 293.2 min,34.5-fold that of wild-type, and its T5015 increasedto 64.8℃.The effective working arranges of pH of mutant 5X-1 extended to 5.0-10.0 from 8.0-9.0 for the wild-type enzyme. Moreover, catalytic activity exhibited by immobilized 5X-1 was two-fold that of immobilized wild-type CESH and the service life of immobilized 5X-1 significantly exceeded wild-type CESH at 37℃, 45℃ and 50℃. The half-life time of immobilized wild-type CESH at 37℃,45℃ and 50℃ was 13 days,5 days and 2 days, whereas, it was 25 days at 50℃,35 days at 45℃ for immobilized mutant 5X-1, and after 38 days, the residual activity of immobilized mutant 5X-1 at 37℃ was still 65%.