[1]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(01):097-104.[doi:10.3969/j.issn.1000-2006.201901018]
 ZHANG Chen,ZANG Ying,XU Qian,et al.Comparison on activities of phenylalanine ammonia-lyase from Populustrichocarpa and its application in trans-cinnamic acid production[J].Journal of Nanjing Forestry University(Natural Science Edition),2020,44(01):097-104.[doi:10.3969/j.issn.1000-2006.201901018]
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毛果杨苯丙氨酸解氨酶活性比较及肉桂酸制备
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《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
44
期数:
2020年01期
页码:
097-104
栏目:
研究论文
出版日期:
2020-01-15

文章信息/Info

Title:
Comparison on activities of phenylalanine ammonia-lyase from Populus trichocarpa and its application in trans-cinnamic acid production
文章编号:
1000-2006(2020)01-0097-08
作者:
张 晨12臧 颖3许 倩12郑兆娟12欧阳嘉12*
(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)
Author(s):
ZHANG Chen12ZANG Ying3XU Qian12ZHENG Zhaojuan12OUYANG Jia12*
(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)
关键词:
毛果杨 苯丙氨酸解氨酶 酶学性质 肉桂酸 原核表达
Keywords:
Populus trichocharpa phenylalanine ammonia lyase enzymological characterization trans-cinnamic acid prolcaryotic expression
分类号:
Q814.9
DOI:
10.3969/j.issn.1000-2006.201901018
文献标志码:
A
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。
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, five PAL 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

参考文献/References:


[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.
[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.
[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.
[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.
[5] CHEMLER J A, KOFFAS M A. Metabolic engineering for plant natural product biosynthesis in microbes[J]. Current Opinion in Biotechnology, 2008, 19(6): 597-605. 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.
[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.
[8] BANG H B, LEE K, LEE Y J, et al. High-level production of trans-cinnamic acid by fed-batch cultivation of Escherichia coli[J]. Process Biochemistry, 2018, 68: 30-36. 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.
[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 synthesis of lignin[J]. China Biotechnology, 2007, 27(3): 120-126. 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.
[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.
[14] SHI R, SUN Y H, LI Q Z, et al. Towards a systems approach for lignin biosynthesis 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.
[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.
[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.
[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 from Olea europaea and its expression in Pichia pastoris[J]. Food Science, 2015, 36(7): 124-130. 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.
[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.
[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.
[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.
[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.
[23] HU G S, JIA J M, HUR Y J, et al. Molecular characterization of phenylalanine ammonia lyase gene from Cistanche deserticola[J]. Molecular Biology Reports, 2011, 38(6): 3741-3750. DOI:10.1007/s11033-010-0489-0.

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备注/Memo

备注/Memo:
收稿日期:2019-01-16 修回日期:2019-04-30基金项目: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)。第一作者:张晨(zhangchen@njfu.edu.cn)。*通信作者:欧阳嘉(hgouyj@njfu.edu.cn),教授,ORCID(0000-0002-3467-253X)。
更新日期/Last Update: 2020-01-15