蜡状芽孢杆菌磷脂酶D的活性形式及HKD活性区域的分子改造对其活性的影响

磷脂酶D是一类特殊的酯键水解酶，它能水解磷脂生成磷脂酸和羟基化合物，并能催化某些含羟基的化合物结合到磷脂的酰基上，形成新的磷脂，在食品和医药领域应用潜力巨大。本研究实现了蜡状芽孢杆菌磷脂酶D的克隆，并在大肠杆菌中成功表达。通常情况下，磷脂酶D存在2个HKD保守序列，以单体形式产生活性；少数原核生物中磷脂酶D只有1个HKD保守序列，以二聚体形式产生活性。通过酵母双杂实验发现，源于蜡状芽孢杆菌的磷脂酶D活性存在形式是单体结构，但其只具有1个HKD保守序列，靠近N端存在1个HRD序列，即HKD中K被R取代。将HRD定点突变为HKD，恢复为经典的2个HKD保守序列，其酶活性提高了10% 左右，蛋白质水平的表达量和稳定性无显著变化。通过定点突变提高磷脂酶D活性，为工业化高效生产新型磷脂奠定了理论基础。

The Active Form of Phospholipase D from Bacillus cereus and the Effect of Molecular Modification of HKD Active Region on its Activity

Phospholipase D, a special kind of ester hydrolase, can hydrolyze phospholipids to generate phosphatidic acid and hydroxyl compounds and catalyze the reaction that some hydroxyl containing compounds bind to phospholipid acyl to form new phospholipids. It shows great potential in food and medicine fields. In this study, the Bacillus cereus phospholipase D was cloned and successfully expressed in Escherichia coli. Typically, phospholipase D has two HKD conserved sequences and elicits activity in monomer. Few prokaryotic phospholipase D has only one conserved HKD sequence and produces activity in dimeric form. With yeast two-hybrid experiment, it was found that the active form of phospholipase D from Bacillus cereus was monomer and has only one HKD conserved sequence. In its N-terminal region, there was a sequence of HRD, in which K (lysine) was replaced by R (Arginine) in HKD. The HRD was then site directed mutated to HKD to restore the classic two conserved HKD sequences. The results showed that enzyme activity of the mutant was increased by 10%, and its protein expression and stability in E.coli had no significant variation after mutation. The present study showed that the activity promotion of phospholipase D could be achieved by site-directed mutagenesis, which provided a possibility to increase industrial production of new phospholipids.