Effect of Nutrients by One Variable At A Time (OVAT) Approach on the Dextransucrase Production from Leuconostoc mesenteroides NRRL B-640
R Purama, A Goyal
dextran, dextransucrase, leuconostoc mesenteroides, tween 80
R Purama, A Goyal. Effect of Nutrients by One Variable At A Time (OVAT) Approach on the Dextransucrase Production from Leuconostoc mesenteroides NRRL B-640. The Internet Journal of Microbiology. 2007 Volume 5 Number 1.
The medium composition including macro and micronutrients were optimized for maximizing the yield of dextransucrase from
There are several reports on sucrose effect on dextransucrase production by various strains of
Various reports on effect of micronutrients on dextransucrase production from
Materials and Methods
Maintenance and Inoculum preparation of
Cultures were maintained in modified MRS agar stab that was prepared by substituting sucrose with glucose as a carbon source . A loop of culture from an agar stab was transferred to 5 ml of sterile medium described by Tsuchiya et al. (1952). 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
Production of dextransucrase
The enzyme was produced using the media described by Tsuchiya et al. (1952) and the enzyme production media contained in (%, w/v) sucrose, 2; yeast extract, 2; K2HPO4, 2; MgSO4.7H2O, 0.02; MnSO4.4H2O, 0.001; FeSO4.7H2O, 0.001; CaCl2.2H2O, 0.001; NaCl, 0.001 and the pH was adjusted to 6.9. Unless stated otherwise, all fermentations were carried out in triplicate sets of 100 ml enzyme production medium (EPM) in 250 ml Erlenmeyer flask at 25°C under shaking condition incubated at 200 rpm. The samples (5 ml) were withdrawn at indicated time intervals and centrifuged at 10,000 rpm for 10 min at 4°C to separate the cells. The supernatant (cell free extract) was analyzed for enzyme activity and protein concentration.
Enzyme activity assay
The assay of dextransucrase was carried out in 1 ml of a reaction mixture in 20 mM sodium acetate buffer, pH 5.4, containing 292 mM sucrose and using the cell free extract (10-20 µl) as 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 by the Nelson-Somogyi procedure [14,22]. 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 (Varian, Cary 100) against a blank using 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. All assays were performed in duplicate sets.
Effect of sucrose on dextransucrase production
The effect of sucrose concentration on dextransucrase production was studied by varying its concentration from 1 to10% in the enzyme production medium by keeping the concentration of other components constant. The medium containing the 2% sucrose was considered as control.
Effect of yeast extract and KHPO on dextransucrase production
The effect of yeast extract was studied in combination with the phosphate concentration. The yeast extract concentration was varied from 1.5% to 4%, where the control was 2% yeast extract. The effect of phosphate on the dextransucrase production was studied by varying the concentration from 1.5% to 3%, where the control was 2% phosphate.
Effect of peptone, beef extract and Tween 80 on dextransucrase production
The effects of peptone and beef extract on dextransucrase production were studied separately in addition to the presence of yeast extract. The effect of peptone was studied by varying the concentration form 0.1% to 1.5%, whereas the beef extract was varied from 0.5% to 2% taking the medium as described by Tsuchiya et al.
Effect of MgSO, MnSO, NaCl and CaCl on dextransucrase production
The effects of MgSO4 and MnSO4 on dextransucrase production were studied separately by varying the concentrations from 0.02 to 0.06% and 0.001 to 0.005%, respectively. The medium described by Tsuchiya et al.
Effect of sucrose
There was a steep rise (5 fold increase) in the dextransucrase production and activity from
Effect of yeast extract and KHPO
An increase in yeast extract concentration from 1.5% to 4% caused an increase in dextransucrase activity at all concentrations of K2HPO4 used (Fig. 2). The increase in activity was approximately 10% at all K2HPO4 concentrations used. The increase in K2HPO4 concentration form 1.5% to 3% resulted in significant increase (approx. 20%) in dextransucrase activity at all yeast extract concentrations (Fig. 2). The maximum activity of 5.9 U/ml was achieved at a combination of 4% yeast extract and 3% K2HPO4. However, for large scale production of enzyme it would be economical to use a combination of 2% yeast extract and 3% K2HPO4 that gave 5.6 U/ml which is 15% higher than the control 4.8 U/ml containing 2% of K2HPO4 and 2% of yeast extract that will also save 2% of yeast extract.
Effect of peptone, beef extract and Tween 80
The effect of peptone on dextransucrase production was studied by varying the concentration from 0.1% to 1.5%. The addition of only 0.1% peptone to control medium showed 17% increase in dextransucrase activity (Fig. 3A). Further increase in peptone concentration beyond 0.5%, did not favor the enzyme production, rather a decrease in enzyme production was observed. This might be due to effect of certain trace elements present in the peptone. Effect of beef extract on dextransucrase production was studied by varying its concentration from 0.5% to 2%. With an increase in the beef extract from 0.5% to 1.5% an increase in enzyme production was observed (Fig. 3B). The addition of 1.5% beef extract gave 15% increase in enzyme production over control medium. Further increase in beef extract concentration beyond 1.5% decreased the enzyme production. Addition of Tween 80 to the medium stimulated the production of dextransucrase. The production of dextransucrase increased with increase in concentration of Tween 80 (Fig. 3C). 0.1% Tween 80 gave a 10% increase in enzyme activity that was saturated at higher concentrations, 0.5% (Fig. 3C). This result is similar to the earlier reports [19,25]. They showed that addition of Tween 80 to the enzyme production medium altered the fatty acid composition of the membrane thus enhancing the secretion of the dextransucrase and its activity [19,25].
Effect of MgSO, MnSO, NaCl and CaCl
The effect of MgSO4 on dextransucrase was studied by increasing the concentration in the production medium from 0.02% to 0.05%. Dextransucrase production enhanced with the increase in MgSO4 from 0.02 % (4.8 U/ml) to 0.04% (5.3 U/ml) (Fig. 4A) showing a 10% increase in the enzyme production (Table 1). The magnesium ions are reported to play role in the signal transduction by enhancing the enzyme production and its release in to the medium .
Sucrose is the known inducer of dextransucrase . Although, some
Tsuchiya et al.
The increase of K2HPO4 from 2% to 3% gave a 15% higher enzyme activity in the medium by
The addition of 0.1% peptone to control medium with 2% yeast extract showed 17% increase in dextransucrase activity from
Dextransucrase production from
A 12% increase in the enzyme production
The authors thank Indian Institute of Technology Guwahati, Guwahati, India, for providing the experimental facilities and Ministry of Human Resource Development, Government of India, for providing a research fellowship to RKP.