毛果杨苯丙氨酸解氨酶活性比较及肉桂酸制备

doi:10.3969/j.issn.1000-2006.201901018

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南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (1): 97-104.doi: 10.3969/j.issn.1000-2006.201901018

毛果杨苯丙氨酸解氨酶活性比较及肉桂酸制备

张晨¹^,²(), 臧颖³, 许倩¹^,², 郑兆娟¹^,², 欧阳嘉¹^,²^,^*()

1.南京林业大学,江苏省林业资源高效加工利用协同创新中心,江苏南京 210037
2.南京林业大学化学工程学院,江苏南京 210037
3.南京林业大学林学院,江苏南京 210037

收稿日期:2019-01-16 修回日期:2019-04-30 出版日期:2020-02-08 发布日期:2020-02-02
通讯作者: 欧阳嘉
作者简介:张晨( zhangchen@njfu.edu.cn)。
基金资助:
国家重点研发计划(2017YFD0600205);江苏省高校自然科学研究重大项目(16KJA220004)

Comparison on activities of phenylalanine ammonia-lyase from Populus trichocarpa and its application in trans-cinnamic acid production

ZHANG Chen¹^,²(), ZANG Ying³, XU Qian¹^,², ZHENG Zhaojuan¹^,², OUYANG Jia¹^,²^,^*()

1. Jiangsu Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, China
2. College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
3. College of Forestry, Nanjing Forestry University, Nanjing 210037, China

Received:2019-01-16 Revised:2019-04-30 Online:2020-02-08 Published:2020-02-02
Contact: OUYANG Jia

摘要/Abstract

摘要：

【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。

关键词: 毛果杨, 苯丙氨酸解氨酶, 酶学性质, 肉桂酸, 原核表达

Abstract:

【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).

Key words: Populus trichocharpa, phenylalanine ammonia lyase, enzymological characterization, trans-cinnamic acid, prolcaryotic expression

中图分类号:

Q814.9

张晨,臧颖,许倩,等. 毛果杨苯丙氨酸解氨酶活性比较及肉桂酸制备[J]. 南京林业大学学报（自然科学版）, 2020, 44(1): 97-104.

ZHANG Chen, ZANG Ying, XU Qian, ZHENG Zhaojuan, OUYANG Jia. Comparison on activities of phenylalanine ammonia-lyase from Populus trichocarpa and its application in trans-cinnamic acid production[J].Journal of Nanjing Forestry University (Natural Science Edition), 2020, 44(1): 97-104.DOI: 10.3969/j.issn.1000-2006.201901018.

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基因号 gene ID	外显子 exon	基因位置 gene location	编码蛋白 protein	蛋白名称 name	长度/氨基酸 length
LOC18100180	2	chromosome 6, NC_037290.1 (10249310..10252657)	XP_006381441.1	PtrPAL1	714
LOC7479878	2	chromosome 8, NC_037292.1 (2124062..2128290)	XP_002312013.1	PtrPAL2	711
LOC7486485	2	chromosome 16, NC_037300.1 (7482830..7486161)	XP_002322884.2	PtrPAL3	715
LOC7463441	2	chromosome 10, NC_037294.1 (20815412..20819188)	XP_002315308.1	PtrPAL4	711
LOC7463442	2	chromosome 10, NC_037294.1 (20822241..20825776)	XP_002315309.3	PtrPAL5	707

重组蛋白 recombinant protein	底物 substrate	K_m/ (μmol·L^-1)	V_max/ (μmol·min^-1·mg^-1)	k_cat/ (min^-1)	k_cat/K_m/ (μmol·min^-1··L^-1)
rePtrPAL3 (55 ℃)	L-Phe	87.436	4.044	1 277.904	14.615
rePtrPAL3 (55 ℃)	L-Tyr	140.680	17.374×10^-3	5.490	39.025×10^-3
rePtrPAL4 (60 ℃)	L-Phe	170.227	4.029	1 273.164	7.478
rePtrPAL4 (60 ℃)	L-Tyr	268.573	6.720×10^-3	2.124	7.910×10^-3
SvPALs^[19] (40 ℃)	L-Phe	32.4~88.4	0.428~1.629	501.0~1 697.4	5.667~10.108
NtPALs^[20] (30 ℃)	L-Phe	36.4~59.8	0.588~1.176	48~90	0.803~2.473
AtPALs^[21] (31 ℃、37 ℃)	L-Phe	64~2 560	0.024~0.630	6~192	0.002~3.000
ZmPALs^[18] (40 ℃)	L-Phe	270~2 492		10.6

处理时间/h treatment time	rePtrPAL3				rePtrPAL4
处理时间/h treatment time	L-Phe残余量/(mmol·L^-1) residual L-Phe		t-ca浓度/(mmol·L^-1) the concentration of t-ca		L-Phe残余量/(mmol·L^-1) residual L-Phe		t-ca浓度/(mmol·L^-1) the concentration of t-ca
8	6.732±1.618	65.735±3.040		13.245±0.402		55.828±3.097

毛果杨苯丙氨酸解氨酶活性比较及肉桂酸制备

Comparison on activities of phenylalanine ammonia-lyase from Populus trichocarpa and its application in trans-cinnamic acid production

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