Purification And Enzyme Characteristics Of Wheat Bran Esterase

Esterase is the hydrolysis of ester bond acid and alcohol produce a large class of enzymes. The plant esterase belongs to the carboxylic acid ester hydrolytic enzymes, exists in different parts of plants, different development period of the cells, the active organophosphate and carbamate pesticides can be suppressed. Plant esterase plant-esterase can hydrolyzel-Naphthyl acetate into a-naphthol and hydrochloric acid. A color reaction can take place between a-naphthol and Fast Blue B and magenta will be seen.Wheat bran was chosen as the resource of plant esterase in the paper, the extraction and purification technology of wheat bran esterase were studied, and some properties of purified enzyme liquid was studied. The main research results are as follows:1.Select a-naphthyl acetate as substrate, the solid blue B salt as chromogenic reagent to wheat bran esterase activity unit(U/mL) as an indicator to study the absorption wavelength, buffer pH, temperature, SDS concentration, substrate concentration, reagent concentration and amount of six parameters. Determined by single factor experiment and response surface best wheat bran esterase reaction conditions:pH of buffer fluid of 7.2, substrate concentration addition amount of 8×10-mol/L, constant wall temperature condition of 40℃, SDS concentration of 4.25%, under the optimal conditions the unit of wheat bran esterase enzyme activity reached 16194.35 U/mL. These values were experimentally verifed, and good agreement between experimental and predicted values was obtained.2.In NaNO3- phosphate buffer as solvent for extracting wheat bran esterase standing bath, analysis of pH buffer, and the concentration of salt solution, extraction time, extraction temperature, solid-liquid ratio, extraction factors influencing wheat bran esterase enzyme activity unit(U/mL). Box-Behnken experimental use to further study the NaNO3 concentration, extraction time, extraction temperature and the interaction of three factors that affect wheat bran esterase enzyme activity. The optimum extraction conditions were confirmed as follow:NaNO3 concentration of 0.15mol/L, extraction temperature 35℃, extraction time of 60min, under this condition, the unit activity of the predictive value of 23338U/mL, verify the value 23018.7U/mL,98.63% to reach the predicted value.3.The extraction of wheat bran esterase by aqueous two-phase system composed of PEG/(NH4)2SO4 was studied. The effects of molecular weight and concentration of polyethylene glycol(PEG), the concentration of (NH4)2SO4 concentration, pH value and NaCl addition etc. On the distribution coefficient, activity yield and purification factor were examined. Response surface methodology was employed to optimize this system. The results showed that the optimum conditions were PEG1000 21%(m/m), (NH.4)2SO4 18%(m/m) and pH at 4.4. Under these conditions, the purification factor could reach to 2.9208, the average degree of purification factor of three times experiment reach to 2.9190,99.93% to reach the predicted value.4.Segmented by ammonium sulfate precipitation, dialysis and SephadexG-200 molecular sieve column chromatography, the enzyme solution was collected and concentrated by ultrafiltration, coomassie brilliant blue G-250 method to measure protein content. The specific activity of the enzyme is 289.14 U/mg·Pr. Recovery of activity is 47.7%, the purify multiple times is 16.17. The purity to the electrophoretically pure, its molecular weight is 26.0 KDa by SDS-PAGE.5.Wheat bran esterase for purified enzymatic properties were studied, experimental results show that:maximum ultraviolet absorption peak for 224nm and 280nm, the optimum temperature is 30℃, temperature stability range of 25～40℃, the optimum pH was 8.0, pH stable range of 4.0-8.0, Cu2+, Mg2+, Ca2+, Al3+, citric acid sodium, sodium tetraborate were esterase inhibition of wheat bran, and Fe2+, Mn2+, citric acid, potassium aluminum sulfate to the enzyme activation, wheat bran to α-naphthyl acetate esterase kinetic parameters for substrate when, Km and Vmax values were 0.85mg·ml-1 and 0.93mg·mL-1·min-1.4℃, wheat bran esterase half-life of 10.4 days, and 25℃, esterase half-life of 8.6 days.