Purification Of Endo-arabinase Secreted From Cellulomonose Bacterial Strain

Arabinogalactan (AG), consisted with arabinan and galactan, is theimportant component of mycobacterial cell wall. Because the complexlybranched structure of AG is difficultly revealed by typically chemicalanalysis, the polysaccharide structure has not been discovered exactly. Thus,AG is degraded into small fragments by specific endo-enzyme, identifyingthe portions structures by regular chemical analysis, which is assumed abetter way to analyze the complex molecular structure. First of all, we haveto achieve the suitable endo-arabinase. A species of Cellulomonas was isolated from soil by Department ofMicrobiology in Colorado State University in 1994, shown to secreteextracellular enzymes capable of degrading AG into a series ofoligosaccharide regions. Undoubtedly, the organism from soil will offer agroup of hydrolases for the determination of polysaccharide structure.Therefore, we choose the endo-arabinase capable of degrading AG intohexa-arabinoside as the goal of the research, and hope to achieve thepurified protein. Since the concerned genetic information and amino acidsequence have not been reported recently, separation and purificationbecome the only way to identify the exactly structure of the protein. At the beginning of the experiment, the tested and confirmedmeasurement of detection of enzyme activity was set up. Then, a series ofseparation and purification methods were used into separating the crudeenzyme, in order to select the effective chromatography. Finally, threeefficient chromatography were arranged to separate the crude enzyme andthe following results have been drawn. 1. The thin layer chromatography was used as the tested andconfirmed measurement of the detection of enzyme activity. 2. Different typical column chromatography were run for purificationof crude enzyme. Hydrophobic interaction chromatography (HIC), gelfiltration chromatography and affinity chromatography were suitable toseparation, however, ion exchange chromatography and absorptionchromatography were unsuitable. 3. Salt precipitation was applied for concentration of crude enzymecontaining extremely low protein, then HIC, Sephacryl S-300 and affinitychromatography were used to primarily purification of AG hydrolases.According to the results of detection of enzymatic activity and SDS-PAGE,most proteins among the crude enzyme were eliminated. Ultimately twobands were purified, and the molecular masses were estimated to be 75kDaor 25kDa.