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首页> 外文期刊>RSC Advances >Integrating error-prone PCR and DNA shuffling as an effective molecular evolution strategy for the production of α-ketoglutaric acid by l-amino acid deaminase
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Integrating error-prone PCR and DNA shuffling as an effective molecular evolution strategy for the production of α-ketoglutaric acid by l-amino acid deaminase

机译：将易于PCR和DNA洗牌整合为通过L-氨基酸脱氨酰酶生产α-酮戊酸的有效分子演化策略

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L -Amino acid deaminases (LAADs; EC 1.4.3.2) belong to a family of amino acid dehydrogenases that catalyze the formation of α-keto acids from L -amino acids. In a previous study, a whole cell biocatalyst with the L -amino acid deaminase (pm1) from Proteus mirabilis was developed for the one-step production of α-ketoglutarate (α-KG) from L -glutamic acid, and the α-KG titer reached 12.79 g L ~(?1) in a 3 L batch bioreactor. However, the product α-KG strongly inhibited pm1 activity, and the titer of α-KG was comparatively lower than expected. Therefore, in this study, multiple rounds of error-prone polymerase chain reaction (PCR) and gene shuffling were integrated for the molecular engineering of pm1 to further improve the catalytic performance and α-KG titer. A variant (pm1338g4), which contained mutations in 34 amino acid residues, was found to have enhanced catalytic efficiency. In a batch system, the α-KG titer reached 53.74 g L ~(?1) when 100 g of monosodium glutamate was used as a substrate. Additionally, in a fed-batch biotransformation system, the maximum α-KG titer reached 89.11 g L ~(?1) when monosodium glutamate was continuously fed at a constant rate of 6 g L ~(?1) h ~(?1) (from 4 to 23 h) with an initial concentration of 50 g L ~(?1) . Analysis of the kinetics of the mutant variant showed that these improvements were achieved due to enhancement of the reaction velocity (from 56.7 μM min ~(?1) to 241.8 μM min ~(?1) ) and substrate affinity (the K _(m) for glutamate decreased from 23.58 to 6.56 mM). A possible mechanism for the enhanced substrate affinity was also evaluated by structural modeling of the mutant. Our findings showed that the integration of error-prone PCR and gene shuffling was an effective method for improvement of the catalytic performance of industrial enzymes.
机译：l-咪啶酸脱胺酶（Laads; Ec 1.4.3.2）属于催化来自L-氨基酸的α-酮酸的氨基酸脱氢酶系列。在先前的研究中，开发了来自蛋白质mirabilis的L-氨基酸脱氨酶（PM1）的全细胞生物催化剂用于从L-乙酸的α-酮戊酸（α-Kg）和α-kg的一步生成滴度在3L批量生反应器中达到12.79g l〜（？1）。然而，产物α-kg强烈抑制PM1活性，α-kg的滴度比预期相对低。因此，在该研究中，为PM1的分子工程集成了多轮错误易于聚合酶链反应（PCR）和基因洗涤，以进一步改善催化性能和α-Kg滴度。发现含有34个氨基酸残基的突变的变体（PM1338G4）具有增强的催化效率。在批量系中，当使用100g谷氨酸氧氨酸钠作为基质时，α-kg滴度达到53.74g L〜（α1）。另外，在联合批次的生物转化系统中，当谷氨酸单钠以6g l〜（α1）h〜（？1）连续喂食时，最大α-kg滴度达到89.11g l〜（α1） （从4至23小时），初始浓度为50g l〜（？1）。对突变体变体的动力学的分析表明，由于增强反应速度（56.7μmmin〜（α1）至241.8μmmin〜（α1））和底物亲和力（k _（m），实现了这些改进）对于谷氨酸从23.58降至6.56mm）。还通过突变体的结构建模评估增强底物亲和性的可能机制。我们的研究结果表明，易于易于PCR和基因洗涤的整合是改善工业酶催化性能的有效方法。

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