Fe(Ⅱ)/2-酮戊二酸依赖型双加氧酶重组大肠杆菌全细胞催化合成4-羟基异亮氨酸
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摘要
以L-异亮氨酸(L-ILe)为底物,以异源表达Fe(Ⅱ)/2-酮戊二酸依赖型双加氧酶的重组大肠杆菌BL21/pET28a-ido全细胞作为催化剂,催化合成4-羟基异亮氨酸(4-HIL).基于重组异亮氨酸双加氧酶(IDO)催化异亮氨酸羟基化的性质和条件,在摇瓶水平上对辅因子亚铁离子、α-酮戊二酸(α-KG)及底物浓度进行了单因素优化.获得的最佳反应条件为2 g/L FeSO_4·7H_2O,底物与α-KG摩尔比为1∶1,该条件下反应8 h可生成190 mmol/L 4-HIL.结合摇瓶水平的最优条件,在反应器水平上继续对搅拌速度、菌体浓度等进行优化,实现了对高底物浓度下催化反应的连续调控.在50 g/L湿菌体及400 r/min转速下可一次转化合成400mmol/L 4-HIL,建立了全细胞催化合成4-羟基异亮氨酸的工艺流程.
4-Hydroxyisoleucine(4-HIL) was synthesised using L-isoleucine(L-Ile) as substrate, the recombinant Escherichia coli BL21/pET28 a-ido heterologously expressing Fe(Ⅱ)/2-ketoglutarate-dependent dioxygenase,and the whole cell as a catalyst. Based on the catalytic properties and conditions of L-Ile dioxygenase catalyzing isoleucine hydroxylation,the optimization of single factors included ferrous sulfate(FeSO_4·7H_2O),α-ketoglutaric acid(α-KG) and substrate concentration. As a result,the optimized yield of 190 mmol/L of 4-HIL was obtained in the 50 mmol/L trimthylolamine hydrochloride(Tris-HCl) buffer system comprising FeSO_4·7H_2O(2 g/L),with the molar ratio of substrate to α-KG molarity as 1 ∶ 1. Associated with the optimized conditions in shake flask level,the stirring speed and the cell concentration were further optimized at the reactor level,to achieve continuous regulation of isoleucine hydroxylation at high substrate concentration.Consequently,a whole-cell transformation system and process was established to produce 400 mmol/L 4-HIL,with the conditions of FeSO_4·7H_2O(2 g/L),molar concentration ratio of substrate and α-KG 1 ∶ 1,wet bacteria 50 g/L,and rotation speed 400 r/min.
4-Hydroxyisoleucine(4-HIL) was synthesised using L-isoleucine(L-Ile) as substrate, the recombinant Escherichia coli BL21/pET28 a-ido heterologously expressing Fe(Ⅱ)/2-ketoglutarate-dependent dioxygenase,and the whole cell as a catalyst. Based on the catalytic properties and conditions of L-Ile dioxygenase catalyzing isoleucine hydroxylation,the optimization of single factors included ferrous sulfate(FeSO_4·7H_2O),α-ketoglutaric acid(α-KG) and substrate concentration. As a result,the optimized yield of 190 mmol/L of 4-HIL was obtained in the 50 mmol/L trimthylolamine hydrochloride(Tris-HCl) buffer system comprising FeSO_4·7H_2O(2 g/L),with the molar ratio of substrate to α-KG molarity as 1 ∶ 1. Associated with the optimized conditions in shake flask level,the stirring speed and the cell concentration were further optimized at the reactor level,to achieve continuous regulation of isoleucine hydroxylation at high substrate concentration.Consequently,a whole-cell transformation system and process was established to produce 400 mmol/L 4-HIL,with the conditions of FeSO_4·7H_2O(2 g/L),molar concentration ratio of substrate and α-KG 1 ∶ 1,wet bacteria 50 g/L,and rotation speed 400 r/min.
引文
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[18]Liu X.H.,Li H.X.,Chen Y.,Cao Y.S.,Sci.Technol.Food Ind.,2011,32(11),475-479(刘晓华,李海星,陈燕,曹郁生.食品工业科技,2011,32(11),475-479)
[2]Fowden L.,Pratt H.M.,Smith A.,Phytochemistry,1973,12(7),1707-1711
[3]Broca C.,Manteghetti M.,Gross R.,Baissac Y.,Jacob M.,Petit P.,Sauvaire Y.,Ribes G.,Eur.J.Pharmacol.,2000,390(3),339-345
[4]Broca C.,Gross R.,Petit P.,Sauvaire Y.,Manteghetti M.,Tournier M.,Masiello P.,Gomis R.,Ribes G.,Am.J.Physiol.,1999,277(4),E617-623
[5]Balant L.,Clin.Pharmacokinet,1981,6(3),215-241
[6]Jennings A.M.,Wilson R.M.,Ward J.D.,Diabetes Care,1989,12(3),203-208
[7]Ogawa J.,Kodera T.,Smirnov S.V.,Hibi M.,Samsonova N.N.,Koyama R.,Yamanaka H.,Mano J.,Kawashima T.,Yokozeki K.,Shimizu S.,Appl.Microbiol.Biotechnol.,2011,89(6),1929-1938.
[8]Hossain G.S.,Li J.,Shin H.D.,Chen R.R.,Du G.,Liu L.,Chen J.,J.Biotechnol.,2014,169(9),112-120
[9]Xu C.,Yin X.H.,Zhang C.,Chen H.Y.,Huang H.,Hu Y.,Chem.Res.Chinese Universities,2018,34(2),279-284
[10]HaefeléC.,Bonfils C.,Sauvaire Y.,Phytochemistry,1997,44(4),563-566
[11]Kodera T.,Smirnov S.V.,Samsonova N.N.,Kozlov Y.I.,Koyama R.,Hibi M.,Ogawa J.,Yokozeki K.,Shimizu S.,Biochem.Biophys.Res.Commun.,2009,390(3),506-510
[12]Zhang C.,Ma J.,Li Z.,Liang Y.,Xu Q.,Xie X.,Chen N.,Bioengineered,2018,9(1),72-79
[13]Fu M.J.,Nie Y.,Mu X.Q.,Xu Y.,Xiao R.,Chem.Ind.Engineering Prog.,2014,33(11),3037-3044(付敏杰,聂尧,穆晓清,徐岩,肖荣.化工进展,2014,33(11),3037-3044)
[14]Hoffart L.M.,Barr E.W.,Guyer R.B.,Bollinger J.M.Jr.,Krebs C.,Proc.Natl.Acad.Sci.USA,2006,103(40),14738-14743
[15]Smirnov S.V.,Kodera T.,Samsonova N.N.,Kotlyarova V.A.,Rushkevich N.Y.,Kivero A.D.,Sokolov P.M.,Hibi M.,Ogawa J.,Shimizu S.,Appl.Microbiol.Biotechnol.,2010,88(3),719-726
[16]Doucette C.D.,Schwab D.J.,Wingreen N.S.,Rabinowitz J.D.,Nat.Chem.Biol.,2011,7(12),894-901
[17]Schiefelbein S.,Fr9hlich A.,John G.T.,Beutler F.,Wittmann C.,Becker J.,Biotechnol.Lett.,2013,35(8),1223-1230
[18]Liu X.H.,Li H.X.,Chen Y.,Cao Y.S.,Sci.Technol.Food Ind.,2011,32(11),475-479(刘晓华,李海星,陈燕,曹郁生.食品工业科技,2011,32(11),475-479)