DOI QR코드

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Microbial β-Galactosidase of Pediococcus pentosaceus ID-7: Isolation, Cloning, and Molecular Characterization

  • Lee, Ji-Yeong (Department of Bio and Fermentation Convergence Technology, BK21 PLUS Project, Kookmin University) ;
  • Kwak, Mi-Sun (BioLeaders Corporation) ;
  • Roh, Jong-Bok (BioLeaders Corporation) ;
  • Kim, Kwang (BioLeaders Corporation) ;
  • Sung, Moon-Hee (Department of Bio and Fermentation Convergence Technology, BK21 PLUS Project, Kookmin University)
  • 투고 : 2016.11.07
  • 심사 : 2016.12.16
  • 발행 : 2017.03.28

초록

Pediococcus pentosaceus ID-7 was isolated from kimchi, a Korean fermented food, and it showed high activity for lactose hydrolysis. The ${\beta}$-galactosidase of P. pentosaceus ID-7 belongs to the GH2 group, which is composed of two distinct proteins. The heterodimeric LacLM type of ${\beta}$-galactosidase found in P. pentosaceus ID-7 consists of two genes partially overlapped, lacL and lacM encoding LacL (72.2 kDa) and LacM (35.4 kDa). In this study, Escherichia coli MM294 was used for the production of LacL, LacM, and LacLM. These three types of recombinant proteins were expressed, purified, and characterized. The specific activities of LacLM and LacL were 339 and 31 U/mg, respectively. However, activity was not detected with LacM alone. The optimal pH of LacLM and LacL was pH 7.5 and pH 7.0, and the optimal temperature of LacLM and LacL was $40^{\circ}C$ and $50^{\circ}C$, respectively. The optimal temperature changes indicate that LacLM is able to achieve higher activity at a relatively lower temperature. LacLM was strongly activated by $Mg^{2+}$, $Mn^{2+}$, and $Zn^{2+}$, which was not true for LacL. Consistent with this, EDTA strongly inactivated LacLM and LacL, but the presence of reducing agents did not dramatically alter the activity. Taken together, multiple alignment of amino acid sequences and phylogenetic analysis results of LacL and LacM of P. pentosaceus ID-7 suggest the evolution of LacL into LacLM and that the use of divalent metal ions results in higher activity.

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참고문헌

  1. Ustok FI, Tari C, Harsa S. 2010. Biochemical and thermal properties of ${\beta}$-galactosidase enzymes produced by artisanal yoghurt cultures. Food Chem. 119: 1114-1120. https://doi.org/10.1016/j.foodchem.2009.08.022
  2. Macfarlane G, Steed H, Macfarlane S. 2008. Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics. J. Appl. Microbiol. 104: 305-344.
  3. Rastall RA, Maitin V. 2002. Prebiotics and synbiotics: towards the next generation. Curr. Opin. Biotechnol. 13: 490-496. https://doi.org/10.1016/S0958-1669(02)00365-8
  4. Gopal PK, Sullivan PA, Smart JB. 2001. Utilisation of galacto-oligosaccharides as selective substrates for growth by lactic acid bacteria including Bifidobacterium lactis DR10 and Lactobacillus rhamnosus DR20. Int. Dairy J. 11: 19-25. https://doi.org/10.1016/S0958-6946(01)00026-7
  5. Fernandez Murga M, Hebert E, Savoy de Giori G, Font De Valdez G. 1997. Beta-galactosidase activity in thermophilic lactobacilli. their potential use as dietary adjuct. Milchwissenschaft 52: 316-318.
  6. Boon M, Janssen A, Van't Riet K. 2000. Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Enzyme Microb. Technol. 26: 271-281. https://doi.org/10.1016/S0141-0229(99)00167-2
  7. Playne M, Crittenden R. 1996. Commercially available oligosaccharides. Bulletin-FIL-IDF (Belgium) FAO Agris.
  8. Henrissat B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280: 309-316. https://doi.org/10.1042/bj2800309
  9. Chen W, Chen H, Xia Y, Yang J, Zhao J, Tian F, et al. 2009. Immobilization of recombinant thermostable ${\beta}$-galactosidase from Bacillus stearothermophilus for lactose hydrolysis in milk. J. Dairy Sci. 92: 491-498. https://doi.org/10.3168/jds.2008-1618
  10. Hidaka M, Fushinobu S, Ohtsu N, Motoshima H, Matsuzawa H, Shoun H, Wakagi T. 2002. Trimeric crystal structure of the glycoside hydrolase family 42 ${\beta}$-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose. J. Mol. Biol. 322: 79-91. https://doi.org/10.1016/S0022-2836(02)00746-5
  11. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  12. Kishore D, Kayastha AM. 2012. A ${\beta}$-galactosidase from chick pea (Cicer arietinum) seeds: its purification, biochemical properties and industrial applications. Food Chem. 134: 1113-1122. https://doi.org/10.1016/j.foodchem.2012.03.032
  13. Nguyen T-T, Nguyen HA, Arreola SL, Mlynek G, Djinovic Carugo K, Mathiesen G, et al. 2012. Homodimeric ${\beta}$-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: expression in Lactobacillus plantarum and biochemical characterization. J. Agric. Food Chem. 60: 1713-1721. https://doi.org/10.1021/jf203909e
  14. Goulas TK, Goulas AK, Tzortzis G, Gibson GR. 2007. Molecular cloning and comparative analysis of four ${\beta}$-galactosidase genes from Bifidobacterium bifidum NCIMB41171. Appl. Microbiol. Biotechnol. 76: 1365-1372. https://doi.org/10.1007/s00253-007-1099-1
  15. Hung M-N, Xia Z, Hu N-T, Lee BH. 2001. Molecular and biochemical analysis of two ${\beta}$-galactosidases from Bifidobacterium infantis HL96. Appl. Environ. Microbiol. 67: 4256-4263. https://doi.org/10.1128/AEM.67.9.4256-4263.2001
  16. Vaillancourt K, Moineau S, Frenette M, Lessard C, Vadeboncoeur C. 2002. Galactose and lactose genes from the galactose-positive bacterium Streptococcus salivarius and the phylogenetically related galactose-negative bacterium Streptococcus thermophilus: organization, sequence, transcription, and activity of the gal gene products. J. Bacteriol. 184: 785-793. https://doi.org/10.1128/JB.184.3.785-793.2002
  17. Craven GR, Steers E, Anfinsen CB. 1965. Purification, composition, and molecular weight of the ${\beta}$-galactosidase of Escherichia coli K12. J. Biol. Chem. 240: 2468-2477.
  18. Juajun O, Nguyen T-H, Maischberger T, Iqbal S, Haltrich D, Yamabhai M. 2011. Cloning, purification, and characterization of ${\beta}$-galactosidase from Bacillus licheniformis DSM 13. Appl. Microbiol. Biotechnol. 89: 645-654. https://doi.org/10.1007/s00253-010-2862-2
  19. Karasova-Lipovova P, Strnad H, Spiwok V, Mala S, Kralova B, Russell NJ. 2003. The cloning, purification and characterisation of a cold-active ${\beta}$-galactosidase from the psychrotolerant Antarctic bacterium Arthrobacter sp. C2-2. Enzyme Microb. Technol. 33: 836-844. https://doi.org/10.1016/S0141-0229(03)00211-4
  20. Nguyen T-H, Splechtna B, Steinbock M, Kneifel W, Lettner HP, Kulbe KD, Haltrich D. 2006. Purification and characterization of two novel ${\beta}$-galactosidases from Lactobacillus reuteri. J. Agric. Food Chem. 54: 4989-4998. https://doi.org/10.1021/jf053126u
  21. Nguyen T-H, Splechtna B, Krasteva S, Kneifel W, Kulbe KD, Divne C, Haltrich D. 2007. Characterization and molecular cloning of a heterodimeric ${\beta}$-galactosidase from the probiotic strain Lactobacillus acidophilus R22. FEMS Microbiol. Lett. 269: 136-144. https://doi.org/10.1111/j.1574-6968.2006.00614.x
  22. Maischberger T, Leitner E, Nitisinprasert S, Juajun O, Yamabhai M, Nguyen TH, Haltrich D. 2010. ${\beta}$-Galactosidase from Lactobacillus pentosus: purification, characterization and formation of galacto-oligosaccharides. Biotechnol. J. 5: 838-847. https://doi.org/10.1002/biot.201000126
  23. Iqbal S, Nguyen T-H, Nguyen TT, Maischberger T, Haltrich D. 2010. ${\beta}$-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides. Carbohydr. Res. 345: 1408-1416. https://doi.org/10.1016/j.carres.2010.03.028
  24. Juers DH, Matthews BW, Huber RE. 2012. LacZ ${\beta}$-galactosidase: structure and function of an enzyme of historical and molecular biological importance. Protein Sci. 21: 1792-1807. https://doi.org/10.1002/pro.2165
  25. Nguyen T-H, Splechtna B, Yamabhai M, Haltrich D, Peterbauer C. 2007. Cloning and expression of the ${\beta}$-galactosidase genes from Lactobacillus reuteri in Escherichia coli. J. Biotechnol. 129: 581-591. https://doi.org/10.1016/j.jbiotec.2007.01.034
  26. Bork P, Sander C, Valencia A. 1993. Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases. Protein Sci. 2: 31-40.
  27. Hildebrand A, Remmert M, Biegert A, Soding J. 2009. Fast and accurate automatic structure prediction with HHpred. Proteins 77: 128-132. https://doi.org/10.1002/prot.22499
  28. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  29. Manchenko GP. 2002. Handbook of Detection of Enzymes on Electrophoretic Gels. CRC Press, Boca Raton, FL. USA.
  30. Lim K, Chae C-B. 1989. A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase. Biotechniques 7: 576-579.
  31. Lo S, Dugdale ML, Jeerh N, Ku T, Roth NJ, Huber RE. 2010. Studies of Glu-416 variants of ${\beta}$-galactosidase (E. coli) show that the active site $Mg^{2+}$ is not important for structure and indicate that the main role of $Mg^{2+}$ is to mediate optimization of active site chemistry. Protein J. 29: 26-31. https://doi.org/10.1007/s10930-009-9216-x
  32. He X, Han N, Wang Y-P. 2016. Cloning, purification, and characterization of a heterodimeric ${\beta}$-galactosidase from Lactobacillus kefiranofaciens ZW3. J. Microbiol. Biotechnol. 26: 20-27. https://doi.org/10.4014/jmb.1507.07013
  33. Hug LA, B ak er BJ, Anantharaman K, B rown CT, P robst AJ, Castelle CJ, et al. 2016. A new view of the tree of life. Nat. Microbiol. 1: 16048. https://doi.org/10.1038/nmicrobiol.2016.48
  34. Bartesaghi A, Merk A, Banerjee S, Matthies D, Wu X, Milne JLS, Subramaniam S. 2015. 2.2 A resolution cryo-EM structure of ${\beta}$-galactosidase in complex with a cell-permeant inhibitor. Science 348: 1147-1151. https://doi.org/10.1126/science.aab1576

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