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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (6): 20-29    DOI: 10.13523/j.cb.2202025
研究报告     
解淀粉芽孢杆菌Q-426酚酸脱羧酶的克隆表达及酶学性质鉴定*
任明杰1,王路路2,申纪辉1,范若辰2,许永斌1,张丽影1,郑维1,权春善1,2,**()
1.大连民族大学生命科学学院 生物技术与资源利用教育部重点实验室 大连 116600
2.大连理工大学生物工程学院 大连 116024
Cloning, Expression and Characterization of Phenolic Acid Decarboxylase from Bacillus amyloliquefaciens Q-426
REN Ming-jie1,WANG Lu-lu2,SHEN Ji-hui1,FAN Ruo-chen2,XU Yong-bin1,ZHANG Li-ying1,ZHENG Wei1,QUAN Chun-shan1,2,**()
1. Key Laboratory of Biotechnology and Bioresources of Ministry of Education, College of Life Seiences, Dalian Minzu University, Dalian 116600, China
2. School of Bioengineering, Dalian University of Technology, Dalian 116024, China
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摘要：

目的:生物法脱羧制备4-乙烯基衍生物具有诸多优势和良好的发展前景,研究解淀粉芽孢杆菌Q-426酚酸脱羧酶(BaPAD-Q-426)的酶学性质,为其进一步应用提供理论基础。方法:从解淀粉芽孢杆菌中克隆酚酸脱羧酶基因;以pET-28a(+)为载体,将重组质粒转化至E. coli BL21(DE3)中,实现酚酸脱羧酶BaPAD-Q-426的高效表达,利用Ni-NTA亲和层析进行纯化,并进行酶学性质鉴定。结果:酚酸脱羧酶BaPAD-Q-426在pH 7.0~9.0范围内保持良好的pH稳定性,最适pH为8.0;在25~40℃范围内保持着较高的酶活性,最适温度为35℃,在4℃时保持30 min后该酶依然保持95%以上的酶活性;K+对BaPAD-Q-426的酶活具有明显促进作用,酶活力提高60%;该酶在石油醚中具有良好的耐受能力,在40%石油醚存在下,仍保留50%以上的酶活力;BaPAD-Q-426的最适底物为阿魏酸,酶活力达到19.5 IU/mL。结论:与其他来源的酚酸脱羧酶相比,BaPAD-Q-426在低温时具有更好的稳定性,在弱碱性环境下对阿魏酸的催化脱羧能力最强。
关键词： 酚酸脱羧酶;  解淀粉芽孢杆菌;  酶学性质    
Abstract:

Objective: The preparation of 4-vinyl derivatives by biological decarboxylation has many advantages and promising prospects. In this study, the enzymatic properties of Bacillus amyloliquefaciens Q-426 phenolic acid decarboxylase (BaPAD-Q-426) were studied in detail to provide theoretical basis for its future application. Methods: In this study, the phenolic acid decarboxylase gene was cloned from Bacillus amyloliquefaciens. Using pET-28a (+) as vector, the recombinant plasmid was transformed into E.coli BL21 (DE3), to achieve high expression of BaPAD-Q-426. It was purified by Ni-NTA affinity chromatography, and the enzymatic properties were identified. Results: BaPAD-Q-426 maintained good pH stability in the range of pH 7.0~9.0, and the optimum pH was 8.0. The enzyme maintained high enzyme activity in the range of 25~40℃, and the optimum temperature was 35℃. After holding at 4℃ for 30 minutes, the enzyme still maintained more than 95% enzyme activity. K+ significantly promoted the enzyme activity of BaPAD-Q-426 with an increase of 60%. This enzyme was well tolerated in petroleum ethers and retained more than 50% of the enzyme activity in the presence of 40% petroleum ethers. The optimum substrate of BaPAD-Q-426 was ferulic acid, and its enzyme activity reached 19.5 IU/mL. Conclusion: Compared with phenolic acid decarboxylases from other sources, BAPAD-Q-426 has better stability at low temperature and has the strongest catalytic decarboxylation of ferulic acid in weakly alkaline environment.
Key words: Phenolic acid decarboxylase    Bacillus amyloliquefaciens    Enzyme characterization
收稿日期: 2022-02-18 出版日期: 2022-07-07
ZTFLH:  Q786  
基金资助: *辽宁省教育厅重点实验室(LS2010049)
通讯作者: 权春善     E-mail: mikyeken@dlnu.edu.cn
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任明杰
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许永斌
张丽影
郑维
权春善

引用本文:

任明杰,王路路,申纪辉,范若辰,许永斌,张丽影,郑维,权春善. 解淀粉芽孢杆菌Q-426酚酸脱羧酶的克隆表达及酶学性质鉴定*[J]. 中国生物工程杂志, 2022, 42(6): 20-29.

REN Ming-jie,WANG Lu-lu,SHEN Ji-hui,FAN Ruo-chen,XU Yong-bin,ZHANG Li-ying,ZHENG Wei,QUAN Chun-shan. Cloning, Expression and Characterization of Phenolic Acid Decarboxylase from Bacillus amyloliquefaciens Q-426. China Biotechnology, 2022, 42(6): 20-29.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2202025    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I6/20

图1  酚酸脱羧酶基因PCR扩增产物琼脂糖凝胶电泳
图2  质粒载体pET-28a(+)-BaPAD-Q-426的构建
图3  酚酸脱羧酶与其他脱羧酶的氨基酸序列比对
图4  BaPAD-Q-426蛋白亲和层析纯化
图5  BaPAD-Q-426最适pH及其稳定性
图6  BaPAD-Q-426最适温度及其稳定性
图7  金属离子对BaPAD-Q-426酶活的影响
图8  有机溶剂对BaPAD-Q-426的影响
图9  不同体积分数有机溶剂对BaPAD-Q-426的影响
图10  BaPAD-Q-426底物特异性
图11  底物化学结构
脱羧酶名称 菌株来源 最适温度/℃ 最适pH 最适底物 参考文献
BaPAD-Q-426 Bacillus amyloliquefaciens Q-426 35 8.0 FA -
AIPAD Aspergillus luchuensis 40 5.7 FA [15]
CjPAD Conocephalum japonicum 25 5.5 pCA [17]
BLPAD Bacillus licheniformis 37 6.0 pCA [18-19]
GAD Lactobacillus plantarum 40 4.8 - [20]
BsPAD Bacillus subtilis - - FA [21]
L. brevis PAD Lactobacillus brevis RM84 30 6.5 FA [22]
表1  酚酸脱羧酶与其他脱羧酶的酶学性质比较
[1] 夏瑛, 李良, 董孝元, 等. 源于解淀粉芽孢杆菌酚酸脱羧酶的克隆与表达. 微生物学通报, 2020, 47(7): 2060-2071.
Xia Y, Li L, Dong X Y, et al. Cloning and expression of phenolic acid decarboxylase from Bacillus amyloliquefaciens. Microbiology China, 2020, 47(7): 2060-2071.
[2] 何雅静, 张群琳, 谷利伟, 等. 柑橘中酚酸类化合物及其生物活性与机理的研究进展. 食品与发酵工业, 2020, 46(15): 301-306.
He Y J, Zhang Q L, Gu L W, et al. Research progress on phenolic acids in citrus and their biological activities and mechanisms. Food and Fermentation Industries, 2020, 46(15): 301-306.
[3] 李菊, 李玉梅, 苟亚妮, 等. 酚酸类物质代谢及其化感效应研究进展. 黑龙江农业科学, 2019(8): 175-182.
Li J, Li Y M, Gou Y N, et al. Research progress in phenolic acid metabolism and allelopathic effect. Heilongjiang Agricultural Sciences, 2019(8): 175-182.
[4] Gu W, Li X M, Huang J W, et al. Cloning, sequencing, and overexpression in Escherichia coli of the Enterobacter sp. Px6-4 gene for ferulic acid decarboxylase. Applied Microbiology and Biotechnology, 2011, 89(6): 1797-1805.
doi: 10.1007/s00253-010-2978-4
[5] Santamaría L, Reverón I, de Felipe F L, et al. Ethylphenol formation by Lactobacillus plantarum: identification of the enzyme involved in the reduction of vinylphenols. Applied and Environmental Microbiology, 2018, 84(17): e01064-e01018.
[6] Terpinc P, Polak T, Šegatin N, et al. Antioxidant properties of 4-vinyl derivatives of hydroxycinnamic acids. Food Chemistry, 2011, 128(1): 62-69.
doi: 10.1016/j.foodchem.2011.02.077 pmid: 25214330
[7] Li L L, Wu X, Long L K, et al. Functional autodisplay of phenolic acid decarboxylase using a GDSL autotransporter on Escherichia coli for efficient catalysis of 4-hydroxycinnamic acids to vinylphenol derivatives. Catalysts, 2019, 9(8): 634.
doi: 10.3390/catal9080634
[8] Natella F, Nardini M, di Felice M, et al. Benzoic and cinnamic acid derivatives as antioxidants: structure-activity relation. Journal of Agricultural and Food Chemistry, 1999, 47(4): 1453-1459.
pmid: 10563998
[9] Sheng X, Lind M E S, Himo F. Theoretical study of the reaction mechanism of phenolic acid decarboxylase. The FEBS Journal, 2015, 282(24): 4703-4713.
doi: 10.1111/febs.13525
[10] Kitaoka N, Nomura T, Ogita S, et al. Bioproduction of 4-vinylphenol and 4-vinylguaiacol β-primeverosides using transformed bamboo cells expressing bacterial phenolic acid decarboxylase. Applied Biochemistry and Biotechnology, 2021, 193(7): 2061-2075.
doi: 10.1007/s12010-021-03522-y pmid: 33544364
[11] Jung D H, Choi W, Choi K Y, et al. Bioconversion of p-coumaric acid to p-hydroxystyrene using phenolic acid decarboxylase from B. amyloliquefaciens in biphasic reaction system. Applied Microbiology and Biotechnology, 2013, 97(4): 1501-1511.
doi: 10.1007/s00253-012-4358-8
[12] Zago A, Degrassi G, Bruschi C V. Cloning, sequencing, and expression in Escherichia coli of the Bacillus pumilus gene for ferulic acid decarboxylase. Applied and Environmental Microbiology, 1995, 61(12): 4484-4486.
doi: 10.1128/aem.61.12.4484-4486.1995 pmid: 8534115
[13] Salgado J M, Rodríguez-Solana R, Curiel J A, et al. Production of vinyl derivatives from alkaline hydrolysates of corn cobs by recombinant Escherichia coli containing the phenolic acid decarboxylase from Lactobacillus plantarum CECT 748T. Bioresource Technology, 2012, 117: 274-285.
doi: 10.1016/j.biortech.2012.04.051 pmid: 22621808
[14] Huang H K, Chen L F, Tokashiki M, et al. An endogenous factor enhances ferulic acid decarboxylation catalyzed by phenolic acid decarboxylase from Candida guilliermondii. AMB Express, 2012, 2(1): 4.
doi: 10.1186/2191-0855-2-4
[15] Maeda M, Tokashiki M, Tokashiki M, et al. Characterization and induction of phenolic acid decarboxylase from Aspergillus luchuensis. Journal of Bioscience and Bioengineering, 2018, 126(2): 162-168.
doi: 10.1016/j.jbiosc.2018.02.009
[16] Mpofu E, Chakraborty J, Suzuki-Minakuchi C, et al. Biotransformation of monocyclic phenolic compounds by Bacillus licheniformis TAB7. Microorganisms, 2019, 8(1): 26.
doi: 10.3390/microorganisms8010026
[17] Gao S, Yu H N, Wu Y F, et al. Cloning and functional characterization of a phenolic acid decarboxylase from the liverwort Conocephalum japonicum. Biochemical and Biophysical Research Communications, 2016, 481(3-4): 239-244.
doi: 10.1016/j.bbrc.2016.10.131
[18] 胡宏飞. 有机溶剂耐受性酚酸脱羧酶及其在生产4-乙烯基酚类物质上的应用.南京: 南京林业大学, 2015.
Hu H F. An organic solvent-tolerant phenolic acid decarboxylase BLPAD and its application in the bioproduction of 4-vinyl phenol derivatives.Nanjing: Nanjing Forestry University, 2015.
[19] Hu H F, Li L L, Ding S J. An organic solvent-tolerant phenolic acid decarboxylase from Bacillus licheniformis for the efficient bioconversion of hydroxycinnamic acids to vinyl phenol derivatives. Applied Microbiology and Biotechnology, 2015, 99(12): 5071-5081.
doi: 10.1007/s00253-014-6313-3
[20] 陈静. 高产GABA乳酸菌的筛选鉴定、发酵优化及谷氨酸脱羧酶基因的分子改造. 自贡: 四川轻化工大学, 2019.
Chen J. Screening, identification, fermentation optimization and molecular modification of glutamate decarboxylase gene of high GABA-producing lactic acid bacteria. Zigong: Sichuan University of Science & Engineering, 2019.
[21] 巩园园, 毛豪, 晋湘宜, 等. 产4-乙烯基愈创木酚细菌的筛选及酚酸脱羧酶基因的克隆与表达. 中国酿造, 2021, 40(7): 160-164.
Gong Y Y, Mao H, Jin X Y, et al. Screening of 4-vinylguaiacol-producing bacteria and cloning and expression of phenolic acid decarboxylase gene. China Brewing, 2021, 40(7): 160-164.
[22] Landete J M, Rodríguez H, Curiel J A, et al. Gene cloning, expression, and characterization of phenolic acid decarboxylase from Lactobacillus brevis RM84. Journal of Industrial Microbiology and Biotechnology, 2010, 37(6): 617-624.
doi: 10.1007/s10295-010-0709-6 pmid: 20333439
[23] 张庆菲. 计算机辅助分子设计提高谷氨酸脱羧酶热稳定性. 杭州: 浙江工业大学, 2020.
Zhang Q F. Computer-aided molecular design to improve the thermotability of glutamate decarboxylase. Hangzhou: Zhejiang University of Technology, 2020.
[24] Barthelmebs L, Diviès C, Cavin J F. Expression in Escherichia coli of native and chimeric phenolic acid decarboxylases with modified enzymatic activities and method for screening recombinant E. coli strains expressing these enzymes. Applied and Environmental Microbiology, 2001, 67(3): 1063-1069.
pmid: 11229892
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