Biomaterial Database

Expression of a Trichoderma reesei beta-xylanase gene (XYN2) in Saccharomyces cerevisiae. 1996

D C la Grange, and I S Pretorius, and W H van Zyl

Department of Microbiology, University of Stellenbosch, South Africa.

The XYN2 gene encoding the main Trichoderma reesei QM 6a endo-beta-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 beta-xylanase was recognized and cleaved at the Kex2-like Lys-Arg residues, enabling the efficient secretion and glycosylation of the heterologous beta-xylanase. The molecular mass of the recombinant beta-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 beta-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 degrees C and retained more than 90% of its activity after 60 min at 50 degrees C.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012441	Saccharomyces cerevisiae	A species of the genus SACCHAROMYCES, family Saccharomycetaceae, order Saccharomycetales, known as "baker's" or "brewer's" yeast. The dried form is used as a dietary supplement.	Baker's Yeast,Brewer's Yeast,Candida robusta,S. cerevisiae,Saccharomyces capensis,Saccharomyces italicus,Saccharomyces oviformis,Saccharomyces uvarum var. melibiosus,Yeast, Baker's,Yeast, Brewer's,Baker Yeast,S cerevisiae,Baker's Yeasts,Yeast, Baker
D014242	Trichoderma	A mitosporic fungal genus frequently found in soil and on wood. It is sometimes used for controlling pathogenic fungi. Its teleomorph is HYPOCREA.	Trichodermas
D014995	Xylosidases	A group of enzymes that catalyze the hydrolysis of alpha- or beta-xylosidic linkages. EC 3.2.1.8 catalyzes the endo-hydrolysis of 1,4-beta-D-xylosidic linkages; EC 3.2.1.32 catalyzes the endo-hydrolysis of 1,3-beta-D-xylosidic linkages; EC 3.2.1.37 catalyzes the exo-hydrolysis of 1,4-beta-D-linkages from the non-reducing termini of xylans; and EC 3.2.1.72 catalyzes the exo-hydrolysis of 1,3-beta-D-linkages from the non-reducing termini of xylans. Other xylosidases have been identified that catalyze the hydrolysis of alpha-xylosidic bonds.	Xylobiases,Xylan Hydrolases,Hydrolases, Xylan
D015971	Gene Expression Regulation, Enzymologic	Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control of gene action in enzyme synthesis.	Enzymologic Gene Expression Regulation,Regulation of Gene Expression, Enzymologic,Regulation, Gene Expression, Enzymologic