Complex Modification Technique By High Deamidation Alkali Protease And Transglutaminase On Glycinin

Based on the theories from Plant Protein Technology, Protein Chemistry, Biochemistry, Physical Chemistry and Modern Instrumental Analysis, Mutation breeding of strains producing alkaline protease with high deamidation, identification of the mutant SDL-9, enzyme production conditions, as well as the enzymatic properties were systematically studied in this article. By comparison of the ability to deamidation and peptide bond hydrolysis from fermentation production of3lichens Bacillus (Bacillus licheniformis No.1, Bacillus licheniformis No.2and Bacillus licheniformis No.3), B.licheniformis No.2was identified as the starting strain for mutation.The best exposure time was determined as30-40s, using the rate of death as an index, by mutation B.licheniformis No.2under the condition of ultraviolet irradiation. Under these conditions of dealing with the UV-induced mutation, we could get a high-degree (27.96%) deamidation strains of SDZ-61, which increased56.6percent after mutation, while the peptide bond hydrolysis degree unchanged. Then, by using nitrogen ion implantation technology, under the condition of the determined best injection of energy (15keV) to mutant the original strain SDZ-61, Mutant SDL-9could be chosen, which deamidation capacity reached as high as48.99percent to raise the85.57percent, while capacity of the peptide bond hydrolysis is relatively weak. It could be found that the strain SDL-9had a good genetic stability. The strain SDL-9was identified to belong to licheniformis (B.licheniformis) and named as B. licheniformis SDL-9, by identification of physiological, biochemical characteristics and16S rDNA.In order to further enhance the yield capacity of high deamidation alkaline protease from SDL-9fermentation, enzyme production conditions was optimized, the results showed that:the best carbon source for SDL-9-fermentation was2%of the corn meal, The best nitrogen source was2%of soy protein,0.04%of Fe3+has a clear role in promoting enzyme production, and addition0.9percent of glutamine had a significant role in inducing the production of the deamidation alkaline protease. The best fermentation conditions are as follows:2%of the inoculation amount, starting pH7.0, fermentation temperature35℃, liquid volume35mL/100mL, rotation speed200r/min.Then, enzymatic properties of the alkaline protease with high deamidation from SDL-9strains was preliminarily studied, by using deamidation capacity as the indicators, the results showed that:40℃was the optimal temperature, and the enzyme to have a higher deamidation capacity between35℃to45℃; the optimal pH9, the enzyme had a higher deamidation capacity under alkaline conditions (pH8～11); thermal stability of the enzyme was in general, its activity fell to52.3%under the condition of50℃for2h, and it activity only had5.4percent under the condition of60℃for2h; pH stability was better, it activity could keep up90%, in the pH7～10; Metal ions had a larger influence on the enzyme's ability to deamidation, Ca2+had a strong activation on the enzyme; Tween-80had a clear-promoting effect on the enzyme's deamidation ability.A semi-works production method which was improved by the method of isoelectric point and cooling precipitation was developed for extraction and isolation of11S fractions from low-temperature defatted soybean draffs, based on the index of the extraction yield which was determined by the Kjeldahl method and purity which was determined by SDS-PAGE. The optimal isolation parameters of11S were as follows:pH6.2, sedimentation time12h, the cooling temperature4℃, the stirring rate of acid30r/min, its purity and yield were76.14%and40.8%respectively. It has many advantages over traditional methods.According to literature report, proteases on the degree of hydrolysis and deamidation ability of11S globulin are different. The higher degree of hydrolysis and deamidation ability are profit to polymerizing substrate for transglutaminase. In this study, neutral protease, alkaline proteinase and SDL-9alkaline proteinase were selected to enzymolysis11S globulin respectively. The degree of hydrolysis and deamidation ability were identified as the best protease which enzymolysis11S globulin.Using the neutral protease enzymolysis11S globulin, and the optimum enzymolysis conditions were:time2h, temperature42℃, addition dosage of the enzyme4000U/g and pH7.5, and the degree of deamidation39.6%and degree of hydrolysis7.1%under these conditions. The optimum enzymolysis conditions for alkaline proteinase:enzymolysis time2.5h, enzymolysis temperature45℃, addition dosage of the enzyme200U/g and pH7.5, and the degree of deamidation41.7%and degree of hydrolysis7.9%under these conditions. The optimum enzymolysis conditions for SDL-9alkaline proteinase enzymolysis were: enzymolysis time2.5h, enzymolysis temperature45℃, addition dosage of the enzyme200U/g and pH7.5, and the degree of deamidation was44.7%and degree of hydrolysis was9.2%under these optimum conditions. Among these conditions, the degree of deamidation and degree of hydrolysis of SDL-9alkaline proteinase were the highest. Therefore, SDL-9alkaline proteinase which enzymolysis on soybean11S globulin were selected in this experiment.Taking the surface hydrophobicity as index, hydrolyzates of soybean11S globulin were polymerized by transglutaminase. The optimum conditions of polymerizing were:reaction temperature was35℃, addition dosage of the enzyme was21.12U/g, reaction time was2.4h, and pH was6.12by single factor experiments and response surface optimization designs. Surface hydrophobicity was the lowest and reached to20under these reaction conditions. Eventually, solubility, liquid-binding power, emulsibility, emulsification stability, sulfhydryl and disulfide bond of modificated anteroposterior soybean11S globulin were determined respectively, at the same time modificated anteroposterior structural changes by Fourier infrared spectrum and electron microscope scanning were also analyzed. The results showed that the solubility of soybean11S globulin after modifying was increased to above95%, the emulsibility stability were increased from36.3%to68.1%and emulsification also increased from47.2%to87.1%, and the liquid-binding power was decreased plentiful from5.1mg/L to3.26mg/L, the disulfide bond was decreased from37.57μmol/g to9.86μmol/g,but the sulfhydryl was increased from204.63μmol/g to259.78μmol/g. The intermolecular and intramolecular disulfide bond of soybean11S globulin after modifying had mostly been broken and formed sulfhydryl. The secondary structure of soybean11S globulin after modifying were changed from the virgin rule curl into random coil structure, which can be discerned by Fourier infrared spectrum and electron microscope scanning.