Separation, Purification, Properties And Application Research Of Beta Amylase From Soybean Whey Wastewater

As an important food raw material, soybean isolate protein is widely used in food industry. With the rapid development of soy protein processing industry,35-65 tons whey wastewater were produced in the process of 1 ton soybean protein isolated production by using alkali-solution and acid-isolation method, w Through the composition analysis of whey wastewater, large amounts of beta amylase were found. Beta amylase has higher plant sources and microbial sources, compared with the microbal sources of beta amylase, plant sources of high beta amylase performs high enzyme activity, good heat resistance and wide pH range, which can have a better application in the food industry production process. Study of soybean whey waste water in the beta amylase is a meaningful topic, not only can increase the economic efficiency, and can extend the bean products industry chain.In this paper, first using MALDI - TOF/TOF mass spectrometry to identify the soybean whey protein components. Then using Hitrap QFF anion chromatographic column and HiPrep 26/60 Sephacryl S - 100 HR gel filtration chromatography column for beta amylase purification. Studying the properties of beta amylase, according to the properties, industrialized production of beta amylase method was carried out. Finally this paper made a comparison of beta amylase enzyme liquid with commercially available beta amylase enzyme liquid on the application. The main research results are as follows:1、According to the composition identification of soybean whey proteins by MALDI - TOF/TOF, we found there were four major proteins:lipoxygenase, beta amylase, soybean agghitinin, trypsin inhibitors.2、Through the purification exploration and optimization of beta amylase using Hitrap QFF anion exchange chromatographic column, the buffer pH is 8.0,flow rate is 0.8ml/min, using the 0.12M NaCI step elution to elute the beta amylase,1M NaCI step elution. to elute the miscellaneous protein. After the anionic purification, we use HiPrep: 26/60 Sephacryl S-100 HR gel filtration chromatographic column for further purification, the mobile phase is 50 mM NaAC buffer (pH6.0), flow rate is 0.8ml/min,. Compared with the beta amylase of soy whey waste water, the enzyme was purified 16.49 fold with 17.32% yield, giving a final specific activity of 29735.92 U/mg.3、Through the enzymology properties analysis of beta amylase, we get the protein was shown to be a monomeric protein of 55892kDa and the pI of 4.87. Optimal temperature for the enzyme was 55℃ and was stable under low temperatures. Optimal pH for the enzyme activity was 6.0. The enzyme has a wide pH range and was stable at a pH range of 5.0-8.0. Through the role of metal ions, surfactant, inhibitor we found K +、Ba2+、Ca2+、Na+ have no significant effect on beta amylase activity (P>0.05) EDTA，Mn2+，Fe3+ have certain inhibition on beta amylase activity (P<0.05), while Cu2+、surfactant SDS and inhibitorβ-mercaptoethanol significantly inhibited the enzyme activity (P<0.01). Zn2+、Mg2+、Li+ have certain activation function on beta amylase activity (P<0.05),while the enzyme activity was strongly enhanced in the presence of inhibitor PMSF and surfactant Tween-20, Tween-40, Tween-60, Tween-80 and Tritonx-100 (P< 0.01). By measuring the relative activity in different substrate, choosing the potato starch as the optimum substrate for beta amylase. The purified enzyme exhibited a Km and Vmax value for potato starch as high as 4.8mg/ml and 12.10 units/minute/ml respectively.4、According to the enzymatic properties of beta amylase, industrial production of beta amylase was studied. Combined the uhrafiltration method with tannic acid precipitatfon method, we get the 61.88% yield of enzyme activity and specific enzyme activity of 3138.92 U/mg. Combined the ultrafiltration method with ethanol precipitation, we get the 77.54% yield of enzyme activity and specific enzyme activity of 5052.21 U/mg. Using the method of ethanol precipitation after ultrafiltration can effectively realize the industrial production of beta amylase from soy whey wastewater, the unit of enzyme activity can reach 150000u/mL5、Through the comparison of soybean beta amylase and commercial beta amylase,the optimum reaction temperature of soybean beta amylase is 55℃ while commercial beta amylase is 50℃. Commercial beta amylase displays poorer stability than soybean beta amylase under high temperatures (50-70℃), while placing 1 h under 70℃ both beta amylase activity completely lost. The difference in optimum reaction temperature and the temperature on the stability also leads to different reactions in the application effect. Using the two kinds of beta amylase on flour produce for resisting retrogradation processing, for flour products hardness index. In terms of enzyme action temperature at 55℃、60℃、65℃, spybean beta amylase in the inhibition retrogradation of flour products has obvious advantage.