Journal of Microbiology and Biotechnology

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

Xylan Hydrolysis by Treatment with Endoxylanase and $\beta$-Xylosidase Expressed in Yeast

  • Heo, Sun-Yeon (Department of Biotechnology and bioengineeing, Dong-Eui University) ;
  • Kim, Joong-Kyun (Department of Biotechnology and Bioengineering, Pukyong National University) ;
  • Kim, Young-Man (Department of Biotechnology and Bioengineering, Pukyong National University) ;
  • Nam, Soo-Wan (Department of Food Science and Nutrition, Oriental Biotech., Co., Dong-Eui University)
  • Published : 2004.02.01
Download PDF
⟨ Previous Next ⟩

Abstract

The endoxylanase (642 bp; 213 amino acids) and $\beta$-xylosidase (1,602 bp; 533 amino acids) genes from Bacillus sp. were amplified by PCR and separately inserted into the downstream of the yeast ADH1 promoters, resulting in the pAEDX-1 (7.63 kb) and pAEX (8.47 kb) plasmids, respectively. When the yeast transformants, S. cerevisiae SEY2102 harboring pAEDX-1 or pAEX, were grown on YPD medium, the total activities of the enzymes were approximately 9.8 unit/ml for endoxylanase and 2.9 unit/m1 for $\beta$-xylosidase. When the three kinds of xylan from oat spelts, birch wood, and corncob were hydrolyzed by treating with recombinant endoxylanase and $\beta$-xylosidase, it was found that xylose, xylobiose, and xylotriose were produced. To efficiently hydrolyze xylan, various reaction conditions such as amount of enzymes, substrate type, substrate concentration, temperature, and reaction time were examined. The optimized conditions for the hydrolysis of xylan were as follows: amount of endoxylanase, 10 units; amount of $\beta$-xylosidase, 10 units; temperature, $50^\circ{C}$; substrate type, oat spelts xylan; substrate concentration, 6%; reaction time, 1 h. Under the optimal condition, xylose was mainly produced from oat spelts xylan by cooperative action of endoxylanase and $\beta$-xylosidase.

Keywords

References

  1. Andries, J. C. and S. Isak. 1991. Co-expression of a Saccharomyces diastaticus glucoamylase-encoding gene and a Bacillus amyloliquefaciens-amylase-encoding gene in Saccharomyces cerevisiae. Gene 100: 85-93
  2. Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3: 286-290
  3. Beg, Q. K., M. Kapoor, L. Mahajan, and G. S. Hoondal. 2001. Microbial xylanase and their industrial application: A review. Appl. Microbiol. Biotechnol. 56: 326-338
  4. Chung, D. K., D. H. Shin, B. W. Kim, J. K. Nam, I. S. Han, and S. W. Nam. 1997. Expression and secretion of Clostridium thermocellum endoglucanase A gene (celA) in different Saccharomyces cerevisiae strains. Biotechnol. Lett. 19: 503-506
  5. Chun, Y. C., K. H. Jung, J. C. Lee, S. H. Park, H. K. Chung, and K. H. Yoon. 1998. Molecular cloning and the nucleotide sequence of a Bacillus sp. KK-1 b-xylosidase gene. J. Microbiol. Biotechnol. 8: 28-33
  6. Emr, S. D., R. Schekman, M. C. Flessel, and J. Thorner. 1983. An MF 1-SUC2 (a-factor-invertase) gene fusion for study of protein localization and gene expression in yeast. Proc. Natl. Acad. Sci. USA 80: 7080-7084
  7. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163-168
  8. Jeong, K. J., P. C. Lee, I. Y. Park, M. S. Kim, and S. C. Kim. 1998. Molecular cloning and characterization of an endoxylanase gene of Bacillus sp. in Escherichia coli. Enz. Microb. Technol. 22: 599-605
  9. Jeong, K. J., I. Y. Park, M. S. Kim, and S. C. Kim. 1998. High-level expression of an endoxylanase gene from Bacillus sp. in Bacillus subtilis DB104 for the production of xylobiose from xylan. Appl. Microbiol. Biotechnol. 50: 113-118
  10. Jeong, T. H., H. O. Kim, J. N. Park, H. J. Lee, D. J. Shin, H. B. Lee, S. B. Chun, and S. Bai. 2001. Cloning and sequencing of the b-amylase gene from Paenibacillus sp. and its expression in Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 11: 65-71
  11. Kang, S. C., H. J. Kim, S. W. Nam, and D. K. OH. 2002. Surface immobilization on silica of endoxylanase production from recombinant Bacillus subtilis. J. Microbiol. Biotechnol. 12: 766-772
  12. Kim, H. J., J. N. Park, H. O. Kim, D. J. Shin, J. E. Chin, H. B. Lee, S. B. Chun, and S. Bai. 2002. Cloning and expression of a Paenibacillus sp. neopullulanase gene in Saccharomyces cerevisiae producing Schwanniomyces occidentalis glucoamylase. J. Microbiol. Biotechnol. 12: 340-344
  13. Kim, J. H., B. W. Kim, K. H. Yoon, and S. W. Nam. 2000. Expression of Bacillus sp. b-xylosidase gene (xylB) in Saccharomyces cerevisiae. Biotechnol. Lett. 22: 1025-1029
  14. La Grange, D. C., I. S. Pretorius, and W. H. Van Zyl. 1997. Cloning of the Bacillus pumilius $\beta$-xylosidase gene (xynB) and its expression in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 47: 262-266 https://doi.org/10.1007/s002530050924
  15. La Grange, D. C., I. S. Pretorius, M. Claeyssens, and W. H. Van Zyl. 2001. Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger TEX>$\beta$-xylosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes. Appl. Environ. Microbiol. 67: 5512-5519
  16. Lee, O. S., C. S. Choi, J. H. Choi, G. J. Joo, and I. K. Rhee. 2001. Production of xylooligosaccharides with thermostable xylanase from the streptomyces Thermocyaneo violaceus. Kor. J. Microbiol. Biotechnol. 29: 221-226
  17. 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
  18. Nam, S. W., K. Yoda, and M. Yamasaki. 1993. Secretion and localization of invertase and inulinase in recombinant Saccharomyces cerevisiae. Biotechnol. Lett. 15: 1049-1054
  19. Reilly, P. J. 1981. Xylanase: structure and function. Basic Life Sci. 18: 111-129
  20. Rhew, B. K., J. W. Lee, C. S. Lee, S. I. Hyun, Y. J. Park, J. B. Ahn, C. K. Yang, and S. W. Lee. 2002. Effect of xylooligosaccharides on the growth of intestinal microflora. Kor. J. Microbiol. Biotechnol. 30: 380-387
  21. Romanos, M. A., C. A. Scorerand, and J. J. Clare. 1992. Foreign gene expression in yeast: a review. Yeast 8: 423- 488
  22. Sun, J. H. and Y. J. Choi. 1996. Synergism among endoxylanase, $\beta$-xylosidase, and acetyl xylan esterase from Bacillus stearothermophilus. J. Microbiol. Biotechnol. 6: 173-178
  23. Sun, J. H., S. S. Cho, and Y. J. Choi. 1996. Synergic effects among endo-xylanase, $\beta$-xylosidase, and $\alpha$-Larabinofuranosidase from Bacillus stearothermophilus. J. Microbiol. Biotechnol. 6: 179-183
  24. Thomson, J. A. 1993. Molecular biology of xylan degradation. FEMS Microbiol. Rev. 104: 65-82 https://doi.org/10.1111/j.1574-6968.1993.tb05864.x
  25. Vernet, T., D. Dignard, and D. Y. Thomas. 1987. A family of yeast expression vectors containing the phage f1 intergenic region. Gene 52: 225-233
  26. Wong, K. K. Y., L. U. L. Tan, and J. N. Saddler. 1988. Multiplicity of $\beta$-1,4-xylanase in microorganism: Function and application. Microbiol. Rev. 52: 305-317