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

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_max of 20.04 μmol/h and a K_m 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.