TY - JOUR
T1 - Expression of a Trichoderma reesei β;-xylanase gene (XYN2) in Saccharomyces cerevisiae
AU - La Grange, Daniël C.
AU - Pretorius, Isak S.
AU - Van Zyl, Willem H.
PY - 1996/3
Y1 - 1996/3
N2 - The XYN2 gene encoding the main Trichoderma reesei QM 6a endo-β-1,4-xylanase was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 699-bp open reading frame that encodes a 223-amino-acid propeptide. The XYN2 gene, located on URA3-based multicopy shuttle vectors, was successfully expressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II (ADH2) and phosphoglycerate kinase (PGK1) gene promoters and terminators, respectively. The 33-amino-acid leader peptide of the Xyn2 β-xylanase was recognized and cleaved at the Kex2-like Lys-Arg residues, enabling the efficient secretion and glycosylation of the heterologous β-xylanase. The molecular mass of the recombinant β-xylanase was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 27 kDa. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of the URA3-based XYN2 shuttle vectors in nonselective complex medium. These autoselective S. cerevisiae strains produced 1,200 and 160 nkat of β-xylanase activity per ml under the control of the ADH2 and PGK1 promoters in rich medium, respectively. The recombinant enzyme showed highest activity at pH 6 and 60°C and retained more than 90% of its activity after 60 min at 50°C.
AB - The XYN2 gene encoding the main Trichoderma reesei QM 6a endo-β-1,4-xylanase was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 699-bp open reading frame that encodes a 223-amino-acid propeptide. The XYN2 gene, located on URA3-based multicopy shuttle vectors, was successfully expressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II (ADH2) and phosphoglycerate kinase (PGK1) gene promoters and terminators, respectively. The 33-amino-acid leader peptide of the Xyn2 β-xylanase was recognized and cleaved at the Kex2-like Lys-Arg residues, enabling the efficient secretion and glycosylation of the heterologous β-xylanase. The molecular mass of the recombinant β-xylanase was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 27 kDa. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of the URA3-based XYN2 shuttle vectors in nonselective complex medium. These autoselective S. cerevisiae strains produced 1,200 and 160 nkat of β-xylanase activity per ml under the control of the ADH2 and PGK1 promoters in rich medium, respectively. The recombinant enzyme showed highest activity at pH 6 and 60°C and retained more than 90% of its activity after 60 min at 50°C.
UR - http://www.scopus.com/inward/record.url?scp=0030051146&partnerID=8YFLogxK
M3 - Article
C2 - 8975597
AN - SCOPUS:0030051146
SN - 0099-2240
VL - 62
SP - 1036
EP - 1044
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
IS - 3
ER -