Enhancing The Activity Of HheA_AM By Molecular Modification To Prepare Tert-butyl?3R,5R?-6-cyano-3,5-dihydroxyhexanoate

tert-butyl(3R,5R)-6-cyano-3,5-dihydroxyhexanoate(A7)is key chiral synthon of a cholesterol-lowering drug of atorvastatin.It's potential to prepare A7 by halohydrin dehalogenases catalyzing dehalogenation and cyanogenation of tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate(D3).Nevertheless,the main problems are lack of halohydrin dehalogenases,low catalytic activity and little research on molecular modification to improve their activity towards D3.In this study,halohydrin dehalogenases were screened and modified towards D3,and the main results were as follows:(1)A halohydrin dehalogenase library was established by gene mining,and HheAAM from Agromyces mediolanus sp.was obtained.The whole-cell activity of HheAAM was 13 U·L-1,and its specific activity was 69.06 U·g-1,higher than the reported HheCs(HHDH and Mut-HheC2360).(2)HheAM was modified by directed evolution and semi-rational design.Six positive mutants contained 84/88 mutation were obtained by error-prone PCR,and 9 positive mutants were obtained by site-saturation mutation of site 84 and 88,and their activity were 1.32-3.46 times of Wt-HheAAM.According to the results of molecular docking,site-saturation mutation of the sites near the substrate binding region was carried out,and 4 positive mutants contained 136/144 mutation were obtained,and their activity were 1.78-3.13 times of Wt-HheAAm.(3)14 positive mutants were obtained by applying Iterative Saturation Mutagenesis at the four sites,their activity were 1.78-15.19 times of Wt-HheAAAM.And 19 positive mutants were obtained by applying Combinatorial Active Site Testing at the four sites,their activity were 1.03-11.98 times of Wt-HheAAM.The best mutant M4-1(84S-88L-136T-144C)was obtained with 13-fold enhancement in specific activity(887.80 U·g-1),which was improved significantly.At the same time,the optimum pH of M4-1 changed from 7.5 to 8.0,the optimum temperature changed from 55? to 60?,and t1/2,50? changed from 0.87 h to 2.38 h.