Production, characterization and applications of two distinct phytases from Thermomyces lanuginosus SSBP

Abstract

Phosphorous (P) is an essential element which is primarily stored as phytic acid in plants and exits as dominating form of organic P in soil. The phytate anion has strong chelation properties and can bind with essential metal ions, proteins, starch and several biomolecules under physiological conditions, and accumulates in soil due to strong binding with soil solid phase. Phytases are enzymes that hydrolyse phytate in stepwise manner to release inositol phosphates and orthophosphate, thereby releasing the chelated molecule. In this study, we report two phytases (Tlphy1 and Tlphy2) from a thermophilic compost-dwelling filamentous fungus, Thermomyces lanuginosus SSBP. The total phytase production was enhanced by 8.56-fold in submerged fermentation after statistical optimization, which was further improved in fed-batch cultivations. Tlphy1 and Tlphy2 were purified to homogeneity with an overall purification of 24.7- and 18.7-fold with 5.16% and 6.4% yield, respectively. Tlphy1 was estimated to be 49 kDa and was optimally active at 55°C and pH 5.0, while Tlphy2 was purified as a 60 kDa protein showing optimal activity at 50°C and pH 2.5. Tlphy1 and Tlphy2 were stable over wide ranges of pH and temperature with a half-life of 138.6 and 58.72 min, respectively at 70°C. The applicability of these phytases was investigated for the improvement of bioethanol production using Colocasia esculenta, dephytinisation of a local non-alcoholic beverage, mageu and in improving growth promotion of common bean plants. Tlphy1 reduced phytate content in Colocasia esculenta starch from 1.43 mg/g to 0.05 mg/g that resulted in an improvement in the availability of fermentable sugars with a concomitant reduction in viscosity and 1.59-fold improvement in ethanol production. Supplementation of 50 U Tlphy2 in 100 g ical industries.of mageu efficiently reduced the total fermentation time from 24 h to 16 h with significant dephytinisation and marked enhancement of essential metal ions. Additionally, Tlphy2 also promoted germination of bean seeds, while an enzyme dose of 25 U markedly improved root and shoot length of bean plants. Overall, the two distinct phytases from T. lanuginosus SSBP have multifarious applicability, and therefore can be of major interest to several biotechnology.