Application of response surface methodology for glucosyltransferase production and conversion of sucrose into isomaltulose using free Erwinia sp. cells
Financial support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Financiadora de Estudos e Projetos em Pesquisa (FINEP) and GETEC Guanabara Química Industrial S.A.
Keywords: batch process, Erwinia sp., free cells, glucosyltransferase, isomaltulose, response surface methodology.
is a structural isomer of sucrose commercially used in food industries.
Glucosyltransferase produced by Erwinia sp. D12 catalyses an
intramolecular transglucosylation of sucrose giving isomaltulose.
The Experimental design and response surface methodology were applied
for the optimization of the nutrient concentration in the culture
medium for the enzyme production in shaken flasks at 200 rpm and
The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-α-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by an intramolecular transglucosylation (Kakinuma et al. 1998). 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).
Chemical synthesis of isomaltulose is very difficult but a small number of bacterial strains can convert sucrose into isomaltulose: Protaminobacter rubrum (Kakinuma et al. 1998), Erwinia rhapontici (Cheetham et al. 1985), Serratia plymuthica (McAllister et al. 1990), Klebsiella planticola (Huang et al. 1998), Klebsiella sp. (Park et al. 1996) and Klebsiella singaporensis sp. (Li et al. 2004). 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.
application of experimental design and 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
sp. D12 producer of glucosyltransferase isolated from the Laboratory
of Biochemistry, Department of Food Science,
23-factorial central composite designs (23-FCCD)
were carried out in order to identify optimum parameter levels for
the glucosyltransferase production. The parameters (or independent
variables) studied were: sugar cane molasses (Companhia Energética Santa Elisa), bacteriological
peptone (Biobrás) and yeast
extract Prodex Lac SD® (Prodesa produtos especiais para
where α is the distance of the axial points and n is the number of independent variables. All data were treated with STATISTICA® 5.0 from Statsoft Inc.
inoculum had the same composition of the production medium which they
are shown in Table 1, Table 2 and Table
glucosyltransferase activity was performed by the increase of the
reducing power from a solution containing sucrose, described by Park
et al. (1996) with modifications. For the extraction of intracellular
enzyme, the cell mass was washed twice with distilled water and then
suspended in 50 mL citrate-phosphate buffer
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 (New
Brunswick Scientific, Edison, N.J., USA). 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
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
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 (Satorius AG, Goettingen, GE). The pH of
the culture medium was measured with an Orion model 710A potentiometer
(Orion Research Inc,
First experimental design. The experimental results of glucosyltransferase production by the first 23-FCCD are shown in Table 1. The effect estimates for each variable: sugar cane molasses (SCM), bacteriological peptone (BP) and yeast extract Prodex Lac SD® (YEP), as well as the interaction between them, were determined and reported in Table 4. Both the t-test and p-value statistical parameters are used to confirm the significance of factors studied. All the independent variables had significant influence in glucosyltransferase activity (p < 0.05), as well as the interactions between them. The increase in BP concentration from 10 to 50 g/L led to an increase in enzyme activity.
The parameter SCM (L) showed higher effect than SCM (Q) indicating that the increase the concentration from 100 to 250 g/L led to a decreased in glucosyltransferase production. The independent variable ELP showed the same effect. A model fitting was accomplished for the first 23-FCCD in Table 1. The independent (glucosyltransferase activity) and the dependent variables were fitted to the second-order model equation and examined in terms of the goodness of fit. The analysis of variance (ANOVA) was used to evaluate the adequacy of the fitted model. The R-square value (determination coefficient) provided a measure of how much of the variability in the observed response values could be explained by the experiment factors and their interactions.
the basis of ANOVA, as shown in Table
where y is the predicted response (glucosyltransferase activity) and x1, x2 and x3 are the coded values of sugar cane molasses, bacteriological peptone and yeast extract Prodex Lac SD®, respectively. As can be seen in Figure 1 and Figure 2 an increase in sugar cane molasses and yeast extract Prodex Lac SD® concentrations led to an decrease in enzyme activity. In the second experimental design, the concentration ranges of SCM, BP and YEP were decreased.
Second experimental design. The trials and results for the second 23-FCCD are shown in Table 2. The effect estimates were determined and reported in Table 4. The analysis of the effects shows that an increase in the concentration of bacteriological peptone from 5 to 35 g/L and concentration of yeast extract from 10 to 50 g/L led to a decrease in glucosyltransferase activity. All interactions between SCM, BP and YEP were statistically significant for enzyme activity. To test the fit of the model the regression equation and determination coefficient (R-square) were calculated. The model presented high determination coefficient (R-square = 0.90) explaining 90% of the variability in the response (Table 6). The ANOVA of quadratic regression model demonstrates that the model is highly significant, as is evident from the F-test.
Based on these results the model can be utilized to generate response surfaces and contour curves for the analysis of the variable effects on glucosyltransferase activity. The response surface and contour curves were obtained using Equation 3:
It can be seen that an decrease in YEP led to an increase in enzyme activity and the best concentrations for SCM and BP were 135 to 190 g/L and 5 to 30 g/L, respectively.
Third experimental design. The trials and results for the third 23-FCCD are shown in Table 3. 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.
the basis of the ANOVA, shown in Table
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 5 and Figure 6 were obtained using Equation 4. It can be see that the maximum glucosyltransferase activity point is situated close to the central point.
model predicted the maximum activity in culture medium composed by
SCM (160 g/L), BP (20 g/L) and YEP (15 g/L) in the conditions studied.
It was obtained 7.26 U/mL (average of the central points) after 8
hrs fermentation at
After optimization, fermentation kinetics was determined at the optimized conditions, as observed in Figure 7. Glucosyltransferase production and growth characteristics of Erwinia sp. D12 using culture medium optimized are illustrated. 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.
activity value of 29.88 U/mL obtained from Erwinia sp D12 cells
is approximately ten times higher than the one produced by Park et
al. (1996) using strain Klebsiella sp. in a culture
medium composed by 1% bacteriological peptone, 0.4% beef extract powder
and 4% sucrose when they obtained 2.95 U/mL. Huang et al. (1998) examined
the effects of carbon sources, inorganic salt and supplemental nitrogen
sources on intracellular glucosyltransferase activity of Klebsiella
planticola CCRC 19112. It was obtained a maximum glucosyltransferase
activity of 11.08 U/mL using a culture medium composed by 1% bacto-tryptone,
7% sucrose, 3% tryptic soy broth and 0.5% NaCl. Li et al. (2004) using the strain Klebsiella sp. LX3 cultured aerobically in
the culture medium composed by 4% sucrose, 1% bacteriological peptone
and 0.4% yeast extract. The authors determined the glucosyltransferase
activity in cell culture, supernatant, cell-free extract and cell-debris
fractions. The enzyme activity of the cell-debris fraction (19.2 U/mL)
was almost identical to that of cell culture fraction (20.1 U/mL).
This result suggesting that the glucosyltransferase is a cell wall
bound enzyme. Moraes et al. (2005) obtained 15.6 U/mL of glucosyltranferase
activity from Erwinia sp. cells incubated in 3.0-L fermenter
containing culture medium composed by 12% sugar cane molasses, 4%
bacteriological peptone and 0,4% beef extract, at
data obtained for 15 cycles of repeated batch operation shown in Table
8 and Figure 8 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
the aid of the experimental design and 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
We thank the Prodesa Produtos Especiais para Alimentos S/A for the supply of the yeast extract Prodex Lac SD®.
AHN, Seung-Joon; YOO, Ji-Hyun; LEE, Hyeon-Cheol; KIM, Sang-Yong; NOH, Bong-Soo; KIM, Jung-Hoe and LEE, Jung-kul. Enhanced conversion of sucrose to isomaltulose by a mutant of Erwinia rhapontici. Biotechnology Letters, July 2003, vol. 25, no. 14, p. 1179-1183. [CrossRef]
CHEETHAM, Peter S.J.; IMBER, Carol E. and ISHERWOOD, Jamie. The formation of isomaltulose by immobilized Erwinia rhapontici. Nature, October 1982, vol. 299, no. 5884, p. 628-631. [CrossRef]
CHEETHAM, Peter S.J.; GARRETT, Christine and CLARK, Jeremy. Isomaltulose production using immobilized cells. Biotechnology and Bioengineering, April 1985, vol. 27, no. 4, p. 471-481. [CrossRef]
KAKINUMA, Hiroyuki; YUASA, Hideya and HASHIMOTO, Hironobu. Glycosyltransfer mechanism of α-glucosyltransferase from Protaminobacter rubrum. Carbohydrate Research, November 1998, vol. 312, no. 3, p. 103-115. [CrossRef]
KRASTANOV, Albert and YOSHIDA, Toshiomi. Production of palatinose using Serratia plymuthica cells immobilized in chitosan. Journal of Industrial Microbiology and Biotechnology, October 2003, vol. 30, no. 10, p. 593-598. [CrossRef]
LI, Xianzhen; ZHANG, Daohai; CHEN, Feng; MA, Jie; DONG, Yihu and ZHANG, Lianhui. Klebsiella singaporensis sp. nov., a novel isomaltulose-producing bacterium. International Journal of Systematic and Evolutionary Microbiology, November 2004, vol. 54, no. 6, p. 2131-2136. [CrossRef]
MCALLISTER, M.; KELLY, C.T.; DOYLE, E. and FOGARTY, W.M. The isomaltulose synthesing enzyme of Serratia plymuthica. Biotechnology Letters, September 1990, vol. 12, no. 9, p. 667-672. [CrossRef]
MORAES, Ana Lúcia Leite; STECKELBERG, Claúdia; SATO, Hélia Harumi and PINHEIRO, Andrelina. Produção de isomaltulose a partir da transformação enzimática da sacarose, utilizando-se Erwiniasp D12 imobilizada com alginato de cálcio. Ciência e Tecnologia de Alimentos, January-March 2005, vol. 25, no. 1, p. 95-102.
PARK, Y.K.; UEKANE, R.T. and SATO, H.H. Biochemical characterization of a microbial glucosyltransferase that converts sucrose to patatinose. Revista de Microbiologia, April-June 1996, vol. 27, p. 131-136.
RAO, K. Jagannadha; KIM, Chul-Ho and RHEE, Sang-Ki. Statistical optimization of medium for the production of recombinant hirudin from Saccharomyces cerevisiae using response surface methodology. Process Biochemistry, February 2000, vol. 35, no. 7, p. 639-647. [CrossRef]
K.; SUGITANI, T.; MIYATA, Y.; EBASHI, T. and NAKAJIMA, Y. Isolation
and characterization of Isomaltulose-and trehalulose-producing bacteria
WU, L. and BIRCH, R.G. Characterization of Pantoeae dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesis. Journal of Applied Microbiology, July 2004, vol. 97, no. 1, p. 93-103. [CrossRef]
ZHAO, C.; ZHANG, D. and LI, X. Substrate induction of isomaltulose synthase in a newly isolated Klebsiella sp. LX3. Journal of Applied Microbiology, September 2003, vol. 95, no. 3, p. 521-527. [CrossRef]
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