Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Chemically Modified Sepharose as Support for the Immobilization of Cholesterol Oxidase

  • Yang, Hailin (The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University) ;
  • Chen, Yi (The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University) ;
  • Xin, Yu (The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University) ;
  • Zhang, Ling (The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University) ;
  • Zhang, Yuran (The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University) ;
  • Wang, Wu (The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University)
  • Received : 2013.04.02
  • Accepted : 2013.05.23
  • Published : 2013.09.28
Download PDF
⟨ Previous Next ⟩

Abstract

Because the cholesterol oxidase from Brevibacterium sp. M201008 was not as stable as the free enzyme form, it had been covalently immobilized onto chemically modified Sepharose particles via N-ethyl-N'-3-dimethylaminopropyl carbodiimide. The optimum immobilization conditions were determined, and the immobilized enzyme activity obtained was 12.01 U/g Sepharose-ethylenediamine. The immobilization of the enzyme was characterized by Fourier transform infrared spectroscopy. The immobilized enzyme exhibited the maximal activity at $35^{\circ}C$ and pH 7.5, which was unchanged compared with the free form. After being repeatedly used 20 times, the immobilized enzyme retained more than 40.43% of its original activity. The immobilized enzyme showed better operational stability, including wider thermal and pH ranges, and retained 62.87% activity after 20 days of storage at $4^{\circ}C$, which was longer than the free enzyme.

Keywords

References

  1. Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC. 1974. Enzymatic determination of total serum cholesterol. Clin. Chem. 20: 470-475.
  2. Chen Y, Xin Y, Yang HL, Zhang L, Zhang YR, Xia XL, et al. 2013. Immobilization and stabilization of cholesterol oxidase on modified Sepharose particles. Int. J. Biol. Macromol. 56: 6-13. https://doi.org/10.1016/j.ijbiomac.2013.01.026
  3. Gilles K, Joseph I, McCarty G. 2005. Examination of cholesterol oxidase attachment to magnetic nanoparticles. J. Nanobiotechnology 3: 1-9. https://doi.org/10.1186/1477-3155-3-1
  4. Hu B, Pan J, Yu HL, Liu JW, Xu JH. 2009. Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate. Process Biochem. 44: 1019-1024. https://doi.org/10.1016/j.procbio.2009.05.001
  5. Kreit J, Germain P, Lefebvre G. 1992. Extracellular cholesterol oxidase from Rhodococcus sp. cells. J. Biotechnol. 24: 177-188. https://doi.org/10.1016/0168-1656(92)90121-O
  6. Kang K, Kan CY, Yeung A, Liu D. 2006. The immobilization of trypsin on soap-free P (MMA-EA-AA) latex particles. Mater. Sci. Eng. C 26: 664-669. https://doi.org/10.1016/j.msec.2005.07.020
  7. Kuo CH, Liu YC, Chang CMJ, Chen JH , Chang C, Shieh CJ. 2012. Optimum conditions for lipase immobilization on chitosan-coated $Fe_3O_4$ nanoparticles. Carbohydr. Polym. 87: 2538-2545. https://doi.org/10.1016/j.carbpol.2011.11.026
  8. Lario IP, Sampson N, Vrielink A. 2003. Sub-atomic resolution crystal structure of cholesterol oxidase; what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity. J. Mol. Biol. 326: 1635-1650. https://doi.org/10.1016/S0022-2836(03)00054-8
  9. Liang HC, Chang WH, Liang HF, Lee MH, Sung HW. 2003. Crosslinking structures of gelatin hydrogels crosslinked with genipin or a water-soluble carbodiimide. J. Appl. Polym. Sci. 91: 4017-4026.
  10. Lee JM, Edwards HHL, Pereira C, Samii SI. 1996. Crosslinking of tissue-derived biomaterials in 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). J. Mater. Sci. Mater. Med. 7: 531-541. https://doi.org/10.1007/BF00122176
  11. Lee DG, Ponvel KM, Kim M, Hwang S, Ahn IS, Lee CH. 2009. Immobilization of lipase on hydrophobic nano-sized magnetite particles. J. Mol. Catal. B Enzym. 57: 62-66. https://doi.org/10.1016/j.molcatb.2008.06.017
  12. Matsumoto I, Mizuno Y, Seno N. 1979. Activation of Sepharose with epichlorohydrin and subsequent immobilization of ligand for affinity adsorbent. J. Biochem. 85: 1091-1098. https://doi.org/10.1093/oxfordjournals.jbchem.a132417
  13. Pollegioni L, Wels G, Pilone MS, Ghisla S. 1999. Kinetic mechanism of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum. Eur. J. Biochem. 264: 140-151. https://doi.org/10.1046/j.1432-1327.1999.00586.x
  14. Rhee HI, Jeong KJ, Park BK, Hoi YS, Lee SY. 1991. One step purification of cholesterol oxidase from culture broth of a Pseudomonas sp. using a novel affinity chromatography method. J. Gen. Microbiol. 137: 1213-1214. https://doi.org/10.1099/00221287-137-5-1213
  15. Richmond W. 1992. Analytical reviews in clinical biochemistry: the quantitative analysis of cholesterol. Ann. Clin. Biochem. 29: 577-597. https://doi.org/10.1177/000456329202900601
  16. Richmond W. 1973. Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin. Chem. 19: 1350.
  17. Smith M, Zahnley J, Pfeifer D, Goff D. 1995. Growth and cholesterol oxidation by Mycobacterium species in Tween 80 medium. Appl. Environ. Microbiol. 59: 1425-1429.
  18. Suman S, Solanki PR, Pandey MK, Malhotra BD. 2006. Covalent immobilization of cholesterol esterase and cholesterol oxidase on polyaniline films for application to cholesterol biosensor. Anal. Chim. Acta 568: 126-132. https://doi.org/10.1016/j.aca.2005.10.008
  19. Tuzmena N, Kalburcub T, Denizlic A. 2012. $\alpha$-Amylase immobilization onto dye attached magnetic beads: optimization and characterization. J. Mol. Catal. B Enzym. 78: 16-23. https://doi.org/10.1016/j.molcatb.2012.01.017
  20. Tardioli PW, Vieira MF, Vieira AMS, Zanin GM, Betancor L, Mateo C, et al. 2011. Immobilization-tabilization of glucoamylase: chemical modification of the enzyme surface followed by covalent attachment on highly activated glyoxyl-agarose supports. Process. Biochem. 46: 409-412. https://doi.org/10.1016/j.procbio.2010.08.011
  21. Uwajima T, Yagi H, Nakamura S, Terada O. 1973. Isolation and crystallisation of extracellular 3-hydroxysteroid oxidase of Brevibacterium sterolicum. Agric. Biol. Chem. 37: 2345-2350. https://doi.org/10.1271/bbb1961.37.2345
  22. Vaidya BK, Ingavle GC, Ponrathnam S, Kulkarni BD, Nene SN. 2008. Immobilization of Candida rugosa lipase on poly (allyl glycidyl ether-co-ethylene glycol dimethacrylate) macroporous polymer particles. Bioresour. Technol. 99: 3623-3629. https://doi.org/10.1016/j.biortech.2007.07.035
  23. Wilmanska D, Sedlaczek L. 1988. The kinetics of biosynthnisis and some properties of an extracellular cholesterol oxidase produced by Arthrobacter sp. IM 79. Acta Microbiol. Polon. 37: 45-51.
  24. Wang SQ, Li SP, Yu YT. 2004. Immobilization of cholesterol oxidase on cellulose acetate membrane for free cholesterol biosensor development. Artif. Cells Blood Substit. Immobil. Biotechnol. 32: 413-425. https://doi.org/10.1081/BIO-200027479
  25. Wang L, Wang W. 2007. Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli. Biotechnol. Lett. 29: 761-766. https://doi.org/10.1007/s10529-006-9295-0
  26. Xin Y, Yang HL, Xia XL. 2011. Affinity purification of a cholesterol oxidase expressed in Escherichia coli. J. Chromatogr. B 879: 853-858. https://doi.org/10.1016/j.jchromb.2011.02.025
  27. Xin Y, Yang HL, Xia XL. 2011. Preparation and characterization of affinity sorbents based on isoalloxazine-like ligands for separation of flavoenzymes. J. Sep. Sci. 34: 2940-2949. https://doi.org/10.1002/jssc.201100474
  28. Yotova LK, Ivanov IP. 2000. Kinetic studies and analytical application of cholesterol oxidase and peroxidase immobilized to synthetic polymer. Appl. Biochem. Biotechnol. 87: 141-151. https://doi.org/10.1385/ABAB:87:2:141