HUNGARIAN JOURNAL OF INDUSTRY AND CHEMISTRY Vol. 47(2) pp. 5–10 (2019) hjic.mk.uni-pannon.hu DOI: 10.33927/hjic-2019-14 PRODUCTION OF THE ENZYME CYCLODEXTRIN GLYCOSYLTRANS- FERASE USING DIFFERENT FERMENTATION TECHNIQUES RÉKA CZINKÓCZKY1 AND ÁRON NÉMETH *1 1Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Műegyetem rkp. 3, Budapest, 1111, HUNGARY Cyclodextrins produced by cyclodextrin glycosyltransferase (CGTase) are widely used in the pharmaceutical industry to improve the solubility of drug substances as well as protect them against oxidation. The use of this enzyme in the cos- metics industry is also significant. CGTase is an enzyme that belongs to the α-amylase family, which is part of the group of non-Leloir glycosyltransferases. Enzyme-catalysed transglycosylation reactions may involve cyclization, coupling and disproportionation processes. The enzyme CGTase is mostly used to produce cyclodextrins (CDs). CGTase can produce α-, β- and γ-CDs during transglycosylation reactions, depending on the number of glucopyranose units involved (6, 7 or 8). The enzyme CGTase can also be used for enzymatic bioconversion, e.g., in the development of alternative sweeten- ers, where the bitter aftertaste of the product is reduced during the enzymatic bioconversion of steviol glycosides, thereby obtaining an even sweeter and more advantageous material. In our research, the enzyme CGTase was produced using different fermentation techniques to compare the activity and amount of CGTase produced by each process and optimize the subsequently planned scale-up. In our studies, the strain DSM 13 of Bacillus licheniformi s was used, which produced CGTase extracellularly. During the experiments the batch, fed-batch and semi-continuous fermentation techniques were compared in terms of enzymatic production. All cultivation processes were carried out in a desktop lab scale fermenter. Keywords: Cyclodextrin glycosyltransferase, fermentation, cyclodextrins, Bacillus licheniformis 1. Introduction Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is a starch-degrading enzyme, which is a member of the α-amylase family. The formal name of CGTase is [1,4-α- D-glucan 4-α-D-(1,4-α-glucano)-transferase(cyclizing). Kuriki et al. [1] reported that CGTase has the same four highly conserved regions as the α-amylases. CGTase catalyses four kinds of transglycosylation reactions (Fig. 1): cyclization, coupling, disproportionation and hydrol- ysis. These reactions are all transglycosylations, in which cyclization is intramolecular, coupling and disproportion- ation are intermolecular, and hydrolysis is the conversion of sugar to H2O [2]. The formation of cyclodextrin (CD) by the enzyme CGTase is an intermolecular transglyco- sylation reaction [3]. Many microorganisms are capable of producing CG- Tase, e.g., Bacillus macerans [4, 5], Bacillus amyloliq- uefaciens [6], Bacillus clarkii [7], Bacillus megaterium [8], Bacillus subtilis [9], Bacillus licheniformis [10, 11], Bacillus firmus [12,13], Bacillus circulans [14,15], Bacil- lus ohbensis [16, 17], Geobacillus stearothermophilus [18], Thermoanaerobacter sp. [19], Klebsiella pneumo- niae, and Klebsiella oxytoca [20]. *Correspondence: naron@f-labor.mkt.bme.hu Figure 1: CGTase-catalysed reactions: A: cyclization, B: coupling, C: disproportionation, D: hydrolysis https://doi.org/10.33927/hjic-2019-14 mailto:naron@f-labor.mkt.bme.hu 6 CZINKÓCZKY AND NÉMETH Figure 2: General structure of cyclodextrins (n: number of glucopyranose units, n = 6 α-CD, n = 7 β-CD, n = 8 γ-CD) (Figure adapted from: https://commons.wikimedia.org/wiki/File:Cyclodextrin.svg CC BY-SA 3.0) (08.07.2019) ) The molecular weight of CGTases may vary from 60 to 110 kDa, typically its proteins have a mass of 75 kDa [21]. The most important demand of metal ions for them is Ca2+, which protects the protein against heat denatura- tion. Most CGTases are strongly inhibited by Zn2+, Cu2+ and Fe2+ [22]. Cyclodextrins, produced from starch or its deriva- tives via enzymatic conversion, proceed through an in- tramolecular transglycosylation reaction using CGTases and to a lesser extent α-amylases [3]. They are cyclic oligosaccharides composed of α-1,4-glycosidic-linked glucosyl residues [23]. Three different types of cyclodex- trins exist and are characterised according to the num- ber of glucosyl residues in the molecule: α-, β- and γ- cyclodextrins consist of 6, 7 and 8 glucose units, respec- tively (Fig. 2). Cyclodextrins are cyclic molecules with a hydrophilic exterior and a hydrophobic cavity that en- ables them to form specific inclusion complexes with small hydrophobic molecules [24]. Cyclodextrins are chi- ral non-reducing oligosaccharides. Glucose is the decom- position product of all cyclodextrins in acidic solutions. The rate of hydrolysis follows the order of γ > β > α. Under acidic conditions, cyclodextrins are more slowly hydrolyzed than maltooligosaccharides. The gly- cosidic bonds in the cyclodextrins can be hydrolyzed by α-amylase, but β-amylase is unable to perform this hy- drolysis. The rate of enzymatic hydrolysis is the fastest for γ-CD, followed by β- then α-CD. All CDs are very stable and soluble in alkaline solutions at high pH. CDs are more resistant to acid or alkaline degradation than starch. CDs do not even degradate at temperatures as high as that of caramelization (> 200 ◦C, sterilization) under both dry or aqueous conditions of between pH 2 and 12. They are also stable up to 250 ◦C under an inert atmo- sphere of, for example, nitrogen [20, 25, 26]. The widespread use of cyclodextrins is due to their specific structure. Since each guest molecule is uniquely surrounded by the CD (or one of its derivatives), it is microencapsulated from a molecular microscopic point of view. This can result in beneficial changes to the chemical and physical properties of guest molecules, e.g., light- or oxygen-sensitive materials can be stabilized; very volatile substances fixed; the chemical reactivity of molecules modified; the solubility of materials improved; changes between phases achieved from liquid substances into powders; degradation of microorganisms avoided; bad smells and tastes masked; and pigments or colors of materials coated. As a result of these characteristics, CDs (and their derivatives) can be used in analytical chemistry, agriculture, the pharmaceutical as well as food industries and other masking areas. CGTase can be used for the transglycosylation of stevioside to rebaudioside through which the edulcorant quality can also be improved by increasing the substitution of steviol glycoside with the help of cornstarch hydrolyzate and CGTase [27–30]. 2. Materials and Methods 2.1 Cultivation of the bacteria The applied bacterial strain was Bacillus licheniformis B.01470 (DSM 13) purchased from the National Col- lection of Agricultural and Industrial Microorganisms in Hungary. Bacillus licheniformis is a Gram-positive, rod- shaped, endospore forming, facultatively anaerobic bac- teria. Nutrient agar was used to maintain the bacterium in Petri dishes [31]. In our research, three types of fermenta- tion techniques for the production of CGTase were com- pared: batch, fed-batch, and semi-continuous fermenta- tion techniques. All cultivation processes were carried out in a benchtop lab scale fermenter (Fig. 3) (Biostat Q, B. Braun Biotech International, Germany). In the fed-batch fermentation, after 24 hours 15 v/v % of the medium was fed into the bioreactor. During the semi-continuous fer- mentation at the end of each cycle, 80 % of the broth was replaced by fresh media. For the experiments in the bioreactor, Horikoshi II medium was used for the cultivation of bacteria contain- ing 1.0 % soluble starch, 0.5 % peptone, 0.5 % yeast ex- tract, 0.1 % K2HPO4, 0.02 % MgSO4 • 7 H2O, and 1.0 % Na2CO3 (all concentrations are given in w/v in distilled water) [32]. Hungarian Journal of Industry and Chemistry https://creativecommons.org/licenses/by-sa/3.0/deed.en PRODUCTION OF THE ENZYME CYCLODEXTRIN GLYCOSYLTRANSFERASE 7 Figure 3: The bioreactor used in the experiments 2.2 The modelling of microbial growth In order to monitor the growth of bacterial cells, samples were taken during fermentations and the optical density (OD) measured at 600 nm. Microbial growth is described by µx = 1 x dx dt , (1) where µx is the specific growth rate of the microbe. It was evaluated through fitting the generalized logistic function Z = Zmax 1 + exp(a + bt + ct2 + dt3) (2) to the measured cell dry weight (CDW) values (calculated from at OD600). To fit the curve, SigmaPlot Version 12.0 software was applied. If the coefficient of determination (R2) was not high enough, the last two members of the generalized logistic function were omitted resulting in Z = Zmax 1 + exp(a + bt) , (3) Figure 4: Colorimetric analysis of CGTase activity which also corresponds to the modified Monod model. The derivative of the fitted function is dZ dt = −Z ( 1 − Z Zmax ) dH dt (4) where dH dt = b + 2ct + 3dt2 (5) is the derivative of the internal function. The auxiliary variable Z in Eq. 2, 3, and 4 was x (biomass in g/L), S (substrate in g/L), and Pi (product in g/L), respectively, while Zmax was xmax, S0 and Pi,max, respectively. If the fit was successful, then, by using determined constants of the model, the velocities and specific growth rates could be calculated by derivation from Eq. 4. 2.3 Measurement of CGTase activity During the fermentations, samples were regularly ex- tracted into Eppendorf tubes, which were centrifuged at 12,000 rpm for 6 minutes, then the cell-free supernatants were used to determine the enzyme activity. The measurement of extracellular CGTase activity was adapted from the method of Kaneko et al. [33] with slight modifications (with a reduced concentration of phenolphtalein). The colorimetric reaction (Fig. 4) was measured by a spectrophotometer at 550 nm. The experiments were conducted in 15 ml centrifuge tubes in a water bath at 40 ◦C. First, 4.5 ml 50 mM Tris- HCl buffer (pH = 9) was added containing a 1 % (w/v) water-soluble starch suspension, then 0.5 ml of cell-free supernatant containing the extracellular CGTase enzyme was introduced and homogenized thoroughly with a vor- tex mixer. Then four 0.5 ml samples were taken from each tube which were boiled for 5 minutes to inactivate the en- zyme. The boiled samples were transferred into 2 ml cu- vettes that contained a staining solution (1.2 ml 0.06 mM phenolphthalein in 0.5 M Na2CO3 solution). Four ab- sorbances at 550 nm of a given assay were plotted against time and the gradient (mmol/min) converted into enzyme activity with the help of a molar extinction coefficient (32,263 M−1cm−1) resulting in the CGTase activity in unit/ml supernatant. 47(2) pp. 5–10 (2019) 8 CZINKÓCZKY AND NÉMETH Figure 5: Microbial growth during the batch fermentation Figure 6: Microbial growth during the fed-batch fermen- tation 3. Results and Discussion 3.1 Batch fermentation Fig. 5 shows the microbial growth during the batch fer- mentation and also represents the changes in the specific growth rate. The maximum value of the specific growth rate was 0.53 1/h. At the end of the fermentation, the fi- nal activity of CGTase was 0.3 U/ml and the productivity was 11.8 mU/(ml h). 3.2 Fed-batch fermentation Fig. 6 represents microbial growth during the fed-batch fermentation. Unfortunately, due to poorly scheduled sampling, it was not possible to adjust the generalized lo- gistic equation, therefore, it was impossible to calculate the specific growth rate. A fresh medium of 15 % was in- jected after 24 hours. The final enzyme activity was 0.5 U/ml and the enzyme productivity was 12.3 mU/(ml h). 3.3 Semi-continuous fermentation The semi-continuous fermentation is shown in Fig. 7, which consisted of 3 cycles. The highest value of the maximum growth rate was during the first cycle (0.5 1/h). As the fermentation progressed, the maximum specific growth rate decreased. Figure 7: Optical density and specific growth rate changes during the semi-continuous fermentation Figure 8: CGTase activities at the end of each cycle Fig. 8 shows that the enzyme activity of CGTase in- creased as the fermentation progressed. 3.4 Comparison of the different fermentation techniques Table 1 summarizes the enzyme activities and productiv- ities achieved by each fermentation technique. The maxi- mum specific growth rates reached in the batch and semi- continuous fermentations were approximately the same, which is characteristic of when the microorganism can multiply. The enzyme activities at the end of the fermentations Table 1: Comparison between the results of the different fermentation techniques Type µmax [1/h] Final enzyme activity [U/ml] Productivity [mU/(ml h)] Batch 0.53 0.3 11.8 Fed-batch n.d. 0.5 12.3 Semi- continuous 0.50 2.4 29.95 ± 0.3 Hungarian Journal of Industry and Chemistry PRODUCTION OF THE ENZYME CYCLODEXTRIN GLYCOSYLTRANSFERASE 9 rose as the complexity of the fermentation technique in- creased. While the fed-batch fermentation elongated the declining phase of the microbial growth cycle, in the semi-continuous fermentation technique an attempt was made to operate in the exponential growth phase. It is as- sumed that this difference caused the higher activity and productivity in the case of the semi-continuous fermenta- tion. 4. Conclusion In our experiments, the effect of the fermentation tech- nique on the activity of the produced enzyme CGTase was investigated. There was no significant difference between the activities of the produced CGTase and productivities of the systems when the batch and fed-batch fermenta- tions were compared. In contrast, bacteria produced a much more active enzyme during the semi-continuous fermentation, moreover, the productivity of this system was also significantly higher than that of the other two fermentation techniques. From the results, it can be assumed that the microbes produce the enzyme during the exponential growth phase, since no significant difference was observed between the batch and fed-batch fermentations. Meanwhile, a repeated exponential growth phase resulted in a much higher activity and productivity. This suggests that CG- Tase production follows growth associated-type product formation. Acknowledgement The research was supported by the Gedeon Richter’s Talentum Foundation, founded by Gedeon Richter Plc. (Gedeon Richter PhD fellowship). REFERENCES [1] Kuriki, T.; Imanaka, T.: The concept of the α- amylase family: Structural similarity and common catalytic mechanism, J. Biosci. Bioeng. 1999 87(5), 557–565 DOI: 10.1016/S1389-1723(99)80114-5 [2] Kobayashi, S.: Cyclodextrin producing enzyme (CGTase) in Park, K.-H.; Robyt, J. F.; Choi, Y.- D. (eds.): Enzymes for carbohydrate engineering (Progress in Biotechnology) 1996 12 23–41 (Else- vier, Amsterdam, The Netherlands) ISBN: 978-0-444- 82408-0 DOI: 10.1016/S0921-0423(96)80360-1 [3] van der Veen, B. A.; van Alebeek, G.-J. W. M.; Uitdehaag, J. C. M.; Dijkstra, B. W.; Dijkhuizen, L.: The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms Eur. J. Biochem. 2000 267(3) 658–665 DOI: 10.1046/j.1432-1327.2000.01031.x [4] Isao, K.; Yoshida, N.: Method of producing β- cyclodextrin 1996 US 5556775, United States Patent and Trademark Office https://patents. google.com/patent/US5556775A/en [5] Shieh, W. J.; Hedges, A. R.: Process for producing alpha-cyclodextrin using cyclomaltodextrin glucan- otransferase in presence of cyclohexane 1994 US 5326701, United States Patent and Trademark Of- fice https://patents.google.com/patent/US5326701A/ en [6] Yu, E. K. C.; Aoki, H.; Misawa, M.: Specific alpha-cyclodextrin production by a novel ther- mostable cyclodextrin glycosyltransferase Appl. Microbiol. Biotechnol. 1988 28(4–5) 377–379 DOI: 10.1007/BF00268199 [7] Wu, D.; Chen, S.; Wang, N.; Chen, J.; Wu, J.: Gamma-cyclodextrin production using cyclodex- trin glycosyltransferase from Bacillus clarkii 7364 Appl. Biochem. Biotechnol. 2012 167(7) 1954–1962 DOI: 10.1007/s12010-012-9741-5 [8] Pishtiyski, I.; Popova, V.; Zhekova, B; Characteri- zation of cyclodextrin glucanotransferase produced by Bacillus megaterium Appl. Biochem. Biotechnol. 2008 144 263–272 DOI: 10.1007/s12010-007-8009-y [9] Cheirsilp, B.; Kitcha, S.; Maneerat, S.: Kinetic characteristics of β-cyclodextrin production by cy- clodextrin glycosyltransferase from newly isolated Bacillus sp. C26 Electron. J. Biotechnol. 2010 13(4) DOI: 10.2225/vol13-issue4-fulltext-6 [10] Bonilha, P. R. M.; Menocci, V.; Goulart, A. J.; Polizeli, M. L. T. M.; Monti, R.: Cyclodextrin gly- cosyltransferase from Bacillus licheniformis: Opti- mization of production and its properties Braz. J. Microbiol. 2006 37(3) 317–323 DOI: 10.1590/S1517- 83822006000300022 [11] Thombre, R. S.; Kanekar, P. P.: Synthesis of β- Cyclodextrin by Cyclodextrin glycosyl transferase produced by Bacillus licheniformis MCM–B1010 J. Microbiol. Biotech. Res. 2013 3(1) 57–60 [12] Matioli, G.; Zanin, G. M.; De Moraes, F. F.: In- fluence of substrate and product concentrations on the production of cyclodextrins by cgtase of Bacil- lus firmus, strain No. 37 Appl. Biochem. Biotechnol. 2002 98(1–9) 947–961 DOI: 10.1385/ABAB:98-100:1- 9:947 [13] Gawande, B. N.; Goel, A.; Patkar, A. Y.; Nene, S. N.: Purifcation and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus Appl. Microbiol. Biotechnol. 1999 51 504–509 DOI: 10.1007/s002530051424 [14] Pinto, F. S. T.; Flôres, S. H.; Ayub, M. A. Z.; Hertz, P. F.: Production of cyclodextrin glycosyltransferase by alkaliphilic Bacillus circulans in submerged and solid-state cultivation Bioprocess Biosyst. Eng. 2007 30(5) 377–382 DOI: 10.1007/s00449-007-0134-z [15] Iyer, J. L.; Shetty, P.; Pai, J. S.: Immobilisa- tion of cyclodextrin glucanotransferase from Bacil- lus circulans ATCC 21783 on purified seasand J. Ind. Microbiol. Biotechnol. 2003 30(1) 47–51 DOI: 10.1007/s10295-002-0009-x 47(2) pp. 5–10 (2019) https://doi.org/10.1016/S1389-1723(99)80114-5 https://doi.org/10.1016/S0921-0423(96)80360-1 https://doi.org/10.1046/j.1432-1327.2000.01031.x https://patents.google.com/patent/US5556775A/en https://patents.google.com/patent/US5556775A/en https://patents.google.com/patent/US5326701A/en https://patents.google.com/patent/US5326701A/en https://doi.org/10.1007/BF00268199 https://doi.org/10.1007/BF00268199 https://doi.org/10.1007/s12010-012-9741-5 https://doi.org/10.1007/s12010-007-8009-y https://doi.org/10.2225/vol13-issue4-fulltext-6 https://doi.org/10.1590/S1517-83822006000300022 https://doi.org/10.1590/S1517-83822006000300022 https://doi.org/10.1385/ABAB:98-100:1-9:947 https://doi.org/10.1385/ABAB:98-100:1-9:947 https://doi.org/10.1007/s002530051424 https://doi.org/10.1007/s002530051424 https://doi.org/10.1007/s00449-007-0134-z https://doi.org/10.1007/s10295-002-0009-x https://doi.org/10.1007/s10295-002-0009-x 10 CZINKÓCZKY AND NÉMETH [16] Nishida, T.; Nakamura, A.; Masaki, H.; Uozumi, T.: Regulation of cyclodextrin glucanotransferase synthesis in Bacillus ohbensis FEMS Microbiol. Lett. 1997 149(2) 221–226 DOI: 10.1111/j.1574- 6968.1997.tb10332.x [17] Cami, P. H.; Majou, D. B.: Process for production of cyclodextrins 1994 US 5376537, United States Patent and Trademark Office https://patents. google.com/patent/US5376537A/en [18] Shiosaka, M.: Heat stable cyclodextrin glycosyl- transferase 1976 US 3988206, United States Patent and Trademark Office https://patents.google.com/ patent/US3988206A/en [19] Martín, M. T.; Plou, F. J.; Alcade, M.; Balles- teros, A.: Immobilization on Eupergit C of cy- clodextrin glucosyltransferase ( CGTase ) and prop- erties of the immobilized biocatalyst J. Mol. Catal. B Enzym. 2003 21(4-6) 299–308 DOI: 10.1016/S1381- 1177(02)00264-3 [20] Wimmer, T.: Cyclodextrins in Ullmann’s Ency- clopedia of Industrial Chemistry 2012 11 DOI: 10.1002/14356007.e08_e02 [21] Uitdehaag, J. C. M.; Kalk, K. H.; van der Veen, B. A.; Dijkhuizen, L.; Dijkstra, B. W.: The cycliza- tion mechanism of cyclodextrin glycosyltransferase (CGTase) as revealed by a γ-cyclodextrin-CGTase complex at 1.8-Å resolution J. Biol. Chem. 1999 274(49) 34868–34876 DOI: 10.1074/jbc.274.49.34868 [22] Thangadurai, D.; Sangeetha, J. (eds.): Industrial Biotechnology, Sustainable Production and Biore- source Utilization 2017 (Apple Academic Press, Oakville, Canada), ISBN: 978-1-771-88262-0 [23] Biwer, A.; Antranikian, G.; Heinzle, E.: Enzy- matic production of cyclodextrins Appl. Microbiol. Biotechnol. 2002 59(6) 609–617 DOI: 10.1007/s00253- 002-1057-x [24] Szejtli, J.: Introduction and general overview of cy- clodextrin chemistry Chem. Rev. 1998 98(5) 1743– 1753 DOI: 10.1021/cr970022c [25] Saenger, W.: Cyclodextrin inclusion compounds in research and industry Angew. Chem. Int. Ed. Engl. 1980 19(5) 344–362 DOI: 10.1002/anie.198003441 [26] Szejtli, J.: The cyclodextrins and their applications in biotechnology Carbohydr. Polym. 1990 12(4) 375–392 DOI: 10.1016/0144-8617(90)90088-A [27] Del Valle, E. M. M.: Cyclodextrins and their uses: a review Process Biochem. 2004 39(9) 1033–1046 DOI: 10.1016/S0032-9592(03)00258-9 [28] Singh, M.; Sharma, R.; Banerjee, U. C.: Biotech- nological applications of cyclodextrins Biotech- nol. Adv. 2002 20(5–6) 341–359 DOI: 10.1016/S0734- 9750(02)00020-4 [29] Li, S.; Li, W.; Xiao, Q. Y.; Xia, Y.: Transglycosyla- tion of stevioside to improve the edulcorant quality by lower substitution using cornstarch hydrolyzate and CGTase Food Chem. 2013 138(2–3) 2064–2069 DOI: 10.1016/j.foodchem.2012.10.124 [30] Kochikyan, V. T.; Markosyan, A. A.; Abelyan, L. A.; Balayan A. M.; Abelyan, V. A.: Combined en- zymatic modification of stevioside and rebaudioside A Appl. Biochem. Microbiol. 2006 42(1) 37–43 DOI: 10.1134/S0003683806010030 [31] DSMZ Nutrient agar (n.d.) Retrieved from https://www.dsmz.de/microorganisms/medium/pdf/ DSMZ_Medium1.pdf, date accessed: 2019.10 [32] Park, C. S.; Park, K. H.; Kim, S. H.: A rapid screen- ing method for alkaline β-cyclodextrin glucan- otransferase using phenolphthalein-methyl orange- containing solid medium Agric. Biol. Chem. 1989 53(4) 1167–1169 DOI: 10.1080/00021369.1989.10869443 [33] Kaneko, T.; Kato, T.; Nakamura, N.; Horikoshi, K.: Spectrophotmetric determination of cyclization ac- tivity of β-cyclodextrin-forming cyclomaltodextrin glucanotransferase J. Jpn. Soc. Starch Sci. 1987 34(1) 45–48 DOI: 10.5458/jag1972.34.45 Hungarian Journal of Industry and Chemistry https://doi.org/10.1111/j.1574-6968.1997.tb10332.x https://doi.org/10.1111/j.1574-6968.1997.tb10332.x https://patents.google.com/patent/US5376537A/en https://patents.google.com/patent/US5376537A/en https://patents.google.com/patent/US3988206A/en https://patents.google.com/patent/US3988206A/en https://doi.org/10.1016/S1381-1177(02)00264-3 https://doi.org/10.1016/S1381-1177(02)00264-3 https://doi.org/10.1002/14356007.e08_e02 https://doi.org/10.1002/14356007.e08_e02 https://doi.org/10.1074/jbc.274.49.34868 https://doi.org/10.1007/s00253-002-1057-x https://doi.org/10.1007/s00253-002-1057-x https://doi.org/10.1021/cr970022c https://doi.org/10.1002/anie.198003441 https://doi.org/10.1016/0144-8617(90)90088-A https://doi.org/10.1016/S0032-9592(03)00258-9 https://doi.org/10.1016/S0734-9750(02)00020-4 https://doi.org/10.1016/S0734-9750(02)00020-4 https://doi.org/10.1016/j.foodchem.2012.10.124 https://doi.org/10.1134/S0003683806010030 https://doi.org/10.1134/S0003683806010030 https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium1.pdf https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium1.pdf https://doi.org/10.1080/00021369.1989.10869443 https://doi.org/10.5458/jag1972.34.45 Introduction Materials and Methods Cultivation of the bacteria The modelling of microbial growth Measurement of CGTase activity Results and Discussion Batch fermentation Fed-batch fermentation Semi-continuous fermentation Comparison of the different fermentation techniques Conclusion