重组Taxus chinensis苯丙氨酸变位酶性质表征及R-β-芳香丙氨酸合成

Characterization of Recombinant Phenylalanine Aminomutase from Taxus chinensis and Its Application for Synthesis of R-β-Arylalanine

为实现酶法合成高附加值的β-苯丙氨酸,首先化学合成来源于紫衫(Taxus chinensis)的苯丙氨酸变位酶(phenylalanine aminomutase,PAM)基因(Tcpam),构建大肠杆菌重组质粒Pet-sumo-Tcpam,转入大肠杆菌中进行异源诱导表达,采用镍柱亲和层析制备电泳纯的重组酶TcPAM,用于催化合成R-β-苯丙氨酸。结果表明:重组质粒Pet-sumo-Tcpam成功在大肠杆菌中实现高效表达,获得可溶性的重组TcPAM,经过亲和层析制备出电泳纯的重组TcPAM。质谱和圆二色谱检测分析结果表明,TcPAM能够催化α-苯丙氨酸异构化为R构型的β-苯丙氨酸。TcPAM在最适温度30℃、pH 9的条件下,酶活力达到4.11 U/mg,金属离子K^(+)、Fe^(2+)和Ca^(2+)对TcPAM的活性影响较小,而Cu^(2+)和Zn^(2+)有强烈抑制性,表面活性剂十六烷基三甲基溴化铵、十二烷基硫酸钠、Triton X-100和Tween 80对重组酶活力影响较小,相对酶活力保持在90%以上。进一步利用TcPAM催化α-芳香丙氨酸异构合成β-芳香丙氨酸,结果表明:苯环上携带不同基团的α-芳香丙氨酸为底物时,苯环上携带供电子基团比吸电子基团的底物转化率更高,底物的α-氨基容易转移至β位,其中4-MeO-β-苯丙氨酸的产率最高,达到45%,为建立酶法合成R-β-芳香丙氨酸提供了参考。

In order to synthesize high value-added β-phenylalanine,the gene encoding phenylalanine aminomutase from Taxus chinensis was cloned and expressed in Escherichia coli.The expression vector Pet-sumo-Tcpam was successfully constructed and transferred into E.coli BL21 to express the recombinant enzyme(TcPAM).Electrophoretically pure TcPAM was obtained using affinity chromatography.The results of mass spectrometry(MS)and circular dichroism(CD)spectroscopy showed that TcPAM could catalyze isomerization ofα-phenylalanine to R-β-phenylalanine.Its activity was 4.11 U/mg under the optimum conditions of 30℃and pH 9.The metal ions K^(+),Fe^(2+)and Ca^(2+)as well as the surfactants cetrimonium bromide(CTAB),sodium dodecyl sulphate(SDS),Triton X-100,and Tween 80 had little effect on its activity,resulting in retention of about 90%of the initial activity,while Cu^(2+)and Zn^(2+)had strong inhibitory effect on TcPAM,Furthermore,when TcPAM was used to catalyze isomerization ofα-arylalanines with different groups on the benzene ring to R-β-phenylalanine,the presence of electron-donating groups on the benzene ring promoted transfer of theα-amino group to theβsite as compared to the presence of electron-withdrawing groups,improving the substrate conversion rate and the highest value of 45%was obtained when the R group was 4-MeO or 4-Me.This study provides the basis for enzymatic synthesis of β-arylalanine.