| Microbial alkaline protease hydrolysis of the protein can not only peptide bond, but also for the protein has a certain ability to amide, 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, enzyme immobilization, and enzymatic properties of molecules has been systematically studied.Pairs produced from Bacillus licheniformis (B·licheniformis) 2709 alkaline protease, by ethanol precipitation, salting, DEAE anion-exchange chromatography, gel chromatography, four-step purification and, ultimately, pure enzyme electrophoresis. In order to go to amido degrees and the enzyme activity as an index of the alkaline protease purification conditions were optimized. The determination of the purified alkaline protease to the amide degree 20.9%, protease activity:total activity 121397U, specific activity 61069U·mg-1. And 2709 alkaline protease enzyme characteristics of a preliminary study results show that:the role of such an alkaline protease optimum pH was 10.0, the optimum reaction temperature of 50℃.40℃insulation 2h maintain more than 80% enzyme activity, at pH8～11 between the high pH stability.To further enhance the thermal stability of the enzyme, using activated single-methoxy polyethylene glycol succinimidyl ester on the surface of alkaline protease amino 2709 was chemically modified using TNBS method of modifying enzymes to the average degree of amino-modified Measurement results show that the modification degree with the reaction time and the mass ratio increases. Infrared spectra of the products on the modified enzyme and the structure of non-modifying enzymes were identified, compared with non-modified enzyme-modifying enzymes in the infrared spectra of the 1749.37cm-1 occurring at the obvious peaks, indicating the surface of the introduction of a single enzyme molecule A oxy PEG-succinimidyl ester. Modifying enzymes as well as the basic enzymatic properties were studied, and in the free enzyme were compared. With non-modified 2709 compared to alkaline protease by MPEG-modified enzymes in thermal stability and pH stability, all have significantly increased, mainly as a modifier MPEG connected with the protease resulting in the formation of a protease outside the protective film, to some extent to resist heat, acid-base damage.Final choice UV-induced poly-HEMA membrane immobilization method of the alkaline protease enzyme membrane immobilized by the polymerization conditions and the immobilized enzyme activity, thermal stability, pH stability, and Km the value of research, to be an ideal membrane enzyme immobilization methods, the system was immobilized membrane. The results showed that:The UV-induced graft reaction of the fixed 2709 alkaline protease, the first step light reaction 6min, monomer concentration of 20%, the second step illumination time of 25min, the grafting yield up to 35.2%; HMDA concentration 25%, amine alkylation time 150min, amine alkylation temperature of 60℃; glutaraldehyde concentration of 3%, glutaraldehyde role of the time,45min; enzyme concentration 10mg/mL, immobilization time of 20h, a fixed temperature of 4℃, immobilized enzyme membrane activity up to 45% of free enzyme.And enzymatic properties of immobilized enzyme membrane made preliminary study results show that:it’s optimum temperature is 55℃, thermal stability of about 40～60℃, through the determination of enzyme membrane half-life at 55℃for 6h; optimum pH value of 9, in the pH7～11 within the relatively stable; through its continuous determination of activity of 30d and found that the remaining activity of about 75%. After comparison with the relevant data and found that the immobilized enzyme membranes possess enzymatic properties can be applied to hydrolyzed soy protein isolate to the study of modification. |
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