The Semi-Rational Directed Evolution Of (+)-Gamma-Lactamase From Sulfolobus Solfataricus P2

(+)Gamma-lactamase from Sulfolobus solfataricus P2 (Sslact) is an ideal biocatalyst for racemic gamma-lactam hydrolysis reaction, which can generate optically pure (-)gamma-lactam, an important pharmaceutical intermediate for synthesizing anti-AIDS drug abacavir.Protein molecule structure of Sslact was constructed by homology modeling and molecular dynamic simulation. Catalytic traid, hydrophobic binding pocket and the three residues responsible for active site folding architecture (3RFA) were predicted by structure analysis. The distance 1j between hydroxyl hydrogen of Ser195 and carbonyl oxygen of substrate and distance d2 between carbonyl oxygen of Cys145 and amido hydrogen of substrate were thought to be two important parameters for predicting mutants that can generate high catalytic activity. Amino acid substititions in hydrophobic pocket and 3RFA were implemented in silico by computer-aided mutation design, whose non-variability were estimated by distance evaluation hypothesis and verified by experiment. we finally selected amino acids Val203, Pro377 and Tyr388 that was far away from active site as mutational hot spots to achive positive mutants.Several mutants were constructed by directed evolution including error-prone PCR (epPCR), site-directed mutagenesis (sdm) and site-saturation mutagenesis (ssm) and among of them, V203N, V203Q and Y388H were positive mutants, their enzyme activities were 418.20 U/mg,373.35 U/mg, and 1132.87 U/mg respectively, higher than that of WT (202.97 U/mg). After analyzing the relationship of structure and activity of these mutants, the reliability of computer-aided mutation design would be verified from different aspects.In order to achieved higher active mutant, double mutant V203N/Y388H was generated by combining two mutational sites. Its specific activity was 1188.69 U/mg, catalytic efficiency was 2.85-fold higher than that of the wild type and Michaelis constant Km was 2.20×10-2mM. Moreover, its was able to maintain relative high thermostability from 50? to 90?. Metallic ions Na+, K+, Ca2+, Mg2+ and Fe3+ could improve the enzymatic reaction.