Cloning of a β-Glucosidase Gene (BGL1) from Traditional Starter Yeast Saccharomycopsis fibuligera BMQ 908 and Expression in Pichia pastoris
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.
[1] A manual of methods for expression of recombinant proteins in Pichia
pastoris. Pichia Expression Kit (2005), Catalog K1740-01, pp. 1-56.
[2] 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.
[3] Bhatia Y., Mishra S., Bisaria V.S. (2002) "Microbial β-glucosidases:
cloning, properties, and applications", Crit. Rev. Biotechnol. 22:375-
407.
[4] 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.
[5] Esen A. (1993) "β-glucosidases: overview" in β-Glucosidases:
Biochemistry and Molecular Biology, Esen A., Ed., American Chemical
Society, Washington DC, 1-14.
[6] 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.
[7] 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.
[8] 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.
[9] 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.
[1] A manual of methods for expression of recombinant proteins in Pichia
pastoris. Pichia Expression Kit (2005), Catalog K1740-01, pp. 1-56.
[2] 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.
[3] Bhatia Y., Mishra S., Bisaria V.S. (2002) "Microbial β-glucosidases:
cloning, properties, and applications", Crit. Rev. Biotechnol. 22:375-
407.
[4] 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.
[5] Esen A. (1993) "β-glucosidases: overview" in β-Glucosidases:
Biochemistry and Molecular Biology, Esen A., Ed., American Chemical
Society, Washington DC, 1-14.
[6] 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.
[7] 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.
[8] 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.
[9] 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.
@article{"International Journal of Biological, Life and Agricultural Sciences:53898", author = "Le Thuy Mai and Vu Nguyen Thanh", title = "Cloning of a β-Glucosidase Gene (BGL1) from Traditional Starter Yeast Saccharomycopsis fibuligera BMQ 908 and Expression in Pichia pastoris", abstract = "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.", keywords = "β-Glucosidase, Pichia pastoris, Saccharomycopsisfibuligera, recombinant enzyme.", volume = "4", number = "1", pages = "58-5", }
