Journal of Microbiology and Biotechnology

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

DOI QR Code

Purification and Characterization of Beta-Glucosidase from Weissella cibaria 37

  • Lee, Kang Wook (Division of Applied Life Science (BK21), Graduate School, Gyeongsang National University) ;
  • Han, Nam Soo (Department of Food Science and Technology, BK21 Education and Research Center for Advanced Bio-Agriculture Technology, Chungbuk National University) ;
  • Kim, Jeong Hwan (Division of Applied Life Science (BK21), Graduate School, Gyeongsang National University)
  • Received : 2012.06.04
  • Accepted : 2012.08.02
  • Published : 2012.12.28
Download PDF
⟨ Previous Next ⟩

Abstract

A gene encoding ${\beta}$-glucosidase was cloned from Weissella cibaria 37, an isolate from human feces. Sequence analysis showed that the gene could encode a protein of 415 amino acids in length, and the translated amino acid sequence showed homology (34-31%) with glycosyl hydrolase family 1 ${\beta}$-glucosidases. The gene was overexpressed in E. coli BL21(DE3) using pET26b(+) and a 50 kDa protein was overproduced, which matched well with the calculated size of the enzyme, 49,950.87 Da. Recombinant ${\beta}$-glucosidase was purified by using a his-tag affinity column. The purified ${\beta}$-glucosidase had an optimum pH and a temperature of 5.5 and $45^{\circ}C$, respectively. Among the metal ions (5mM concentration), $Ca^{2+}$ slightly increased the activity (108.2%) whereas $Cu^{2+}$ (46.1%) and $Zn^{2+}$ (56.7%) reduced the activity. Among the enzyme inhibitors (1 mM concentration), SDS was the strongest inhibitor (16.9%), followed by pepstatin A (45.2%). The $K_m$ and $V_{max}$ values of purified enzyme were 4.04 mM and 0.92 ${\mu}mol/min$, respectively, when assayed using pNPG (p-nitrophenyl-${\beta}$-D-glucopyranoside) as the substrate. The enzyme liberated reducing sugars from carboxymethyl cellulose (CMC).

Keywords

References

  1. Bauer, M. W., E. J. Bylina, R. V. Swanson, and R. M. Kelly. 1996. Comparison of a ${\beta}$-glucosidase and a ${\beta}$-mannosidase from the hyperthermophilic Archaeon Pyrococcus furiosus. J. Biol. Chem. 271: 23749-23755. https://doi.org/10.1074/jbc.271.39.23749
  2. Belancic, A., Z. Gunata, M. J. Vallier, and E. Agosin. 2003. Beta-glucosidase from the grape native yeast Debrayomyces; purification, characterization, and its effect on monoterpene content of a muscat grape juice. J. Agric. Food Chem. 51: 1453-1459. https://doi.org/10.1021/jf025777l
  3. Benoliel, B., M. J. Pocas-Fonseca, F. A. G. Torres, and L. M. P. de Moraes. 2010. Expression of a glucose-tolerant ${\beta}$-glucosidase from Humicola grisea var. thermoidea in Saccharomyces cerevisiae. Appl. Biochem. Biotechnol. 160: 2036-2044. https://doi.org/10.1007/s12010-009-8732-7
  4. Bhatia, Y., S. Mishra, and V. S. Bisaria. 2002. Microbial ${\beta}$- glucosidases: Cloning, properties, and applications. Crit. Rev. Biotechnol. 22: 375-407. https://doi.org/10.1080/07388550290789568
  5. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  6. Chi, H. and G.-E. Ji. 2005. Transformation of ginsenosides Rb1 and Re from Panax ginseng by food microorganisms. Biotechnol. Lett. 27: 765-771. https://doi.org/10.1007/s10529-005-5632-y
  7. Coulon, S., P. Chemardin, Y. Gueguen, A. Arnaud, and P. Galzy. 1998. Purification and characterization of an intracellular ${\beta}$-glucosidase from Lactobacillus casei ATCC393. Appl. Biochem. Biotechnol. 74: 105-114. https://doi.org/10.1007/BF02787177
  8. Fang, Z., W. Fang, J. Liu, Y. Hong, H. Peng, X. Zhang, et al. 2010. Cloning and characterization of a ${\beta}$-glucosidase from marine microbial metagenome with excellent glucose tolerance. J. Microbiol. Biotechnol. 20: 1351-1358. https://doi.org/10.4014/jmb.1003.03011
  9. Georgiou, G. and P. Valax. 1996. Expression of correctly folded proteins in Escherichia coli. Curr. Opin. Biotechnol. 7: 190-197. https://doi.org/10.1016/S0958-1669(96)80012-7
  10. Gueguen, Y., P. Chemardin, P. Labrot, A. Arnaud, and P. Galzy. 1997. Purification and characterization of an intracellular ${\beta}$- glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava. J. Appl. Microbiol. 82: 469-476. https://doi.org/10.1046/j.1365-2672.1997.00136.x
  11. Hernandez, L. F., J. C. Espinosa, M. Fernandez-Gonzalez, and A. Briones. 2003. ${\beta}$-Glucosidase activity in a Saccharomyces cerevisiae wine strain. Int. J. Food Microbiol. 80: 171-176. https://doi.org/10.1016/S0168-1605(02)00149-6
  12. Izumi, T., M. K. Piskula, S. Osawa, A. Obata, K. Tobe, M. Saito, et al. 2000. Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. J. Nutr. 130: 1695-1699.
  13. Kang, S. W., E. H. Ko, J. S. Lee, and S. W. Kim. 1999. Overproduction of ${\beta}$-glucosidase by Aspergillus niger mutant from lignocellulosic biomass. Biotechnol. Lett. 21: 647-650. https://doi.org/10.1023/A:1005556523241
  14. Kim, D. S., S. H. Choi, D. W. Kim, S. H. Nam, R. N. Kim, A. Kang, et al. 2011. Genome sequence of Weissella cibaria KACC 11862. J. Bacteriol. 193: 797-798. https://doi.org/10.1128/JB.01342-10
  15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685. https://doi.org/10.1038/227680a0
  16. Lee, K. W., J. Y. Park, H. R. Jeong, H. J. Heo, N. S. Han, and J. H. Kim. 2012. Probiotic properties of Weissella strains isolated from human faeces. Anaerobe 18: 96-102. https://doi.org/10.1016/j.anaerobe.2011.12.015
  17. Michlmayr, H., C. Schumann, N. M. Barreira Braz da Silva, K. D. Kulbe, and A. M. del Hierro. 2010. Isolation and basic characterization of a ${\beta}$-glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture. J. Appl. Microbiol. 108: 550-559. https://doi.org/10.1111/j.1365-2672.2009.04461.x
  18. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428. https://doi.org/10.1021/ac60147a030
  19. Park, J. K., L.-X. Wang, H. V. Patel, and S. Roseman. 2002. Molecular cloning and characterization of a unique ${\beta}$-glucosidase from Vibrio cholerae. J. Biol. Chem. 277: 29555-29560. https://doi.org/10.1074/jbc.M202978200
  20. Sestelo, A. B. F., M. Poz, and T. G. Villa. 2004. ${\beta}$-Glucosidase activity in a Lactobacillus plantarum wine strain. World J. Microbiol. Biotechnol. 20: 633-637.
  21. Wang, L., Q.-M. Liu, B.-H. Sung, D.-S. An, H.-G. Lee, S.-G. Kim, et al. 2011. Bioconversion of ginsenosides Rb1, Rb2, Rc and Rd by novel ${\beta}$-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: Cloning, expression, and enzyme characterization. J. Biotechnol. 156: 125-133. https://doi.org/10.1016/j.jbiotec.2011.07.024
  22. Wulff-Strobel, C. R. and D. B. Wilson. 1995. Cloning, sequencing, and characterization of a membrane-associated Prevotella ruminicola B14 ${\beta}$-glucosidase with cellodextrinase and cyanoglycosidase activities. J. Bacteriol. 177: 5884-5890.

Cited by

  1. Why Are Weissella spp. Not Used as Commercial Starter Cultures for Food Fermentation? vol.3, pp.3, 2012, https://doi.org/10.3390/fermentation3030038
  2. In silico Approach to Elucidate Factors Associated with GH1 β-Glucosidase Thermostability vol.13, pp.4, 2012, https://doi.org/10.22207/jpam.13.4.07