Conversion of Sucrose into Palatinose with Immobilized Serratia Plymuthica Cells

7 Bulgarian Journal of Agricultural Science, (6), 7-76 National Centre for Agrarian Sciences Conversion of Sucrose into Palatinose with Immobilized Serratia Plymuthica Cells A. KRASTANOV, D. BLAZHEVA and Toshiomi YOSHIDA University of Food Technology, Department of Biotechnology, BG - 4 Plovdiv, Bulgaria ICBiotech, Osaka University, Osaka, Japan Abstract KRASTANOV, A., D. BLAZHEVA and Toshiomi YOSHIDA, 6. Conversion of sucrose into palatinose with immobilized Serratia plymuthica cells. Bulg. J. Agric. Sci., : 7-76 Palatinose (isomaltulose) can be used as a substitute of sucrose. Here we describe a simple and effective method of converting concentrated sucrose solutions into palatinose using immobilized Serratia plymuthica cells. The cells were immobilized in alginate beads and on chitin. A conversion rate of up to 99 % was achieved. The process was carried out in a batch and continuous mode in a column. The effect of temperature and flow rate on the conversion rate and the productivity of the column was investigated. Key words: Palatinose, Serratia plymuthica, immobilized cells, alginate, chitin, conversion rate, productivity Introduction Palatinose (isomaltulose, 6-O-αglucopiranosyl-D-fructose) is a structural isomer of sucrose with similar physicochemical, chemical and organoleptic properties. It is not metabolized by the bacteria causing caries and after its consumption the levels of the blood monosaccharides remain low (Goda et al., 983 and Minami et al., 99). Due to this facts palatinose is successfully applied as a sucrose alternative and the interest towards its production on an industrial scale is growing. Lately, the technologies employing immobilized cells for conversion of sucrose into paltinose have almost completely replaced the conventional chemical and enzyme technologies, and the annual production of palatinose exceeds 5 t. At present, as potential microorganisms, which can be used in an immobilized form for the conversion of sucrose into palatinose, are thoroughly studied Protaminobacter rubrum (Mizutani, 99) and Erwinia rhapontici (Cheetham et al., 985). Taking into consideration the investigations of a number of scientists

7 (McAllister et al., 99 and Veronese et al., 999) on the biosynthesis and the properties of α-glucosyltransferase (sucrose isomerase) from Serratia plymuthica, a matter of interest was the application of immobilized cells of this microorganism for bioconversion of sucrose into palatinose. Materials and Methods A. Krastanov, D. Blazheva and T. Yoshida The standard inoculum of Serratia plymuthica ATCC 598 cells was prepared in 5 cm 3 nutrient medium with the following composition (%): sucrose - 5., beef extract -.3, bacto peptone -., yeast extract -.5, NaCl -.3, Na HPO 4 -. until the culture reached OD 6 =.4-.. The inoculum (%) was then transferred into cm 3 nutrient medium with the same composition (ph 6.8) and the cells were cultivated at 8 C. The cell suspension was centrifuged at 8 rpm for min. The biomass was analyzed for enzyme activity and immobilized in alginate and on the surface of chitin. The immobilization in alginate (3%) was realized through adding of the alginate dropwise in a CaCl solution. The immobilization on the surface of chitin was carried out through mixing of the cell suspension (.5 g/cm 3 ) with chitin and glutraldehyde, which was followed by several hours of stirring at room temperature and ph 6.. The conversion of sucrose into palatinose with the immobilized in alginate cells was accomplished in a batch enzyme reactor, and with those immobilized on chitin - in a tubular reactor (H=3 mm, D=5mm, V=5cm 3 ) with equipment for temperature and flow rate control. The enzyme activity was determined through the incubation of cm 3 enzyme solution (or a definite quantity of immobilized preparation) with cm 3 (5 cm 3 for the immobilized preparations) 4% sucrose solution in 5 mm phosphate buffer with ph 6. at temperature 3 C. The palatinose content was measured by the Somogy-Nelson method as a reducing sugar. The activity of the free cells was analyzed after their ultrasonic disintegration and the determination of the sucrose isomerase activity of the liquid phase. The conversion of sucrose into palatinose was studied through thin layer chromatography by the Adachi method (Adachi, 965). Results and Discussion Initially our attention was directed to some aspects of the biosynthesis of intracellular sucrose isomerase by Serratia plymuthica ATCC 598. The data presented on Figure shows, that the enzyme is synthesized along with the biomass until the 8 h of the development of the cells. After that its production was maintained at the same level until the 48 h and decreases after further cultivation. On the basis of this results were immobilized S. plymuthica cells in different growth phases of their development. The immobilized preparations, which were obtained from cell cultivated for -4 h had the highest specific enzyme activity (Figure ). It is interesting, that the specific activity of the cells cultivated for 4 h was lower. This is probably due to some structural changes in the cell wall, which hinder the diffusion of the substrate to the enzyme. In order to increase the specific activity of the immobilized in alginate preparations they were placed in liquid nutrient medium with the same composition and in the same conditions as those during the

Conversion of Sucrose into Palatinose with Immobilized Serratia Plymuthica Cells 73 4 8 9 Biomass, g/l (ww) 8 6 4 4 6 Time, h Biomass, g/l (ww) Activity, U/g (ww) 6 4 8 6 4 Activity, mg (Palatinose)/g(gel).min 8 7 6 5 4 3 6h h 4h 4h Activity of the immobilized cells Activity of the immobilized cells after Fig.. Activity of the immobilized preparations with different cell age Fig.. Microbial growth and biosynthesis of intracellular sucrose isomerase during the cultivation of Serratia plymuthica ATCC 598 cultivation of the free cells. Therefore, for 8 h of treatment the specific activity increased with 5-3% (Figure ). Further treatment led to the development of cells outside the gel, which was especially well exhibited during the immobilization of cells, cultivated for 4 h. These preparations showed the biggest increase of specific activity (resulting from the increase of the viable cell counts in the gel matrix), but they proved to be instable because of disintegration of the gel beads. With the obtained immobilized cells was conducted a successful conversion of sucrose (% solution) into palatinose in a batch reactor. The results are presented on Figure 3 and Figure 4. A dependency was determined between the conversion rate and the gel: substrate solution proportion (m/v). With the decrease of the quantity of the substrate solution in relation to the weight of the immobilized preparation a higher conversion rate could be achieved for a shorter reaction time. When this proportion was :, the gel beads were - mm in diameter, at the optimal temperature 4-43 C, for 6 min was reached 99% conversion rate (Figure 4). The described immobilized preparations demonstrated good operational stability. The activity of the immobilized cells

74 A. Krastanov, D. Blazheva and T. Yoshida :7 :4 :4 after 6h Conversion rate, % 45 4 Palatinose content, mg/ml 35 3 5 5 8 99 5 5 Time, min 75 5 Fig. 3. Conversion of sucrose into palatinose with immobilized in alginate beads cells at different gel: substrate solution proportion, m/v Fig. 4. Conversion of sucrose into palatinose with immobilized in alginate beads cells at gel: substrate solution proportion (m/v) : (- 3 min reaction time; - 5 min reaction time) did not change significantly after 6 h of continuous conversion of sucrose into palatinose (Figure 5). The conversion of sucrose into palatinose with immobilized on the surface of chitin S. plymuthica cells was conducted in a continuous tubular reactor. A flow rate of ml/min (. column volumes/ h) provided simultaneously the best productivity and the highest content of palatinose in the reaction medium (Figure 6). The obtained conversion rate, palatinose content in the eluate respectively, was higher than the described in literature analogous systems, employing immobilized Erwinia rhapontici cells (Cheetham, 98). The influence of temperature and sucrose concentration on the conversion rate and the productivity of the column with immobilized on chitin S. plymuthica cells was investigated. Maximal productivity and conversion rate were detected at 4-43 o C. The conversion rate of the % sucrose solutions was almost two times bigger than that of the 4% solutions and consequently the concentration of the sucrose solutions had comparatively weak effect on the productivity of the column (Figure 7). A better stability

Conversion of Sucrose into Palatinose with Immobilized Serratia Plymuthica Cells 75 6 8 5 4 Productivity, mg/min Palatinose content, mg/ml Activity, % 6 4 3 5 3 35 4 45 5 Temperature, o C.5.5.5 Flow rate, ml/min Fig. 5. Temperature profile of the immobilized in alginate Serratia plymuthica cells Fig. 6. Influence of the flow rate on the productivity of the column reactor and the palatinose content in the outlet solution after the immobilization Serratia plymuthica cells on chitin Conversion rate, % 5 45 4 35 3 5 5 5 4 6 8 Productivity, g/l 3.5 3.5.5.5 4 6 8 Temperature, o C % sucrose solution Temperature, o C % sucrose solution 4% sucrose solution 4% sucrose solution a b Fig. 7. Influence of the temperature on the conversion rate (a) and the productivity (b) of a column reactor with immobilized on chitin Serratia plymuthica cells

76 of the productivity was observed when the process was conducted with more concentrated sucrose solutions at temperatures higher than the optimal. Conclusions As a result of these experiments can be concluded, that the described preparations of immobilized in alginate and on chitin S. plymuthica cells can successfully be used for production of palatinose from concentrated sucrose solutions (up to 4%) in batch and continuous enzyme reactors. The immobilization methods are easy to execute and the produced preparations are stable and have high specific activity. This enables the realization of the conversion process at comparatively high temperatures, which given the specific character of the employed products, has a significant technological importance. Acknowledgement The National Science Fund, Bulgaria sponsored this work. References Adachi, S., 964. Thin-layer chromatography of carbohydrates in the presence of bi-sulfite. Journal of Chromatgraphy, 7: 95-99. A. Krastanov, D. Blazheva and T. Yoshida Cheetham, P., C. E. Imber, and J. Isherwood, 98. The formation of isomaltulose by immobilized Erwinia rhapontici. Nature, 99: 68-63. Cheetham, P., C. Garrett and J. Clark, 985. Isomaltulose production using immobilized cells. Biotechnology and Bioengineering, 7: 47-48. Goda, T. and N. Hosoya, 983. Hydrolysis of palatinose by rat intestinal sucraseisomaltase complex. Journal of Japanese Society of Nutrition and Food Science, 36: 69-73. McAllister, M, C. T. Kelly, E. Doyle and W. M. Fogarty, 99. The isomaltulose synthesizing enzyme of Serratia plymuthica. Biotechnology Letters, (9): 667-67. Minami, T, T. Fujiwara, T. Ooshima, Y. Nakajima and S. Hamada, 99. Interaction of structural isomers of sucrose in the reaction between sucrose and glucosyltransferases from mutans streptococci. Oral Microbiology and Immunology, 5: 89-94. Mitzutani, T., 99. Preparation and use of palatinose oligosaccharides. New Food Industry, 33: 9-. Veronese, T and P. Perlot, 999. Mechanism of sucrose conversion by the sucrose isomerase of Serratia plymuthica ATCC 598. Enzyme and Microbial Technology, 4: 63-69. Received June,, 6; accepted September, 8, 6.