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Alpha-amino ester hydrolase (АЕН, EC 3.1.1.43) is new alternative to penicillin acylase for synthesis of alpha-amino-beta-lactam antibiotics. Use of AEH of esters as acylation agents instead of amides for penicillin acylase should facilitate production of acyl-enzyme covalent complex and whole process of antibiotic synthesis through acyl transfer reaction. We cloned the gene of alpha-amino ester hydrolase from Xanthomonas rubrilineans VKPM В-9915. The recombinant XrAEH were overexpressed in E. coli cells. The enzyme was purified and tested in synthesis of some alpha-amino penicillins and cephalosporins. Model structure of XrAEH was built and docking of substrates and products in active site was done. Recombinant as well as native alpha-amino ester hydrolase from Xanthomonas rubrilineans VKPM В-9915 (XrАЕН, EC 3.1.1.43) was tested for synthesis of amino-beta-lactam antibiotic cephalexin. It was shown that recombinant enzyme rXrАЕН produced by Escherichia сoli VKPM В-11246 is more efficient compared to native one, wt-XrАЕН prepared from mutant strain Xanthomonas rubrilineans VKPM В-9915. Addition of ethylene glycol to reaction mixture (33 v/v%) results in increase of maximum yield of cephalosporin from 75 to 95% using as biocatalyst rXrAEH. At optimal synthesis conditions change of rXrAEH with wt-XrАЕН results in decrease of cephalosporin maximum yield from 95 to 85%. This effect is caused by presence of beta-lactamase side activity in wt-XrАЕН preparation.