Study On Separation And Purification And Immobilization Membrane Of The Transglutaminase

Microbial alkaline protease hydrolysis of the protein has a certain ability to polymerization,and for the protein and has wide variety of sources of raw materials, and cheap, with dramatic effect, high safety, etc. so in the field of modified soy protein has broad application prospects. In this paper, a high Deamidation Activity of alkaline protease purification, molecular modification (product corss-linked Enzyme crystals), enzyme crystal immobilization, and enzymatic properties of molecules has been systematically studied.The transglutaminase was purified by alcohol precipitation, ammonium sulfate fractionation precipitation, ultrafiltration, SephadexG-100gel chromatography,and the apparent purity was examined by SDS-PAGE. Study on the separation purification condition of the transglutaminase,The result was that the saturation degree of ammonium sulfate was 65% may make the Zymoprotein became Precipitation, pressure of ultra filter was 0.2MPa may made 80.2% zymoproteins through the ultra filter, the speed of Sephadex G-100 chromatographic analysis was 1mL/min, after eluting 60min, may obtain the enzyme fluid which had higher enzyme vigor.The results indicated that purification multiple was 10.42, The recovery of lactoperoxidase activity was 27.3%,The enzyme properties of transglutaminase were also studied,The result was the optimal temperature and pH were4 0℃and 6.0 respectively,.The pure enzyme was stable between 30℃and 40℃,and the enzyme was relatively stable at pH5-7. Metal ions of Ca2+,Mg2+and Ba2+ had little effect on the activity of transglutaminase, but Fe3+,Zn2+ and Cu2+ could inhibit the enzyme and Fe3+ had serious effect on enzyme activity.To further enhance the thermal stability of the enzyme, The enzyme crystals obtained by the vapor diuffsion method ndependently using 2-methyl-2,4-pentnaediol as a preeipitant. The better crystallize conditions are as follow:the concentration of 2-methyl-2,4-pentanediol 50%,pH 8,crystallize temperature 10℃,crystallize time 5 day,enzyme concentration 12mg/ml. The crystals of transglutaminase cross-likned with a biufnetional agent such as glutaraldehyde. The conditions are as follow:the concentration of glutaraldehyde 1%,pH6.0,tempearture 4℃and reaetive time 40min. Infrared spectra of the products on the free enzyme and the corss-linked enzyme crystals were identified,the result showed enzyme structure had changed which namely a-helical structure intoβ-folding tructure and produced random curing.It could show that free enzymes in the crystallization of the above conditions generated good character crystals.The corss-linked Enzyme crystals properties of transglutaminase were also studied,and and in the free enzyme were compared. The results indicated that with free enzyme compared to a The corss-linked transglutaminase enzyme crystals not only enhance thermal stability and pH stability but also attain the activity in aqueous-ogrnaie mixtures such as 50% terthaydrofuran,Main reason is the crystal state of substances and enzyme molecules in the results of chemical cross-linking.Finally,choice UV-induced poly-HEMA membrane immobilization method of the transglutaminase crystal membrane immobilized by the polymerization conditions and the immobilized enzyme activity, optimal temperature and pH, thermal stability, pH stability, storage and operational stability of research, to be an ideal membrane enzyme crystal immobilization methods, the system was immobilized membrane. The results showed that:The UV-induced graft reaction of the fixed transglutaminase crystal, the first step light reaction 12min, light distance 10cm, monomer concentration of 20%, the second step illumination time of 20min, the grafting yield up to 34.56%; HMDA concentration 25%, amine alkylation time 90min, amine alkylation temperature of 50℃;glutaraldehyde concentration of 2%, glutaraldehyde role of the time,40min; enzyme concentration 15mg/mL, immobilization time of 24h, a fixed temperature of 4℃, immobilized enzyme membrane activity up to 31.2 U/g. And enzymatic properties of immobilized enzyme crystal membrane made preliminary study results show that:it's optimum temperature is 50℃, good thermal stability and store 2.5h at 50℃, enzyme activity retention maintain 80%,through the determination of enzyme membrane half-life at 50℃for 6.5h,optimum pH value of 6, in the pH4～8 within the relatively stable; through its continuous determination of activity of 30d and found that the remaining activity of about 70%.At 40℃and 50℃, after continuous reaction 10h, activity of immobilized enzyme crystal membrane retention rate at 60% and 35%, and the appearance is still very smooth, no injury, no fracture, the operation stable was so good.After comparison with the relevant data and found that the immobilized enzyme crystal membranes possess enzymatic properties can be applied to ploumerizated whey soy protein isolate to the study of modification.