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

摘要:

为合成高附加值β-苯丙氨酸，以重组苯丙氨酸氨基变位酶为催化剂，转化α-苯丙氨酸生成β-苯丙氨酸。首先克隆来源于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1626 bp，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.coli BL21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α-苯丙氨酸为β-苯丙氨酸，在最适条件下（30 ℃、pH 9、1.5 mol/L NH4+），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+、Mg2+、Ca2+、Fe3+对PaPAM的活性影响较小，表面活性剂SDS和Triton 100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 %。

Abstract:

In order to synthesize high value-added β-phenylalanine, the phenylalanine aminomutase was used to convert α-phenylalanine to β-phenylalanine. The phenylalanine aminomutase gene (pam) from Pantoea agglomerans was cloned, the expression vector was constructed for heterologous expression. The results showed that the gene pam with1626 bp encoding 541 amino acids of PaPAM was successfully cloned. The expression vector pET28a-pam was constructed and transferred into E. coli BL21 for induced expression using isopropy-β-D-thiogalactoside (IPTG). The electrophoretically pure recombinant phenylalanine aminomutase (PaPAM) was produced by affinity chromatograph. The product of β-phenylalanine was detected using 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 and surfactants have different effects on enzyme activity. Na+, Mg2+, Ca2+, and Fe3+ have little effect on PaPAM activity, while SDS and Triton 100 can strongly inhibit activity. Under optimal condition, the conversion rate of α-phenylalanine was reached 92 %.