Purification And Partial Characterization Of Cattle Hyaluronidase

Hyaluronidases are classes of glycosidases that predominately degrade hyaluronic acid.They are widely distributed in nature，it has a variety of functions, for example, when use it inophthalmology, surgical and dental surgery anesthesia, can accelerate the spread of the localanesthetic; dissipating local edema and hematoma after surgery and trauma, et al. Theresearch used bovine testicular as raw material, using single factor experiment andPlackett-Burman experiment to screen significant factors that effect the extraction ofhyaluronidase; on this basis, using surface analysis to optimize the significant factors, toobtain the best parameters of extraction; by screening some parameters, such as filler, saltconcentration, elution mode, flow rate, et al, to get the best purification process; by using theultraviolet wavelength scanning and the SDS-PAGE, to identify the enzyme and molecularweight; hyaluronidase after identification was studied on its enzymatic characteristics. Thusaim to improve the utilization rate of the cattle by-products, and provide a theory basis andreference for the further research of bovine testicular hyaluronidases. The test results are asfollows:1. The extraction of cattle hyaluronidase: The extraction conditions of hyaluronidasewere researched by using single factor experiment, Plackett-Burman experiment and surfaceanalysis. The results showed that the significant factors include pH of extraction buffersolution, NaCl concentration and solid-liquid ratio. The primary and secondary order was asfollows: pH of extraction buffer solution, NaCl concentration and solid-liquid ratio. Theoptimum extraction parameters were: extraction buffer solution is pH5.71, the concentrationof NaCl is0.14mol/L and solid-liquid ratio is1:1.74. Under these conditions, specific activityof the enzyme was118mU/mg, and extraction rate was0.938%.2. The purification of cattle hyaluronidase: Using ion exchange chromatographypreliminarily purify hyaluronidase, the optimum parameters were: the SP Sepharose FastFlow was choosed as the main ion exchange filling, meanwhile, the step elution way wasapplied to elution, the concentration of NaCl was0,0.1,0.2,0.3,0.4,0.5mol/L. Under theseconditions, purification fold was7.53and recovery rate was67.87%. Using Gel filtrationchromatography futher purify hyaluronidase, the optimum parameters were: the SephacrylS-100was choosed as the main gel filtration filling, collecting hyaluronidases with the flowrate of0.5mL/min. Ultimately, the purification fold of enzyme was26.11and recovery ratewas12.93%.3. The identification of cattle hyaluronidase: N-acety-D-glucosam and the resultant from hyaluronidase were detected by the wavelength scanning, the results showed that the twocurves were basic consistent, and there were two high point in544nm and585nm wavelength,thus to determine the purified enzyme is bovine testicular hyaluronidase. Using SDS-PAGEpreliminarily determine molecular weight, the results revealed two close bands withapproximate molecular weights of60kDa and67kDa, respectively.4. Enzymatic characteristics of cattle hyaluronidase: The results showed that the pHoptimum for the enzyme was pH4.5and maximum activity was obtained at40℃. It hasbetter stability at pH4~5and20~40℃. The enzyme also required NaCl at a concentration of0.3mol/L. The hyaluronidase was inhibited by Fe3+, Mn2+, Zn2+, Al3+, Ca2+, K+and Mg2+invarious degree. The Km value for hyaluronidase was0.106mg/mL.