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

国家林草科技领军期刊
中国精品科技期刊
中国高校百佳科技期刊
江苏省新闻出版政府奖期刊奖
RCCSE林学权威期刊（A+）
CSCD核心期刊
Scopus数据库收录期刊
中文核心期刊
SCD核心期刊

作者加群：102861116

微信公众号：南京林业大学学报

高级检索

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.

图/表 10

表1

图1

图2

图3

图4

图5

表2

图6

表3

图7

参考文献 23

[1]	欧阳光察, 薛应龙. 植物苯丙烷类代谢的生理意义及其调控[J]. 植物生理学报, 1988, 24(3):9-16.
	OUYANG G C, XUE Y L. Physiological role and regulation of phenylpropanoid metabolism in plant[J]. Plant Physiology Communications, 1988, 24(3):9-16. DOI: 10.13592/j.cnki.ppj.1988.03.003. doi: 10.13592/j.cnki.ppj.1988.03.003
[2]	贺立红, 张进标, 宾金华. 苯丙氨酸解氨酶的研究进展[J]. 食品科技, 2006, 31(7):31-34.
	HE L H, ZHANG J B, BIN J H. Research progress of phenylalanine ammonia-lyase[J]. Food Science and Technology, 2006, 31(7):31-34. DOI: 10.3969/j.issn.1005-9989.2006.07.009. doi: 10.3969/j.issn.1005-9989.2006.07.009
[3]	KOSUGE T, CONN E E. The metabolism of aromatic compounds in higher plants, V: purification and properties of dihydrocoumarin hydrolase of Melilotus alba[J]. Journal of Biological Chemistry, 1962, 237(5):1653-1656. DOI: 10.1021/ic00086a018. doi: 10.1021/ic00086a018
[4]	杜欣谊. 苯丙氨酸解氨酶的研究进展[J]. 现代化农业 2016(7):24-26.
	DU X Y. Research advances on plant phenylalanine ammonia-lyase gene[J]. Modernizing Agriculture, 2016(7):24-26. DOI: 10.3969/j.issn.1001-0254.2016.07.017. doi: 10.3969/j.issn.1001-0254.2016.07.017
[5]	CHEMLER J A, KOFFAS M A. Metabolic engineering for plant natural product biosynjournal in microbes[J]. Current Opinion in Biotechnology, 2008, 19(6):597-605. DOI: 10.1016/j.copbio.2008.10.011. doi: 10.1016/j.copbio.2008.10.011
[6]	JOHNSON , JOHN R. The Perkin reaction and related reactions[J]. Organic Reactions 2011(1):210-265. DOI: 10.1002/0471264180.or001.08. doi: 10.1002/0471264180.or001.08
[7]	LANGER B, LANGER M, RÉTEY J. Methylidene-Imidazolone (MIO) from histidine and phenylalanine ammonia-lyase[J]. Advances in Protein Chemistry, 2001, 58(58):175-214. DOI: 10.1016/s0065-3233(01)58005-5. doi: 10.1016/s0065-3233(01)58005-5
[8]	BANG H B, LEE K, LEE Y J, et al. High-level production of trans-cinnamic acid by fed-batch cultivation ofEscherichia coli[J]. Process Biochemistry, 2018, 68:30-36. DOI: 10.1016/j.procbio.2018.01.026. doi: 10.1016/j.procbio.2018.01.026
[9]	ZANG Y, JIANG T, CONG Y, et al. Molecular characterization of a recombinant Zea mays phenylalanine ammonia-Lyase (ZmPAL2) and its application in trans-cinnamic acid production from l-phenylalanine[J]. Applied Biochemistry and Biotechnology, 2015, 176(3):924-937. DOI: 10.1007/s12010-015-1620-4. doi: 10.1007/s12010-015-1620-4
[10]	刘晓娜, 刘雪梅, 杨传平, 等. 木质素合成研究进展[J]. 中国生物工程杂志, 2007, 27(3):120-126.
	LIU X N, LIU X M, YANG C P, et al. Advance in the synjournal of lignin[J]. China Biotechnology, 2007, 27(3):120-126. DOI: 10.3969/j.issn.1671-8135.2007.03.021. doi: 10.3969/j.issn.1671-8135.2007.03.021
[11]	薛永常. 杨树木质素合成酶PAL基因的克隆、鉴定及其表达载体的构建[D]. 北京: 中国林业科学研究院, 2001.
	XUE Y C. Cloning, identification of poplar phenylalanine ammonia-lyase gene and the construction of its expressional vectors[D]. Beijing: Chinese Academy of Forestry, 2001.
[12]	冯洁, 陈其煐. 棉株体内几种生化物质与抗枯萎病之间关系的初步研究[J]. 植物病理学报, 1991, 21(4):291-297.
	FENG J, CHEN Q Y. On the biochemical substances in cotton plants related to the resistance to Fusarium wilt [J]. Acta Phytopathologica Sinica, 1991, 21(4):291-297. DOI: 10.13926/j.cnki.apps.1991.04.016. doi: 10.13926/j.cnki.apps.1991.04.016
[13]	SUBRAMANIAM R, REINOLD S, MOLITOR E K, et al. Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa × Populus deltoides) phenylalanine ammonia-lyase genes[J]. Plant Physiology, 1993, 102(1):71-83. DOI: 10.1104/pp.102.1.71. doi: 10.1104/pp.102.1.71
[14]	SHI R, SUN Y H, LI Q Z, et al. Towards a systems approach for lignin biosynjournal in Populus trichocarpa: transcript abundance and specificity of the monolignol biosynthetic genes[J]. Plant and Cell Physiology, 2010, 51(1):144-163. DOI: 10.1093/pcp/pcp175. doi: 10.1093/pcp/pcp175
[15]	D’CUNHA G B, SATYANARAYAN V, MADHUSUDANAN NAIR P. Purification of phenylalanine ammonia lyase from Rhodotorula glutinis[J]. Phytochemistry, 1996, 42(1):17-20. DOI: 10.1016/0031-9422(95)00914-0. doi: 10.1016/0031-9422(95)00914-0
[16]	HSIEH L S, HSIEH Y L, YEH C S, et al. Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii[J]. Molecular Biology Reports, 2011, 38(1):283-290. DOI: 10.1007/s11033-010-0106-2. doi: 10.1007/s11033-010-0106-2
[17]	陈文拴, 黄乾明, 陈华萍, 等. 油橄榄苯丙氨酸解氨酶基因的克隆及其在毕赤酵母中的表达[J]. 食品科学, 2015, 36(7):124-130.
	CHEN W S, HUANG Q M, CHEN H P, et al. Cloning of phenylalanine ammonia lyase gene fromOlea europaea and its expression in Pichia pastoris [J]. Food Science, 2015, 36(7):124-130. DOI: 10.7506/spkx1002-6630-201507023. doi: 10.7506/spkx1002-6630-201507023
[18]	ROSLER J, KREKEL F, AMRHEIN N, et al. Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity[J]. Plant Physiology, 1997, 113(1):175-179. DOI: 10.1104/pp.113.1.175. doi: 10.1104/pp.113.1.175
[19]	DE JONG F, HANLEY S J, BEALE M H, et al. Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar[J]. Phytochemistry, 2015, 117:90-97. DOI: 10.1016/j.phytochem.2015.06.005. doi: 10.1016/j.phytochem.2015.06.005
[20]	REICHERT A I, HE X Z, DIXON R A. Phenylalanine ammonia-lyase (PAL) from tobacco (Nicotiana tabacum): characterization of the four tobacco PAL genes and active heterotetrameric enzymes[J]. Biochemical Journal, 2009, 424(2):233-242. DOI: 10.1042/bj20090620. doi: 10.1042/bj20090620
[21]	COCHRANE F C, DAVIN L B, LEWIS N G. The Arabidopsis phenylalanine ammonia lyase gene family: kinetic characterization of the four PAL isoforms[J]. Phytochemistry, 2004, 65(11):1557-1564. DOI: 10.1016/j.phytochem.2004.05.006. doi: 10.1016/j.phytochem.2004.05.006
[22]	宛国伟, 董娟娥, 梁宗锁, 等. 培养条件对离体丹参根苯丙氨酸解氨酶和多酚氧化酶活性的影响[J]. 西北植物学报, 2007, 27(12):2471-2477.
	WAN G W, DONG J E, LIANG Z S, et al. Phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) activities of in vitro root under different culturing conditions in Salvia miltiorrhiza [J]. Acta Botanica Boreali-Occidentalia Sinica, 2007, 27(12):2471-2477. DOI: 10.3321/j.issn:1000-4025.2007.12.019. doi: 10.3321/j.issn:1000-4025.2007.12.019
[23]	HU G S, JIA J M, HUR Y J, et al. Molecular characterization of phenylalanine ammonia lyase gene fromCistanche deserticola[J]. Molecular Biology Reports, 2011, 38(6):3741-3750. DOI: 10.1007/s11033-010-0489-0. doi: 10.1007/s11033-010-0489-0

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

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 23

相关文章 15

编辑推荐

Metrics

本文评价

作者

读者

审稿

[1]	高源, 孙佳彤, 周晨光, 姜立泉, 李伟, 李爽. LBD12转录因子调控毛果杨木材形成研究[J]. 南京林业大学学报（自然科学版）, 2024, 48(1): 29-38.
[2]	孙佳彤, 国艳娇, 李爽, 周晨光, 姜立泉, 李伟. 基于CRISPR/Cas9的毛果杨bHLH106转录因子的功能研究[J]. 南京林业大学学报（自然科学版）, 2021, 45(6): 15-23.
[3]	王竹雯, 国艳娇, 李爽, 周晨光, 姜立泉, 李伟. 基于CRISPR/Cas9的毛果杨PtrHBI1基因功能解析[J]. 南京林业大学学报（自然科学版）, 2021, 45(6): 31-39.
[4]	王佳懿,李泰仑,王天女,卢磊. 短小芽孢杆菌LC01的鉴定及其芽孢漆酶性质的研究[J]. 南京林业大学学报（自然科学版）, 2018, 42(05): 113-120.
[5]	李丹蕾,王峰,陈俏丽,张瑞芝,王佳楠. 美洲黑杨×毛果杨NDR1基因表达对E4锈菌侵染的响应[J]. 南京林业大学学报（自然科学版）, 2018, 42(01): 21-26.
[6]	王浩然,李爽爽,乐丽娜,匡华琳,黄敏仁,陈英. miR164a及其靶基因PeNAC1相互作用研究[J]. 南京林业大学学报（自然科学版）, 2016, 40(05): 29-33.
[7]	汤叶根,施泉,林新春,徐英武. 雷竹SOC1蛋白原核表达及纯化[J]. 南京林业大学学报（自然科学版）, 2016, 40(03): 41-46.
[8]	李迅,顾华祥,王飞. 痤疮丙酸杆菌亚油酸异构酶的克隆表达及酶学性质研究[J]. 南京林业大学学报（自然科学版）, 2014, 38(06): 105-109.
[9]	李琦,赵东霞,刘世萍,柴常升,赵林果. 不同来源重组漆酶的酶学特性[J]. 南京林业大学学报（自然科学版）, 2014, 38(03): 93-97.
[10]	张冰,向冰冰,崔岱宗,赵敏. 枯草芽孢杆菌WD-23 β-甘露聚糖酶的纯化及其酶学性质[J]. 南京林业大学学报（自然科学版）, 2014, 38(03): 88-92.
[11]	朱煜,谭梦月,邹爱兰,张文举,戚金亮,杨永华. 拟南芥、水稻和毛果杨中CesA基因的进化和表达分析[J]. 南京林业大学学报（自然科学版）, 2013, 37(03): 11-16.
[12]	徐勇，华凤飞，朱均均，勇强，余世袁. 里氏木霉液体发酵选择性合成果胶酶[J]. 南京林业大学学报（自然科学版）, 2012, 36(01): 93-96.
[13]	陈英,王浩然,许庆,黄敏仁*. ptr-MIR156a启动子克隆及特征分析[J]. 南京林业大学学报（自然科学版）, 2011, 35(06): 1-5.
[14]	李治林1，李迅1，王丽丽1，王飞1*，蒋剑春2. 米根霉产脂肪酶的条件及酶学性质[J]. 南京林业大学学报（自然科学版）, 2009, 33(06): 103-.
[15]	田晓明，谭晓风*，胡孝义，刘巧，罗茜. 油茶苯丙氨酸解氨酶基因的cDNA克隆与序列分析[J]. 南京林业大学学报（自然科学版）, 2009, 33(04): 24-28.