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One-pot conversion of cephalosporin C by using an optimized two-enzyme process

机译：使用优化的两酶过程对头孢菌素C进行一锅转化

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摘要

The main industrial process for producing 7-aminocephalosporanic acid (7-ACA), a precursor of semi-synthetic cephalosporin antibiotics, is the two-step enzymatic route based on D-amino acid oxidase and glutaryl acylase working in separate reactors. Taking advantage of the recently developed variants of cephalosporin C acylase from Pseudomonas N176 (named VAC) and the optimized recombinant overproduction of the two enzymes, we set up a one-pot system to directly convert cephalosporin C into 7-ACA. We optimized the process by identifying the most favorable operational conditions, substrate, and enzyme concentrations. Among the VAC variants employed, our results indicated that HS-HS-F72 beta R VAC is the best choice because of the high activity on both substrates (glutaryl-7-ACA and cephalosporin C) and the absence of substrate and product inhibition effects. Under optimized conditions and by adding further aliquots of the biocatalysts, >98% of cephalosporin C was converted, yielding 7-ACA as the main reaction product (oxo-7-ACA was below the detection level and glutaryl-7-ACA was <1 mM). At the 20 mL bioconversion scale, approximately 81 mg of 7-ACA are produced in 41 hours from 15 mM cephalosporin C. The low cost of the one-pot enzymatic production of 7-ACA is the main advantage of the proposed method, which is further strengthened by the good purity of the final product.
机译：产生7-氨基孢子丙酸的主要工业过程（7-ACA）是半合成头孢菌素抗生素的前体，是基于D-氨基酸氧化酶和在单独反应器中工作的D-氨基酸氧化酶和谷胱甘肽酰基酶的两步酶促途径。利用新近开发的头孢菌素C酰基酶从假单胞菌N176（命名为VAC）和两种酶的优化重组过量产生的变体，我们建立了一个单盘系统，以直接将头孢菌素C转换为7-ACA。我们通过确定最有利的操作条件，底物和酶浓度来优化该过程。在所采用的VAV变体中，我们的结果表明，HS-HS-F72βRVAC是最佳选择，因为对底物的活性很高（肝脏-7-ACA和头孢菌素C），并且缺乏底物和底物抑制作用。在优化的条件下并添加进一步的生物催化剂等分试样，将> 98％的头孢菌素C转化，作为主反应产物，产生7-ACA（Oxo-7-ACA（Oxo-7-ACA）低于检测水平，谷胱甘肽-7-ACA为<1 <1毫米）。在20毫升生物转化量表上，从15 mM头孢菌素C中产生了约81 mg的7-ACA。最终产品的良好纯度进一步加强。

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