漆酶是一种多铜氧化酶,在食品、纺织、环境修复和造纸等领域有重要应用。为提高漆酶的发酵水平,该研究将枯草芽孢杆菌(Bacillus subtilis)来源的漆酶(CotA)表达于大肠杆菌(Escherichia coli)BL21(DE3)中,并分别考察了核糖体结合位点、诱导表达条件及共表达裂解蛋白对其表达效率的影响。结果显示,最优核糖体结合位点(ribosome binding site,RBS)重组菌CotA-RBS5胞内CotA表达水平达到1 236 U/L,较优化前提高1.75倍。基于正交实验设计确定了最佳诱导表达条件为:待菌体浓度OD600达到0.8时,加入终浓度为2 mmol/L Cu2+及0.1 mmol/L 异丙基-β-D-硫代半乳糖苷后,于30 ℃诱导。在该条件下,重组菌CotA-RBS5的胞内酶活力提升至7 926 U/L,较优化前提升了5.4倍;同时,胞外酶活力达到4 984 U/L。共表达裂解蛋白E促进了细胞的裂解。在菌体浓度OD600达到3.5时诱导裂解蛋白E表达,胞外CotA酶活力在发酵30 h达到10 283 U/L。研究结果将为漆酶的工业化生产提供了重要的基础数据。
Laccase is a multi-copper oxidase, which has important applications in food, textile, environmental remediation, and papermaking. To improve the fermentation level of laccase, Bacillus subtilis laccase (CotA) was expressed in Escherichia coli BL21 (DE3), and the effects of ribosome binding site, induction expression condition, and co-expression of lysis protein on its expression efficiency were investigated. The results showed that the recombinant strain with the optimal ribosome binding site (CotA-RBS5) achieved 1 236 U/L of intracellular CotA, 1.75 times higher than that of before optimization. Based on the orthogonal experimental, the optimal condition of induction expression was as follows: the final concentration of 2 mmol/L Cu2+ and 0.1 mmol/L IPTG were added when the biomass OD600 reached 0.8; then, induction condition was performed at 30℃. Under the optimal condition, the intracellular enzyme activity of the recombinant strain increased to 7 926 U/L, 5.40 times higher than that of before optimization; meanwhile, the extracellular enzyme activity reached 4 984 U/L. Co-expression of lysis protein E promoted cell lysis. With the expression of cleavage protein E was induced at 3.5 of OD600, the extracellular CotA activity reached 10 283 U/L as it fermented to 30 h. The results will provide important basic data for the industrial production of laccase.
[1] AUSEC L, BERINI F, CASCIELLO C, et al.The first acidobacterial laccase-like multicopper oxidase revealed by metagenomics shows high salt and thermo-tolerance[J].Applied Microbiology and Biotechnology, 2017, 101(15):6 261-6 276.
[2] GUAN Z B, LUO Q, WANG H R, et al.Bacterial laccases:Promising biological green tools for industrial applications[J].Cellular and Molecular Life Sciences:CMLS, 2018, 75(19):3 569-3 592.
[3] HECK T, FACCIO G, RICHTER M, et al.Enzyme-catalyzed protein crosslinking[J].Applied Microbiology and Biotechnology, 2013, 97(2):461-475.
[4] NAVAS L E, CARBALLO R, LEVIN L, et al.Fast decolorization of azo dyes in alkaline solutions by a thermostable metal-tolerant bacterial laccase and proposed degradation pathways[J].Extremophiles:Life Under Extreme Conditions, 2020, 24(5):705-719.
[5] DAI X L, LV J, YAN G X, et al.Bioremediation of intertidal zones polluted by heavy oil spilling using immobilized laccase-bacteria consortium[J].Bioresource Technology, 2020, 309:123305.
[6] SHARMA D, CHAUDHARY R, KAUR J, et al.Greener approach for pulp and paper industry by xylanase and laccase[J].Biocatalysis and Agricultural Biotechnology, 2020, 25:101604.
[7] FENG S Z, SU Y R, DONG M Z, et al.Laccase activity is proportional to the abundance of bacterial laccase-like genes in soil from subtropical arable land[J].World Journal of Microbiology & Biotechnology, 2015, 31(12):2 039-2 045.
[8] BRIJWANI K, RIGDON A, VADLANI P V.Fungal laccases:Production, function, and applications in food processing[J].Enzyme Research, 2010, 2010:149748.
[9] DAVARPANAH S J, AHN J W, KO S M, et al.Stable expression of a fungal laccase protein using transplastomic tobacco[J].Plant Biotechnology Reports, 2012, 6(4):305-312.
[10] LI Q, WANG X H, KORZHEV M, et al.Potential biological role of laccase from the sponge Suberites domuncula as an antibacterial defense component[J].Biochimica et Biophysica Acta (BBA)-General Subjects, 2015, 1850(1):118-128.
[11] NG I S, ZHANG X, ZHANG Y, et al.Molecular cloning and heterologous expression of laccase from Aeromonas hydrophila NIU01 in Escherichia coli with parameters optimization in production[J].Applied Biochemistry and Biotechnology, 2013, 169(7):2 223-2 235.
[12] LIU Y H, HUANG L, GUO W, et al.Cloning, expression, and characterization of a thermostable and pH-stable laccase from Klebsiella pneumoniae and its application to dye decolorization[J].Process Biochemistry, 2017, 53:125-134.
[13] TAN S I, NG I S, YU Y J.Heterologous expression of an acidophilic multicopper oxidase in Escherichia coli and its applications in biorecovery of gold[J].Bioresources and Bioprocessing, 2017, 4:20.
[14] ECE S L, LAMBERTZ C, FISCHER R, et al.Heterologous expression of a Streptomyces cyaneus laccase for biomass modification applications[J].AMB Express, 2017, 7(1):86.
[15] WANG T N, ZHAO M.A simple strategy for extracellular production of CotA laccase in Escherichia coli and decolorization of simulated textile effluent by recombinant laccase[J].Applied Microbiology and Biotechnology, 2017, 101(2):685-696.
[16] WANG T N, ZHAO M.A simple strategy for extracellular production of CotA laccase in Escherichia coli and decolorization of simulated textile effluent by recombinant laccase[J].Applied Microbiology and Biotechnology, 2017, 101(2):685-696.
[17] SALIS H M, MIRSKY E A, VOIGT C A.Automated design of synthetic ribosome binding sites to control protein expression[J].Nature Biotechnology, 2009, 27(10):946-950.
[18] 彭政. 芽孢杆菌降解羽毛角蛋白关键步骤解析及角蛋白酶的高效表达[D].无锡:江南大学, 2020.
PENG Z.Analysis of key steps in degradation of feather keratin by Bacillus and efficient expression of keratinase[D].Wuxi:Jiangnan University, 2020.
[19] 魏伟伟, 王阳, 堵国成, 等.大肠杆菌四氢嘧啶合成途径的构建与优化[J].食品与生物技术学报, 2021, 40(7):30-41.
WEI W W, WANG Y, DU G C, et al.Construction and optimization of ectoine synthesis pathway in Escherichia coli[J].Journal of Food Science and Biotechnology, 2021, 40(7):30-41.
[20] 吴家鑫, 王玉, 李政, 等.细菌菌蜕的研究进展[J].北京联合大学学报, 2013, 27(1):73-75;81.
WU J X, WANG Y, LI Z, et al.Progress in research on bacterial ghosts[J].Journal of Beijing Union University, 2013, 27(1):73-75;81.
[21] 张宁. 大肠杆菌产CotA蛋白的高效诱导表达、分离纯化及酶学性质的研究[D].哈尔滨:东北林业大学, 2012.
ZHANG N.The induction, purification and characterization of protein CotA from Escherichia coli[D].Harbin:Northeast Forestry University, 2012.
[22] 常美会, 初晓宇, 伍宁丰.翻译速率对重组蛋白表达影响的研究进展[J].中国农业科技导报, 2017, 19(4):45-50.
CHANG M H, CHU X Y, WU N F.Progress on the influence of translation rate on the expression of recombinant proteins[J].Journal of Agricultural Science and Technology, 2017, 19(4):45-50.
[23] TULLER T, ZUR H.Multiple roles of the coding sequence 5′ end in gene expression regulation[J].Nucleic Acids Research, 2014, 43(1):13-28.
[24] BALDRIAN P, GABRIEL J.Copper and cadmium increase laccase activity in Pleurotus ostreatus[J].FEMS Microbiology Letters, 2002, 206(1):69-74.
[25] DING J J, LIANG G H, ZHANG K, et al.Extra metabolic burden by displaying over secreting:Growth, fermentation and enzymatic activity in cellobiose of recombinant yeast expressing β-glucosidase[J].Bioresource Technology, 2018, 254:107-114.
