黑曲霉单宁酶基因Tan2克隆与表达

doi:10.13560/j.cnki.biotech.bull.1985.2020-0800

摘要/Abstract

摘要：

单宁酶（Tannase,EC 3.1.1.20）能水解单宁中的酯键和羧酚酸键,产生没食子酸以及对应醇,在食品、饮料、饲料、制药、医药、化妆品等各类工业中应用广泛,也在普洱茶发酵中具有重要作用。从普洱茶发酵中分离的黑曲霉菌株PU001中克隆得到单宁酶基因Tan2,并连接到表达载体pCold-Ⅰ构建BL21-pCold Ⅰ原核冷诱导表达系统,转化至感受态大肠杆菌BL21中,SDS-PAGE与质谱鉴定均表明单宁酶Tan 2表达成功,旨在为进一步研究该酶在普洱茶发酵中的作用机制以及其他领域的应用奠定基础。

关键词: 单宁酶, 黑曲霉, 普洱茶

Abstract:

Tannase（EC 3.1.1.20）can hydrolyze the ester bond and carboxyphenolic acid bond in tannins to produce gallic acid and corresponding alcohols. It is widely used in food,beverage,feed,pharmacy,medicine,cosmetics and other industries,and also plays an important role in the fermentation of Pu-erh tea. In this work,tannase gene Tan 2 was cloned from Aspergillus niger PU001,which was previously isolated from fermentation in Pu-erh tea. It was ligated with expression vector pCold-I to construct the prokaryotic cold inducible expression system of BL21-pCold I,which was transformed into Escherichia coli BL21. SDS-PAGE and mass spectrometry identification showed that tannase Tan 2 was expressed successfully. This result provides a basis for further survey on the role of tannase in the fermentation of Pu-erh tea and application in other fields.

Key words: tannase, Aspergillus niger, Pu-erh tea

李红叶, 陈立佼, 刘明丽, 郭天杰, 王道平, 潘映红, 赵明. 黑曲霉单宁酶基因Tan2克隆与表达[J]. 生物技术通报, 2021, 37(3): 44-52.

LI Hong-ye, CHEN Li-jiao, LIU Ming-li, GUO Tian-jie, WANG Dao-ping, PAN Ying-hong, ZHAO Ming. Cloning and Expression of Tannase Gene Tan2 from Aspergillus niger[J]. Biotechnology Bulletin, 2021, 37(3): 44-52.

图/表 10

参考文献 26

[1]	Ni H, Chen F, Jiang ZD, et al. Biotransformation of tea catechins using Aspergillus niger tannase prepared by solid state fermentation on tea byproduct[J]. LWT - Food Science and Technology, 2015,60(2):1206-1213. doi: 10.1016/j.lwt.2014.09.010 URL
[2]	Ichikawa K, Shiono Y, Shintani T, et al. Efficient production of recombinant tannase in Aspergillus oryzae using an improved glucoamylase gene promoter[J]. Journal of Bioscience and Bioengineering, 2020,129(2):150-154. doi: S1389-1723(19)30599-7 pmid: 31492608
[3]	Dhiman S, Mukherjee G, Singh AK. Recent trends and advancements in microbial tannase-catalyzed biotransformation of tannins:a review[J]. Int Microbiol, 2018,21(4):175-195. doi: 10.1007/s10123-018-0027-9 pmid: 30810902
[4]	Baik JH, Shin K, Park Y, et al. Biotransformation of catechin and extraction of active polysaccharide from green tea leaves via simultaneous treatment with tannase and pectinase[J]. Journal of the Science of Food and Agriculture, 2014,95(11):2337-2344. doi: 10.1002/jsfa.6955 URL pmid: 25307474
[5]	Liu ML, Xie HF, Ma Y, et al. High performance liquid chromatography and metabolomics analysis of tannase metabolism of gallic acid and gallates in tea leaves[J]. Journal of Agricultural and Food Chemistry, 2020,68(17):1-40. doi: 10.1021/acs.jafc.9b07943 URL
[6]	李梦迪, 张志萌, 董自星, 等. 黑曲霉单宁酶TahA的克隆表达和酶学特性解析[J]. 食品与发酵工业, 2018,44(11):19-25.
	Li MD, Zhang ZM, Dong ZX, et al. Cloning, expression and biochemical characterization of tannase TahA from Aspergillus niger[J]. Food and Fermentation Industries, 2018,44(11):15-21.
[7]	段颖异, 陈洪章. 固态发酵基质的三相结构变化对其传递性质的影响[J]. 化工学报, 2012,63(4):1204-1210. doi: 10.3969/j.issn.0438-1157.2012.04.030 URL
	Duan YY, Chen HZ. Effect of three-phase structure of solid-state fermentation substrates on its transfer properties[J]. CIESC Journal, 2012,63(4):1204-1210.
[8]	Mónica CG, Luis VRD, Nagamani B, et al. Biotechnological advances and challenges of tannase:an overview[J]. Food & Bioprocess Technology, 2011,5(2):445-459.
[9]	Krensky AM, Clayberger C. Granulysin:a novel host defense molecule[J]. Am J Transplant, 2005,8(5):1789-1792.
[10]	Iwamoto K, Tsuruta H, Nishitaini Y, et al. Identification and cloning of a gene encoding tannase(tannin acylhydrolase)from Lactobacillus plantarum ATCC 14917T[J]. Systematic and Applied Microbiology, 2008,31(4):269-277. doi: 10.1016/j.syapm.2008.05.004 URL pmid: 18653299
[11]	Zhang S, Cao Y, Cheng H. Expression of Aspergillus niger N5-5 in E. coli and purification and identification of products[J]. Saudi Journal of Biological Sciences, 2017,24(8):1842-1848. doi: 10.1016/j.sjbs.2017.11.025 URL pmid: 29551933
[12]	郑重谊, 资云, 张冬雪, 等. 黑曲霉WB-1固态发酵产单宁酶的研究[J]. 化学与生物工程, 2016,32(1):37-39.
	Zheng CY, Zi Y, Zhang DX, et al. Study on the production of tannase from Aspergillus niger WB-1 by solid state fermentation[J]. Chemistry and Bioengineering, 2016,32(1):37-39.
[13]	季爱兵, 龚婉莹, 彭文书, 等. 普洱茶中微生物研究进展[J]. 现代农业科技, 2016(21):253-255.
	Ji AB, Gong WY, Peng WS, et al. Research progress of microorganism in pu-erh tea[J]. Modern Agricultural Sciences and Technology, 2016(21):253-255.
[14]	周才碧, 张敏星, 蒋陈凯, 等. 黑曲霉及其与普洱茶品质关系研究进展[J]. 微生物学杂志, 2014,34(2):88-91.
	Zhou CB, Zhang MX, Jiang CK, et al. Advanced in Aspergillus niger & its relationship with quality of puer tea[J]. Journal of Microbiology, 2014,34(2):88-91.
[15]	高琛, 朱继新, 黄婧, 等. 从黑茶中分离和筛选产单宁酶菌株[J]. 微生物学杂志, 2015,35(5):28-35.
	Gao C, Zhu JX, Huang J, et al. Isolation & screening of tannase producing strains from Chinese dark tea[J]. Journal of Microbiology, 2015,35(5):28-35.
[16]	邓军, 马少敏, 王荣辉. 单宁酶及其在茶饮料中的应用[J]. 轻工科技, 2009,25(3):6-7.
	Deng J, Ma SM, Wang RH. Tannase and its application in tea beverage[J]. Guangxi Journal of Light Industry, 2009,25(3):6-7.
[17]	王征, 谭周进, 杨虹琦. 单宁酶活力测定方法的研究[J]. 生物技术, 2000,10(6):40-42.
	Wang Z, Tan ZJ, Yang HQ. Study on the determination of tannase activity[J]. Biotechnology, 2000,10(6):40-42.
[18]	岳苗苗. 单宁酶基因在黑曲霉中的同源表达研究[D]. 哈尔滨:东北农业大学, 2014.
	Yue MM. Homologous expression of tannase gene in the Aspergillus niger[D]. Harbin:Northeast Agricultural University, 2014.
[19]	Wu M, Peng X, Wen H, et al. Expression, purification, crystallization and preliminary X-ray analysis of tannase from Lactobacillus plantarum[J]. Acta Crystallographica Sect F Struct Biol Cyst Commun, 2013,69(4):456-459.
[20]	Iwamoto K, Tsuruta H, Nishitaini Y, et al. Identification and cloning of a gene encoding tannase(tannin acylhydrolase)from Lactobacillus plantarum ATCC 14917^T[J]. Systematic & Applied Microbiology, 2008,31(4):269-277. doi: 10.1016/j.syapm.2008.05.004 URL pmid: 18653299
[21]	Zhong XF, Peng LS, Zheng SL, et al. Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris[J]. Protein Expr Purif, 2004,36(2):165-169. doi: 10.1016/j.pep.2004.04.016 URL pmid: 15249037
[22]	Erik B, Rüdiger B, Hans-Peter M, et al. Atan1p - an extracellular tannase from the dimorphic yeast Arxula adeninivorans:molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme[J]. Yeast, 2009,26(6):323-337. doi: 10.1002/yea.1669 URL pmid: 19387973
[23]	Yao J, Chen QL, Shen AX, et al. A novel feruloyl esterase from a soil metagenomic library with tannase activity[J]. Journal of Molecular Catalysis B Enzymatic, 2013,95(11):55-61. doi: 10.1016/j.molcatb.2013.05.026 URL
[24]	黄小凤, 韦宇拓, 韦传东, 等. 单宁酶基因在黑曲霉ST31中的克隆与表达[J]. 中国生物工程杂志, 2005,25(9):74-77.
	Huang XF, Wei YT, Wei CD, et al. Cloning and expressing of tannase gene from Aspergillus oryzae in Aspergillus niger ST31[J]. China Biotechnology, 2005,25(9):74-77.
[25]	马凯, 胡红霞, 于婧, 等. 双酶切和同源重组方法构建 pMIR-reporter载体的比较[J]. 中国病原生物学杂志, 2015,10(6):495-499.
	Ma K, Hu HX, Yu J, et al. Comparison of the construction of a pMIR-reporter vector using conventional double enzyme restriction and recombination[J]. Journal of Pathogen Biology, 2015,10(6):495-499.
[26]	Li M, Wang X, Cao Y, et al. Strength comparison between cold-inducible promoters of Arabidopsis cor15a and cor15b genes in potato and tobacco[J]. Plant Physiol Biochem, 2013,71:77-86. doi: 10.1016/j.plaphy.2013.06.021 URL pmid: 23886924

Accession	Blast results（NCBI）
Accession	Description	Organism	Identity
XP_001401809.1	tannase	Aspergillus niger CBS 513.88	100%
EHA20668.1	hypothetical protein	Aspergillus niger ATCC 1015	99.83%
GCB23703.1	tannase	Aspergillus awamori	99.65%
RDK47096.1	tannase and feruloyl esterase	Aspergillus phoenicis ATCC 13157	99.48%
XP_026632695.1	tannase and feruloyl esterase	Aspergillus welwitschiae	99.30%
XP_025515125.1	tannase and feruloyl esterase	Aspergillus piperis CBS 112811	97.91%
XP_025567423.1	tannase and feruloyl esterase	Aspergillus vadensis CBS 113365	97.91%
OJZ87444.1	hypothetical protein	Aspergillus luchuensis CBS 106.47	97.74%
GAA82877.1	tannase and feruloyl esterase	Aspergillus luchuensis IFO 4308	97.56%
OJJ71084.1	hypothetical protein	Aspergillus brasiliensis CBS 101740	97.56%

Accession	Blast results（NCBI）
Accession	Description	Organism	Identity
XP_001401809.1	tannase	Aspergillus niger CBS 513.88	100%
EHA20668.1	hypothetical protein	Aspergillus niger ATCC 1015	99.83%
GCB23703.1	tannase	Aspergillus awamori	99.65%
RDK47096.1	tannase and feruloyl esterase	Aspergillus phoenicis ATCC 13157	99.48%
XP_026632695.1	tannase and feruloyl esterase	Aspergillus welwitschiae	99.30%
XP_025515125.1	tannase and feruloyl esterase	Aspergillus piperis CBS 112811	97.91%
XP_025567423.1	tannase and feruloyl esterase	Aspergillus vadensis CBS 113365	97.91%
OJZ87444.1	hypothetical protein	Aspergillus luchuensis CBS 106.47	97.74%
GAA82877.1	tannase and feruloyl esterase	Aspergillus luchuensis IFO 4308	97.56%
OJJ71084.1	hypothetical protein	Aspergillus brasiliensis CBS 101740	97.56%

	单宁酶/（U·mL^-1）	ΔGA/（mg·mL^-1）	PG/（mg·mL^-1）
样品管	1.02±0.06	0.0034±0.0002	0.0486±0.00115
空白管	—	—	0.0545±0.00309
对照管	—	—	0.0527±0.00274

	单宁酶/（U·mL^-1）	ΔGA/（mg·mL^-1）	PG/（mg·mL^-1）
样品管	1.02±0.06	0.0034±0.0002	0.0486±0.00115
空白管	—	—	0.0545±0.00309
对照管	—	—	0.0527±0.00274