World Academy of Science, Engineering and Technology
Published January 23, 2010 | Version 4787
Journal article Open

Cloning of a β-Glucosidase Gene (BGL1) from Traditional Starter Yeast Saccharomycopsis fibuligera BMQ 908 and Expression in Pichia pastoris

Description

β-Glucosidase is an important enzyme for production of ethanol from lignocellulose. With hydrolytic activity on cellooligosaccharides, especially cellobiose, β-glucosidase removes product inhibitory effect on cellulases and forms fermentable sugars. In this study, β-glucosidase encoding gene (BGL1) from traditional starter yeast Saccharomycosis fibuligera BMQ908 was cloned and expressed in Pichia pastoris. BGL1 of S. fibuligera BMQ 908 shared 98% nucleotide homology with the closest GenBank sequence (M22475) but identity in amino-acid sequences of catalytic domains. Recombinant plasmid pPICZαA/BGL1 containing the sequence encoding BGL1 mature protein and α-factor secretion signal was constructed and transformed into methylotrophic yeast P. pastoris by electroporation. The recombinant strain produced single extracellular protein with molecular weight of 120 kDa and cellobiase activity of 60 IU/ml. The optimum pH of the recombinant β-glucosidase was 5.0 and the optimum temperature was 50°C.

Files

4787.pdf

Files (415.3 kB)

Name Size
md5:3916346a98c02b3910deb1a2160febf7
415.3 kB Preview Download

Additional details

References

  • A manual of methods for expression of recombinant proteins in Pichia pastoris. Pichia Expression Kit (2005), Catalog K1740-01, pp. 1-56.
  • An C.L., Lim W. J., Hong S.Y., Kim E.J., Shin E.C., Kim M.K., Lee J.R., Park S.R., Woo J.G., Lim Y.P., Yun H.D. (2004) "Analysis of bgl operon structure and characterization of β-glucosidase from Pectobacterium carotovorum subsp. carotovorum LY34", Biosci. Biotechnol. Biochem. 68:2270-2278.
  • Bhatia Y., Mishra S., Bisaria V.S. (2002) "Microbial β-glucosidases: cloning, properties, and applications", Crit. Rev. Biotechnol. 22:375- 407.
  • Dan S., Marton I., Dekel M., Bravdo B.A., He S., Withers S.G., Shoseyov O. (2000) "Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger β-glucosidase", J. Biol. Chem. 275:4973-4980.
  • Esen A. (1993) "β-glucosidases: overview" in β-Glucosidases: Biochemistry and Molecular Biology, Esen A., Ed., American Chemical Society, Washington DC, 1-14.
  • Fukuda T., Kato-Murai M., Kadonosono T., Sahara H., Hata Y., Suye S. (2007), "Enhancement of substrate recognition ability by combinatorial mutation of β-glucosidase displayed on the yeast cell surface", Ueda M. Appl. Microbiol. Biotechnol. 76:1027-1033.
  • Gundllapalli S.B., Pretorius I.S., Cordero Otero R.R. (2007) "Effect of the cellulose-binding domain on the catalytic activity of a β-glucosidase from Saccharomycopsis fibuligera", J. Ind. Microbiol. Biotechnol. 34:413-421.
  • Lachance M.A., Bowles J.M., Starmer W.T., Barker J.S.F. (1999) "Kodamaea kakaduensis and Candida tolerans, two new yeast species from Australian Hibiscus flowers", Can. J. Microbiol. 45:172-177.
  • Lieckfeldt E., Meyer W., Börner T. (1993) "Rapid identification and differentiation of yeasts by DNA and PCR fingerprinting", J. Basic Microbiol. 33:413-425. [10] Machida M., Ohtsuki I., Fukui S., Yamashita I. (1988) "Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular β- glucosidases as expressed in Saccharomyces cerevisiae", Appl. Environ. Microbiol. 54:3147-3155. [11] Palmeri R., Spagna G. (2007) "β-Glucosidase in cellular and acellular form for winemaking application", Enzyme Microbial Technol. 40:382- 389. [12] Prakash S., Singhal R.S., Kulkarni P.R. (2002) "Enzymic debittering of Indian grapefruit (Citrus paradisi) juice", J. Sci. Food Agricult. 82: 394- 397. [13] Sambrook J., Fritsch E.F., Mainiatis T., (1989) "Molecular cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York. [14] Thanh V.N., Mai L.T., Tuan D.A. (2008) "Microbial diversity of traditional Vietnamese alcohol fermentation starters (banh men) as determined by PCR-mediated DGGE", Int. J. Food Microbiol. 128:268- 273. [15] Woosowska S., Synowiecki J. (2004) "Thermostable β-glucosidase with a broad substrate specificity suitable for processing of lactosecontaining products". Food Chem. 85:181-187. [16] Yang S., Wang L., Yan Q., Jiang Z., Li L. (2009) "Hydrolysis of soybean isoflavone glycosides by a thermostable β-glucosidase from Paecilomyces thermophila", Food Chem. 115:1247-1252.