| Abalone, ranking the first place among the four major seafoods (namely sea cucumber, shark's fin, fish maw and abalone), is not only nutritious, but also of a high medicinal value. However, the viscera which accounted for about 30% to 40% of a total weight of abalone soft tissue, is a non-edible part of a large number of discarded or processed into low-value products, which greatly reduces the value of the abalone and at the same time causes serious environmental pollution. Abalone viscera was used as raw materials in this thesis, with the supplement of ultrasonic using conventional methods extraction abalone viscera crude ?-glucosidase solution, exploring the best extraction process, and studying its optimal assay conditions of enzyme activity. The crude P-glucosidase in the crude enzyme solution by salting-out, dialysis, freeze-drying was made abalone viscera crude ?-glucosidase powder and it was to do the physicochemical analysis and enzymology characteristics research. The main conclusions of the thesis were as follow:1. A set of ultrasonic assisted extraction process of abalone viscera crude P-glucosidase solution was determined through the single factor and orthogonal experiments of conventional extraction process, and the single factor experiment of ultrasonic assisted extraction process. The effects of various factors of conventional extraction method on the enzyme activity: solid-liquid ratio, buffer pH had a significant effect on the extraction (P<0.05), but extraction time, type of buffer, centrifugation speed had no significant effect on the extraction. Ultrasonic treatment was conducive to extraction of the abalone viscera crude P-glucosidase solution could be seen by comparing the conventional extraction and ultrasonic assisted extraction methods. The optimum extraction conditions:buffer was 0.1mol/L pH 3.5 acetic acid-sodium acetate, solid-liquid ratio was 1:4, extraction time was 4h, ultrasonic time was 20min, ultrasonic power was 200 W.2. The optimal assay conditions of abalone viscera ?-glucosidase homogenate's enzyme activity:the detection wavelength was 400nm, the buffer was pH 3.50.2mol/L disodium hydrogen phosphate-0.1mol/L citric acid, the concentration of the substrate was 35mmol/L, the reaction temperature was 50?, the reaction time was 30min.3. Ammonium sulfate was determined as the salting-out agent for the extraction and preliminary purification of abalone viscera ?-glucosidase through the effects of different salting-out agents on the salting-out effect of the abalone viscera homogenate. A better separation effect of P-glucosidase could be obtained through adopting the step-by-step salting method which selected ammonium sulphate a saturation of 30%, the second saturation of 80%.4.Abalone viscera P-glucosidase homogenate through preliminary separation and purification steps such as step-by-step salting, supplemented by dialysis, there was a certain improvement of specific activity. The technical route is easy to operate, high recovery, and it is a design reasonable route of separation and purification of ?-glucosidase. Methods of different purification achieving the purification effect are different, if you want to achieve higher purification factor can be further separated and purified by ion exchange column chromatography and gel filtration column chromatography.5.After treated by free-drying, the abalone viscera crude p-glucosidase powder was pale yellow. The P-glucosidase powder ingredients included:crude protein 60.58%, soluble protein 29.56%, polysaccharide 16.34%, moisture 10.69%, lipid and ash almost non-existent. The cause of the polysaccharide content higher was abalone viscera contains more sugar protein, salting process along with ?-glucosidase precipitated.6.After treated by freeze-drying, abalone viscera crude P-glucosidase powder which the optimum temperature was 50?, the optimum pH was 3.5, had good thermal stability and pH stability in the range of below 50? and pH 3.0 to 6.0, it could be stored for a long period in the refrigerator at 4?. Zn2+, Cu2+, Ag+had a significant inhibitory effect on enzyme activity of abalone viscera P-glucosidase; Na+, K+, Mg2+, Ca2+, Fe2+had no significant effect on enzyme activity; Ba2+, EDTA, Mn2+had activation effect on enzyme activity, which, the role of activation of Ba2+, EDTA was weak, Mn2+strong activation. p-NPG as substrate, the kinetic constants were Km= 4.66 mmol/L, and Vmax= 3.79U/L.7.By SDS-PAGE electrophoresis, abalone viscera P-glucosidase after dialysis could achieve higher purity, the molecular weight was about 54.0?76.0 kD. |
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