Amylase production by Preussia minima, a fungus of endophytic origin: optimization of fermentation conditions and analysis of fungal secretome by LC-MS.

BACKGROUND: Environmental screening programs are used to find new enzymes that may be utilized in large-scale industrial processes. Among microbial sources of new enzymes, the rationale for screening fungal endophytes as a potential source of such enzymes relates to the hypothesised mutualistic relationship between the endophyte and its host plant. There is a need for new microbial amylases that are active at low temperature and alkaline conditions as these would find industrial applications as detergents. RESULTS: An α-amylase produced by Preussia minima, isolated from the Australian native plant, Eremophilia longifolia, was purified to homogeneity through fractional acetone precipitation and Sephadex G-200 gel filtration, followed by DEAE-Sepharose ion exchange chromatography. The purified α-amylase showed a molecular mass of 70 kDa which was confirmed by zymography. Temperature and pH optima were 25°C and pH 9, respectively. The enzyme was activated and stabilized mainly by the metal ions manganese and calcium. Enzyme activity was also studied using different carbon and nitrogen sources. It was observed that enzyme activity was highest (138 U/mg) with starch as the carbon source and L-asparagine as the nitrogen source. Bioreactor studies showed that enzyme activity was comparable to that obtained in shaker cultures, which encourages scale-up fermentation for enzyme production. Following in-gel digestion of the purified protein by trypsin, a 9-mer peptide was sequenced and analysed by LC-ESI-MS/MS. The partial amino acid sequence of the purified enzyme presented similarity to α-amylase from Magnaporthe oryzae. CONCLUSIONS: The findings of the present study indicate that the purified α-amylase exhibits a number of promising properties that make it a strong candidate for application in the detergent industry. To our knowledge, this is the first amylase isolated from a Preussia minima strain of endophytic origin.

Optimization of protease production by endophytic fungus, Alternaria alternata, isolated from an Australian native plant.

Endophytes are recognised as potential sources of novel secondary metabolites, including enzymes and drugs, with applications in medicine, agriculture and industry. There is a growing need for new enzymes, including proteases, for use in industry that can function under a variety of conditions. In this study, three fungal endophytes (Alternaria alternata, Phoma herbarum and an unclassified fungus), were isolated from the Australian native plant, Eremophilia longifolia, and assessed for production of proteases. The lyophilised growth media obtained after fungal fermentation were analysed for protease production using enzyme activity assays. Protease production was optimised by assessing the effects of temperature, pH, carbon source and nitrogen source on activity. A. alternata showed the greatest protease activity in a wide range of pH (3-9). The broadest activity between 9 and 50 °C was observed at pH 7, suggesting a neutral protease. Overall, the optimum conditions were 37 °C and pH 7 with a maximum specific activity value of 69.86 BAEE units/mg. The characteristics demonstrated by this fungal endophyte showed that it is a potential source of an enzyme with particular application in the dairy industry. However, further studies of the tolerance to higher temperatures and pH will indicate whether the enzyme is suitable to such applications.