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

Characterization of Phenylalanine Aminomutase from Pantoea Agglomerans and Synthesis of β-phenylalanine

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 synthesizeβ-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 NH4+.The enzyme exhibited high stability at 30~50℃and pH 8~10.Metal ions Na+,Mg2+,Ca2+and Fe3+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 ofα-phenylalanine reached 92%.