嗜酸喜温硫杆菌硫加氧还原酶基因的克隆、表达及酶活性研究

doi:10.13560/j.cnki.biotech.bull.1985.2015.11.024

生物技术通报 ›› 2015, Vol. 31 ›› Issue (11): 186-194.doi: 10.13560/j.cnki.biotech.bull.1985.2015.11.024

嗜酸喜温硫杆菌硫加氧还原酶基因的克隆、表达及酶活性研究

李凌凌, 吕早生, 左振宇, 李尧益

武汉科技大学化学工程与技术学院，武汉 430081

收稿日期:2015-01-28 出版日期:2015-11-26 发布日期:2015-11-26
作者简介:李凌凌，女，博士，副教授，研究方向: 微生物浸矿技术；E-mail: moonletsmile@163.com
基金资助:
湖北省科技厅基金项目(2009CDA006)，湖北省自然科学基金项目(2014CFB802)

Cloning and Expression of Sulfur Oxygenase Reductase Gene from Acidithiobacillus caldus and the Study of the Recombinant Enzyme Activity

Li Lingling, Lü Zaosheng, Zuo Zhenyu, Li Yaoyi

College of Chemical Engineering and Technology，Wuhan University of Science and Technology，Wuhan 430081

Received:2015-01-28 Published:2015-11-26 Online:2015-11-26

摘要/Abstract

摘要： 旨为研究嗜酸喜温硫杆菌硫加氧还原酶的生化特性，以嗜酸喜温硫杆菌TST3的基因组DNA为模板，PCR扩增出硫加氧还原酶基因(sor)，构建了表达载体pET-sor，转化到Escherichia coli BL21(DE3)后，获得了重组菌株E.coli BL21(pET-sor2)。SDS-PAGE实验证明，经IPTG诱导，该重组菌可以表达目的蛋白SOR。对诱导条件进行优化，并在最优的条件下诱导SOR酶表达，再经超声波破碎重组菌，上清液通过Ni⁺-NTA亲和层析柱纯化获得重组酶，其催化氧化反应的比酶活、K_m值和V_max分别为0.70 U/mg，15.672×10^-2g/mL和12.755×10^-5 mol/ (L·min)，而催化的还原反应的比酶活、K_m值和V_max分别为2.21 U/mg，0.507×10^-2g/mL和4.876×10^-5 mol/ (L·min)。

关键词: 生物浸矿, 嗜酸喜温硫杆菌, 硫加氧还原酶, 酶活

Abstract: In order to investigate biochemical properties of sulfur oxygenase reductase from Acidithiobacillus caldus(AcSOR), the sor gene was amplified by PCR with the extracted A. caldus TST3 genomic DNA as template, which was inserted into vector pET-28a to construct recombinant plasmid pET-sor. And the plasmid was then transformed into Escherichia coli BL21(DE3)to obtain recombinant strain E.coli BL21(pET-sor2). SDS-PAGE showed that the target enzyme SOR could be expressed in this recombinant strain after induction by IPTG. Induction conditions were optimized. The recombinant AcSOR was purified with Ni⁺-NTA column from the supernatant of the sonicated E.coli BL21(pET-sor2)induced under the optimal conditions. For the oxidation reaction, specific activity, K_m and V_max of the purified AcSOR were 0.70 units of oxidase/mg of protein, 15.672×10^-2g/mL and 12.755×10^-5 mol/(L·min), respectively. Furthermore, specific activity, K_m and V_max of AcSOR for the reduction reaction were 2.21 units of reductase/mg of protein, 0.507×10^-2 g/mL and 4.876×10^-5 mol/(L·min), respectively.

Key words: bioleaching, Acidithiobacillus caldus, sulfur oxygenase reductase, enzyme activity

李凌凌, 吕早生, 左振宇, 李尧益. 嗜酸喜温硫杆菌硫加氧还原酶基因的克隆、表达及酶活性研究[J]. 生物技术通报, 2015, 31(11): 186-194.

Li Lingling, Lü Zaosheng, Zuo Zhenyu, Li Yaoyi. Cloning and Expression of Sulfur Oxygenase Reductase Gene from Acidithiobacillus caldus and the Study of the Recombinant Enzyme Activity[J]. Biotechnology Bulletin, 2015, 31(11): 186-194.

参考文献

[1]Urich T, Gomes CM, Kletzin A, et al. X-ray structure of a self-compartmentalizing sulfur cycle metalloenzyme[J]. Science, 2006, 311(5763): 996-1000.
[2]Chen LX, Ren YL, Lin JQ, et al. Acidithiobacillus caldus sulfur oxidation model based on transcriptome analysis between the wild type and sulfur oxygenase reductase defective mutant[J]. PLoS One, 2012, 7(9): e39470.
[3] Veith A, Urich T, Seyfarth K, et al. Substrate pathways and mechanisms of inhibition in the sulfur oxygenase reductase of Acidianus ambivalens[J]. Frontiers in Microbiology, 2011, 2: 37.
[4] Veith A, Botelho HM, Kindinger F, et al. The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme[J]. Journal of Bacteriology, 2012, 194(3): 677-685.
[5]吴为荣, 郑志宏, 刘金辉, 等. 氧化硫硫杆菌的耐氟驯化试验研究[J]. 有色金属: 冶炼部分, 2007(3): 38-40.
[6]沈萍, 范秀容, 李广武. 微生物学实验[M]. 第3版. 北京: 高等教育出版社, 1999.
[7]吕早生, 关海燕, 李凌凌, 等. 一株高浸磷嗜酸氧化硫硫杆菌的分离与鉴定[J]. 应用与环境生物学报, 2011, 17: 326-329.
[8]李凌凌, 吕早生, 吴敏, 等. 二苯并噻吩降解菌的筛选鉴定及煤脱硫的研究[J]. 环境工程学报, 2010, 4(10): 2391-2396.
[9]李凌凌, 吕早生, 吴敏, 等. 重组的葡萄糖脱氢酶催化辅酶的再生性质[J]. 华中科技大学学报: 自然科学版, 2010, 38(3): 112-115.
[10]Kletzin A. Coupled enzymatic production of sulfite, thiosulfate and hydrogen sulfide from sulfur: purification and properties of a sulfur oxygenase reductase from the facultatively anaerobic archaebacterium Desulfurolobus ambivalens[J]. Journal of Bacteriology, 1989, 171(3): 1638-1643.
[11]Rabinowitz JC. Analysis of acid-labile sulfide and sulfhydryl groups[J]. Methods Enzymol, 1978, 53: 275-277.
[12]管启旺, 马江锋, 贺爱永, 等. 敏捷嗜酸菌腈水解酶基因在大肠杆菌中的表达[J]. 生物技术通报, 2015, 31(1): 181-185.
[13]Emmel T, Sand W, K?nig WA, et al. Evidence for the existence of a sulphur oxygenase in Sulfolobus brierleyi[J]. Journal of General Microbiology, 1986, 132(12): 3415-3420.
[14]He ZG, Li YQ, Zhou PJ, et al. Cloning and heterologous expression of a sulfur oxygenase/reductase gene from the thermoacidophilic archaeon Acidianus sp. S5 in Escherichia coli[J]. FEMS Microbiology Letters, 2000, 193(2): 217-221.
[15]Urich T, Bandeiras TM, Leal SS, et al. The sulphur oxygenase reductase from Acidianus ambivalens is a multimeric protein containing a low-potential mononuclear non-haem iron centre[J]. Biochem J, 2004, 381(Pt1): 137-146.
[16]Chen ZW, Jiang CY, She QX, et al. Key role of cysteine residues in catalysis and subcellular localization of sulfur oxygenase-reductase of Acidianus tengchongensis[J]. Applied and Environmental Microbiology, 2005, 71(2): 621-628.
[17]Sun CW, Chen ZW, He ZG, et al. Purification and properties of the sulfur oxygenase/reductase from the acidothermophilic archaeon, Acidianus strain S5[J]. Extremophiles, 2003, 7(2): 131-134.
[18] Chen ZW, Liu YY, Wu JF, et al. Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates[J]. Appl Microbiol Biotechnol, 2007, 74(3): 688-698.
[19]Pelletier N, Leroy G, Guiral M, et al. First characterisation of the active oligomer form of sulfur oxygenase reductase from the bacterium Aquifex aeolicus[J]. Extremophiles, 2008, 12(2): 205-215.
[20]Zhang HJ, Guo WB, Xu CA, et al. Site-specific mutagenesis and functional analysis of active sites of sulfur oxygenase reductase from Gram-positive moderate thermophile Sulfobacillus acidophilus TPY[J]. Microbiological Research, 2013, 168(10): 654-660.
[21]You XY, Meng Z, Chen DW, et al. Structural basis for the thermostability of sulfur oxygenase reductase[J]. Chinese Journal of Chemical Engineering, 2012, 20(1): 52-61.

嗜酸喜温硫杆菌硫加氧还原酶基因的克隆、表达及酶活性研究

Cloning and Expression of Sulfur Oxygenase Reductase Gene from Acidithiobacillus caldus and the Study of the Recombinant Enzyme Activity

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	杨志晓, 侯骞, 刘国权, 卢志刚, 曹毅, 芶剑渝, 王轶, 林英超. 不同抗性烟草品系Rubisco及其活化酶对赤星病胁迫的响应[J]. 生物技术通报, 2023, 39(9): 202-212.
[2]	饶紫环, 谢志雄. 一株Olivibacter jilunii 纤维素降解菌株的分离鉴定与降解能力分析[J]. 生物技术通报, 2023, 39(8): 283-290.
[3]	赵忠娟, 杨凯, 扈进冬, 魏艳丽, 李玲, 徐维生, 李纪顺. 盐胁迫条件下哈茨木霉ST02对椒样薄荷生长及根区土壤理化性质的影响[J]. 生物技术通报, 2022, 38(7): 224-235.
[4]	袁存霞, 李艳楠, 张肖冲, 杨瑞, 刘建利, 李靖宇. As³⁺胁迫下Bacillus sp. ZJS3菌株的生理生化响应特性[J]. 生物技术通报, 2022, 38(7): 236-246.
[5]	王新光, 田磊, 王恩泽, 钟成, 田春杰. 玉米秸秆高效降解微生物复合菌系的构建及降解效果评价[J]. 生物技术通报, 2022, 38(4): 217-229.
[6]	王小琴, 黄银萍, 王蔚倩, 吴萍, 全舒. 含非天然氨基酸定点突变的MLL3_SET蛋白表达与纯化[J]. 生物技术通报, 2022, 38(3): 194-202.
[7]	贾海红, 李冰清. 超氧化物歧化酶翻译后修饰的研究进展[J]. 生物技术通报, 2022, 38(2): 237-244.
[8]	武杞蔓, 田诗涵, 李昀烨, 潘英杰, 张颖. 微生物菌肥对设施黄瓜生长、产量及品质的影响[J]. 生物技术通报, 2022, 38(1): 125-131.
[9]	袁媛, 王蕾, 石亚伟. 微生物来源碱性蛋白酶活性提高策略的研究进展[J]. 生物技术通报, 2021, 37(5): 231-236.
[10]	陈晓雨, 张建, 张新亚, 唐雨婷, 邵钰晨, 罗志丹, 卢辰. 一种快速精确测定Tth DNA聚合酶活性的方法[J]. 生物技术通报, 2021, 37(5): 281-286.
[11]	谢果珍, 唐圆, 吴仪, 黄莉莉, 谭周进. 七味白术散总苷对菌群失调腹泻小鼠肠道微生物及酶活性的影响[J]. 生物技术通报, 2021, 37(12): 124-131.
[12]	陶治东, 何艳慧, 邓子禾, 孙琳琳, 武占省. 香菇菌渣高效纤维素降解菌的筛选及产酶优化[J]. 生物技术通报, 2021, 37(11): 158-165.
[13]	田庚, 高伟强, 陈晓波, 张春晓. 地衣芽孢杆菌KD-1β-甘露聚糖酶定点突变提高酶活性及稳定性[J]. 生物技术通报, 2021, 37(10): 100-109.
[14]	袁亮. 微生物碳酸酐酶诱导CaCO₃沉淀的影响因素及生成机理[J]. 生物技术通报, 2020, 36(8): 79-68.
[15]	李鹏昊, 梁严予, 王彦伟, 关洋, 逄文强, 田克恭. 非洲猪瘟病毒K196R和A240L蛋白的可溶性表达及酶活力分析[J]. 生物技术通报, 2020, 36(11): 70-75.