黑曲霉单宁酶TahA的克隆表达和酶学特性解析
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摘要
单宁酶在茶饮料和医药等行业的应用受到了许多因素的限制,包括酶学性质研究的缺乏和较高的生产成本等。为此,该研究通过基因工程技术将黑曲霉CICIM F0510的单宁酶基因tah A在毕赤酵母中进行了克隆表达,构建获得了重组酶GS115 (pPIC-tahA)。摇瓶水平上,重组菌的单宁酶酶活为1. 04 U/mL。酶学性质的解析表明,重组酶TahA的最适作用温度和pH值分别为30℃和4. 5;分别在20～50℃和pH值3. 5～5. 0孵育1 h和2 h后,TahA的相对酶活仍能保持在60%以上; Cu~(2+)、Mn~(2+)和K~+对重组酶的活性有明显的促进作用,而Co~(2+)、Fe~(3+)、Ca~(2+)、Mg~(2+)、Zn~(2+)、Sn~(2+)、EDTA和SDS对其酶活有不同程度的抑制作用;以没食子酸甲酯为底物时,重组酶的Vmax和Km值分别为0. 436μmol/(mL·min)和5. 143 mmol/L。此外,该重组酶还可以水解茶叶提取物中的酯型儿茶素。这些良好的生化特征为重组单宁酶TahA在茶饮料行业中的应用奠定了坚实的基础。
The practical use of tannase in tea beverage and pharmaceutical industries has been limited by a number of factors,including insufficient knowledge about its properties and high production costs. In the present study,gene tahA encoding tannase of Aspergillus niger CICIM F0510 was successfully cloned and expressed in Pichia pastoris,generating the recombinant strain GS115( pPIC-tahA). In shaking flask experiments,the activity of the recombinant enzyme was 1. 04 U/mL. The temperature and pH optima of TahA was shown to be 30 ℃ and 4. 5,respectively.After incubation at 20-50 ℃ or pH 3. 5-5. 0 for 1 h and 2 h,respectively,the recombinant enzyme still retained more than 60% of its initial activity. Cu~(2+),Mn~(2+)and K~+significantly enhanced the activity of TahA,whereas Co~(2+),Fe~(3+),Ca~(2+),Mg~(2+),Zn~(2+),Sn~(2+),EDTA and SDS exerted different inhibitory effects on its activity. The Vmaxand Km of TahA toward methyl gallate were determined to be 0. 436 μmol/( mL·min) and 5. 143 mmol/L. Besides,TahA hydrolyzed the ester catechins in tea extracts. All these distinct biochemical properties lay a solid foundation for the application of TahA in tea beverages.
The practical use of tannase in tea beverage and pharmaceutical industries has been limited by a number of factors,including insufficient knowledge about its properties and high production costs. In the present study,gene tahA encoding tannase of Aspergillus niger CICIM F0510 was successfully cloned and expressed in Pichia pastoris,generating the recombinant strain GS115( pPIC-tahA). In shaking flask experiments,the activity of the recombinant enzyme was 1. 04 U/mL. The temperature and pH optima of TahA was shown to be 30 ℃ and 4. 5,respectively.After incubation at 20-50 ℃ or pH 3. 5-5. 0 for 1 h and 2 h,respectively,the recombinant enzyme still retained more than 60% of its initial activity. Cu~(2+),Mn~(2+)and K~+significantly enhanced the activity of TahA,whereas Co~(2+),Fe~(3+),Ca~(2+),Mg~(2+),Zn~(2+),Sn~(2+),EDTA and SDS exerted different inhibitory effects on its activity. The Vmaxand Km of TahA toward methyl gallate were determined to be 0. 436 μmol/( mL·min) and 5. 143 mmol/L. Besides,TahA hydrolyzed the ester catechins in tea extracts. All these distinct biochemical properties lay a solid foundation for the application of TahA in tea beverages.
引文
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[22] SELWAL M K,YADAV A,SELWAL K K,et al. Tannase production by Penicillium Atramentosum Km under SSF and its applications in wine clarification and tea cream solubilization[J]. Brazilian Journal of Microbiology,2011,42(1):374-387.
[23] BELUR P D,MUGERAYA G,KUPPALU N R. Temperature and p H stability of a novel cell-associated tannase of Serratia ficaria DTC[J]. International Journal of Biotechnology&Biochemistry,2010,6(5):667-674.
[24] DAS MOHAPATRA P K,MAITY C,RAO R S,et al.Tannase production by Bacillus licheniformis KBR6:Opti-mization of submerged culture conditions by Taguchi DOE methodology[J]. Food Research International,2009,42(4):430-435.
[25]王赞苗,李永刚.单宁酶对酯型儿茶素作用研究[J].中国食品添加剂,2005(1):42-44+37.
[26] ZHANG Ying-na,YIN Jun-feng,CHEN Jian-xin,et al.Improving the sweet aftertaste of green tea infusion with tannase[J]. Food Chemistry,2016,192:470-476.
[27] LU M J,CHEN C. Enzymatic modification by tannase increases the antioxidant activity of green tea[J]. Food Research International,2008,41(2):130-137.
[28] ONG C B,ANNUAR M S M. Polyphenolic composition and invitro antioxidant activities of native-and tannasetreated green tea extracts[J]. International Journal of Food Science&Technology,2017,52(3):1-9.
[2] CHAVEZ-GONZALEZ M, RODRIGUEZ-DURAN L V,BALAGURUSAMY N, et al. Biotechnological advances and challenges of tannase:An overview[J]. Food and Bioprocess Technology,2012,5(2):445-459.
[3]王挥.黑曲霉产单宁酶与固定化酶制备没食子酸及其丙酯的研究[D].长沙:中南林业科技大学,2012.
[4] YAO J,GUO G S,REN G H,et al. Production,characterization and applications of tannase[J]. Journal of Molecular Catalysis B-Enzymatic,2014,101:137-147.
[5]邓军,马少敏,王荣辉.单宁酶及其在茶饮料中的应用[J].广西轻工业,2009,25(3):6-7+17.
[6]保玉心,邱树毅,李秧针,等.固态发酵黑曲霉产单宁酶发酵条件研究[J].食品与发酵工业,2008,34(6):54-56+61.
[7]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.
[8] VEANA F,DURON-VAZQUEZ R,GUERRERO-OLAZARAN M,et al. Tannase production by the xerophilic Aspergillus niger GH1 strain and partial isolation of the tannase gene[J]. Revista Mexicana De Ingenieria Quimica,2016,15(1):51-56.
[9] FUENTES-GARIBAY J A,AGUILAR C N,RODRIGUEZHERRERA R,et al. Tannase sequence from a xerophilic Aspergillus niger Strain and production of the enzyme in Pichia pastoris[J]. Molecular Biotechnology,2015,57(5):439-447.
[10] ZHANG Shuai,CUI Feng-chao,CAO Yong,et al. Sequence identification,structure prediction and validation of tannase from Aspergillus niger N5-5[J]. Chinese Chemical Letters,2016,27(7):1 087-1 090.
[11]张帅,曹庸,梁晓莹,等.黑曲霉N5-5单宁酶的纯化及酶学性质测定[J].食品科学,2017,38(06):142-146.
[12] KAZEMI A,ZADE M A,JAFARI A. Identification,isolation,cloning and sequencing of tannase gene from Aspergillus niger[J]. Jundishapur Journal of Microbiology,2011,4:S27-S34.
[13] RAMIREZ-CORONEL M A,VINIEGRA-GONZALEZ G,DARVILL A,et al. A novel tannase from Aspergillus niger with beta-glucosidase activity[J]. Microbiology,2003,149(Pt 10):2 941-2 946.
[14]董自星,肖华,王静培,等.黑曲霉果糖基水解酶的重组表达及酶学特征分析[J].食品工业科技,2017,38(10):178-184.
[15]诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社,1994:413-450.
[16]奥斯伯F,金斯顿R,塞德曼J,等.精编分子生物学实验指南[M].北京:科学出版社,2008:42-50.
[17] SHARMA S,BHAT T K,DAWRA R K. A spectrophotometric method for assay of tannase using rhodanine[J].Analytical Biochemistry,2000,279(1):85-89.
[18] BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72(1/2):248-254.
[19]蔡明瑛,倪辉,李利君,等.液相色谱检测乌龙茶水浸提物及其单宁酶水解产物中的儿茶素[J].中国食品学报,2013,13(5):211-219.
[20] RIUL A J,GONCALVES H B,JORGE J A,et al. Characterization of a glucose-and solvent-tolerant extracellular tannase from Aspergillus phoenicis[J]. Journal of Molecular Catalysis B-Enzymatic,2013,85-86:126-133.
[21]朱继新,杨民和.微生物单宁酶研究现状[J].微生物学杂志,2013,33(1):75-81.
[22] SELWAL M K,YADAV A,SELWAL K K,et al. Tannase production by Penicillium Atramentosum Km under SSF and its applications in wine clarification and tea cream solubilization[J]. Brazilian Journal of Microbiology,2011,42(1):374-387.
[23] BELUR P D,MUGERAYA G,KUPPALU N R. Temperature and p H stability of a novel cell-associated tannase of Serratia ficaria DTC[J]. International Journal of Biotechnology&Biochemistry,2010,6(5):667-674.
[24] DAS MOHAPATRA P K,MAITY C,RAO R S,et al.Tannase production by Bacillus licheniformis KBR6:Opti-mization of submerged culture conditions by Taguchi DOE methodology[J]. Food Research International,2009,42(4):430-435.
[25]王赞苗,李永刚.单宁酶对酯型儿茶素作用研究[J].中国食品添加剂,2005(1):42-44+37.
[26] ZHANG Ying-na,YIN Jun-feng,CHEN Jian-xin,et al.Improving the sweet aftertaste of green tea infusion with tannase[J]. Food Chemistry,2016,192:470-476.
[27] LU M J,CHEN C. Enzymatic modification by tannase increases the antioxidant activity of green tea[J]. Food Research International,2008,41(2):130-137.
[28] ONG C B,ANNUAR M S M. Polyphenolic composition and invitro antioxidant activities of native-and tannasetreated green tea extracts[J]. International Journal of Food Science&Technology,2017,52(3):1-9.