Purified Dextransucrase from Leuconostoc mesenteroides NRRL B-640 Exists as Single Homogeneous Protein: Analysis by Non-denaturing Native-PAGE
R Purama, A Goyal
dextran, dextransucrase, glucosyltransferase, glycoside hydrolase, polyethylene glycol
R Purama, A Goyal. Purified Dextransucrase from Leuconostoc mesenteroides NRRL B-640 Exists as Single Homogeneous Protein: Analysis by Non-denaturing Native-PAGE. The Internet Journal of Microbiology. 2008 Volume 6 Number 1.
The extracellular dextransucrase from
The extracellular dextransucrase from
Materials and Methods
The microorganism and reagents
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
The assay of dextransucrase was carried out in 1 ml of a reaction mixture in 20 mM sodium acetate buffer, pH 5.4, containing 146 mM (5%) sucrose and using the cell free supernatant (10- 20 µl) for the enzyme source. The reaction mixture was incubated at 30°C for 15 min. The enzyme activity was measured by estimating the liberated reducing sugar (20,21). Aliquots (0.2 ml), from the reaction mixture were analyzed for reducing sugar concentration. The absorbance was measured at 500 nm using a UV-visible spectrophotometer (Cary100 Bio, Varian Inc., USA) against a blank with D-fructose as a standard. One unit (U) of dextransucrase activity is defined as the amount of enzyme that liberates 1 µmol of reducing sugar per min at 30°C in 20 mM sodium acetate buffer, pH 5.4. The total protein content of the cell free supernatant was estimated by the method of Lowry
Purification of dextransucrase by PEG fractionation
The polyethylene glycol PEG-400 pre chilled to 0°C was added to 200 ml cell free supernatant to obtain the final concentrations of 20, 25, 33, 40 and 50 (%, v/v). The mixture was incubated for 12h at 4°C to allow the dextransucrase to precipitate. The mixture was centrifuged at 12,000
Non-denaturing native -PAGE analysis of purified enzyme
The non-denaturing native-polyacrylamide gel electrophoresis was performed with a vertical slab mini gel unit (BioRad, USA) using 1.5 mm thick gels, following the method described elsewhere (12) using only 7.5% (w/v) acrylamide resolving gel prepared in 0.125 M Tris-HCl pH 8.8. The protein samples were prepared in 0.0625 M Tris-HCl buffer (pH 6.8) containing glycerol, 5% (w/v) and 0.05% (w/v) bromophenol blue but with out 2-mercaptoethanol and SDS. The enzyme samples from all PEG-400 purified fractions were loaded on 7.5% acrylamide gel without heating. The electrophoresis was carried out using Tris-Glycine buffer (0.025M Tris and 0.192 M Glycine) pH 8.3 with a current of 2mA per lane. The protein bands were fixed with solution containing acetic acid (5%, v/v) for 5 min, then stained for 30 min with 0.25% (w/v) Coomassie Brilliant Blue, and destained by repeated washing using a solution containing 20% methanol and 10% (v/v) acetic acid. Although for native-PAGE markers are not used but the molecular mass marker (Myosin from Rabbit Muscle, 205000; Phosphorylase b 97400; Bovine serum albumin, 66000; Ovalbumin, 43000; Carbonic anhydrase, 29000 Da purchased from Genei, India) along with BSA sample with out boiling were added to the gel for reference.
Results and Discussion
Dextransucrase fractionation by PEG 400
The culture broth obtained after 12 h growth of
Analysis of purification by non denaturing Native-PAGE
The dextransucrase samples obtained with PEG-400 fractionation were analyzed by SDS-PAGE to check the purity of dextransucrase. The results showed the presence of multiple protein bands however there was a prominent protein band of approximately 180 kDa molecular size obtained from all concentrations of PEG-400 as shown earlier (12). From SDS-PAGE results dextransucrase appeared to exist in multiple molecular forms, however, the same samples showed predominantly a single, intact and homogeneous protein band on non-denaturing native-PAGE (Fig. 3). This showed that dextransucrase remains in native state after it is purified and dialysed and shows multiple forms only in under denaturing conditions when enzyme sample was heated before loading and when it contained 2-mercaptoethanol. The non-denaturing native-PAGE of dextransucrase samples purified by higher than optimum concentration of 25% PEG-400 showed presence of other contaminating non-dextransucrase protein(s) with increasing protein band intensities (Fig. 3) which were not visible in SDS-PAGE, owing to low protein content and being denatured or proteolysed under denaturing conditions (12). The native-PAGE revealed that a concentration of PEG-400 lower than optimum (25%) fails to fractionate most of dextransucrase and so specific activity was low and the higher concentrations of PEG-400 fractionated also other proteins which accounted for decreased specific activity of dextransucrase (Table 1),
The authors thank Indian Institute of Technology Guwahati, Guwahati, India, for providing experimental facilities and Ministry of Human Resource Development, Government of India, for providing a PhD fellowship to RKP.