Isolation of thermotolerant phytase producing fungi and optimisation of phytase production by Aspergillus niger NRF9 in solid state fermentation using response surface methodology

Abstract

Phytase is an enzyme that hydrolyses phytic acid into myo-inositol and inorganic phosphate. One hundred thirteen fungi were isolated from 58 soil samples collected from different sites with high organic matter content. They were screened for phytate hydrolysing activity on phytase screening medium (PSM). Clear zones were formed around the colonies of 72 isolates on Ca-containing PSM. Solid state fermentation was done for the selected fungal isolates for production of the phytase enzyme. Among the isolates, NRF9 showed maximum enzyme activity (39.2 U/gds) upon quantitative screening after 5 days of incubation at 30℃. The fungus grew vigorously and secreted enzyme at temperatures of 30, 37, and 45℃. The maximum phytase production was obtained at 30℃; the next-best production was obtained at 37℃. The isolate was identified and characterised as Aspergillus niger NRF9 on the basis of 18S rDNA sequencing. Various process parameters were optimised using a one-variable-at-a-time approach’. The significant factors for phytase production were identified as incubation time, solid:liquid ratio and glucose concentration. These factors were further optimised using a Box-Behnken design. Maximum phytase production (112.1 U/gds) was observed at 5 days of incubation time, a 1:2 solid:liquid ratio and a 1% glucose concentration. Validation of these optimised variables was done in shake flasks. Optimisation resulted in a 2.9-fold increase in phytase production compared to unoptimised cultivation conditions.