产磷脂酶D工程菌构建及发酵条件优化  

Engineering Phospholipase D-Producing Strains of Bacillus subtilis and Optimization of Fermentation Conditions

作　　者：刘琦 张海洋 李雪晗 刘振 孙建安[1] 毛相朝[1,2] LIU Qi;ZHANG Hai-yang;LI Xue-han;LIU Zhen;SUN Jian-an;MAO Xiang-zhao(College of Food Science and Engineering,Ocean University of China,Qingdao 266003,Shandong,China;Qingdao National Pilot Laboratory for Marine Science and Technology/Laboratory for Marine Drugs and Bioproducts,Qingdao 266003,Shandong,China)

机构地区：[1]中国海洋大学食品科学与工程学院,山东青岛266003 [2]青岛海洋科学与技术国家试点实验室/海洋药物与生物制品功能实验室,山东青岛266003

出　　处：《食品研究与开发》2022年第11期26-34,共9页Food Research and Development

基　　金：国家重点研发计划(2019YFD0901901);山东省自然科学基金(ZR2020JQ15)。

摘　　要：该研究构建产磷脂酶D(phospholipase D,PLD)的枯草芽孢杆菌(Bacillus subtilis)工程菌,比较4个单启动子(P_(HpaⅡ)、_(Pylb(F4))、P_(2069m)、P_(43))对PLD酶活的影响,摇瓶发酵表明启动子P_(HpaⅡ)酶活最高,为0.32 U/mL。进一步以此工程菌作为发酵菌株,通过单因素试验和响应面试验,对该菌株产PLD的发酵条件进行优化。结果表明最佳产酶条件为接种量2.5 mL/dL、发酵温度37℃、硫酸铵质量浓度0.8 g/dL、甘油质量浓度1.94 g/dL、酵母粉质量浓度1.93 g/dL,在此发酵条件下发酵12.5 h,PLD的酶活达到0.85 U/mL,比出发菌株提高62.5%。同时,以环戊基甲醚作为有机相,柠檬酸-柠檬酸钠为水相,利用双相反应体系合成磷脂酰葡萄糖苷(phosphatidyl-glucose,PtdGlc)、磷脂酰丝氨酸(phosphatidylserine,PS)和磷脂酰甘油(phosphatidylglycerol,PG),转化率分别为94.8%、94.0%和87.1%。结果表明,该研究构建的枯草芽孢杆菌工程菌经过发酵条件优化后,制备的PLD具有较好的转酯活性,显示出良好的应用潜力。In this study,engineered strains of Bacillus subtilis producing phospholipase D(PLD)were constructed and the effects of four individual promoters(P_(HpaⅡ),P_(ylb(F4)),P_(2069m) and P_(43))on the expression level of PLD were compared.Shake-flask fermentation revealed that the expression level of the promoter PHpaⅡ was the highest(0.32 U/mL).Therefore,the engineered strain with the PHpaⅡ promoter was used as the fermentation strain,and the fermentation conditions of PLD were optimized using the single factor and response surface experiments.The optimal enzyme production conditions were as follows:an inoculum volume concentration of 2.5 mL/dL,fermentation temperature of 37℃,and mass concentrations of ammonium sulfate,glycerol,and yeast powder of 0.8,1.94 g/dL and 1.93 g/dL,respectively.Under these conditions,the fermentation time was 12.5 h and the hydrolysis activity of PLD reached 0.85 U/mL,which was 62.5%higher than that of the starter culture.Additionally,phosphatidyl-glucose(PtdGlc),phosphatidylserine(PS),and phosphatidylglycerol(PG)were successfully synthesized using the biphasic reaction system with cyclopentyl methyl ether as the organic phase and citric acid-sodium citrate as the aqueous phase.The conversion rates of PtdGlc,PSand PG were 94.8%,94.0%and 87.1%,respectively.These results showed that the engineered strains constructed in this study possess good transphosphatidylation activity under optimized fermentation conditions and offer potential applicability in the large scale production of PLD.

关 键 词：磷脂酶D 枯草芽孢杆菌 启动子 发酵优化 转磷脂酰化反应 

分 类 号：TQ925[轻工技术与工程—发酵工程]