磷脂酶D的大肠杆菌表达系统及其转酰基催化活性  

The E.coli expression system of phospholipase D and its catalytic activity for transacylation

作　　者：程青 谢志鹏 CHENG Qing;XIE Zhipeng(Pharmaceutical Biotechnology Institute,School of Medicine,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学医学院药物生物技术研究所,浙江杭州310058

出　　处：《浙江大学学报（理学版）》2023年第3期351-359,共9页Journal of Zhejiang University（Science Edition）

摘　　要：链霉菌(Streptomyces)来源的磷脂酶D(phospholipase D,PLD)在食品、保健品、医药等行业的应用潜力巨大。目前用大肠杆菌异源表达PLD的方法较常见,但其具有发酵周期长、分离纯化步骤烦琐、培养成本高等缺点。为此,对比研究了大肠杆菌胞内表达、分泌表达和表面展示3种方式对PLD酶活及其磷脂酰丝氨酸(phosphatidylserine,PS)催化合成效率的影响。采用单因素法获得PLD表达的最佳诱导条件为温度20℃、OD600=0.7、IPTG浓度0.3 mmol·L^(-1)。发酵时间曲线表明,在诱导表达8 h时,3种方式的PLD水解酶活均达最高值,分别为0.35,0.23,0.11 U·mL^(-1)。扫描电镜结果表明,大肠杆菌表达PLD后,其细胞结构有一定程度破坏,其中分泌表达菌株的变形塌陷情况最为严重。在水-乙酸乙酯两相体系中,以表面展示菌株全细胞催化磷脂酰胆碱(phosphatidylcholine,PC)方式制备PS的产率最高,可达59.1%,显示了大肠杆菌表面展示表达PLD具有大大降低生物催化转化成本的巨大潜力。Phospholipase D(PLD)derived from Streptomyces has great application potential in food,health products,medicine and other industries,and the commonly used heterologous expression host is E.coli.However,there are problems such as long fermentation time,cumbersome separation and purification steps,and high cultivation cost.Therefore,the effects of intracellular expression,secretory expression and surface display of E.coli on PLD enzyme activity and phosphatidylserine(PS)catalytic synthesis efficiency were comparatively studied.A single factor method was used to derive the optimal induction conditions for PLD expression modes,specifically,the bacterial concentration OD600 was 0.7,IPTG concentration was 0.3 mmol·L^(-1)and the temperature was 20℃.The fermentation time curve under optimal conditions showed that the PLD enzyme activity by the above three methods reached the highest value when the expression was induced for 8 h,which were 0.35,0.23,and 0.11 U·mL^(-1),respectively.Scanning electron microscopy showed that the structure of E.coli cells expressing PLD has been damaged to a certain extent.Among them,the deformation and collapse of the cell structure of the secretory expression strain is the most serious.In the water-ethyl acetate biphase catalyzed phosphatidylcholine(Phosphatidylcholine,PC)system,the PLD surface display strain in the form of a whole cell obtained the highest PS molar conversion rate of 59.1%,compared with the intracellular expression strain and secretion expression strain in the form of sonicated crude enzyme solution,showing that the expression of the E.coli surface display system PLD has great potential for whole-cell catalytic synthesis of PS with reducing production costs.

关 键 词：链霉菌 磷脂酶D(PLD) 表面展示 磷脂酰丝氨酸 

分 类 号：Q7[生物学—分子生物学]