DOI QR코드

DOI QR Code

Galactooligosaccharide Synthesis by Active ${\beta}$-Galactosidase Inclusion Bodies-Containing Escherichia coli Cells

  • Lee, Sang-Eun (Department of Biotechnology, Chungju National University) ;
  • Seo, Hyeon-Beom (Department of Biotechnology, Chungju National University) ;
  • Kim, Hye-Ji (Department of Biotechnology, Chungju National University) ;
  • Yeon, Ji-Hyeon (Department of Biotechnology, Chungju National University) ;
  • Jung, Kyung-Hwan (Department of Biotechnology, Chungju National University)
  • Received : 2011.05.13
  • Accepted : 2011.07.23
  • Published : 2011.11.28

Abstract

In this study, a galactooligosaccharide (GOS) was synthesized using active ${\beta}$-galactosidase (${\beta}$-gal) inclusion bodies (IBs)-containing Escherichia coli (E. coli) cells. Analysis by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry revealed that a trisaccharide was the major constituent of the synthesized GOS mixture. Additionally, the optimal pH, lactose concentration, amounts of E. coli ${\beta}$-gal IBs, and temperature for GOS synthesis were 7.5, 500 g/l, 3.2 U/ml, and $37^{\circ}C$, respectively. The total GOS yield from 500 g/l of lactose under these optimal conditions was about 32%, which corresponded to 160.4 g/l of GOS. Western blot analyses revealed that ${\beta}$-gal IBs were gradually destroyed during the reaction. In addition, when both the reaction mixture and E. coli ${\beta}$-gal hydrolysate were analyzed by high-performance thin-layer chromatography (HP-TLC), the trisaccharide was determined to be galactosyl lactose, indicating that a galactose moiety was most likely transferred to a lactose molecule during GOS synthesis. This GOS synthesis system might be useful for the synthesis of galactosylated drugs, which have recently received significant attention owing to the ability of the galactose molecules to improve the drugs solubility while decreasing their toxicity. ${\beta}$-Gal IB utilization is potentially a more convenient and economic approach to enzymatic GOS synthesis, since no enzyme purification steps after the transgalactosylation reaction would be required.

Keywords

References

  1. Ahmed, S. A. and L. A. Smith. 2000. Light chain of botulinum A neurotoxin expressed as an inclusion body from a synthetic gene is catalytically and functionally active. J. Protein Chem. 19: 475-487. https://doi.org/10.1023/A:1026549431380
  2. Arie, J. P., M. Miot, N. Sassoon, and J. M. Betton. 2006. Formation of active inclusion bodies in the periplasm of Escherichia coli. Mol. Microbiol. 62: 427-437. https://doi.org/10.1111/j.1365-2958.2006.05394.x
  3. Botelho-Cunha, V. A., M. Mateus, J. C. C. Petrus, and M. N. de Pinho. 2010. Tailoring the enzymatic synthesis and nanofiltration fractionation of galacto-oligosaccharides. Biochem. Eng. J. 50: 29-36. https://doi.org/10.1016/j.bej.2010.03.001
  4. Cardelle-Cobas, A., M. Villamiel, A. Olano, and N. Corzo. 2008. Study of galacto-oligosaccharide formation from lactose using Pectinex Ultra SP-L. J. Sci. Food Agric. 88: 954-961. https://doi.org/10.1002/jsfa.3173
  5. Crittenden, R. G. and M. J. Playne. 1996. Production, properties and applications of food-grade oligosaccharides. Trends Food Sci. Technol. 7: 353-361. https://doi.org/10.1016/S0924-2244(96)10038-8
  6. del-Val, M. I., C. G. Hill Jr., J. Jiménez-Barbero, and C. Otero. 2001. Selective enzymatic synthesis of 6'-galactosyl lactose by Pectinex Ultra SP in water. Biotechnol. Lett. 23: 1921-1924. https://doi.org/10.1023/A:1013794019371
  7. Ebrahimi, M., L. Placido, L. Engel, K. S. Ashaghi, and P. Czermak. 2010. A novel ceramic membrane reactor system for the continuous enzymatic synthesis of oligosaccharides. Desalination 250: 1105-1108. https://doi.org/10.1016/j.desal.2009.09.118
  8. Garcia-Fruitos, E., A. Aris, and A. Villaverde. 2007. Localization of functional polypeptides in bacterial inclusion bodies. Appl. Environ. Microbiol. 73: 289-294. https://doi.org/10.1128/AEM.01952-06
  9. Garcia-Fruitos, E., M. M. Carrio, A. Aris, and A. Villaverde. 2005. Folding of a misfolding-prone $\beta$-galactosidase in absence of DnaK. Biotechnol. Bioeng. 90: 869-875. https://doi.org/10.1002/bit.20496
  10. Garcia-Fruitos, E., N. Gonzalez-Montalban, M. Morell, A. Vera, R. M. Ferraz, A. Aris, et al. 2005. Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microb. Cell Fact. 4: 27. https://doi.org/10.1186/1475-2859-4-27
  11. Gosling, A., G. W. Stevens, A. R. Barber, S. E. Kentish, and S. L. Gras. 2010. Recent advances refining galactooligosaccharide production from lactose. Food Chem. 121: 307-318. https://doi.org/10.1016/j.foodchem.2009.12.063
  12. Jaskolla, T., B. Fuchs, M. Karas, and J. Schiller. 2009. The new matrix 4-chloro-$\alpha$-cyanocinnamic acid allows the detection of phosphatidylethanolamine chloramines by MALDI-TOF mass spectrometry. J. Am. Soc. Mass Spectrom. 20: 867-874. https://doi.org/10.1016/j.jasms.2008.12.028
  13. Jung, K.-H. 2008. Enhanced enzyme activities of inclusion bodies of recombinant $\beta$-galactosidase via the addition of inducer analog after L-arabinose induction in the araBAD promoter system of Escherichia coli. J. Microbiol. Biotechnol. 18: 434- 442.
  14. Jung, K.-H., J.-H. Yeon, S.-K. Moon, and J. H. Choi. 2008. Methyl $\alpha$-D-glucopyranoside enhances the enzymatic activity of recombinant $\beta$-galactosidase inclusion bodies in the araBAD promoter system of Escherichia coli. J. Ind. Microbiol. Biotechnol. 35: 695-701. https://doi.org/10.1007/s10295-008-0329-6
  15. Li, Z., M. Xiao, L. Lu, and Y. Li. 2008. Production of nonmonosaccharide and high-purity galactooligosaccharides by immobilized enzyme catalysis and fermentation with immobilized yeast cells. Process Biochem. 43: 896-899. https://doi.org/10.1016/j.procbio.2008.04.016
  16. Lotti, M. 2011. Bacterial inclusion bodies as active and dynamic protein ensembles. FEBS J. 278: 2407. https://doi.org/10.1111/j.1742-4658.2011.08162.x
  17. Mahoney, R. R. 1998. Galactosyl-oligosaccharide formation during lactose hydrolysis: A review. Food Chem. 63: 147-154. https://doi.org/10.1016/S0308-8146(98)00020-X
  18. Martínez-Villaluenga, C., A. Cardelle-Cobas, N. Corzo, A. Olano, and M. Villamiel. 2008. Optimization of conditions for galactooligosaccharide synthesis during lactose hydrolysis by bgalactosidase from Kluyveromyces lactis (Lactozym 3000 L HP G). Food Chem. 107: 258-264. https://doi.org/10.1016/j.foodchem.2007.08.011
  19. Nahalka, J., A. Vikartovska, and E. Hrabarova. 2008. A crosslinked inclusion body process for sialic acid synthesis. J. Biotechnol. 134: 146-153. https://doi.org/10.1016/j.jbiotec.2008.01.014
  20. Nakkharat, P. and D. Haltrich. 2006. Lactose hydrolysis and formation of galactooligosaccharides by a novel immobilized $\beta$-galactosidase from the thermophilic fungus Talaromyces thermophilus. Appl. Biochem. Biotechnol. 129-132: 215-225.
  21. Neri, D. F. M., V. M. Balcäo, R. S. Costa, I. C. A. P. Rocha, E. M. F. C. Ferreira, D. P. M. Torres, et al. 2009. Galactooligosaccharides production during lactose hydrolysis by free Aspergillus oryzae $\beta$-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol. Food Chem. 115: 92-99. https://doi.org/10.1016/j.foodchem.2008.11.068
  22. Neri, D. F. M., V. M. Balcäo, F. O. Q. Dourado, J. M. B. Oliveira, L. B. Carvalho Jr., and J. A. Teixeira. 2009. Galactooligosaccharides production by $\beta$-galactosidase immobilized onto magnetic polysiloxane-polyaniline particles. React. Funct. Polym. 69: 246-251. https://doi.org/10.1016/j.reactfunctpolym.2009.01.002
  23. Oleg Shadyro, O., I. Yurkova, M. Kisel, O. Brede, and J. Arnhold. 2004. Formation of phosphatidic acid, ceramide, and diglyceride on radiolysis of lipids; identification by MALDITOF mass spectrometry. Free Radical Biol. Med. 36: 1612- 1624. https://doi.org/10.1016/j.freeradbiomed.2004.03.013
  24. Park, A. R. and D. K. Oh. 2010. Galacto-oligosaccharide production using microbial $\beta$-galactosidase: Current state and perspectives. Appl. Microbiol. Biotechnol. 85: 1279-1286. https://doi.org/10.1007/s00253-009-2356-2
  25. Pocedicova, K., L. Curda, D. Misu, A. Dryakova, and L. Diblíkova. 2010. Preparation of galacto-oligosaccharides using membrane reactor. J. Food Eng. 99: 479-484. https://doi.org/10.1016/j.jfoodeng.2010.02.001
  26. Rabiu, B. A., A. J. Jay, G. R. Gibson, and R. A. Rastall. 2001. Synthesis and fermentation properties of novel galactooligosaccharides by $\beta$-galactosidases from Bifidobacterium species. Appl. Environ. Microbiol. 67: 2526-2530. https://doi.org/10.1128/AEM.67.6.2526-2530.2001
  27. Robyt, J. F. and R. Mukerjea. 1994. Separation and quantitative determination of nanogram quantities of maltodextrins and isomaltodextrins by thin-layer chromatography. Carbohydr. Res. 251: 187-201. https://doi.org/10.1016/0008-6215(94)84285-X
  28. Sakai, T., H. Tsuji, S. Shibata, K. Hayakawa, and K. Matsumoto. 2008. Repeated-batch production of galactooligosaccharides from lactose at high concentration by using alginate-immobilized cells of Sporobolomyces singularis YIT 10047. J. Gen. Appl. Microbiol. 54: 285-293. https://doi.org/10.2323/jgam.54.285
  29. Shoaf, K., G. L. Mulvey, G. D. Armstrong, and R. W. Hutkins. 2006. Prebiotic galactooligosaccharides reduce adherence of enteropathogenic Escherichia coli to tissue culture cells. Infect. Immun. 74: 6920-6928. https://doi.org/10.1128/IAI.01030-06
  30. Sinclair, H. R., J. de Slegte, G. R. Gibson, and R. A. Rastall. 2009. Galactooligosaccharides (GOS) inhibit Vibrio cholerae toxin binding to its GM1 receptor. J. Agric. Food Chem. 57: 3113-3119. https://doi.org/10.1021/jf8034786
  31. Tanabe, S. and S. Hochi. 2010. Oral administration of a galactooligosaccharide preparation inhibits development of atopic dermatitis-like skin lesions in NC/Nga mice. Int. J. Mol. Med. 25: 331-336.
  32. Tokatlidis, K., P. Dhurjati, J. Millet, P. Beguin, and J. P. Aubert. 1991. High activity of inclusion bodies formed in Escherichia coli overproducing Clostridium thermocellum endoglucanase D. FEBS Lett. 282: 205-208. https://doi.org/10.1016/0014-5793(91)80478-L
  33. Tsukahara, T., R. Inoue, N. Shimojo, K. Nakayama, S. Saito, T. Sato, T. Itoh, K. Fujita, and K. Ushida. 2010. Alpha-linked galactooligosaccharide suppresses small intestinal eosinophil infiltration and improves growth performance in weaning pigs. J. Vet. Med. Sci. 72: 547-553. https://doi.org/10.1292/jvms.09-0462
  34. van Dyck, S., P. Gerbaux, and P. Flammang. 2009. Elucidation of molecular diversity and body distribution of saponins in the sea cucumber Holothuria forskali (Echinodermata) by mass spectrometry. Comp. Biochem. Physiol. B 152: 124-134. https://doi.org/10.1016/j.cbpb.2008.10.011
  35. Vulevic, J., A. Drakoularakou, P. Yaqoob, G. Tzortzis, and G. R. Gibson. 2008. Modulation of the fecal microflora profile and immune function by a novel trans-galactooligosaccharide mixture (B-GOS) in healthy elderly volunteers. Am. J. Clin. Nutr. 88: 1438-1446.
  36. Worrall, D. M. and N. H. Goss. 1989. The formation of biologically active $\beta$-galactosidase inclusion bodies in Escherichia coli. Aust. J. Biotechnol. 3: 28-32.
  37. Yakup, A. and A. Tanryseven. 2007. Immobilization of Pectinex Ultra SP-L to produce galactooligosaccharides. J. Mol. Catal. B Enzym. 45: 73-77. https://doi.org/10.1016/j.molcatb.2006.12.005
  38. Yeon, J.-H. and K.-H. Jung. 2010. Operation of packed-bed immobilized cell reactor featuring active $\beta$-galactosidase inclusion body-containing recombinant Escherichia coli cells. Biotechnol. Bioprocess Eng. 15: 822-829. https://doi.org/10.1007/s12257-010-0034-y
  39. Yeon, J.-H. and K.-H. Jung. 2011. Repeated-batch operation of immobilized $\beta$-galactosidase inclusion bodies-containing Escherichia coli cell reactor for lactose hydrolysis. J. Microbiol. Biotechnol. 21: 972-978. https://doi.org/10.4014/jmb.1104.04029
  40. Zheng, P., H. Yu, Z. Sun, Y. Ni, W. Zhang, Y. Fan, and Y. Xu. 2006. Production of galacto-oligosaccharides by immobilized recombinant $\beta$-galactosidase from Aspergillus candidus. Biotechnol. J. 1: 1464-1470. https://doi.org/10.1002/biot.200600100

Cited by

  1. Long-term Repeated-Batch Operation of Immobilized Escherichia coli Cells to Synthesize Galactooligosaccharide vol.22, pp.11, 2011, https://doi.org/10.4014/jmb.1204.04020
  2. β-Galactosidase-Catalyzed Synthesis of Galactosyl Chlorphenesin and Its Characterization vol.171, pp.6, 2011, https://doi.org/10.1007/s12010-013-0213-3
  3. Production of Chlorphenesin Galactoside by Whole Cells of ${\beta}$-Galactosidase-Containing Escherichia coli vol.23, pp.6, 2011, https://doi.org/10.4014/jmb.1211.11009
  4. Synthesis of Galactooligosaccharides in the Cheese Whey-based Medium by a Lactase from Lactobacillus paracasei YSM0308 vol.33, pp.5, 2011, https://doi.org/10.5851/kosfa.2013.33.5.565
  5. Enzymatic Synthesis of 2-Phenoxyethanol Galactoside by Whole Cells of ${\beta}$-Galactosidase-Containing Escherichia coli vol.24, pp.9, 2011, https://doi.org/10.4014/jmb.1404.04004
  6. Analysis, structural characterization, and bioactivity of oligosaccharides derived from lactose vol.35, pp.11, 2014, https://doi.org/10.1002/elps.201300567
  7. Escherichia coli β-galactosidase-catalyzed synthesis of 2-phenoxyethanol galactoside and its characterization vol.38, pp.2, 2011, https://doi.org/10.1007/s00449-014-1276-4
  8. 베타-갈락토시데이즈를 이용하여 합성한 1, 2-Hexanediol Galactoside의 보습력과 항균력에 대한 연구 vol.43, pp.4, 2011, https://doi.org/10.15230/scsk.2017.43.4.373