Molecular characterization and expression of a phytase gene from the thermophilic fungus Thermomyces lanuginosus

• Published in: Applied and Environmental Microbiology Vol. 64; no. 11; pp. 4423 - 4427
• Main Authors: Berka, R.M. (Novo Nordisk Biotech, Davis, CA.), Rey, M.W, Brown, K.M, Byun, T, Klotz, A.V
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.11.1998
• Subjects: 6-Phytase - biosynthesis; 6-Phytase - chemistry; 6-Phytase - genetics; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; AMINO ACID SEQUENCES; Binding Sites; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Catalytic Domain; CHEMICAL COMPOSITION; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; DNA Primers; ENZYMIC ACTIVITY; Enzymology and Protein Engineering; Fundamental and applied biological sciences. Psychology; Fungi; Fusarium - genetics; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Genes; GENESEQN/T90070; Hot Temperature; Microbiology; Miscellaneous; Mission oriented research; Mitosporic Fungi - enzymology; Mitosporic Fungi - genetics; Mitosporic Fungi - growth & development; MOLECULAR SEQUENCE DATA; NUCLEOTIDE SEQUENCE; Polymerase Chain Reaction; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; SECUENCIA NUCLEOTIDICA; Sequence Alignment; Sequence Homology, Amino Acid; SEQUENCE NUCLEOTIDIQUE; Transformation, Genetic; Purification; Nucleotide sequence; Enzyme; 3-Phytase; Phosphoric monoester hydrolases; Environmental factor; Homology; Gene organization; Thermophily; Esterases; Gene expression; Thermal stability; Fungi; Enzymatic activity; Hydrolases; Fungi Imperfecti; Recombinant protein; Molecular cloning; Physicochemical properties; Aminoacid sequence; Thallophyta
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.64.11.4423-4427.1998

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 micromoles 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