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

doi:10.3969/j.issn.1000-2006.201901018

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2020, Vol. 44 ›› Issue (1): 97-104.doi: 10.3969/j.issn.1000-2006.201901018

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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 E-mail:zhangchen@njfu.edu.cn;hgouyj@njfu.edu.cn

Abstract

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

CLC Number:

Q814.9

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, 2020, 44(1): 97-104.

Figures/Tables 10

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References 23

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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

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

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