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首页> 外文期刊>Journal of Biotechnology >Saturation mutagenesis reveals the importance of residues alphaR145 and alphaF146 of penicillin acylase in the synthesis of beta-lactam antibiotics
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Saturation mutagenesis reveals the importance of residues alphaR145 and alphaF146 of penicillin acylase in the synthesis of beta-lactam antibiotics

机译：饱和诱变揭示了青霉素酰化酶残基alphaR145和alphaF146在合成β-内酰胺抗生素中的重要性

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摘要

Penicillin acylase (PA) from Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived beta-lactam nuclei, a conversion that can be used for the industrial synthesis of beta-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues alphaR145 and alphaF146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5-15-fold. Moreover, due to increased tendency of the acyl-enzyme intermediate to react with beta-lactam nucleophile instead of water, mutants alphaR145G, alphaR145S and alphaR145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic beta-lactam antibiotics.
机译：大肠杆菌的青霉素酰化酶（PA）可以催化酰基与青霉素和头孢菌素衍生的β-内酰胺核的偶联，这种转化可用于工业合成β-内酰胺抗生素。野生型酶的适度合成性质使得工程改造改良的突变体是合乎需要的。对PA晶体结构的分析表明，残基alphaR145和alphaF146在大配体与活性位点结合后会发生广泛的重新定位，这表明这些残基可能是诱变的好靶标，旨在提高PA的催化性能。因此，在两个位置上都进行了位点饱和诱变，并对全部38个变体的完整集进行了快速HPLC筛选，以改善氨苄青霉素的合成。不少于33个突变体显示出改进的合成，表明突变残基在PA催化的酰基转移动力学中的重要性。在几个底物浓度低的突变体中，氨苄西林产量的最高水平提高到1.5倍，合成速率与水解速率之比提高了5-15倍。而且，由于酰基酶中间体与β-内酰胺亲核试剂而不是与水反应的趋势增强，因此在高底物浓度下，突变体αR145G，αR145S和αR145L的合成产率高于野生型PA。这伴随着6-APA向氨苄青霉素的转化增加以及酰基供体的不希望的水解降低。因此，这些突变体是酶促生产半合成β-内酰胺类抗生素的有趣候选者。

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