Preparation Of High F Value Yeast Oligopeptide And Immobilization Of Double Enzyme

High F value yeast oligopeptides are small mixture peptides and full of branched chain amino acids.It has many biological activities,including protecting liver,treating phenylketonuria,anti-fatigue and so on.It is one of the hotspots in the field of active peptides at present.In this study,active dry yeast was used as the initial raw material for preparing high F value oligopeptides.The cell wall of the yeast was broken by ultrasonic,and the crushing process was optimized,and the ultrasonic conditions were obtained:the ultrasonic power was400 W and the time was 20 minutes.Alkaline protease was selected from the four common endoglucanases as the optimum yeast extracted enzyme to obtain the yeast protein.The single factor optimization of alkaline protease extracting yeast cell conditions were found as follows:the material liquid ratio 1:6,the temperature 50℃,the enzyme addition amount 1000 U·g^-1,the pH 8.0 and the enzymolysis time 6 h.The yeast extracts were concentrated by nanofiltration operation and the free amino acid was removed,so that we could obtain yeast crude peptides.The nanofiltration operating conditions were optimized by the use of 500 Da nanofiltration membrane to concentrate threefold yeast extracts at 0.3 MPa.The yeast crude peptides were orientedly hydrolyzed byα-chymotrypsin and carboxypeptidase A,so that the aromatic amino acid was free from the yeast crude peptides.The two enzymes were subjected to single factor and orthogonal preferred experiments.The optimum conditions for the optimal enzymatic hydrolysis ofα-chymotrypsin were obtained.The enzyme amount was8000 U·g^-1,the temperature was 40℃,the pH was 8.0 and the time was 5 h.The optimum conditions of carboxypeptidase A were found as follows:the enzyme amount 4 U·mL^-1,the temperature 40℃,the pH 8.0,the time 4 h.The activated carbon was used to adsorb the free aromatic amino acid,so that high F value oligopeptids were obtained.The adsorption conditions were optimized by single factor:the adsorption time was 2 h,the adsorption temperature was 30℃,the adsorption pH was 2.5 and the adsorption carbon ratio was 1:10.In this integration process,a number of high F value yeast oligopeptides with F values between 30-40 were obtained successfully,the yield of the whole process polypeptide was14.1%and the proportion of oligopeptides with molecular weight below 1000 Da were 95%.In order to reduce the production cost of high F value oligopeptides,a composite magnetic nano-particle with three-dimensional structure was prepared by solvothermal method.Scanning electron microscopy,transmission electron microscope and specific surface analyzer,X-ray diffractometer,Fourier transform infrared spectroscopy and zeta potential were used to characterize the properties.The results indicated that the average particle size of the prepared magnetic nanoparticles was 12 nm.It had a Fe₃O₄ spinel structure with a specific surface area of 78.9377 m2·g^-1 and full of different functional groups.Theα-chymotrypsin was immobilized on magnetic nanoparticles.It was found that the immobilized enzyme could be reused and had better stability of pH and thermal than free enzyme.After 8 cycles of immobilizedα-chymotrypsin（40℃,pH 8.0）,the catalytic activity of the immobilizedα-chymotrypsin could be kept at 65%of the initial enzyme activity.Moreover,the storage stability ofα-chymotrypsin was improved by immobilization.After 30 days’storage at room temperature,the initial activity of immobilizedα-chymotrypsin was kept at 60%.The magnetic nanoparticles were firstly immobilized withα-chymotrypsin and then coated with sodium alginate of mixed carboxypeptidase A to form the magnetic gel particles.The embedding conditions were optimized by single factor.The concentration of sodium alginate was 2.5%（w/v）,the concentration of CaCl₂ solution was 5%（w/v）,the amount of carboxypeptidase A was 500 U and the immobilization time was 2 h.After that,the two enzymes were confirmed to bind to the magnetic gel particles by fluorescence labeling.The yeast crude peptides were hydrolyzed by immobilized enzyme,and the residual rate of enzyme activity was 30%after 5 times of enzymatic hydrolysis.By immobilization of two kinds of enzymes with high F value oligopeptides and applied to actual enzymatic hydrolysis,it was found that the two enzymes could be reused after immobilization,although the efficiency of enzymatic hydrolysis was decreased,co-immobilization of two enzymes had certain application prospects.