Screening Of High-yield Neutral Protease-producing Strains And Study On Fermentation Characteristics

The neutral protease （NPR） is a protease whose optimum catalytic pH is6.0-7.5. It cancatalyze the cleavage of peptide bonds in proteins and is widely used in food, leather andpharmacy industries.A series of mutations and screenings for higher enzyme producers were performed usingthe ultra violet （UV） and diethyl sulphate （DES） from parent strain Bacillus subtilis SHB2010-1. One high-yield neutral protease-producing strain named as DES-59was selected andenzyme activity reached7088U/mL, which is59.65%higher than the parent strain.The characteristics of protease production and feeding strategies were studied in thisresearch. Corn flour was chosen as the best carbon source and the best nitrogen source wassoybean meal. Addition of phosphate increased the protease production. When Na₂HPO₄andKH₂PO₄were not added in the medium, Ca²⁺and Mg²⁺at the given concentration inhibitedprotease production. Ca²⁺and Mg²⁺at1mmol/L resulted in12%and8%increase in theenzyme production, respectively, with the addition of Na₂HPO₄and KH₂PO₄in the medium;However, higher concentration of Ca²⁺and Mg²⁺had an inhibitory effect on the enzymeproduction. The pH control didn’t stop sharp decrease in neutral protease production at theend of fermentation. Feeding of corn flour （10g/L） and soybean meal （7.5g/L） at11、18and24h resulted in96%and112%increase in protease yield and protease productivity,respectively when compared to the values obtained for batch culture.The fermentation medium and culture conditions for the production of neutral proteasewas optimized by response surface methodology （RSM）, using cassava pulp as carbon sourceand B. subtilis DES-59as strain. The Plackett-Burman design was applied to evaluate theeffects of variables such as the concentration of substrates （cassava pulp, soybean meal andwheat bran）, initial pH, sharker’s rotating speed, temperature, inoculum size and incubationperiod on the production of protease. Among the eight parameters, three significant variables（cassava pulp, soybean meal and inoculum size） were selected for the subsequentoptimization study, in which a central composite design （CCD） was used to optimize theconcentrations of cassava pulp, soybean meal and inoculum size, and the interactive effects of the three variables were investigated. The optimum concentrations obtained from RSM were37.78g/L of cassava pulp,15g/L of soybean meal and6.5%（V/V） of inoculum size,respectively, resulting in a maximum neutral protease activity of3849U/mL which wasclosed to the predicted value, confirming the validity of the established model.The extraction and purification of the neutral protease were investigated. The culturebroth was centrifuged to separate solids. Solid ammonium sulfate was added to supernate with40%saturation and the precipitate was removed as the impurity proteins. Solid ammoniumsulfate was added to the supernate with60%saturation. The precipitate was collected bycentrifugation, and then dried by vacuum freeze dryer to obtain the solid crude powder of theneutral protease. The specific activity of neutral protease reached3191U/mg which was3.29-fold compared to the broth. Recovery rate of the protease was59.79%. The activity ofthe crude product was230000U/g.This thesis had done some researchs on the B. subtilis including the screening ofhigh-yield neutral protease-producing strains using different mutations, fermentationcharacteristics, different feeding strategies, optimization on the fermentation medium andculture conditions using cassava pulp as carbon source and the extraction and purificationprocess, which could be a foundation of industrial production of neutral protease.