体外催化花生四烯酸生产前列腺素F_(2α)  

In vitro catalytic production of prostaglandin F_(2α)using arachidonic acid

作　　者：范潇 何国威 黄薇 童建华[1] 卢向阳[1] 田云[1] 刘虎虎[1] FAN Xiao;HE Guowei;HUANG Wei;TONG Jianhua;LU Xiangyang;TIAN Yun;LIU Huhu(College of Bioscience and Biotechnology,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南农业大学生物科学技术学院,长沙410128

出　　处：《中国油脂》2024年第1期114-119,共6页China Oils and Fats

基　　金：国家自然科学基金(21808052);湖南农业大学“双一流”建设项目(SYL201802002);长沙市杰出创新青年培养计划(kq2106049)。

摘　　要：前列腺素F_(2α)(PGF_(2α))具有广泛的生理活性,是一种重要的脂质介质。为了实现PGF_(2α)的绿色生物合成,构建了pET30a-TbPGFS、pET30a-MmPGHS、pET30a-GvPGHS载体并在大肠杆菌BL21(DE3)中分别表达,同时对异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达条件进行了优化以提高重组蛋白在大肠杆菌中的表达量。另外,以最佳诱导表达条件下得到的重组蛋白为催化剂,催化花生四烯酸合成PGF_(2α)。结果表明:MmPGHS蛋白在大肠杆菌中没有表达;GvPGHS蛋白的最佳诱导表达条件为诱导剂IPTG浓度0.2 mmol/L、诱导温度30℃、诱导时间2 h,TbPGFS蛋白的最佳诱导表达条件为诱导剂IPTG浓度0.2 mmol/L、诱导温度30℃、诱导时间6 h;在最佳诱导表达条件下,由GvPGHS和TbPGFS的粗酶液构成的双酶催化未能检测到产物PGF_(2α),而在酶偶联化学催化时,GvPGHS能将花生四烯酸转化为PGH_(2),PGH_(2)被SnCl_(2)进一步还原为PGF_(2α)。综上,通过体外酶促反应结合化学法可高效转化花生四烯酸生产PGF_(2α)。As the important lipid mediators,prostaglandin F_(2α)(PGF_(2α))performs a wide range of physiological activities.To achieve the green biosynthesis of PGF_(2α),pET30a-TbPGFS,pET30a-MmPGHS and pET30a-GvPGHS vectors were constructed and expressed in Escherichia coli BL21(DE3),respectively,and the induction expression conditions of isopropyl-β-D-thiogalactopyranoside(IPTG)were optimized to improve the expression of the recombinant protein in E.coli.In addition,the recombinant protein obtained under the optimal conditions were used as a catalyst to catalyze the synthesis of PGF_(2α)from arachidonic acid.The results showed that MmPGHS protein was not expressed in E.coli.However,GvPGHS protein performed the optimized expression under the conditions of IPTG concentration 0.2 mmol/L,induction temperature 30℃and induction time 2 h,the TbPGFS protein performed the optimized expression under the conditions of IPTG concentration 0.2 mmol/L,induction temperature 30℃and induction time 6 h.Under the above optimized conditions,PGF_(2α)was not detected by the two-enzyme coupled catalysis system composed of crude extract of GvPGHS and TbPGFS.However,arachidonic acid was converted into PGH_(2)by GvPGHS,which was further reduced to PGF_(2α)by SnCl_(2)under the enzyme-coupled chemical catalysis.In conclusion,In vitro enzymatic reaction combined with chemical method can efficiently convert arachidonic acid to produce PGF_(2α).

关 键 词：花生四烯酸 前列腺素F_(2α) 体外催化 前列腺素H合成酶 

分 类 号：Q782[生物学—分子生物学] TQ645.63[化学工程—精细化工]