He Ying, Huang Ao, Liu Yun
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
Appl Biochem Biotechnol. 2025 May 20. doi: 10.1007/s12010-025-05278-1.
Phospholipase D (PLD) is a valuable enzyme in industrial processes for converting phosphatidylcholine (PC) to phosphatidylserine (PS). In this study, a strain of Bacillus cereus was isolated from soil and identified through 16S rRNA sequencing. To enhance PLD activity, various mutagenesis strategies-including chemical treatment, irradiation, and their combinations-were employed, resulting in four high-activity positive mutants (C-7, I-12, CI 7-12, and IC 13-14). Among these, the CI 7-12 mutant exhibited a significantly higher enzymatic activity, showing a 3.12-fold increase (312.2%) compared to the wild-type strain. Fermentation conditions were optimied using response surface methodology (RSM), achieving a PLD activity of 35 U/mL. The enzyme demonstrated stability over a wide temperature range (30-60 °C) and pH range (6-10), with a half-life of 128 days. Kinetic analysis revealed a V of 20.04 μmol/h and a K of 7.13 μmol/mL, indicating efficient activity. In bioconversion experiments, the PLD-enriched fermentation broth catalyzed the conversion of PC to PS, achieving a 53.0% conversion rate and a 92.3% selectivity for PS in a two-phase system. These findings expand the potential sources of PLD and underscore its applicability for PS production in biotechnological applications.
磷脂酶D（PLD）是工业生产中一种有价值的酶，可将磷脂酰胆碱（PC）转化为磷脂酰丝氨酸（PS）。在本研究中，从土壤中分离出一株蜡样芽孢杆菌，并通过16S rRNA测序进行鉴定。为提高PLD活性，采用了各种诱变策略，包括化学处理、辐照及其组合，获得了四个高活性阳性突变体（C-7、I-12、CI 7-12和IC 13-14）。其中，CI 7-12突变体表现出显著更高的酶活性，与野生型菌株相比提高了3.12倍（312.2%）。使用响应面法（RSM）优化发酵条件，PLD活性达到35 U/mL。该酶在较宽的温度范围（30-60°C）和pH范围（6-10）内表现出稳定性，半衰期为128天。动力学分析显示V为20.04 μmol/h，K为7.13 μmol/mL，表明活性高效。在生物转化实验中，富含PLD的发酵液在两相体系中催化PC转化为PS，转化率达到53.0%，对PS的选择性为92.3%。这些发现扩展了PLD的潜在来源，并强调了其在生物技术应用中生产PS的适用性。
