Study On Properties And Application Of Proteases In Ethanol Solution

Many proteins that occur naturally in plant materials can dissolve in aqueous/organic media, and they exhibit high insolubility in aqueous media. It is necessary to utilize ethanol solution as medium and modification by enzymes. But most of enzyme will denature in ethanol solution. In this paper, in order to apply the proteases in the development of prolamine, properties and the relationship between structure and function of proteases in ethanol solution were researched.Gliadine is the ideal material for preparing functional glutamine-bioactive peptides. The glutamine-bioactive peptides were applied to medicine and factional beverage.In this paper, firstly the activity of several proteases in ethanol solution was investigated. The properties and conformation of papain and Alcalase were determined. Secondly, wheat gluten was hydrolyzed in ethanol solution and glutamine-bioactive peptide was prepared. In addition, the separation and purification of glutamine-bioactive peptides were also investigated.(1) Activities of six proteases in 30% and 50% ethanol solution were investigated and the residual activity rates of these proteases in descending order was papain, Alcalase, pepsin, bromelin, trypsinase, neutral protease.(2) The optimum temperatures of alkaline protease and papain in ethanol solutions reduced with the increase of ethanol concentration and the range of temperature descent was 5-10℃. The optimum pH in ethanol solutions had little difference comparing with buffer solution. With the increase of ethanol concentration, the stability of alkaline protease and papain were affected. Especially, when the concentration of ethanol solutions was up to 60%, the stability of alkaline protease and papain decreased rapidly. The stability of papain was higher than alkaline protease in the same ethanol concentration and the half-life of papain was 21 times of alkaline protease in 60% ethanol.(3) The Circular dichroism spectra and fluorescence spectra showed that the secondary and tertiary structure of alkaline protease and papain were altered markedly.. The absorption peak of Alcalase has a redshift in ethanol solution and papain assumes a blueshift with different degrees. The fluorescence intensity of alkaline protease and papain in ethanol solutions were both weakened obviously.(4) Glutamine-bioactive peptides were prepared by hydrolyzing wheat gluten with gradient dilution thrice to descending the concentration of ethanol. The optimum conditions of enzymatic hydrolysis were obtained as follows: Alcalase at E%=0.32 (w/w) and S%=14% (w/v) with gradient dilution thrice, DH=15.34%, the content of peptides is 25.26mg/mL, the content of amide is 2.93mmol/g protein. The content of free amino acid in this hydrolysate is 1.87% and much lower than that of it obtained in aqueous solution.(5) Three methods (DA201-C macroporous absorbent resin, gel filtration chromatography and RP-HPLC) for the separation of glutamine-bioactive peptides from the hydrolysate were adopted in this paper. The results showed that the best separation effect was obtained by RP-HPLC, and the content of peptides in highest-amide component is 1.99mg/mL and the content of amide is 3.21mmol/g protein.