Purification,Characterization And Modification Of Groups Of The Extracellular Ribonuclease From Bacillus Megaterium

Ribonucleases (RNases) are types of nucleases that catalyze the degradation of RNA into smaller components, which exist in various organisms ranging from microbes to animals and plants. This kind of enzyme participates in RNA metabolism, cell maturation and apoptosis, angiogenesis, and host defense mechanism against RNA virus. Ribonuclease widely used as biochemical models of enzymology in molecular biological study and are important analytical enzymes in the food and pharmaceutical industries. In addition, it’s also used to produce nucleotide and flavoring. Microbial RNases have special biological function, such as antiviral, antifungal and antiproliferative activity.Bacillus megaterium belongs to Bacillus spp., and it’s widely distributed in nature. It has ad-vantage of genetic stability, secreting production, simple fermentation technology, and it’s widely used in producting industrial enzymes such as amylase, penicillin amide enzyme and glucose isom-erase. Our study showed that Bacillus megaterium can efficiently secrete extracellular ribonuclease, and as a fermented products, the type and content of proteins from Bacillus megaterium is less than animal and plant materials, and the purification is simpler so that can reduce the cost in getting RNase, and could be applied in industrial RNase production. Purification of RNase from Bacillus megaterium has not been reported until now on. In our study, RNase has been separated and purified from Bacillus megaterium, and the properties of the enzyme had been analysed in this paper. Through our hard work, we hope that we could lay out the foundation for development and applica-tion of this kind of enzyme.By a combination of amonanium sulfate precipitation, ion-exchange chromatography on CM-Sepharose column and Superdex-200 gel-filtration, ribonuclease was purified to electrophoretic homogeneity from Bacillus megaterium, and some of these enzymological properties were explored. The enzyme was 606.67-fold purification with specific activity 54272.27U/mg.11.37% of the ribo-nuclease activity was recovered.The molecular weight of this enzyme was 33.3kD. Optimum activity of the enzyme was achieved at 52.5℃and pH8.5. The enzyme displayed a stable characterization in heat condition be-tween 20-40℃, and pH 6-7. The apparent Km values of the enzyme with different concentrations of yeast RNA as the substrate was 2630.02μg/mL at 52.5℃and pH 8.5. The activity of this enzyme could be inhibited by Fe2+, Cu2+, SDS, ascorbic acid and oxalic acid. KSCN, Li+, K+, Mn2+, Ba2+, Co2+, Na+and Zn2+ in low concentration have little inhibition to the enzyme. The organic solvents such as isopropanol, methanol, ethanol, acetonitrile can slightly activate the enzyme.Study of chemical modification shows that Chloramine-T, NAI and PMSF have no influence to the activity of the enzyme. DTT, Maleic anhydride, BD, BrAc and PCMB in low concentration in-hibit the enzyme, but with the increase of inhibitor concentration, the inhibitory effect don’t increase. It is considered that disulfide bond,ε-amino group of lysine, imidazole of histidine and sulfydryl probably effect the activity but are not at the activity center of the enzyme.