Expression, purification, and some enzymatic properties of His-tagged recombinant <b><i>Candida utilis</i></b> uricase _West China Medical Journal

Authors：

WANG Ying ¹ , XIAO Xue ¹ , LI Dan ¹ , ZHONG Chunyan ¹ , FAN Xiang ² , LI Gangrui ² ,  MENG Yao ¹

1. School of Laboratory Medicine, Chengdu Medical College, Chengdu, Sichuan 610500, P. R. China;
2. College of Life Science / Key Laboratory of China Ministry Education for Bio-resources and Eco-environment, Sichuan University, Chengdu, Sichuan 610064, P. R. China;

Corresponding author：

MENG Yao, Email: myaoworks@foxmail.com

Keywords：

Uricase; His-tag; Candida utilis; Inducing fermentation

DOI：

10.7507/1002-0179.201604005

Video：

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ObjectiveTo study the efficient expression conditions, purification, and partial enzymatic properties of His-tagged recombinant Candida utilis uricase.MethodsThe effects of isopropyl β-D-1-thiogalactopyranoside (IPTG) and lactose as inducers which were added in the end logarithmic phase were compared by the method of shake flask culture. The induction culturing time was studied with 50 L fermentor. The protein of interest was purified by Ni-Sepharose and Sephacryl S-200 HR chromatographies, and the optimal pH value, temperature, and thermal stability were also studied.ResultsThe shake flask culturing experiment results showed that IPTG was better than lactose as an inducer. In the fermentor culturing and at the end of the logarithmic growth stage, the enzyme activity of 164 U per gram bacteria and biomass of 23 grams per litter fermented solution were maximal after adding lactose for 7 hours as an inducer, i.e. the enzyme activity could be collected at 3 772 U per liter. The specific activity of purified uricase was 4.5 U/mg and the optimal pH value and temperature were 7.5 and 40℃. Additionally, the enzyme was stable at pH 6.0–10.0 and the thermal stability was below 45℃.ConclusionsIt is better to use lactose as an inducer in the process of culture. The recombinant uricase with His label can be purified by Ni-column affinity chromatography and Sephacryl S-200HR molecular sieve chromatography, and the purification process is easier than ever before. The optimum temperature, pH value, acid-base stability and thermal stability of the purified enzyme have been established to provide some experimental basis for the relationship between structure and function and practical application in the future.

Citation： WANG Ying, XIAO Xue, LI Dan, ZHONG Chunyan, FAN Xiang, LI Gangrui, MENG Yao. Expression, purification, and some enzymatic properties of His-tagged recombinant Candida utilis uricase . West China Medical Journal, 2018, 33(8): 989-993. doi: 10.7507/1002-0179.201604005 Copy

1.	Zhou Y, Zhang M, He D, et al. Uricase alkaline enzymosomes with enhanced stabilities and anti-hyperuricemia effects induced by favorable microenvironmental changes. Sci Rep, 2016, 7: 20136.
2.	Feng J, Li X, Yang XL, et al. A new practical system for evaluating the pharmacological properties of uricase as a potential drug for hyperuricemia. Arch Pharm Res, 2010, 33(11): 1761-1769.
3.	吴双林, 陈斌, 刘成倩, 等. 猪尿酸氧化酶在大肠杆菌中的表达、纯化与部分酶学性质分析. 生物工程学报, 2009, 25(11): 1664-1670.
4.	邓永康, 吴民泸, 刘盛邦, 等. 乳糖诱导重组尿酸酶基因在大肠杆菌中的表达. 中国生物工程杂志, 2009, 29(7): 74-79.
5.	汪家政, 范明. 蛋白质技术手册. 北京: 科学出版社, 2000: 42-46.
6.	Nishiya Y, Hibi T, Oda JL. A purification method of the diagnostic enzyme Bacillus uricase using magnetic beads and non-specific protease. Protein Expr Purif, 2002, 25(3): 426-429.
7.	Liu J, Li G, Liu H, et al. Purification and properties of uricase from Candida sp. and its application in uric acid analysis in serum. Appl Biochem Biotechnol, 1994, 47(1): 57-63.
8.	Chen Z, Wang Z, He X, et al. Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene. Appl Microbiol Biotechnol, 2008, 79(4): 545-554.
9.	Koyama Y, Ichikawa T, Nakano E. Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase). J Biochem, 1996, 120(5): 969-973.
10.	Li J, Chen Z, Hou L, et al. High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli. Protein Expr Purif, 2006, 49(1): 55-59.
11.	Huang SH, Wu TK. Modified colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis. Eur J Biochem, 2004, 271(3): 517-523.
12.	赵明, 何中杰, 王瑞, 等. 重组尿酸酶发酵培养及制备的工艺条件. 食品与生物技术学报, 2015, 34(3): 311-315.
13.	钱文雨, 赵兴秀, 田素华, 等. 产朊假丝酵母( Candida utilis)尿酸酶的基本特性. 兰州大学学报: 自然科学版, 2006, 42(6): 62-64.
14.	Liu X, Wen M, Li J, et al. High-yield expression, purification, characterization, and structure determination of tag-free Candida utilis uricase. Appl Microbiol Biotechnol, 2011, 92(3): 529-537.

1. Zhou Y, Zhang M, He D, et al. Uricase alkaline enzymosomes with enhanced stabilities and anti-hyperuricemia effects induced by favorable microenvironmental changes. Sci Rep, 2016, 7: 20136.
2. Feng J, Li X, Yang XL, et al. A new practical system for evaluating the pharmacological properties of uricase as a potential drug for hyperuricemia. Arch Pharm Res, 2010, 33(11): 1761-1769.
3. 吴双林, 陈斌, 刘成倩, 等. 猪尿酸氧化酶在大肠杆菌中的表达、纯化与部分酶学性质分析. 生物工程学报, 2009, 25(11): 1664-1670.
4. 邓永康, 吴民泸, 刘盛邦, 等. 乳糖诱导重组尿酸酶基因在大肠杆菌中的表达. 中国生物工程杂志, 2009, 29(7): 74-79.
5. 汪家政, 范明. 蛋白质技术手册. 北京: 科学出版社, 2000: 42-46.
6. Nishiya Y, Hibi T, Oda JL. A purification method of the diagnostic enzyme Bacillus uricase using magnetic beads and non-specific protease. Protein Expr Purif, 2002, 25(3): 426-429.
7. Liu J, Li G, Liu H, et al. Purification and properties of uricase from Candida sp. and its application in uric acid analysis in serum. Appl Biochem Biotechnol, 1994, 47(1): 57-63.
8. Chen Z, Wang Z, He X, et al. Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene. Appl Microbiol Biotechnol, 2008, 79(4): 545-554.
9. Koyama Y, Ichikawa T, Nakano E. Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase). J Biochem, 1996, 120(5): 969-973.
10. Li J, Chen Z, Hou L, et al. High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli. Protein Expr Purif, 2006, 49(1): 55-59.
11. Huang SH, Wu TK. Modified colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis. Eur J Biochem, 2004, 271(3): 517-523.
12. 赵明, 何中杰, 王瑞, 等. 重组尿酸酶发酵培养及制备的工艺条件. 食品与生物技术学报, 2015, 34(3): 311-315.
13. 钱文雨, 赵兴秀, 田素华, 等. 产朊假丝酵母( Candida utilis)尿酸酶的基本特性. 兰州大学学报: 自然科学版, 2006, 42(6): 62-64.
14. Liu X, Wen M, Li J, et al. High-yield expression, purification, characterization, and structure determination of tag-free Candida utilis uricase. Appl Microbiol Biotechnol, 2011, 92(3): 529-537.

West China Medical Journal

Expression, purification, and some enzymatic properties of His-tagged recombinant Candida utilis uricase

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