基因拼接型蜡状芽孢杆菌ZY12磷脂酶D的异源表达及转酯活性的研究  

Heterologous Expression and Transesterification Activity of Gene Splicing of Phospholipase D from Bacillus Cereus ZY12

作　　者：赵雨 郭建华[1] 张春枝[2] ZHAO Yu;GUO Jianhua;ZHANG Chunzhi(School of Food and Biological Engineering,Qiqihar University,Qiqihar 160006,China;School of Biological Engineering,Dalian Polytechnic University,Dalian 116034,China)

机构地区：[1]齐齐哈尔大学食品与生物工程学院,黑龙江齐齐哈尔160006 [2]大连工业大学生物工程学院,辽宁大连116034

出　　处：《食品科技》2021年第4期15-20,共6页Food Science and Technology

基　　金：2017年黑龙江省自然科学基金面上项目(C2017066);2019黑龙江省省属高等学校基本科研业务费科研项目(135409221)。

摘　　要：磷脂酶D是一类酯键水解酶,能够水解磷脂生成磷脂酸,并在某些羟基化合物存在的条件下,通过脱水缩合生成新型磷脂。利用磷脂酶D生成新型磷脂的活性,通过生物转化可以制备稀有磷脂如磷酯酰丝氨酸等。因此,磷脂酶D在食品及医疗领域应用潜力巨大。实验室前期发现的蜡状芽孢杆菌ZY12来源新型磷脂酶D具有水解活性,但检测不到转酯活性,将其与链霉菌PMF来源磷脂酶D进行基因拼接,通过pET-30b重组质粒在大肠杆菌BL21(Lyss)中异源表达。实验结果表明,通过基因拼接手段对蜡状芽孢杆菌ZY12磷脂酶D的改造,使该磷脂酶D获得转酯活性。在诱导温度为16 ℃、IPTG添加量0.5 mmol/L、诱导时间12 h,可溶性拼接型磷脂酶D活性最高87 mU/mL。转酯反应温度为45 ℃、pH为4.5、反应时间3 h,生成磷酯酰丝氨酸产量最高7.1 mg/L。Phospholipase D (PLD) is a special kind of ester hydrolase,which hydrolyzes phospholipids to generate phosphatidic acid.In the presence of some hydroxyl compounds,new phospholipids can be produced by dehydration condensation.Therefore phospholipase D has great potential in food and medicine fields.The new phospholipase D from Bacillus cereus ZY12 was found to have hydrolytic activity,but no transesterification activity was detected.The recombinant plasmid pET-30B was heterologously expressed in Escherichia coli BL21(LYSS) after gene splicing with phospholipase D from Streptomyces PMF.The results showed that the phospholipase D of Bacillus cereus ZY12 was modified by gene splicing,and the phospholipase D obtained transesterification activity.The maximum activity of soluble splice phospholipase D was 87 mU/mL at the induction temperature of 16 ℃,IPTG dosage of 0.5 mmol/L and induction time of 12 h.The maximum yield of phosphoesteryl serine was 7.1 mg/L at 45 ℃,pH 4.5 and reaction time 3 h.

关 键 词：磷脂酶D 磷酯酰丝氨酸 蜡状芽孢杆菌 基因拼接 

分 类 号：TS201.3[轻工技术与工程—食品科学]