Suitable medium for production of milk clotting enzyme (MCE) by Bacillus subtilis (natto) Takahashi in submerged liquid-state fermentation was screened, the nutrient factors affecting MCE production was optimized by response surface methodology. The MCE production by B. subtilis (natto) Takahashi was increased significantly by 428% in the optimal medium developed. The MCE was filtered and concentrated by ultrafiltration. The retentate after tandem filtration carried out with the combined membranes of MWCO 50kDa and 5 kDa showed two major bands between 25kDa and 30kDa on SDS-PAGE, and the MCA and MCA/PA improved significantly in comparison with those in the initial broth. The crude enzyme thus obtained showed MCA and MCA/PA ratio of 48,000 SU/g and 6,400, which are commensurate with those (MCA 26,667 SU/g and MCA/PA 6,667) of the commercial rennet. It had optimal pH and temperature at pH 6 and 60°C, and showed excellent pH and thermal stability.
Lignocellulosic materials, mostly from agricultural and forestry residues, provide a potential renewable resource for sustainable biorefineries. Reducing sugars can be produced only after a pre-treatment stage, which normally involves chemicals but can be biological. In this case, two steps are usually necessary: solid-state cultivation of fungi for deconstruction, followed by enzymatic hydrolysis using cellulolytic enzymes. In this research, the utilisation of solid-state bioprocessing using the fungus Trichoderma longibrachiatum was implemented as a simultaneous microbial pretreatment and in-situ enzyme production method for fungal autolysis and further enzyme hydrolysis of fermented solids. Suspending the fermented solids in water at 50°C led to the highest hydrolysis yields of 226mg/g reducing sugar and 7.7mg/g free amino nitrogen (FAN). The resultant feedstock was shown to be suitable for the production of various products including ethanol.
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