Streptomyces septatus磷脂酶D在毕赤酵母中的重组表达及酶学性质分析  

Recombinant Expression of Phospholipase D from Streptomyces septatus in Pichia pastoris and Analysis of Its Enzymatic Properties

作　　者：黄琳[1] 马杰莹 王爽 王楠[1] MORADANA GAMAGE Rasadi Sajeewa Rajakaruna 路福平[1] 刘逸寒[1] HUANG Lin;MA Jieying;WANG Shuang;WANG Nan;MORADANA GAMAGE Rasadi Sajeewa Rajakaruna;LU Fuping;LIU Yihan(Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin Key Laboratory of Industrial Microbiology,College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China)

机构地区：[1]工业发酵微生物教育部重点实验室,天津市工业微生物重点实验室,天津科技大学生物工程学院,天津300457

出　　处：《食品科学》2020年第8期100-107,共8页Food Science

基　　金：国家自然科学基金面上项目(31571805);天津市企业科技特派员项目(19JCTPJC52200);天津市自然科学基金一般项目(17JCYBJC23700)。

摘　　要：以Streptomyces septatus TCCC 21057基因组为模板,扩增得到磷脂酶D(phospholipase D,PLD)基因(pld),经合成获得pld密码子优化基因(pldm),以pPIC9K为表达载体,毕赤酵母(Pichia pastoris)GS115为宿主菌株,构建获得毕赤酵母重组菌株GS115/pPIC9K-pldm,摇瓶发酵后获得的重组PLD(rPLDM)转酯活力达2.37 U/mL;rPLDM经纯化后进行转酯酶学性质分析,其最适作用温度为60℃,最适作用pH值为6.5;在单水相反应体系中,40℃、pH 6.5、Ca2+浓度10 mmol/L、大豆磷脂酰胆碱和L-丝氨酸物质的量比1∶10、rPLDM添加4.0 U/mL,反应6 h后,磷脂酰丝氨酸(phosphatidylserine,PS)的产率可达到33%。本研究通过对rPLDM的重组表达、转酯酶学性质及催化合成PS的研究,为进一步实现酶法合成新资源食品PS奠定了基础。The phospholipase D(PLD)encoding sequence(pld)was amplified from the genomic DNA of Streptomyces septatus TCCC 21057.The optimized pldm sequence was synthesized according to the codon usage bias in Pichia pastoris and then integrated into the expression vector pPIC9K.The resulting recombinant plasmid was then transformed into P.pastoris GS115 competent cells to obtain the recombinant strain GS115/pPIC9K-pldm.The transphosphatidylation activity of recombinant PLD(rPLDM)could reach 2.37 U/mL after shaking flask fermentation.The recombinant enzyme was purified and characterized.Its optimal activity was observed at 60℃and pH 6.5.The yield of phosphatidylserine(PS)could reach 33%after 6 h reaction in a purely aqueous system under the following conditions:molar ratio between the substrates soybean phosphatidylcholine(PC)and L-serine substrate 1:10,rPLDM amount 4.0 U/mL,temperature 40℃,pH 6.5,and Ca2+concentration 10 mmol/L.This study lays a foundation for enzymatic synthesis of PS as a novel food ingredient.

关 键 词：链霉菌 磷脂酶D 毕赤酵母 表达 酶学性质 磷脂酰丝氨酸 

分 类 号：Q814[生物学—生物工程]