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一种新的产磷脂酶D蜡样芽孢杆菌:分类学、诱变、发酵优化及酶特性分析

A New Phospholipase D-Producing Bacillus cereus: Taxonomy, Mutagenesis, Fermentation Optimization and Enzyme Characterization.

作者信息

He Ying, Huang Ao, Liu Yun

机构信息

Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.

出版信息

Appl Biochem Biotechnol. 2025 May 20. doi: 10.1007/s12010-025-05278-1.

Abstract

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.

摘要

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。

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