DOI QR코드

DOI QR Code

효소 특성 개선을 위한 Exiguobacterium sp. β-glucosidase의 키토산 비드에 효소 고정화

Immobilization of β-Glucosidase from Exiguobacterium sp. DAU5 on Chitosan Bead for Improved Enzymatic Properties

  • 투고 : 2010.09.23
  • 심사 : 2010.10.28
  • 발행 : 2010.11.30

초록

Exiguobacterium sp. 유래의 $\beta$-glucosidase 고정을 위하여 글루타르알데하이드를 사용한 키토산 비드를 조제하였다. 키토산 비드의 교차결합 및 고정화의 조건을 최적화하였다. $\beta$-glucosidase 고정화의 최적생산 조건에서 20%의 수율과 5.22 U/g의 효소활성을 나타냈다. 최적 pH 와 온도는 9.0과 $55^{\circ}C$를 나타냈다. 고정된 효소의 안정성은 pH 7.0-10.0에서는 80%, $40^{\circ}C$ 2시간 반응에서는 80% 및 $50^{\circ}C$ 1시간 반응에서는 48%의 활성을 보유하였다. 이러한 결과는 높은 pH와 고온에서 비고정 효소보다 안정성을 보여주었다. 고정된 효소를 가지고 대두 이소플라본 배당체의 높은 가수분해능을 확인하였다. 이상의 결과는 고정화 효소의 다양한 이용 가능성을 시사하였다.

Glutaraldehyde was used to cross-link chitosan beads to immobilize the crude enzyme $\beta$-glucosidase from Exiguobacterium sp. DAU5. The conditions for preparing cross-linking chitosan beads and immobilization such as concentration of glutaradehyde, cross-linking time, immobilization pH and time were optimized. The chitosan beads were cross-linked with 1.5% glutaraldehyde for 1.5 hr. The immobilized $\beta$-glucosidase had an overall yield of 20% and specific activity of 5.22 U/g. The optimized pH and temperature were 9.0 and $55^{\circ}C$, respectively. More than 80% of its activity at pH 7.0-10.0, 80% at $40^{\circ}C$ for 2 hr and 48% at $50^{\circ}C$ for 1 hr, were retained. However, the immobilization product showed higher pH and thermal stabilities than free enzymes. It also showed high hydrolyzing activity on soybean isoflavone glycoside linkage. These results suggest the broad application prospects of immobilization enzymes.

키워드

참고문헌

  1. Abdel-Naby, M. A. 1993. Immobilization of Aspergillus niger NRC 107 xylanase and ${\beta}$-xylosidase, and properties of the immobilized enzymes. Appl. Biochem. Biotechnol. 38, 69-81. https://doi.org/10.1007/BF02916413
  2. Allan, G. G., J. R. Fox, and N. Kong. 1978. pp. 63-78, In Muzzarelli, R. A. A. and E. R. Pariser (eds.), Proceedings of the first international conference on chitin and chitosan. MIT Press, Cambridge, MA.
  3. Brown, J. P. 1998. Hydrolysis of glycosides and esters. pp. 109-144, In: Role of the gut flora in toxicity and cancer (ed.), London: Academic Press., Rowland, I.R.
  4. Chang, M. Y., H. C. Kao, and R. S. Juang. 2008. Thermal inactivation and reactivity of ${\beta}$-glucosidase immobilized on chitosan-clay composite. International J. Biological Macro. 43, 48-53 https://doi.org/10.1016/j.ijbiomac.2007.10.004
  5. Chang, J., I. H. Park, Y. S. Lee, S. C. Ahn, Y. Zhou, and Y. L. Choi. 2010. Cloning, expression, and characterization of ${\beta}$-glucosidase from Exiguobacterium sp. DAU5 and synthesis of lactose oligomer. Bioproc. Biotech. Engin. (in press)
  6. Fenton, D. M. and D. E. Eveleigh. 1981. Purification and mode of action of a chitosanase from Penicillium islandicum. J. Gen. Microbiol. 126, 151-165.
  7. Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280, 309-316.
  8. Krajewska, B. 2004. Application of chitin- and chitosan-based materials for enzyme immobilizations: a review Enzyme Microb. Technol. 35, 126-139. https://doi.org/10.1016/j.enzmictec.2003.12.013
  9. Krajewska, K. 2004. Application of chitin- and chitosan-based materials for enzyme immobilizations: a review Barbara. Enzyme Microb. Technol. 35, 126-139. https://doi.org/10.1016/j.enzmictec.2003.12.013
  10. Krogh, K. B., P. V. Harris, C. L. Olsen, K. S. Johansen, J. Hojer-Pedrsen, J. Borjesson, and L. Olsson. 2009. Characterization and kinetic analysis of a thermostable GH3 ${\beta}$-glucosidase from Penicillium brasilianum. Appl. Microbiol. Biotechnol. 86, 143-154.
  11. Kuo, L. C. and K. T. Lee. 2008. Cloning, expression, and characterization of two beta-glucosidases from isoflavone glycoside-hydrolyzing Bacillus subtilis natto. J. Agric. Food Chem. 56, 119-125. https://doi.org/10.1021/jf072287q
  12. Lowry, O., N. Rosebrough, A. Farr, and R. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275.
  13. Pelletier, A. and J. Sygusch. 1990. Purification and characterization of three chitosanase activities from Bacillus megaterium P1. Appl. Environ. Microbiol. 56, 844-848.
  14. Peter, M. 1995. Applications and environmental aspects of chitin and chitosan. J. Macromol. Sci. Pure Appl. Chem. 32, 629-640. https://doi.org/10.1080/10601329508010276
  15. Rorrer, G. L., T. Y. Hsien, and J. D. Way. 1993. Synthesis of porous-magnetic chitosan beads for removal of cadmium ions from wastewater. Ind. Eng. Chem. Res. 32, 2170-2178. https://doi.org/10.1021/ie00021a042
  16. Roy, P., S. Mishra, and T. K. Chaudhuri. 2005. Cloning, sequence analysis, and characterization of a novel ${\beta}$-glucosidase-like activity from Pichia etchellsii. Biochem. Biophys. Res. Commun. 336, 299-308. https://doi.org/10.1016/j.bbrc.2005.08.067
  17. Setchell, K. D., N. M. Brown, L. Zimmer-Nechemias, W. T. Brashear, B. E. Wolfe, A. S. Kirschner, and J. E. Heubi. 2002. Evidence for lack of absorption of soy isoflavone glycosides in humans, supporting the crucial role of intestinal metabolism for bioavailability. Am. J. Clin. Nutr. 76, 447-453.
  18. Shengtang, Z., S. Gao, and G. Gao. 2010. Immobilization of ${\beta}$-Galactosidase onto magnetic beads. Appl. Biochem. Biotechnol. 160, 1386-1393 DOI 10.1007/s12010-009-8600-5.
  19. Singh, A. and K. Hayashi. 1995. Construction of chimeric ${\beta}$-glucosidases with improved enzymatic properties. J. Biol. Chem. 270, 21928-21933. https://doi.org/10.1074/jbc.270.37.21928
  20. Srere, P. A. and K. Uyeda. 1976. Functional groups on enzymes suitable for binding to matrixes. Method. Enzymol. 44, 11-19. https://doi.org/10.1016/S0076-6879(76)44004-1
  21. Xu, X., K. S. Harris, H. J. Wang, P. A. Murphy, and S. Hendrich. 1995. Bioavailability of soybean isoflavones depends upon gut microflora in women. J. Nutr. 125, 2307-2315.

피인용 문헌

  1. Characterization of β-glucosidase immobilized on chitosan-multiwalled carbon nanotubes (MWCNTS) and their application on tea extracts for aroma enhancement vol.89, 2016, https://doi.org/10.1016/j.ijbiomac.2016.05.008
  2. Immobilization of Trypsin on Chitosan Nonwoven Using Glutaraldehyde vol.37, pp.7, 2013, https://doi.org/10.5850/JKSCT.2013.37.7.852
  3. Physicochemical properties and microencapsulation process of rice fermented with Bacillus subtilis CBD2 vol.22, pp.2, 2015, https://doi.org/10.11002/kjfp.2015.22.2.225
  4. Development of an enzyme-immobilized support using a polyester woven fabric vol.87, pp.1, 2017, https://doi.org/10.1177/0040517515624874