Zhongguo youzhi (Jan 2024)

体外催化花生四烯酸生产前列腺素F2αIn vitro catalytic production of prostaglandin F2α using arachidonic acid

  • 范潇，何国威，黄薇，童建华，卢向阳，田云，刘虎虎 FAN Xiao, HE Guowei, HUANG Wei, TONG Jianhua, LU Xiangyang, TIAN Yun, LIU Huhu

DOI
https://doi.org/10.19902/j.cnki.zgyz.1003-7969.220662
Journal volume & issue
Vol. 49, no. 1
pp. 114 – 119

Abstract

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前列腺素F2α（PGF2α）具有广泛的生理活性，是一种重要的脂质介质。为了实现PGF2α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PGF2α。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PGF2α，而在酶偶联化学催化时，GvPGHS能将花生四烯酸转化为PGH2，PGH2被SnCl2进一步还原为PGF2α。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PGF2α。As the important lipid mediators, prostaglandin F2α（PGF2α）performs a wide range of physiological activities. To achieve the green biosynthesis of PGF2α, 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 PGF2α 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, PGF2α was not detected by the two-enzyme coupled catalysis system composed of crude extract of GvPGHS and TbPGFS. However, arachidonic acid was converted into PGH2 by GvPGHS, which was further reduced to PGF2α by SnCl2 under the enzyme-coupled chemical catalysis. In conclusion, In vitro enzymatic reaction combined with chemical method can efficiently convert arachidonic acid to produce PGF2α.

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