Cloning, expression and biochemical characterization of the cholesterol oxidase CgChoA from Chryseobacterium gleum

BMC Biotechnology

Renate Reiss¹  Greta Faccio¹  Linda Thöny-Meyer¹  Michael Richter¹ 

[1] Empa. Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biomaterials, Lerchenfeldstr. 5, 9014 St. Gallen, Switzerland

关键词: Taurocholate;    Biocatalysis;    Recombinant expression in Escherichia coli;    Cholesterol oxidase;    Chryseobacterium gleum;   

Others  :  834844
DOI  :  10.1186/1472-6750-14-46

 received in 2013-06-07, accepted in 2014-03-25,  发布年份 2014

【 摘 要 】

Background

Cholesterol oxidases are important enzymes for applications such as the analysis of cholesterol in clinical samples, the synthesis of steroid derived drugs, and are considered as potential antibacterial drug targets.

Results

The gene choA encoding a cholesterol oxidase from Chryseobacterium gleum DSM 16776 was cloned into the pQE-30 expression vector and heterologously expressed in Escherichia coli JM109 co-transformed with pRARE2. The N-terminally His-tagged cholesterol oxidase (CgChoA) was assigned to be a monomer in solution by size exclusion chromatography, showed a temperature optimum of 35°C, and a pH optimum at 6.75 using 0.011 M MOPS buffer under the tested conditions. The purified protein showed a maximum activity of 15.5 U/mg. CgChoA showed a Michaelis-Menten like kinetic behavior only when the substrate was dissolved in water and taurocholate (apparent K_m = 0.5 mM). In addition, the conversion of cholesterol by CgChoA was studied via biocatalytic batches at analytical scale, and cholest-4-en-3-one was confirmed as product by HPLC-MS.

Conclusion

CgChoA is a true cholesterol oxidase which activity ranges among the high performing described cholesterol oxidases from other organisms. Thus, the enzyme broadens the available toolbox of cholesterol oxidases for e.g. synthetic and biosensing applications.

【 授权许可】

   
2014 Reiss et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Vrielink A, Ghisla S: Cholesterol oxidase: biochemistry and structural features. Febs Journal 2009, 276(23):6826-6843.
• [2]Dym O, Eisenberg D: Sequence-structure analysis of FAD-containing proteins. Protein Sci 2001, 10(9):1712-1728.
• [3]Aparicio JF, Martin JF: Microbial cholesterol oxidases: bioconversion enzymes or signal proteins? Mol Biosyst 2008, 4(8):804-809.
• [4]Vrielink A: Cholesterol oxidase: structure and function. Subcell Biochem 2010, 51:137-158.
• [5]Kumari L, Kanwar SS: Cholesterol Oxidase and Its Applications. Advances in Microbiology 2012, 2(2):49-65.
• [6]Volonte F, Pollegioni L, Molla G, Frattini L, Marinelli F, Piubelli L: Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli. Bmc Biotechnology 2010, 10:33. BioMed Central Full Text
• [7]Ohta T, Fujishiro K, Yamaguchi K, Uwajima T, Aisaka K, Hasegawa M: Hyperexpression and Analysis of choB Encoding Cholesterol Oxidase of Brevibacterium sterolicum in Escherichia coli and Streptomyces lividans. Biosci Biotechnol Biochem 1992, 56(11):1786-1791.
• [8]Kreit J, Sampson NS: Cholesterol oxidase: physiological functions. Febs Journal 2009, 276(23):6844-6856.
• [9]Caldinelli L, Iametti S, Barbiroli A, Bonomi F, Fessas D, Molla G, Pilone MS, Pollegioni L: Dissecting the structural determinants of the stability of cholesterol oxidase containing covalently bound flavin. J Biol Chem 2005, 280(24):22572-22581.
• [10]Doukyu N: Characteristics and biotechnological applications of microbial cholesterol oxidases. Appl Microbiol Biotechnol 2009, 83(5):825-837.
• [11]Pollegioni L, Piubelli L, Molla G: Cholesterol oxidase: biotechnological applications. Febs Journal 2009, 276(23):6857-6870.
• [12]Corbin DR, Grebenok RJ, Ohnmeiss TE, Greenplate JT, Purcell JP: Expression and chloroplast targeting of cholesterol oxidase in transgenic tobacco plants. Plant Physiol 2001, 126(3):1116-1128.
• [13]Guo LW, Wilson WK, Pang JH, Shackleton CHL: Chemical synthesis of 7- and 8-dehydro derivatives of pregnane-3,17 alpha,20-triols, potential steroid metabolites in Smith-Lemli-Opitz syndrome. Steroids 2003, 68(1):31-42.
• [14]Chaudhari PN, Chaudhari BL, Chincholkar SB: Cholesterol biotransformation to androsta-1,4-diene-3,17-dione by growing cells of Chryseobacterium gleum. Biotechnol Lett 2010, 32(5):695-699.
• [15]Dogra N, Qazi GN: Steroid biotransformation by different strains of Micrococcus sp. Folia Microbiologica 2001, 46(1):17-20.
• [16]Munro AW, Noble MA: Fluorescence analysis of Flavoproteins. In Flavoprotein protocols, Volume 131. Edited by Chapman SK, Reid GA. Totawa, N.J: Humana Press; 1999.
• [17]Gadda G, Wels G, Pollegioni L, Zucchelli S, Ambrosius D, Pilone MS, Ghisla S: Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum. Eur J Biochem 1997, 250(2):369-376.
• [18]Pollegioni L, Gadda G, Ambrosius D, Ghisla S, Pilone MS: Cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum: effect of surfactants and organic solvents on activity. Biotechnol Appl Biochem 1999, 30:27-33.
• [19]Kim KP, Rhee CH, Park HD: Degradation of cholesterol by Bacillus subtilis SFF34 isolated from Korean traditional fermented flatfish. Lett Appl Microbiol 2002, 35(6):468-472.
• [20]Xin Y, Yang HL, Xia XL, Zhang L, Cheng C, Mou GC, Shi JB, Han YF, Wang W: Affinity purification of a cholesterol oxidase expressed in Escherichia coli. J Chromatogr B Analyt Technol Biomed Life Sci 2011, 879(13–14):853-858.
• [21]Doukyu N, Shibata K, Ogino H, Sagermann M: Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase. Appl Microbiol Biotechnol 2009, 82(3):479-490.
• [22]Wang L, Wang W: Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli. Biotechnol Lett 2007, 29(5):761-766.
• [23]Xin Y, Yang HL, Xia XL, Zhang L, Zhang YR, Tong YJ, Chen Y, Wang W: Expression and comparison of recombinant cholesterol oxidases (COD) in Escherichia coli with native cholesterol oxidase expressed in Brevibacterium sp. Afr J Biotechnol 2011, 10(66):14968-14976.
• [24]Coulombe R, Yue KQ, Ghisla S, Vrielink A: Oxygen Access to the Active Site of Cholesterol Oxidase through a Narrow Channel Is Gated by an Arg-Glu Pair. J Biol Chem 2001, 276(32):30435-30441.
• [25]Fujishiro K, Uchida H, Shimokawa K, Nakano M, Sano F, Ohta T, Kayahara N, Aisaka K, Uwajima T: Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10. FEMS Microbiol Lett 2002, 215(2):243-248.
• [26]Doukyu N, Aono R: Purification of Extracellular Cholesterol Oxidase with High Activity in the Presence of Organic Solvents from Pseudomonas sp. Strain ST-200. Appl Environ Microbiol 1998, 64(5):1929-1932.
• [27]Lyubimov AY, Chen L, Sampson NS, Vrielink A: A hydrogen-bonding network is important for oxidation and isomerization in the reaction catalyzed by cholesterol oxidase. Acta Crystallographica Section D-Biological Crystallography 2009, 65:1222-1231.
• [28]Sampson NS, Kass IJ, Ghoshroy KB: Assessment of the Role of an Ω Loop of Cholesterol Oxidase: A Truncated Loop Mutant Has Altered Substrate Specificity†. Biochemistry 1998, 37(16):5770-5778.
• [29]Studier FW: Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 2005, 41(1):207-234.