Purification And Partial Characterization Of Endo-arabinase Secreted From A Strain Of Cellulomonose

Objective: To purify and partial characterize the enzyme (named temporarily endo-arabinase) with the properties of endo-arabinase, secreted from a bacterial of Cellulomonose strain. Arabinogalactan (AG) can be hydrolyzed into hexa-arabinoside by the studied endo-arabinase. The enzyme was produced by an unnamed bacteria, which having similar nature to Cellulomonose. Since the concerned genetic information and amino acid sequence have not been reported recently, separation and purification become the only way to identify the exactly structure of the protein. AG, consisted with arabinan and galactan, is the important component of mycobacterial cell wall. Meanwile AG, used as anti-TB drugs targets because of its unique structure, has complex branches. Recently, the researches on analyzing the structure of AG and looking for an effective way to degrading AG have been focused. Some glycosyl groups of AG always have been changed by typically chemical analysis and the polysaccharide structure has not been discovered exactly. However, substituted by specific enzyme, the native form is preserved. By such sort, AG is degraded into small fragments by specific endo-enzyme, identifying the portions structures by mass spectrum and NMR analysis, and combined the information together into complete AG structure. First of all, we have to achieve the suitable endo-arabinase. The results of preliminary experiments indicated that the enzyme will be used as a new tool to research the structure of Mycobacterial cell wall and hold promise for the study of the concerned enzymes. Methods: The strategy of the research was to purify and partial characterize the endo-arabinase from the fermentation broth of Cellulomonose by several chromatographies. The methods were as follows: 1) to develop a reliable assay for endo-arabinase. Attempt to separate the product from substrate based on the thin layer chromatography (TLC) and do arabinase activity assay with this method, quantitatively analyzed with Quanti-Scan software. 2)To purify endo-arabinase by different chromatography methods (ion-exchange chromatography, sephacryl S-300 chromatography and Blue-Dye affinity chromatography) and to trace the activity by method above. The purified sample was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 3) The molecular mass was conformed by SDS– PAGE. The endo-arabinase activity was detected by TLC assay and Quanti-Scan. The optimal pH and temperature were studied. The enzyme kinetic parameters were determined by Linewear-Burk.Results: 1) By use of the TLC to separate the substrate of AG from arabinase released small molecule products of oligoarabinan which could be quantitated by Quanti-Scan software. How to choose the mobile phase of TLC is the key of success. To obtain the ideal results, ethanol, isopentanol, acetate and n-Butanol were mixed in different proportion. Based on the results, n-Butanol: acetate (1: 1) system was determined. Many samples could be done simultaneously with this assay which was rapid, stable, reliable and quantitative, and was adapted to trace the activity of arabinase during purification. 2) Some components of the culture medium, such as AG and Yeast extraction, retained in the crude enzyme. The residue with high viscosity and the very low concentration of endo-arabinase made following purification more difficultly. Since the physical and chemical properties of endo-arabinase is unknown, many chromatographies were attempted, and several effective methods were chosen. DEAE- Sepharose ion-exchange chromatography was best to be the first step because it could deal with abundant crude sample at very short time to capture target protein steadily and to get rid of a lot of other proteins and impurities. Sephacryl S-300 chromatography separated proteins according to the difference of molecular weight and increased the purity and the specific activity of endo-arabinase. Blue-Dye nonspecific affinity column could increase the purity and specific activity of endo-arabinase dramatically. Combined chromatography methods were used to purify endo-arabinase effectively. The purified sample was separated by SDS-PAGE and then visualized by and silver stain and partial characterized. 3) The molecular mass of endo-arabinase is 45kD. The enzyme had apparent Km values of 2.5 mg/ml and apparent Vmax of 0.083μmol/L·min. The enzyme had optimal activity at pH 8.0 and 40℃, shown stability at pH 6.0-9.0 and below 40℃. The specific activity and purification of endo-arabinase were 45 kD, 15.42×10~3 (U/ug) and 77.1.Conclusion: The TLC assay was rapid, stable, reliable and quantitative. The qualitative and quantitative analysis of the degraded oligosaccharide products could be realized by the combination P-2 column and HPLC , however, the use of TLC and Quanti-Scan was more effective to trace the activity during the separation. Because of the limit culture medium, abundance of crude enzyme could not be obtained, the low concentration endo-arabinase made the purification process difficultly. In order to solve the problem, attempts on purification process, protein concentration assay and enzyme activity assay were practiced, which would contribute to the separation of low concentration proteins. The endo-arabinase, a kind of endo-enzyme of arabinan, was purified and partial characterized. The enzyme could released hexa-arabinoside from the terminal of AG, which was an important component concerned of mycobacterial physiology and disease process. Furthermore, the crucial end played a role in anti-TB agents. In a word, endo-arabinase was not only a powerful tool but also a compare basis on the studied of other AG-degrading-enzyme. In addition, it could provide further insight into the complex structures of mycobacterial cell walls and exploitation of the targets of anti-TB drugs.