成团泛菌苯丙氨酸氨基变位酶的性质表征及用于合成β-苯丙氨酸
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摘要
克隆来源于Pantoea agglomerans的苯丙氨酸氨基变位酶基因(pam),构建表达载体,转入大肠杆菌中进行异源表达,采用亲和层析制备电泳纯的重组酶(PaPAM),用于催化合成β-苯丙氨酸。结果表明:成功克隆得到基因pam,长度为1626bp,编码541个氨基酸长度的PaPAM,构建了大肠杆菌表达载体pET28a-pam,转入E.coliBL21中经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,镍柱亲和层析纯化获得电泳纯的重组PaPAM。MS和NMR表征结果表明,重组PaPAM能异构化a-苯丙氨酸为β-苯丙氨酸,在最适条件下(30℃、pH 9、1.5 mol/L NH_4~+),酶活力达到2.5 kU/g,在30～50℃、pH 8～10下PaPAM具有较高的稳定性,金属离子Na~+、Mg~(2+)、Ca~(2+)、Fe~(3+)对PaPAM的活性影响较小,表面活性剂SDS和Triton100对PaPAM有较强抑制作用,在最佳反应条件下,底物的转化率达到92%。
The gene of phenylalanine aminomutase(pam) was cloned from Pantoea agglomerans and the expression vector was constructed for heterologous expression in E. coli. The electrophoretically pure recombinant phenylalanine aminomutase(PaPAM) obtained by affinity chromatography was used to synthesize β-phenylalanine. The results showed that the gene pam with 1626 bp encoding 541 amino acids of PaPAM was successfully cloned. The expression vector pET28 a-pam was constructed and transferred into E. coli BL21 for induced expression using isopropy-β-D-thiogalactoside(IPTG). The product ofβ-phenylalanine was characterized by MS and NMR. The enzyme activity reached 2.5 k U/g under the optimum conditions of 30 ℃, pH 9.0 and 1.5 mol/L NH_4~+. The enzyme exhibited high stability at 30～50 ℃ and pH 8～10. Metal ions Na~+, Mg~(2+), Ca~(2+) and Fe~(3+) had little effect on the activity of PaPAM, while surfactants SDS and Triton 100 had strong inhibitory effect on PaPAM. Under the optimal reaction condition, the conversion of α-phenylalanine reached 92%.
The gene of phenylalanine aminomutase(pam) was cloned from Pantoea agglomerans and the expression vector was constructed for heterologous expression in E. coli. The electrophoretically pure recombinant phenylalanine aminomutase(PaPAM) obtained by affinity chromatography was used to synthesize β-phenylalanine. The results showed that the gene pam with 1626 bp encoding 541 amino acids of PaPAM was successfully cloned. The expression vector pET28 a-pam was constructed and transferred into E. coli BL21 for induced expression using isopropy-β-D-thiogalactoside(IPTG). The product ofβ-phenylalanine was characterized by MS and NMR. The enzyme activity reached 2.5 k U/g under the optimum conditions of 30 ℃, pH 9.0 and 1.5 mol/L NH_4~+. The enzyme exhibited high stability at 30～50 ℃ and pH 8～10. Metal ions Na~+, Mg~(2+), Ca~(2+) and Fe~(3+) had little effect on the activity of PaPAM, while surfactants SDS and Triton 100 had strong inhibitory effect on PaPAM. Under the optimal reaction condition, the conversion of α-phenylalanine reached 92%.
引文
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[6]Forro E,Fueloep F.Recent lipase-catalyzed hydrolytic approaches to pharmacologically important beta-and gamma-amino acids[J].Current Medicinal Chemistry,2012,19(36):6178-6187.
[7]Grulich M,Brezovsky J,Stepanek V,et al.Resolution of alpha/beta-amino acids by enantioselective penicillin G acylase from Achromobacter sp[J].Journal of Molecular Catalysis B-Enzymatic,2015,122:240-247.
[8]Mathew S,Jeong S S,Chung T,et al.Asymmetric synthesis of aromatic beta-amino acids using omega-transaminase:Optimizing the lipase concentration to obtain thermodynamically unstable beta-keto acids[J].Biotechnology Journal,2016,11(1):185-190.
[9]Mathew S,Bea H,Nadarajan S P,et al.Production of chiralα-amino acids usingω-transaminase from Burkholderia graminis[J].Journal Biotechnology,2015,20(196/197):1-8.
[10]Parmeggiani F,Weise N J,Ahmed S T,et al.Synthetic and therapeutic applications of ammonia-lyases and aminomutases[J].Chemical Reviews,2018,118(1):73-118.
[11]Wybenga G,Szymanski W,Wu B,et al.Structural investigations into the stereochemistry and activity of a phenylalanine-2,3-aminomutase from Taxus chinensis[J].Biochemistry,2014,53(19):3187-3198.
[12]Wu B,Szymanski W,Wybenga G G,et al.Mechanism-inspired engineering of phenylalanine aminomutase for enhanced betaregioselective asymmetric amination of cinnamates[J].Angewandte Chemie-International Edition,2012,51(2):482-486.
[13]Chesters C,Wilding M,Goodall M,et al.Thermal bifunctionality of bacterial phenylalanine aminomutase and ammonia lyase enzymes[J].Angewandte Chemie-International Edition,2012,51(18):4344-4348.
[14]Zhu Longbao(朱龙宝),Tao Yugui(陶玉贵),Ge Fei(葛飞),et al.Production and characterization of phenylalanine aminomutase from Streptomyces Maritimus and synthesis of beta-arylalanine[J].Chemical Journal of Chinese Universities-Chinese(高等学校化学学报),2017,38(2):206-211.
[15]Bradford M M.A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(9):248-254
[16]Heberling M M,Masman M F,Bartsch S,et al.Ironing out their differences:dissecting the structural determinants of a phenylalanine aminomutase and ammonia lyase[J].ACS Chemical Biology,2015,10(4):989-997.
[17]Wu B,Szymanski W,Wietzes P,et al.Enzymatic synthesis of enantiopure alpha-and beta-amino acids by phenylalanine aminomutase-catalysed amination of cinnamic acid derivatives[J].Chembiochem,2009,10(2):338-344.
[18]Walker K D,Klettke K,Akiyama T,et al.Cloning,heterologous expression,and characterization of a phenylalanine aminomutase involved in Taxol biosynthesis[J].Journal Biological Chemistry,2004,279(52):53947-53954.