ИСТИНА

Alpha-amino acid ester hydrolase (EC 3.1.1.43, AEH) is an enzyme catalyzing hydrolysis of alpha-amino acid esters and acyl amide bond in different penicillins and cephalosporins. Also it performs N-acylation of 7-aminocephem and 6-aminopenam compounds by alpha-amino acid esters. Such substrate specificity let it use as biocatalyst in synthesis of penicillins and cephalosporins, containing alpha amino group in acyl moiety(amoxicillin, cephalexin and ets.). Biocatalysis is the perspective alternative to organic synthesis of semisynthetic antibiotics from key intermediate products for production in quantity. Progress in genetic engineering on construction recombinant strains- producer different beta-lactam transformation enzymes create basis for biocatalytical implementation in industrial production of semi-synthetic penicillins and cephalosporins. At the present days penicillin acylase enzyme (PA) is used for b-lactam antibiotics synthesis, but AEH has a number of advantages. Since AEH is hydrolase (in contrast to PA, which is amidase) more reactive esters can be used as donor of acyl moiety. The product of reaction is amide and it is less hydrolyzed by enzyme that is improves synthesis/hydrolysis ratio. On the one hand substrate specifity of AEH is narrow, since the enzymes show a very high selectivity for an amino group on the Ca-position and prefer esters to amides. But on the other hand various beta-lactam nuclei are accepted in hydrolysis and synthesis reactions. Moreover acyl moieties with rings that have a hydroxyl group on the para-position or that are partially unsaturated are accepted both in hydrolysis and in transfer reactions. At the moment in literature there are only two different AEH that described in details. These are enzymes from Xanthomonas citri and Acetobacter turbidans. New unique gene of AEH from X. rubrilineans (VKM B-629) has been cloned. It was performed in two steps. At the first step primers to conservative regions of potential AEH were created. Using PCR from X. rubrilineans chromosomal DNA some fragments were obtained. At the second step, a mini-genomic library of X. rubrilineans was prepared formed and a clone with plasmid with AEH gene was detected. Sequencing of isolated plasmid showed that inset has a full gene of alpha-amino acid ester hydrolase. The AEH gene was subcloned into two the expression vectors pET-24a and pET-28a. The first system produces wild-type enzyme and the second system differs by the presence of His-tag at N-termini. Specific activity (on cephalexin synthesis) of recombinant enzyme in these systems exceeded activity obtained at cultivation of native strain of X. rubrilineans in 50 and 20 times respectively. The three-dimensional structure of X. rubrilineans AEH was simulated by the homology modeling method for rational design experiments. The analysis of active site was performed and its structure was specified. Docking of different substrates in AEH active site was carried out, which allowed to obtain the structures of complexes with ampicillin, amoxicillin, cephalexin, D-phenylglycine and para-oxi-D-phenylglycine methyl ester. It was shown that X. rubrilineans AEH has the highest efficiency in antibiotics synthesis with some substrates, which were determined by modeling of complexes. This work was supported by Russian Foundation for Basic Research (grant 11-04-00962-a).