Improving Catalytic Activity Of Halohydrin Dehalogenase HHDH By Directed Evolution

Using (3^,5S)-ter-Butyl-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) in the preparation of (3R,5R)-ter-Butyl-6-cyano-3,5-dihydroxyhexanoate ((3R,5R)-CDHHB) has great significance, since the later oneis the key intermediate in the preparation of atorvastatin. With good development potential and prospects, the synthesis have become a hot research field of biocatalysis and transformation.Firstly, by means of Error-Prone PCR of HHDH halohydrin dehalogenase,we construct mutant library. In that reaction, we treat (3R,5S)-CDHH as substrate and after several rounds of primary and secondary screenings, several strains with improved catalytic activity were obtained. They were W86R/R87P, A82M/W86R, A82T/W86R and V84T/W86R, with 1.81、1.80.2.27 and 2.42 times higher catalytic activity separately.Based on that by means of site-directed mutagenesis to transform the site W86 to Arg, its Vmax and Kmvalues were 263 μM/min and 4.5 mM. The specific activity of W86I is 3.75 times as large as that of HHDH. Through molecular modeling and docking found that the activation energy has decreased and the steric hindrance is reduced. The combined effect of both above factors increased the enzyme specific activity.Secondly, using saturation mutagenesis and directed evolution methods to constrct mutant library including site86 and key amino acids nearby. We obtained many mutants with greater catalytic activity at site86:W86I, W86L, W86V and W86A, which with 9.42,7.25,6.96 and 3.3 times higher specific activity compared to the WT. One of the best is W86I with specifi activity of 0.65 U/mg and it’s Vmax and Km values were 328μM/min and 3.85 mM. Analysing the results of homology modeling found that activation energy had decreased a lot, reduction of the steric hindrance and the increasing flexibility of mutations both improve the enzyme specific activity.While steric and hydrogen bonding may well explain the activity of several other mutations decrease or disappear.Finally, study about the reaction system of W86I. The catalytic activity was increased to 14.21 U/L as a result of the optimizationon the conditions of producing soluble enzymes in LB medium. Then the kinds of co-solvent were also optimized.