Extraction, Purification and Identification of a Dexamethasone-degrading Enzymes Generated by <i>Pseudomonas Alcaligenes</i>_Journal of Biomedical Engineering

Authors：

ZHULili ¹ ,  YANGZhibang ² , YANGQian ¹ , SHIZhongquan ³ , DENGXichuan ²

1. Department of Basic Medicine, Chongqing Medical University, Chongqing 400016, China;
2. Labortory of Pathogen Biology and Immunology, Experimental Teaching Center of Basic Medicine, Chongqing University of Medical Sciences, Chongqing 400016, China;
3. Department of Pathogen Biology and Immunology, Chongqing Three Gorges Medical College, Chongqing 404120, China;

Corresponding author：

YANGZhibang, Email: dryangfm365@sina.com

Keywords：

Pseudomonas alcaligenes; dexamethasone; degradation enzymes; extraction; purification; identification

DOI：

10.7507/1001-5515.20150185

Video：

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Abstract Full text Figures/Tables Video References Cited by

In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U·mg^-1. Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.

Citation： ZHULili, YANGZhibang, YANGQian, SHIZhongquan, DENGXichuan. Extraction, Purification and Identification of a Dexamethasone-degrading Enzymes Generated by Pseudomonas Alcaligenes. Journal of Biomedical Engineering, 2015, 32(5): 1044-1049. doi: 10.7507/1001-5515.20150185 Copy

1.	BRAGIN E Y, SHTRATNIKOVA V Y, DOVBNYA D V, et al. Comparative analysis of genes encoding key steroid core oxidation enzymes in fast-growing Mycobacterium spp. strains[J]. J Steroid Biochem Mol Biol, 2013, 138:41-53.
2.	SCHRIKS M, VAN LEERDAM J A, VAN DER LINDEN S C, et al. High-resolution mass spectrometric identification and quantification of glucocorticoid compounds in various wastewaters in the Netherlands[J]. Environ Sci Technol, 2010, 44(12):4766-4774.
3.	石中全,周裕珍,杨致邦,等.废水中地塞米松污染的探讨[J].中国医药指南, 2012, 10(9):319-321.
4.	LIU Z H, KANJO Y, MIZUTANI S. Removal mechanisms for endocrine disrupting compounds (EDCs) in wastewater treatment——physical means, biodegradation, and chemical advanced oxidation:a review[J]. Sci Total Environ, 2009, 407(2):731-748.
5.	胡平,丁德玲,孙春宝.医院废水处理中存在的问题及对策[J].环境与可持续发展, 2007, (4):29-30.
6.	WANG Yi, YANG Zhibang, ZHU Lili, et al. Isolation and identification of dexamethasone sodium phosphate degrading Pseudomonas alcaligenes[J].J Basic Microbiol,2015,55(2):262-268.
7.	LIU Yi, WANG Feng, XIA Siqing, et al. Study of 4-t-octylphenol degradation and microbial community in granular sludge[J]. J Environ Sci (China), 2008, 20(2):167-171.
8.	QUAN S, HINIKER A, COLLET J F, et al. Isolation of bacteria envelope proteins[J].Methods Mol Biol, 2013, 966:359-366.
9.	BURGES R R, DEUTSCHER M P. Guide to protein purification[M]. 2nd ed. Singapore:Elsevier, 2013, 258-262.
10.	CAO Wanlu, LI Haixin, ZHANG Juan, et al. Periplasmic expression optimization of VEGFR2 D3 adopting response surface methodology:antiangiogenic activity study[J]. Protein Expr Purif, 2013, 90(2):55-66.
11.	HU Min, QIU Zonghao, ZHOU Peng, et al. Proteomic analysis of ‘Zaosu’ pear (Pyrus bretschneideri Rehd.) and its red skin bud mutation[J]. Proteome Sci, 2012, 10(1):51.
12.	PABST T M, ANTOS D, CARTA G A, et al. Protein separations with induced pH gradients using cation-exchange chromatographic columns containing weak acid groups[J]. J Chromatogr A, 2008, 1181(1/2):83-94.
13.	TANAKA K, BUDD M A, EFRON M L, et al. Isovaleric acidemia:a new genetic defect of leucine metabolism[J]. Proc Natl Acad Sci U S A, 1966, 56(1):236-242.
14.	FÖRSTER-FROMME K, JENDROSSEK D. Biochemical characterization of isovaleryl-CoA dehydrogenase (LiuA) of Pseudomonas aeruginosa and the importance of liu genes fora functional catabolic pathway of methyl-branched compounds[J]. FEMS Microbiol Lett, 2008, 286(1):78-84.
15.	FÖRSTER-FROMME K, CHATTOPADHYAY A, JENDROSSEK D. Biochemical characterization of AtuD from Pseudomonas aeruginosa, the first member of a new subgroup of acyl-CoA dehydrogenases with specificity for citronellyl-CoA[J]. Microbiology, 2008, 154(3):789-796.

1. BRAGIN E Y, SHTRATNIKOVA V Y, DOVBNYA D V, et al. Comparative analysis of genes encoding key steroid core oxidation enzymes in fast-growing Mycobacterium spp. strains[J]. J Steroid Biochem Mol Biol, 2013, 138:41-53.
2. SCHRIKS M, VAN LEERDAM J A, VAN DER LINDEN S C, et al. High-resolution mass spectrometric identification and quantification of glucocorticoid compounds in various wastewaters in the Netherlands[J]. Environ Sci Technol, 2010, 44(12):4766-4774.
3. 石中全,周裕珍,杨致邦,等.废水中地塞米松污染的探讨[J].中国医药指南, 2012, 10(9):319-321.
4. LIU Z H, KANJO Y, MIZUTANI S. Removal mechanisms for endocrine disrupting compounds (EDCs) in wastewater treatment——physical means, biodegradation, and chemical advanced oxidation:a review[J]. Sci Total Environ, 2009, 407(2):731-748.
5. 胡平,丁德玲,孙春宝.医院废水处理中存在的问题及对策[J].环境与可持续发展, 2007, (4):29-30.
6. WANG Yi, YANG Zhibang, ZHU Lili, et al. Isolation and identification of dexamethasone sodium phosphate degrading Pseudomonas alcaligenes[J].J Basic Microbiol,2015,55(2):262-268.
7. LIU Yi, WANG Feng, XIA Siqing, et al. Study of 4-t-octylphenol degradation and microbial community in granular sludge[J]. J Environ Sci (China), 2008, 20(2):167-171.
8. QUAN S, HINIKER A, COLLET J F, et al. Isolation of bacteria envelope proteins[J].Methods Mol Biol, 2013, 966:359-366.
9. BURGES R R, DEUTSCHER M P. Guide to protein purification[M]. 2nd ed. Singapore:Elsevier, 2013, 258-262.
10. CAO Wanlu, LI Haixin, ZHANG Juan, et al. Periplasmic expression optimization of VEGFR2 D3 adopting response surface methodology:antiangiogenic activity study[J]. Protein Expr Purif, 2013, 90(2):55-66.
11. HU Min, QIU Zonghao, ZHOU Peng, et al. Proteomic analysis of ‘Zaosu’ pear (Pyrus bretschneideri Rehd.) and its red skin bud mutation[J]. Proteome Sci, 2012, 10(1):51.
12. PABST T M, ANTOS D, CARTA G A, et al. Protein separations with induced pH gradients using cation-exchange chromatographic columns containing weak acid groups[J]. J Chromatogr A, 2008, 1181(1/2):83-94.
13. TANAKA K, BUDD M A, EFRON M L, et al. Isovaleric acidemia:a new genetic defect of leucine metabolism[J]. Proc Natl Acad Sci U S A, 1966, 56(1):236-242.
14. FÖRSTER-FROMME K, JENDROSSEK D. Biochemical characterization of isovaleryl-CoA dehydrogenase (LiuA) of Pseudomonas aeruginosa and the importance of liu genes fora functional catabolic pathway of methyl-branched compounds[J]. FEMS Microbiol Lett, 2008, 286(1):78-84.
15. FÖRSTER-FROMME K, CHATTOPADHYAY A, JENDROSSEK D. Biochemical characterization of AtuD from Pseudomonas aeruginosa, the first member of a new subgroup of acyl-CoA dehydrogenases with specificity for citronellyl-CoA[J]. Microbiology, 2008, 154(3):789-796.

Journal of Biomedical Engineering

Extraction, Purification and Identification of a Dexamethasone-degrading Enzymes Generated by Pseudomonas Alcaligenes

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