Abstract Enzymatic synthesis of penicillin V (penV) by acylation of 6-aminopenicillanic acid (6-APA) was carried out using methyl phenoxyacetate (MPOA) as activated acyl donor and soluble penicillin acylase from Streptomyces lavendulae (SlPVA) as biocatalyst. The effect of different reaction conditions on penV synthesis was investigated, such as enzyme concentration, pH, molar ratio of 6-APA to MPOA, as well as presence of DMSO as water-miscible co-solvent at different concentrations. Time-course profiles of all reactions followed the typical pattern of kinetically controlled synthesis (KCS) of β-lactam antibiotics: penV concentration reached a maximum (highest yield or Ymax) and then decreased gradually. Such maximum was higher at pH 7.0, observing that final penV concentration was abruptly reduced when basic pH values were employed in the reaction. Under the selected conditions (100 mM Tris/HCl buffer pH 7.0, 30 °C, 2.7% (v/v) DMSO, 20 mM MPOA, 0.3 UI/ml of SlPVA), Ymax was enhanced by increasing the substrate molar ratio (6-APA to MPOA) up to 5, reaching a maximum of 94.5% and a S/H value of 16.4 (ratio of synthetic activity to hydrolytic activity). As a consequence, the use of an excess of 6-APA as nucleophile has allowed us to obtain some of the highest Ymax and S/H values among those reported in literature for KCS of β-lactam antibiotics. Although many penicillin G acylases (PGAs) have been described in kinetically controlled acylations, SlPVA should be considered as a different enzyme in the biocatalytic tool-box for novel potential synthetic processes, mainly due to its different substrate specificity compared to PGAs.

中文翻译：

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淡紫色链霉菌青霉素酰化酶催化的 6-APA 的动力学控制酰化反应：反应条件对青霉素 V 酶促合成的影响

摘要 以苯氧基乙酸甲酯 (MPOA) 作为活化酰基供体，以薰衣草链霉菌 (Streptomyces lavendulae) 的可溶性青霉素酰化酶 (SlPVA) 为生物催化剂，通过 6-氨基青霉酸 (6-APA) 酰化酶促合成青霉素 V (penV)。研究了不同反应条件对 penV 合成的影响，例如酶浓度、pH 值、6-APA 与 MPOA 的摩尔比，以及 DMSO 作为不同浓度的水溶性共溶剂的存在。所有反应的时间过程曲线遵循 β-内酰胺抗生素的动力学控制合成 (KCS) 的典型模式：penV 浓度达到最大值（最高产率或 Ymax），然后逐渐降低。这种最大值在 pH 7.0 时更高，观察到当反应中采用碱性 pH 值时，最终的 penV 浓度突然降低。在选定条件下（100 mM Tris/HCl 缓冲液 pH 7.0、30 °C、2.7% (v/v) DMSO、20 mM MPOA、0.3 UI/ml SlPVA），通过增加底物摩尔比（6 -APA 至 MPOA) 高达 5，最高可达 94.5%，S/H 值为 16.4（合成活性与水解活性的比率）。因此，使用过量的 6-APA 作为亲核试剂使我们能够获得一些最高的 Ymax 和 S/H 值，这些值在文献中报道的 β-内酰胺抗生素的 KCS 值中。尽管许多青霉素 G 酰化酶 (PGA) 已被描述为动力学控制的酰化，但 SlPVA 应被视为生物催化工具箱中的一种不同的酶，用于新的潜在合成过程，