Application of response surface methodology for glucosyltransferase production and conversion of sucrose into isomaltulose using free Erwinia sp. cells

Isomaltulose is a sugar commercially used in food industries. Glucosyltransferase produced by Erwinia sp. D12 catalyses a transglucosylation of sucrose giving isomaltulose. The response surface methodology was applied for the optimization of the nutrient concentration in the culture medium for the enzyme production in shaken flasks at 200 rpm and 30ºC. The three variables involved in this study were sugar cane molasses, bacteriological peptone and yeast extract Prodex Lac SD^®. The highest enzyme activity was observed in culture medium containing sugar cane molasses (160 g/L), bacteriological peptone (20 g/L) and yeast extract Prodex Lac SD^® (15 g/L). Maximum glucosyltransferase activity of 29.88 U/mL was achieved in a 6.6-L fermenter using the optimized medium. Free Erwinia sp. D12 cells were used for isomaltulose production from sucrose during fifteen successive batches. The final isomaltulose concentration of 75.6% obtained in the first batch increased to 77.21% (mean value) in the other fourteen batches.

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose from sucrose by an intramolecular transglucosylation. Isomaltulose is a reducing sugar and it is a structural isomer of sucrose naturally presents in honey in very small quantities. The interest in isomaltulose is due to the low cariogenic, low hydrolysis speed and formation of monosaccharides in the organism, and also due to the possibility of conversion of this sugar to a mixture of sugar-alcohol with low caloric value and non-cariogenic property known as Isomalt^® or Palatinit^®. This disaccharide has a sweet taste and very similar physical and organoleptic properties to sucrose (Krastanov and Yoshida, 2003). The microbial formation of isomaltulose has attracted commercial interest and the production of this sugar has aroused great interest since this structural isomer of sucrose has interesting potential.

The application of response surface methodology in fermentations process can result in improved product yields, reduced process variability and development time and over all costs (Rao et al. 2000). In this work, the effect of both nitrogen source (bacteriological peptone and yeast extract) and carbon source concentrations (sugar cane molasses) on glucosyltransferase production by Erwinia sp. D12 was studied at 30ºC and the optimal conditions for glucosyltransferase production were determined in shaken flasks and on a bioreactor 6.6 litres. The conversion of sucrose into isomaltulose by immobilized cells well knows process (Cheetham et al. 1985; Krastanov and Yoshida, 2003). An attempt was made to study the production of isomaltulose in repeated batch operations using free Erwinia sp. D12 cells.

Microorganism and culture maintenance

Erwinia sp. D12 producer of glucosyltransferase isolated from the Laboratory of Biochemistry, College of Food Engineering/UNICAMP was used in this study. Bacteria were maintained in culture medium containing per litre sterilized water: 6 g sucrose, 4 g peptone, 0.4 g beef extract and 2 g agar. The cultures were kept at 5ºC.

Culture medium optimization using response surface methodology

The 2³-factorial central composite design (2³-FCCD) was used and the variables studied were: sugar cane molasses, bacteriological peptone and yeast extract Prodex Lac SD^®. The variables and their levels are presented in Table 1. The 2³-FCCD contained a total of 17 experimental trials. All data were treated with STATISTICA^® 5.0 from Statsoft Inc.

Cultivation and enzyme production

The inoculum had the same composition of the production medium which they are shown in Table 1. A loop full of cells was inoculated to inoculum in Erlenmeyer flasks of 250 mL containing 50 mL of culture medium and the flasks were incubated in a rotatory shaker (200 rpm) at 30ºC for 15 hrs. Then a 10% (v/v) inoculum was added to production medium. Throughout the work, liquid cultures were incubated at 30ºC and 200 rpm in a rotatory shaker. After 8 hrs of fermentation, the culture was centrifuged at 9,650 x g for 15 min and glucosyltranferase activity was determined.

Glucosyltransferase assay

The glucosyltransferase activity was performed by the increase of the reducing power from a solution containing sucrose, described by Park et al. (1996) with modifications (Kawaguti et al. 2005). Reducing sugars were measured by Somogyi method (Somogyi, 1945) using glucose as standard. One activity unit (U) of glucosyltransferase is defined as the amount of enzyme that liberates one µmol of reducing sugars/minute/mL of the enzyme from sucrose under standard assay conditions.

Production of cell biomass: growth determination and glucosyltransferase production under optimal culture medium in 6.6-L fermenter

Bacterial growth and glucosyltransferase activity were determined under optimal culture medium: sugar cane molasses (160 g/L), bacteriological peptone (20 g/L) and yeast extract Prodex Lac SD (15 g/L) on a bioreactor 6.6-L fermenter New Brunswick Bioflo IIc. Two loop full of culture were inoculated in three 250 mL Erlenmeyer flasks containing 100 mL of culture medium optimized each one and incubated in a rotatory shaker 200 rpm at 30ºC for 15 hrs. An aliquot of 300 mL inoculum was transferred to 2.700 mL of culture medium optimized contained in a 6.6-L fermenter and incubated under the following conditions: temperature 26ºC, initial pH 6.5, aeration rate 1 vvm, and agitation speed 200 rpm. Samples were collected at time-defined intervals and submitted to analysis. Aliquots (20 mL) of the culture broth were centrifuged at 7,850 x g for 15 min, at 5ºC. The cell mass was washed twice with 20 mL of distilled water and re-suspended in 20 mL of distilled water. A Beckman DU 70 spectrophotometer was used to monitor cell growth by measuring the optical density at 660 nm (OD₆₆₀). Glucosyltransferase activity was estimated as described previously. The pH of the culture medium was measured with an Orion model 710A potentiometer.

Performance of repeated batch operations using free cells

Duplicate repeated batch conversion runs were carried out in 250 mL Erlenmeyer flaks containing the mixture of 35% (w/v) sucrose solution and free-cell of Erwinia sp. D12 (sucrose solution:free-cell - 10:1). The flasks were maintained in a rotatory shaker at 150 rpm and 35ºC for 15 min. At the end of each batch, samples were collected and submitted to analysis, the reaction mixture was centrifuged and the free-cells were used for the next batch conversion of sucrose into isomaltulose with the fresh substrate. This process was repeated for several times.

Viable cell numbers, biomass, pH and HPLC-PAD analysis

Samples were submitted to serial dilutions and viable counts were performed by spread plate technique. The biomass (wet cell mass) was measured with a precision balance. The pH of the culture medium was measured with an Orion model 710A potentiometer. The sugars analysis was performed with HPLC system DIONEX DX-600. The carbohydrates were analyzed from their retention times as compared to those of the fructose, glucose, sucrose and isomaltulose standards.

Culture medium optimization using response surface methodology

The trials and results for the 2³-FCCD are shown in Table 1. According to the results obtained, the best conditions for glucosyltransferase production occurred in experiments 15, 16 and 17 corresponding to the central points. These experiments correspond to the cultivation medium composed by sugar cane molasses 160 g/L, bacteriological peptone 20 g/L and yeast extract Prodex Lac SD 15 g/L.

On the basis of the ANOVA, shown in Table 2, a second-order model (Equation 1) was established, describing the enzyme activity as a function of SCM, BP and YEP concentrations:

Based on the F-test the model is predictive, since the F-value calculated is 8.75 higher than the critical F-value and the determination coefficient 0.94 is close to unity. The pure error was very low, indicating a good reproducibility of the experimental data. The response surfaces and contour curves in Figure 1 and Figure 2 were obtained using Equation 2. It can be see that the maximum glucosyltransferase activity point is situated close to the central point. The model predicted the maximum activity in culture medium composed by SCM (160 g/L), BP (20 g/L) and YEP (15 g/L).

Production of cell biomass: growth determination and glucosyltransferase production under optimal culture medium in 6.6-L fermenter

After optimization, fermentation kinetics was determined at the optimized conditions, as observed in Figure 3. The glucosyltransferase production started at exponential growth phase and the enzyme activity was increased at the beginning of the cultivation (2 hrs of fermentation time) and reached a maximum level. Subsequently, the glucosyltransferase activity decreased slowly after 9 hrs of fermentation time. The highest enzyme activity was obtained after 9 hrs (29.88 U/mL) after inoculum and the activity was maintained constant, between 23-25 U/mL, until 14 hrs of fermentation time. Thereafter, the glucosyltransferase production was diminished and after 24 hrs de enzyme activity decreased to 16.06 U/mL. The pH of the culture medium was about 6.5-6.5 during fermentation, suggesting little production of acid as a by-product.

Overall, in this work, sugar cane molasses (agro-industrial residue) and commercial yeast extract were used with the purpose of resulting in a low cost culture media. The cell mass obtained from the bioreactor fermentation was used in further studies to verify the conversion of sucrose into isomaltulose from free-cells.

Performance of repeated batch operations using free cells

The data obtained for 15 cycles of repeated batch operation shown in Figure 4 indicated that free cells were active and could be reused. The biomass (wet cell mass) decreased with the batches of operation for the first five cycles of operation and remained almost steady for the subsequent batches between 3.73 g and 3.33 g of wet cell mass. The pH of the medium reaction remained constantly between 6.0-6.5, however after seventy batches the pH decrease to 5.5 remained in this value suggesting little production of acid as a by-product. It can be observe that the growth as free cells did occur slowly (viable cell number) in the reaction medium between the first and the fifteenth batches. After eight batches the viable cell number diminished gradually. The wet cell mass and the viable cell number decreased but did not affect the conversion of sucrose into isomaltulose.

Figure 4 shows the high conversion of sucrose into isomaltulose between 73-79%. As can be verified the efficiency of production of isomaltulose increased after the first batch, leading to increases in the isomaltulose concentration and productivities (around 1.1 g/L x hr) in the last batches. The highest conversion of sucrose into isomaltulose was 79.2% (batch number eight). The isomaltulose production using free Erwinia sp. cells was very efficient. It was obtained high yield from 35% sucrose solution with high speed about fifty minutes reaction time for complete conversion.

With the aid of the response surface methodology, the optimal concentrations of sugar cane molasses, bacteriological peptone and yeast extract Prodex Lac SD^® for the production of glucosyltransferase by Erwinia sp. D12 were found to be 160 g/L, 20 g/L and 15 g/L, respectively. The highest glucosyltransferase activity was obtained at 30ºC after 8 hrs of fermentation time. The results were satisfactory and the components sugar cane molasses and yeast extract Prodex Lac SD^® are very useful carbon and nitrogen sources, respectively to enzyme production. Maximum glucosyltransferase activity of 29.88 U/mL was achieved in a 6.6-L fermenter using the optimized medium at 26ºC, 200 rpm agitation, and 1 vvm aeration rate. The free Erwinia sp. D12 cells supported high production levels in repeated batch operations and the results showed potential for repeated reuse of free-cells.

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