Purified Dextransucrase from Leuconostoc mesenteroides NRRL B-640 Exists as Single Homogeneous Protein: Analysis by Non-denaturing Native-PAGE

摘要

The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-640 was purified using polyethylene glycol (PEG-400) fractionation. A 25% (v/v) PEG-400 concentration gave dextransucrase with maximum specific activity of 9.2 U/mg with 16 fold purification in a single step. The purified enzyme by PEG-400 showed multiple protein bands on SDS-PAGE with one prominent band corresponding to the size 180 kDa (12). However, the same PEG-$00 fractionated dextransucrase samples showed single, intact and homogeneous band when analyzed on non-denaturing native-PAGE. This showed that dextransucrase remains in single molecular form in the native state and shows multiple forms only under denaturing conditions when it is heated before loading and when it contained SDS or 2-mercaptoethanol. Introduction Leuconostoc mesenteroides NRRL B-640 is known to produce extracellular dextransucrase that gives highly linear dextran (1,2). The linear dextran is water soluble and has numerous applications in pharmaceutical, food and fine chemical industries (3). Several attempts have been made to enhance the production of dextransucrase from Leuconostoc mesenteroides NRRL B-640 (4,5,6,78,9). Various methods have been reported for purification of dextransucrase. The purification methods such as salt, glycerol and alcohol precipitation, fractionation by polyethylene glycol, ultra-filtration, chromatography and phase-partitioning have been standardized and successfully used for purification of dextranucrase from different strains (10). Among various purification methods, polyethylene glycol (PEG) fractionation is a simple and rapid method for dextransucrase purification (11,12,13). Polyethylene glycols are nonionic hydrophilic polymers and are known to selectively precipitate proteins. They also have an advantage of being readily removed by dialysis. The purification by two-phase partitioning using different molecular weight PEGs have also been reported for dextransucrase from various strains of Leuconostoc mesenteroides (14,15,16,17). The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-640 was purified using polyethylene glycol fractionation PEG 400. The purified dextransucrase fractions confirmed their presence by synthesis of dextran, when run on non-denaturing SDS-PAGE gels for in-situ activity detection by Periodic Acid Schiff staining and the activity bands corresponded to the native and active form of the purified dextransucrase of approximately, 180 kDa molecular size, that appeared on the identical denaturing SDS-PAGE gels stained with Coomassie Brilliant Blue (12). The purified enzyme by PEG-400 showed multiple protein bands on SDS-PAGE with one prominent band corresponding to 180 kDa (12). In the present study the native state of purified enzyme by PEG-400 fractionation was analyzed by non-denaturing native polyacrylamide gel electrophoresis. Materials and Methods The microorganism and reagents Leuconostoc mesenteroides NRRL B-640 was procured from Agricultural Research Service (ARS-Culture collection), USDA, Peoria, USA. The culture was maintained in modified MRS (sucrose in place of glucose) agar medium (18) as a stab at 4°C and sub cultured every 2 weeks. The ingredients required for the maintenance and enzyme production media were from Hi-Media Pvt. Ltd., India. All the chemicals required for reducing sugar estimation, protein estimation and buffer preparation were of highest purity grade commercially available. PEG400 (Ranbaxy Pvt. Ltd., India) was used for fractionation of dextransucrase. Production of dextransucrase For enzyme production a loop of culture from modified MRS agar stab was transferred to 5 ml of sterile medium as described by Tsuchiya et al., 1952 (19). The cultures were grown at 25°C with 200 rpm for 12-16h. 1% of the culture inoculum was used for the enzyme production from Leuconostoc meseteroides NRRL B-640. The culture broth was centrifuged at 10,000g for 10 min at 4°C to separate the cells. The cell