Preparation, Stability And Application Of Liquid Neutral Protease

The earliest kind of protease which used in industrial production is neutral protease. It was widely uesd in food, medicine, leather, feed, chemical and waste disposal due to its mild condition, faster catalytic rate. Especially, the neutral protease can hydrolyze protein and decrease the bitter peptides, its application in food industry is broad. Due to the advantages in the culture conditions and production costs of microbial production, microbial source protease becomes more and more improtant in the protease market, and it takes more than 60% of the market share in china. At present, the types of neutral protease in deomestic market are mostly solid powder, liquid types are not common and most of the liquid neutral protease are imported. The stability of liquid enzyme becomes the key point in the production. This paper use the neutral protease fermental broth producted by the marine microorganism strain S-3685 isolated from the South China Sea as the sample, study on the preparation, stability and application of liquid neutral protease.Preparation of liquid neutral protease:Ammonium sulfate precipitation was used to remove other proteins and get enzyme mud, when the saturation of ammonium sufate was 0-45%, the other proteins removal rate was 33% and the recovery rate of enzyme activity was 91%. Compared with activated carbon, H2O2 and resin method, iron exchange resin D380(Cl) demonstrated the highest decoloration efficiency and good protease recovery. Under the optimized dynamic adsorption conditions through single factor method and response surface methodology (pH 6, uploading and elution flow rate of 2.35 BV/h,0.4 BV of 40000 U/mL enzyme solution), the ratios of decoloration and protease yield for it were 86% and 95%, respectively. Used resin could be regenerated easily, and the regenerated resin was as effective as not used one. After 10 times reuses of regenerated resin D380(Cl), the ratios of decoloration and protease yield can also reach 80% and 90%, respectively. It provides a protential approach for large-scale production of neutral protease. The enzyme solution was concentrated by ultrafiltration, and the remaining total enzyme activity was 90% when the concentration ratio was 10. And the final protease yield of the liquid neutral protease was 77.8%.Stability of liquid neutral protease:Use accelerated method and based on the premise that the liquid neutral protease is used in food and medical industry, study on the stability of liquid neutral protease through changing pH value, adding various kinds of metal ions, alcohols, saccharides and other additives. Finally get the fittest recipe(pH 6,20%glycerol,20%propylene glycol,15%glucose,12.5%NaCl,75 mM CaCl2,0.03%VC) which can stailize the liquid neutral protease, under this condition, the liquid enzyme becomes stable in six months at room temperature.Application of liquid neutral protease:Use the cod skin as raw material, optimize the conditions of enzymatic hydrolysis of cod skin through single factor method and response surface methodology(45℃, solid-liquid ratio is 1:15, pH 8.1,6.9 h, the amout of adding enzyme is 15 OOOU/g raw material), the DH of the product is 13.06%. And the hydrolysates under the concentration of 30 mg/mL scavenging capacity on DPPH, OH- and O2- radicals equivalent to 200μg/mL,570μg/mL and 1.2 mg/mL VC solution, has a certain capacity of antioxidation.