转氨酶高效表达重组工程菌的培养基及诱导条件优化
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摘要
通过构建重组菌株PE-ATS(BL21)表达可催化底物合成西格列汀的转氨酶,采用摇瓶正交和单因素实验确定发酵培养基的最佳条件为甘油9.0 g/L,酵母粉16.0 g/L、硫酸铵4.0 g/L、氯化钠6.0 g/L、磷酸氢二钠2.0 g/L、磷酸二氢钾3.0 g/L;最佳表达条件为36℃、0.6 mmol/L IPTG诱导4 h。该条件下,摇瓶培养菌液OD600=6.63,SDS-PAGE分析显示酶表达量约占可溶蛋白总量的50%,酶活为758 U/L。
Through the construction of recombinant strain PE-ATS(BL21) expression of a catalytic substrate for the synthesis of siglitine transaminase, the optimal conditions for fermentation medium were determined by shaking bottle orthogonal and single factor experiment, namely 9.0 g/L of glycerol, 16.0 g/L of peptone, 4.0 g/L of ammonium sulfate, 6.0 g/L of sodium chloride, 2.0 g/L of disodium phosphate, and 3.0 g/L of potassium dihydrogen phosphate. Best expression condition of 36 ℃, tendency for 0.6 mmol/L IPTG induction 4 h. Under the condition, the bacteria solution in shaking bottle was OD600 = 6.63. SDS-PAGE analysis showed that the soluble expression of transaminase accounted for about50% of the total soluble protein, and the enzyme activity was 758 U/L.
Through the construction of recombinant strain PE-ATS(BL21) expression of a catalytic substrate for the synthesis of siglitine transaminase, the optimal conditions for fermentation medium were determined by shaking bottle orthogonal and single factor experiment, namely 9.0 g/L of glycerol, 16.0 g/L of peptone, 4.0 g/L of ammonium sulfate, 6.0 g/L of sodium chloride, 2.0 g/L of disodium phosphate, and 3.0 g/L of potassium dihydrogen phosphate. Best expression condition of 36 ℃, tendency for 0.6 mmol/L IPTG induction 4 h. Under the condition, the bacteria solution in shaking bottle was OD600 = 6.63. SDS-PAGE analysis showed that the soluble expression of transaminase accounted for about50% of the total soluble protein, and the enzyme activity was 758 U/L.
引文
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[11]罗水忠,蔡静,姜绍通,等.重组大肠杆菌产谷氨酰胺转氨酶的发酵条件优化[J].食品科技,2016,37(12):183-189.
[2]CK Savile,JM janey,EC Mundorff,et al.Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture[J].Science,2010,329(5989):305-309.
[3]Lianghui Jia,Hairong Cheng,Hengwei Wang,et al.From Shake Flasks to Bioreactors:Survival of E.coli Cells Harboring pGST-hPTH through Auto-Induction by Controlling Initial Content of Yeast Extract[J].Applied Microbiology and Biotechnology,2011,9(40):1419-1428.
[4]贾红华,朱清禾,李艳,等.ω转氨酶制备手性胺的研究进展[J].现代化工,2012,32(3):16-20.
[5]黄亚杰,王子辉,赵艳伟,等.重组大肠杆菌产谷酰胺转氨酶培养基及发酵条件优化[J].中国酿造,2013,31(4):21-24.
[6]王璐,徐刚,吴坚平,等.转氨酶ATA117基因在大肠杆菌BL21(DE3)中的重组表达及产酶条件优化[J].化学反应工程与工艺,2014,30(6):513-521.
[7]黄宗庆,陆建光,张喜全,等.催化合成西格列汀的转氨酶基因克隆[J].中国医药工业杂志,2015,46(12):1296-130.
[8]陈坚,堵国成.发酵工程原理与技术[M].北京:化工出版社,2017.
[9]夏温娜,孙雨,闵聪,等.转氨酶催化不对称合成芳香族L-氨基酸[J].生物工程学报,2012,28(11):1346-1258.
[10]乔宇,丁红标,闫俊艳,等.重组大肠杆菌产普鲁兰酶的高密度发酵工艺研究[J].生物技术进展,2012,2(3):195-200.
[11]罗水忠,蔡静,姜绍通,等.重组大肠杆菌产谷氨酰胺转氨酶的发酵条件优化[J].食品科技,2016,37(12):183-189.