【目的】苯丙氨酸解氨酶(PAL)在苯丙烷代谢途径中具有重要作用,PtrPAL基因的挖掘、酶学性质分析和应用为毛果杨苯丙烷代谢途径的研究和肉桂酸(t-ca)合成提供参考。【方法】基于基因组注释信息,挖掘毛果杨PtrPAL基因,进行原核表达和酶学性质分析。以L-苯丙氨酸(L-Phe)为底物,通过全细胞催化合成t-ca。【结果】通过挖掘毛果杨基因组得到了5个PtrPAL基因,系统发育树分析表明其在进化上分为两组。PtrPAL3和PtrPAL4来自不同进化分支,选取其进行原核表达和酶学性质分析。镍柱纯化后的重组蛋白rePtrPAL3和rePtrPAL4最适温度分别是55 ℃和60 ℃,最适pH分别是8.5和8.0,比酶活分别为3.363和3.381 U/mg,且均具有微弱的酪氨酸解氨酶活力。重组蛋白的动力学分析表明,rePtrPAL3对L-Phe的亲和力更高。将rePtrPAL3应用于L-Phe脱氨生产t-ca,全细胞催化7 h可产生104 mmol/L t-ca。【结论】具有不同转录特异性的两种rePtrPAL酶学性质接近,但rePtrPAL3表现出更高的催化效率,更适合用于t-ca的生物合成。而且,其产量高于文献报道的28 mmol/L水平,具有良好的应用潜力。

【Objective】 Phenylalanine ammonia-lyase (PAL) plays an important role in the phenylpropanoid metabolism pathway. It would be of importance to determine the PAL genes from Populus trichocarpa, analyze their enzymatic properties, and apply them in biosynthesis. This study provides a reference for the research of its phenylpropanoid metabolic pathway and synthesis of trans-cinnamic acid (t-ca). 【Method】 Based on the genomic annotation information, the PtrPAL genes of P. trichocarpa were searched. Some of them were selected for prokaryotic expression and enzymatic analysis. Finally, t-ca was biosynthesized from L-phenylalanine (L-Phe) by whole-cell catalysis. 【Result】 In this paper, fivePAL genes were predicted in the genome of P. trichocarpa. They were divided into two groups based on the phylogenetic tree analysis. The PtrPAL3 and PtrPAL4, which from different groups, were selected for protein expression in Escherichia coli and enzymatic analysis. The optimum temperature of rePtrPAL3 and rePtrPAL4 was 55 °C and 60 °C, respectively. The optimum pH was 8.5 and 8.0, respectively. The rePtrPAL3 (3.363 U/mg) and rePtrPAL4 (3.381 U/mg) exhibited similar PAL activity and weak tyrosine ammonia-lyase activity. Kinetic analysis indicated that rePtrPAL3 has higher affinity for L-Phe; therefore, it was used to produce t-ca from L-Phe, and the titer reached 104 mmol/L in 7 h by whole-cell catalysis. 【Conclusion】 In spite of different transcriptional specificities, the two PtrPALs displayed similar enzymatic properties. However, rePtrPAL3 showed higher catalytic efficiency. Thus, rePtrPAL3 is more suitable for the biosynthesis of t-ca. In addition, it has good application potential for that the titer of t-ca is higher than other reports (28 mmol/L).