Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Hydrolysis of Egg Yolk Protein in a Packed Bed Reactor by Immobilized Enzyme

충진층 반응기에서 고정화 효소에 의한 난황 단백질의 가수분해

  • 강병철 (동의대학교 화학공학과)
  • Received : 2010.08.18
  • Accepted : 2010.11.11
  • Published : 2010.11.30
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Abstract

Alkaline protease for the hydrolysis of egg yolk protein was immobilized on five carriers - Duolite A568, Celite R640, Dowex-1, Dowex 50W and Silica gel R60. Duolite A568 showed a maximum immobilization yield of 24.7%. Optimum pH for the free and immobilized enzyme was pH 8 and 9, respectively. However, no change was observed in optimum temperature ($50^{\circ}C$). Thermal stability was observed in immobilized enzymes compared to free enzymes. The immobilized enzyme retained 86% activity after 10 cycle operations in a repeated batch process. The effect of flow rate on the stability of enzyme activity in continuous packed-bed reactor was investigated. Lowering flow rate increased the stability of the immobilized enzyme. After 96 hr of continuous operation in a packed-bed reactor, the immobilized enzyme retained 83 and 61% activity when casein and egg yolk were used as a raw materials, respectively.

난황단백질 가수분해를 위한 알칼리성 단백질분해효소를 5가지 담체 Duolite A568, Celite R640, Dowex-1, Dowex 50W 그리고 Silica gel R60 에 고정화하였다. Duolite A568의 경우에 24.7%의 최대 고정화 효율을 나타내었다. 자유 효소와 고정화 효소에 대한 최적의 pH는 각각 8과 9였고, 최적의 pH는 고정화에 의해 염기성으로 1만큼 증가하였다. 그러나 최적 온도는 자유 효소와 고정화 효소 모두 $50^{\circ}C$로 같았다. 고정화 효소가 자유 효소에 비해 높은 열 안정성을 보였다. 재사용 회분식 공정에서 10 cycle 동안 효소활성은 초기 활성의 86%를 유지하였다. 연속 공정을 위한 충진층 반응기에서 여러 유속에 대한 장기 조업에서 효소 활성의 안정성 평가하였는데 낮은 유속일수록 높은 활성을 유지하였다. 연속 조업에서 casein과 난황 단백질을 사용하여 원료에 대한 고정화 효소의 활성에 대한 영향을 조사하였다. 96시간 연속 조업에서 casein의 경우는 초기 활성의 83%를 유지하였고 난황 단백질의 경우는 초기 활성의 61%를 유지하였다.

Keywords

References

  1. Ahmed, S. A., S. A. Saleh, and A. F. Abdel-Fattah. 2007. Stabilization of Bacillus licheniformis ATCC 21415 alkaline protease by immobilization and modification. Aust. J. Basic Appl. Sci. 1, 313-322.
  2. Altun, G. D. and S. A. Cetinus. 2007. Immobilization of pepsin on chitosan beads. Food Chem. 100, 964-971. https://doi.org/10.1016/j.foodchem.2005.11.005
  3. Bayramoglu, G., M. Yılmaz, A. U. Senel, and M. Y. Arica. 2008. Preparation of nanofibrous polymer grafted magnetic poly (GMA-MMA)-g-MAA beads for immobilization of trypsin via adsorption. Biochem. Eng. J. 40, 262-274. https://doi.org/10.1016/j.bej.2007.12.013
  4. Benkhelifa, J., C. Bengoa, C. Larre, E. Guibal, Y. Popineau, and J. Legrand. 2005. Casein hydrolysis by immobilized enzymes in a torus reactor. Process Biochem. 40, 461-467. https://doi.org/10.1016/j.procbio.2004.01.022
  5. Cao, L. 2005. Immobilized enzymes: science or art. Curr. Opin. Chem. Biol. 9, 217-226. https://doi.org/10.1016/j.cbpa.2005.02.014
  6. Gea, S., H. Bai, H. Yuan, and L. Zhang. 1996. Continuous production of high degree casein hydrolysates by immobilized proteases in column reactor. J. Biotechnol. 50, 161-170. https://doi.org/10.1016/0168-1656(96)01561-1
  7. Greenberg, D. M. 1957. Plant proteolytic enzymes. Methods Enzymol. 2, 54-64.
  8. Gutierrez, M. A., T. Mitsuya, H. Hatta, M. Koketsu, R. Kobayashi, L. R. Juneja, and M. Kim. 1998. Comparison of egg-yolk protein hydrolysate and soybean protein hydrolysate in terms of nitrogen utilization. Br. J. Nutr. 80, 477-484.
  9. Haider, T. and Q. Husain. 2008. Concanavalin A layered calcium alginate–starch beads immobilized ${\beta}$-galactosidase as a therapeutic agent for lactose intolerant patients. Int. J. Pharm. 359, 1-6. https://doi.org/10.1016/j.ijpharm.2008.03.013
  10. Hong, J., P. Gong, D. Xu, L. Dong, and S. Yao. 2007. Stabilization of ${\alpha}$-chymotrypsin by covalent immobilization on amine-functionalized superparamagnetic nanogel. J. Biotechnol. 128, 597-605. https://doi.org/10.1016/j.jbiotec.2006.11.016
  11. Juneja, L. R., M. Koketsu, K. Nishimoto, M. Kim, T. Yamamoto, and T. Itoh. 1991. Large-scale preparation of sialic acid from chalaza and egg-yolk membrane. Carbohydr. Res. 214, 179-183. https://doi.org/10.1016/S0008-6215(00)90540-8
  12. Lowry, O. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275.
  13. Mannheim, A. and M. Cheryan. 1981. Continous hydrolysis of milk protein in a membrane reactor. J. Food Sci. 55, 381-385.
  14. Mateo, C., J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan, and R. Fernandez-Lafuente. 2007. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 40, 1451-1463. https://doi.org/10.1016/j.enzmictec.2007.01.018
  15. Ortega, N., M. Perez-Mateos, M. C. Pilar, and M. D. Busto. 2009. Neutrase immobilization on alginate-glutaraldehyde beads by covalent attachment. J. Agric. Food Chem. 57, 109-115. https://doi.org/10.1021/jf8015738
  16. Parkinson, T. L. 1966. The chemical composition of eggs. J. Sci. Food Agric. 17, 101-106. https://doi.org/10.1002/jsfa.2740170301
  17. Potumarthi, R., C. Subhakar, A. Pavani, and A. Jetty. 2008. Evaluation of various parameters of calcium-alginate immobilization method for enhanced alkaline protease production by Bacillus licheniformis NCIM-2042 using statistical methods. Bioresour. Technol. 99, 1776-1786. https://doi.org/10.1016/j.biortech.2007.03.041
  18. Puhl, A. C., C. Giacomini, G. Irazoqui, F. Batista-Viera, A. Villarino, and H. Terenzi. 2009. Covalent immobilization of tobacco-etch-virus NIa protease: a useful tool for cleavage of the histidine tag of recombinant proteins. Biotechnol. Appl. Biochem. 53, 165-174. https://doi.org/10.1042/BA20080063
  19. Roy, I. and M. N. Gupta. 2003. Lactose hydrolysis by Lactozym immobilized on cellulose beads in batch and fluidized bed modes. Process Biochem. 39, 325-332. https://doi.org/10.1016/S0032-9592(03)00086-4
  20. Sharma, S., A. Mittal, V. K. Gupta, and H. Singh. 2007. Improved stabilization of microencapsulated Cathepsin B in harsh conditions. Enzyme Microb. Technol. 40, 337-342. https://doi.org/10.1016/j.enzmictec.2006.04.024
  21. Silva, C., G. Gubitz, and A. Cavaco-Paulo. 2006. Optimization of a serine protease coupling to Eudragit S-100 by experimental design techniques. J. Chem. Technol. Biotechnol. 81, 8-16. https://doi.org/10.1002/jctb.1350
  22. Silva, C., Q. Zhang, J. Shen, and A. Cavaco-Paulo. 2006. Immobilization of proteases with a water soluble/insoluble reversible polymer for treatment of wool. Enzyme Microb. Technol. 39, 634-640. https://doi.org/10.1016/j.enzmictec.2005.11.016
  23. Wang, S., H. Bao, P. Yang, and G. Chen. 2008. Immobilization of trypsin in polyaniline-coated nano-Fe3O4 /carbon nanotube composite for protein digestion. Anal. Chim. Acta 612, 182-189. https://doi.org/10.1016/j.aca.2008.02.035
  24. Yu, X., Y. Li, C. Wang, and D. Wu. 2004. Immobilization of Aspergillus niger tannase by microencapsulation and its kinetic characteristic. Biotechnol. Appl. Biochem. 40, 151-155. https://doi.org/10.1042/BA20030180

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