title: Molecular characterization and expression of a phytase gene from the thermophilic fungus Thermomyces lanuginosus.
abstract: The phyA gene encoding an extracellular phytase from the thermophilic fungus Thermomyces lanuginosus was cloned and heterologously expressed, and the recombinant gene product was biochemically characterized. The phyA gene encodes a primary translation product (PhyA) of 475 amino acids (aa) which includes a putative signal peptide (23 aa) and propeptide (10 aa). The deduced amino acid sequence of PhyA has limited sequence identity (ca. 47%) with Aspergillus niger phytase. The phyA gene was inserted into an expression vector under transcriptional control of the Fusarium oxysporum trypsin gene promoter and used to transform a Fusarium venenatum recipient strain. The secreted recombinant phytase protein was enzymatically active between pHs 3 and 7.5, with a specific activity of 110 micromol of inorganic phosphate released per min per mg of protein at pH 6 and 37 degrees C. The Thermomyces phytase retained activity at assay temperatures up to 75 degrees C and demonstrated superior catalytic efficiency to any known fungal phytase at 65 degrees C (the temperature optimum). Comparison of this new Thermomyces catalyst with the well-known Aspergillus niger phytase reveals other favorable properties for the enzyme derived from the thermophilic gene donor, including catalytic activity over an expanded pH range.
authors.affiliation: Novo Nordisk Biotech, Davis, California 95616-4880, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Berka
authors.forename: M W
authors.initials: MW
authors.lastname: Rey
authors.forename: K M
authors.initials: KM
authors.lastname: Brown
authors.forename: T
authors.initials: T
authors.lastname: Byun
authors.forename: A V
authors.initials: AV
authors.lastname: Klotz
mesh_terms: D010832:6-Phytase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D017931:DNA Primers; D005670:Fusarium / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D006358:Hot Temperature; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D010832:6-Phytase