Selectivity and kinetic modeling of penicillin G acylase variants for the synthesis of cephalexin under a broad range of substrate concentrations.
Selectivity and kinetic modeling of penicillin G acylase variants for the synthesis of cephalexin under a broad range of substrate concentrations.
复制标题
在广泛的底物浓度范围内合成头孢氨苄的青霉素 G 酰基转移酶变体的选择性和动力学模型。
DOI：
10.1002/bit.28214
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发表时间：
2022
影响因子：
3.8
通讯作者：
Bommarius,AndreasS
中科院分区：
文献类型：
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作者：
Harris,PatrickR;Grover,MarthaA;Rousseau,RonaldW;Bommarius,AndreasS
在宽范围的底物浓度下表征了头孢氨苄合成和活化酰基供体前体苯甘氨酸甲酯（PGME）水解的动力学。Youshko-Svedas先前开发的模型涉及酰基-酶复合物的形成，然后结合亲核β-内酰胺供体，该模型无法使用初始速率数据完全估计不同浓度下头孢氨苄合成的最大反应产率。发现7-氨基去乙酰氧基头孢烷酸（7-ADCA）是头孢氨苄水解的有效抑制剂，这可能是模型预测偏离的原因。比较了头孢氨苄合成的三种动力学模型，其中包含7-ADCA引起的竞争性抑制的模型得到最佳拟合。此外，对于评价的浓度范围以及初始速率实验和时程合成实验，与野生型相比，β F24 A变体和Assemblase®在头孢氨苄合成动力学方面未表现出显著差异。最后，使用包含7-ADCA竞争性抑制的模型模拟头孢氨苄合成的连续搅拌釜反应器，在生产率、部分收率和PGME转化率之间观察到明显的权衡。
The kinetics of cephalexin synthesis and hydrolysis of the activated acyl‐donor precursor phenylglycine methyl ester (PGME) were characterized under a broad range of substrate concentrations. A previously developed model by Youshko‐Svedas involving the formation of the acyl‐enzyme complex followed by binding of the nucleophilic β‐lactam donor does not fully estimate the maximum reaction yields for cephalexin synthesis at different concentrations using initial‐rate data. 7‐aminodesacetoxycephalosporanic acid (7‐ADCA) was discovered to be a potent inhibitor of cephalexin hydrolysis, which may account for the deviation from model predictions. Three kinetic models were compared for cephalexin synthesis, with the model incorporating competitive inhibition due to 7‐ADCA yielding the best fit. Additionally, the βF24A variant and Assemblase® did not exhibit significantly different kinetics for the synthesis of cephalexin compared to the wild‐type, for the concentration range evaluated and for both initial‐rate experiments and time‐course synthesis experiments. Lastly, a continuous stirred‐tank reactor for cephalexin synthesis was simulated using the model incorporating competitive inhibition by 7‐ADCA, with clear tradeoffs observed between productivity, fractional yield, and PGME conversion.