Efficient synthesis of phosphatidylglycerol using phospholipase D mutant with enhanced transphosphatidyl activity modified by the semi-rational design strategy

IF 6.6 1区农林科学 Q1 FOOD SCIENCE & TECHNOLOGY

LWT - Food Science and Technology Pub Date : 2025-09-26 DOI:10.1016/j.lwt.2025.118560

Shuizhi Lin , Yizhen Meng , Rongxin Wu , Shuhua Qi , Yinghua Lu , Chuanyi Yao , Cuixue Chen , Xueping Ling

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引用次数: 0

Abstract

Phospholipase D (PLD) enables efficient enzymatic synthesis of phosphatidylglycerol (PG) with high catalytic efficiency and eco-friendliness. A semi-rational design strategy was employed to mutate PLD by reducing its hydrolysis and enhancing transphosphatidylation. The mutant G381A exhibited suppressed hydrolysis, achieving a PG yield of 82.3 %, 36 % higher than the wild-type. In 5-L fermentor cultures, G381A's transphosphatidylation activity reached 35.7 U/mL, a 7.4-fold increase over the shake flask culture. Kinetic analysis revealed decreased Km and elevated Vmax for G381A. Structural analysis suggested that reorientation of loop 379–385 underpins its enhanced activity. Optimized conditions for G381A (32 °C, pH 8.0, anhydrous ether as organic phase, aqueous/organic phase ratio 1:2.5, glycerol/phosphatidylcholine molar ratio 160:1, 0.84 U/mL enzyme, 30 mmol/L Ca²⁺, 3 h) yielded 97.6 % PG, which is the highest reported to date. This work demonstrates directed evolution's utility in developing PLD mutants has industrial potential, offering insights for rational enzyme design and scalable PG synthesis.

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利用半合理设计策略修饰的转磷脂酰活性增强的磷脂酶D突变体高效合成磷脂酰甘油

磷脂酶D （Phospholipase D， PLD）是一种高效的酶促合成磷脂酰甘油（PG）的酶制剂，具有较高的催化效率和生态友好性。采用半合理的设计策略，通过减少PLD的水解和增强转磷脂酰化来突变PLD。突变体G381A表现出抑制水解，达到82.3%的PG产率，比野生型高36%。在5-L发酵罐培养中，G381A的转磷脂酰化活性达到35.7 U/mL，比摇瓶培养提高7.4倍。动力学分析显示G381A降低Km和提高Vmax。结构分析表明，379-385环的重新定向是其活性增强的基础。优化条件为G381A(32℃，pH 8.0，无水醚为有机相，水/有机相比1:25 .5，甘油/磷脂酰胆碱摩尔比160:1，酶0.84 U/mL, 30 mmol/L Ca2+, 3 h)，产率97.6%，为迄今为止报道的最高产率。这项工作证明了定向进化在开发PLD突变体方面的效用具有工业潜力，为合理的酶设计和可扩展的PG合成提供了见解。

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来源期刊

LWT - Food Science and Technology 工程技术-食品科技

CiteScore

11.80

自引率

6.70%

发文量

1724

审稿时长

65 days

期刊介绍： LWT - Food Science and Technology is an international journal that publishes innovative papers in the fields of food chemistry, biochemistry, microbiology, technology and nutrition. The work described should be innovative either in the approach or in the methods used. The significance of the results either for the science community or for the food industry must also be specified. Contributions written in English are welcomed in the form of review articles, short reviews, research papers, and research notes. Papers featuring animal trials and cell cultures are outside the scope of the journal and will not be considered for publication.