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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.

SUBMITTER: Berka RM

PROVIDER: S-EPMC106663 | biostudies-literature | 1998 Nov

REPOSITORIES: biostudies-literature

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

Berka R M RM Rey M W MW Brown K M KM Byun T T Klotz A V AV

Applied and environmental microbiology 19981101 11

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 insert ...[more]

PMID: 9797301

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Project description:Annexins (ANXs) are widely expressed and structurally related proteins which play multiple biological roles in animals, plants, and fungi. Although ANXs have been localized to the cytosol and the cell membrane and the molecular basis of the four annexin repeats is well established, the in vivo roles of these proteins are still far from clear, particularly with regard to the filamentous fungi. Thermomyces lanuginosus, a thermophilic fungus, is widely used in the fermentation industry; however, the role of ANX in this organism is unknown. In this study, a single ANX homologue (ANXC7) was identified and characterized in T. lanuginosus. The expression pattern indicated that ANXC7 is closely associated to conidium development, and it accumulated in the mitochondria of the forming conidia. The deletion of ANXC7 (?ANXC7) resulted in no obvious phenotype related to colony growth on solid CM medium. However, when ?ANXC7 was grown in CM liquid culture, the mycelium masses appeared to be larger and looser compared to the wild-type. Additionally, the dry weight of the mutant mycelia was significantly increased. Under conditions that compromise cell-wall integrity, ?ANXC7 was less vulnerable than the wild-type with regard to such damage. Moreover, based on a surface hydrophobicity test, the ?ANXC7 strain was clearly less hydrophobic. The growth of ?ANXC7 was inhibited when grown under selected stress conditions, particularly with regard to salt stress; however, the oxidative resistance to exogenous H2O2 in ?ANXC7 was increased, and endogenous H2O2 levels within the ?ANXC7 were lower than in the wild-type, thereby suggesting that the ANXC7 specifically controls oxidative resistance. Based on microscopic observation, 4-day-conidia were more prevalent than 5-day conidia on the conidiophore stalk of ?ANXC7, even though the ?ANXC7 demonstrated an increased production of conidia during these days, indicating precocious conidial maturation and shedding from the conidiophore stalk in this strain. Taken together, our data indicate that ANXC7 localizes to the mitochondria and is involved in controlling conidium development and oxidative resistance in T. lanuginosus.

Dataset Information

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

Publications

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

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