Study On Optimal Cultivation Condition And Characterization Of Hyaluronidase From Arthrobacter Nicotinovorous

Hyaluronidase is a group of enzymes which are widely distributed in nature, they are found in a variety of organs and tissues of mammals and micro-organisms. From now on there are in total over forty microorganisms which can express hyaluronidase. With the progress of study, scholars are attaching more and more importance to the biotechnologies which could help us to find new sources of hyaluronidase for separation, purification, cloning of the enzyme. In this study, an effectively hyaluronidase-producing Arthrobacter nicotinovorous which was isolated from polluted sodium hyaluronate solution was experimented on. The ability of producing hyauronidase of the strain, cultivation conditions and enzyme characteristic were studied to provide a basis for exploring new sources of hyaluronidase and industrial applications. The main contents are as follows:1. Identification the enzyme production of Arthrobacter nicotinovorus. In this experiment we separate a hyaluronidase-produce strain and use four methods that were done by some other scientists to verify the ability. The methods include viscosity method, high performance liquid chromatography, plate method, zymography. We detected the viscosity of hyaluronate sodium decreased obviously by ubbelohde viscometer in12hours. At the same time the high molecular weight hyaluronate sodium was degraded to small hyaluronan oligosaccharides. Compared to the two methods metioned above, we can directly see the reaction caused by the enzyme in the plate. Zymography is an effect methof to detect activity of a specific enzyme. The enzyme sample was separated by native page, the polyacrylamide gel incubated with substrate gel for12hours. We can see the area of enzyme after alcian blue staining and decolour.2.Optimization of culture conditions for hyaluronidase from Athrobacter nicotinovorus.Using one-factor-at-a-time methodology and turbiduty method to determine the supernatant hyaluronidase activity, the C source and N source conditions has been settled as:sodium hyaluronate0.5%, KNO30.1%. The clture conditions are settled as:5%inoculum volume,30℃,120rpm,48h incubation, initial pH6.5. The highest enzyme activity is got as64.32U/mL.3. The third part of study is aim to isolate the enzyme and analyze enzyme characteristic. Through all the treatment with centrifugation, ultrafiltration, successive chromatography on EDAE-Sepharose Fast Flow anion-exchange, zymography and SDS-PAGE, the hyaluronidase from Arthrobacter nicotinovorus was obtained. The molecular weight was estimated to be22.1kDa. The result of enzyme characteristic is that the sutiable temperature is37℃, the enzyme keep stable at low temperature, it will lose activity at the temperature over40℃; the optimum pH for enzyme is7.0, and keep high enzyme activity between pH4-7. With the increase of salt concentration in environment, the enzyme activity and stability gradually decreased. In all kinds of metal ions, Ag+, Cu2+can lead to the loss of the enzyme activity; Li+, Na+, Ca2+, Zn2+could inhibit the enzyme activity to a great extent;50mM Ba2+has no effect on the activity of enzyme, concentration at100mM can improve the activity of the enzyme.