Molecular characterization and expression of a phytase gene from the thermophilic fungus Thermomyces lanuginosus.
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.
Berka
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RM
Novo Nordisk Biotech, Davis, California 95616-4880, USA.
Rey
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MW
Brown
K M
KM
Byun
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T
Klotz
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AV
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
D017931:DNA Primers; D011994:Recombinant Proteins; D010832:6-Phytase