Optimization of $\beta$-Galactosidase Production in Stirred Tank Bioreactor Using Kluyveromyces lactis NRRL Y-8279

  • Published : 2009.12.31

Abstract

This paper investigates the production and optimization of $\beta$-galactosidase enzyme using synthetic medium by Kluyveromyces lactis NRRL Y-8279 in stirred tank bioreactor. Response surface methodology was used to investigate the effects of fermentation parameters on $\beta$-galactosidase enzyme production. Maximum specific enzyme activity of 4,622.7 U/g was obtained at the optimum levels of process variables (aeration rate 2.21 vvm, agitation speed 173.4 rpm, initial sugar concentration 33.8 g/L, incubation time 24.0 hr). The optimum temperature and pH of the $\beta$-galactosidase enzyme produced under optimized conditions were $37^{\circ}C$ and pH 7.0, respectively. The enzyme was stable over a pH range of 6.0-7.5 and a temperature range of $25-37^{\circ}C$. The $K_m$ and $V_{max}$ values for O-nitrophenol-$\beta$-D-galactopyranoside (ONPG) were 1.20 mM and $1,000\;{\mu}mol/min{\cdot}mg$ protein, respectively. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in $\beta$-galactosidase enzyme production. Hence, this study fulfills the lack of using mathematical and statistical techniques in optimizing the $\beta$-galactosidase enzyme production in stirred tank bioreactor.

Keywords

References

  1. Cortes G, Trujillo-Roldan MA, Ramirez OT, Galindo E. Production of $\beta$-galactosidase by Kluyveromyces marxianus under oscillating dissolved oxygen tension. Process Biochem. 40: 773-778 (2005) https://doi.org/10.1016/j.procbio.2004.02.001
  2. Boon MA, Riet van't K, Janssen AEM. Enzymatic synthesis of oligosaccharides: Product removal during a kinetically controlled reaction. Biotechnol. Bioeng. 70: 411-420 (2000) https://doi.org/10.1002/1097-0290(20001120)70:4<411::AID-BIT6>3.0.CO;2-W
  3. Albayrak N, Yang ST. Production of galacto-oligosaccharides from lactose by Aspergillus oryzae $\beta$-galactosidase immobilized on cotton cloth. Biotechnol. Bioeng. 77: 8-19 (2002) https://doi.org/10.1002/bit.1195
  4. Shaikh SA, Khire JM, Khan MI. Production of $\beta$-galactosidase from thermophilic fungus Rhizomucor sp. J. Ind. Microbiol. Biot. 19: 239-245 (1997) https://doi.org/10.1038/sj.jim.2900452
  5. Santos A, Ladero M, Garcia-Ochoa F. Kinetic modeling of lactose hydrolysis by a $\beta$-galactosidase from Kluyveromyces fragilis. Enzyme Microb. Tech. 22: 558-567 (1998) https://doi.org/10.1016/S0141-0229(97)00236-6
  6. Dagbagli S, Goksungur Y. Optimization of $\beta$-galactosidase production using Kluyveromyces lactis NRRL Y-8279 by response surface methodology. Electron. J. Biotechnol. [online]. 11(4): 11-12 (2008)
  7. Gao H, Gu WY. Optimization of polysaccharide and ergosterol production from Agaricus brasiliensis by fermentation process. Biochem. Eng. J. 33: 202-210 (2007) https://doi.org/10.1016/j.bej.2006.10.022
  8. Roukas T, Liakopoulou-Kyriakides M. Production of pullulan from beet molasses by Aureobasidium pullulans in a stirred tank fermentor. J. Food Eng. 40: 89-94 (1999) https://doi.org/10.1016/S0260-8774(99)00043-6
  9. Lazaridou A, Roukas T, Billiaderis CG, Vaikousi H. Characterization of pullulan produced from beet molasses by Aureobasidium pullulans in stirred tank reactor under varying agitation. Enzyme Microb. Tech. 31: 122-132 (2002) https://doi.org/10.1016/S0141-0229(02)00082-0
  10. Goksungur Y, Da$\breve{g}$a$\breve{g}$iS, Ucan A, Guvenc U. Optimization of pullulan production from synthetic medium by Aureobasidium pullulans in a stirred tank reactor by response surface methodology. J. Chem. Technol. Biot. 80: 819-827 (2005) https://doi.org/10.1002/jctb.1254
  11. Schneider ALS, Merkle R, Carvalho-Jonas MF, Jonas R, Furlan S. Oxygen transfer on $\beta$-D-galactosidase production by Kluyveromyces marxianus using sugar cane molasses as carbon source. Biotechnol. Lett. 23: 547-550 (2001) https://doi.org/10.1023/A:1010338904870
  12. Furlan S, Schneider ALS, Merkle R, Carvalho-Jonas MF, Jonas R. Formulation of a lactose-free, low cost culture medium for the production of $\beta$-D-galactosidase by Kluyveromyces marxianus. Biotechnol. Lett. 22: 589-593 (2000) https://doi.org/10.1023/A:1005629127532
  13. Barberis S, Gentina JC. Effect of dissolved oxygen level on lactase production by Kluyveromyces fragillis. J. Chem. Technol. Biot. 73: 71-73 (1998) https://doi.org/10.1002/(SICI)1097-4660(199809)73:1<71::AID-JCTB924>3.0.CO;2-D
  14. Pinheiro R, Belo I, Mota M. Growth and $\beta$-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure. Lett. Appl. Microbiol. 37: 438-442 (2003) https://doi.org/10.1046/j.1472-765X.2003.01429.x
  15. Barberis SE, Segovia RF. Dissolved oxygen concentration-controlled feeding of substrate into Kluyveromyces fragilis culture. Biotechnol. Tech. 11: 797-799 (1997) https://doi.org/10.1023/A:1018421123983
  16. He YQ, Tan TW. Use of response surface methodology to optimize culture medium for production of lipase with Candida sp. 99-125. J. Mol. Catal. B-Enzym. 43: 9-14 (2006) https://doi.org/10.1016/j.molcatb.2006.02.018
  17. Liu CH, Lu WB, Chang JS. Optimizing lipase of Burkholderia sp. by response surface methodology. Process Biochem. 41: 1940-1944 (2006) https://doi.org/10.1016/j.procbio.2006.04.013
  18. Battestin V, Macedo GA. Tannase production by Paecilomyces variotii. Bioresource Technol. 98: 1832-1837 (2007) https://doi.org/10.1016/j.biortech.2006.06.031
  19. Uma Maheswar Rao JL, Satyanarayana T. Improving production of hyperthermostable and high maltose-forming $\alpha$-amylase by an extreme thermophile Geobacillus thermoleovorans using response surface methodology and its applications. Bioresource Technol. 98: 345-352 (2007) https://doi.org/10.1016/j.biortech.2005.12.022
  20. Ibrahim HM, Yusoff WMW, Hamid AA, Illias RM, Hassan O, Omar O. Optimization of medium for the production of $\beta$-cyclodextrin glucanotransferase using central composite desing (CCD). Process Biochem. 40: 753-758 (2005) https://doi.org/10.1016/j.procbio.2004.01.042
  21. Naessens M, Vercauteren R, Vandamme EJ. Three-factor response surface optimization of the production of intracellular dextran dextrinase by Gluconobacter oxydans. Process Biochem. 39: 1299-1304 (2004) https://doi.org/10.1016/j.procbio.2003.08.001
  22. Nawani NN, Kapadnis BP. Optimization of chitinase production using statistics based experimental designs. Process Biochem. 40: 651-660 (2005) https://doi.org/10.1016/j.procbio.2004.01.048
  23. Tari C, Gogus N, Tokatli F. Optimization of biomass, pellet size, and polygalacturonase production by Aspergillus sojae ATCC 20235 using response surface methodology. Enzyme Microb. Tech. 40: 1108-1116 (2007) https://doi.org/10.1016/j.enzmictec.2006.08.016
  24. Chen KC, Lee TC, Houng JY. Search method for the optimal medium for the production of lactase by Kluyveromyces fragillis. Enzyme Microb. Tech. 14: 659-664 (1992) https://doi.org/10.1016/0141-0229(92)90043-N
  25. Manera AP, Ores JD, Ribeiro VA, Andre C, Burkert V, Kalil SJ. Optimization of the culture medium for the production of $\beta$-galactosidase from Kluyveromyces marxianus CCT 7082. Food Technol. Biotech. 46: 66-72 (2008)
  26. FCC. Lactase ($\beta$-galactosidase) activity. p. 491. In: Food Chemicals Codex. 3rd ed. National Academy Press, Washington, DC, USA (1993)
  27. Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428 (1959) https://doi.org/10.1021/ac60147a030
  28. Bradford MM. 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 (1976) https://doi.org/10.1016/0003-2697(76)90527-3
  29. Myers RH, Montgomery DD. Response Surface Methodology: Process and Product Optimization Using Designed Experiments. John Wiley&Sons, Inc., New York, NY, USA. p. 700 (1995)
  30. Potumarthi R, Ch S, Jetty A. Alkaline protease production by submerged fermentation in stirred tank reactor using Bacillus licheniformis NCIM-2042: Effect of aretion and agitation regimes. Biochem. Eng. J. 34: 185-192 (2007) https://doi.org/10.1016/j.bej.2006.12.003
  31. Domingues L, Lima N, Teixeira JA. Aspergillus niger $\beta$-galactosidase production by yeast in a continous high cell density reactor. Process Biochem. 40: 1151-1154 (2005) https://doi.org/10.1016/j.procbio.2004.04.016
  32. Linko S, Enwald S, Zhu YH, Mayra-Makinen. Production of $\beta$-galactosidase by Streptococcus salivarius subsp thermophilus 11F. J. Ind. Microbiol. Biot. 20: 215-219 (1998) https://doi.org/10.1038/sj.jim.2900515
  33. Rech R, Cassini CF, Secchi A, Ayub MAZ. Utilization of proteinhydrolyzed cheese whey for production of $\beta$-galactosidase by Kluyveromyces marxianus. J. Ind. Microbiol. Biot. 23: 91-96 (1999) https://doi.org/10.1038/sj.jim.2900692
  34. Goksungur Y, Mantzouridou F, Roukas T, Kotzekidou P. Production of $\beta$-carotene from beet molasses by Blakeslea trispora in stirredtank and bubble column reactors. Appl. Biochem. Biotech. 112: 37-54 (2004) https://doi.org/10.1385/ABAB:112:1:37
  35. Martins DBG, de Souza Jr CG, Simoes DA, de Morais Jr MA. The $\beta$-galactosidase activity in Kluyveromyces marxianus CBS6556 decreases by high concentrations of galactose. Curr. Microbiol. 44: 379-382 (2002) https://doi.org/10.1007/s00284-001-0052-2
  36. Ornelas AP, Silveira WB, Sampaio FC, Passos FML. The activity of $\beta$-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate. J. Appl. Microbiol. 104: 1008-1013 (2008) https://doi.org/10.1111/j.1365-2672.2007.03622.x
  37. Furlan SA, Schneider ALS, Merkle R, Carvalho-Jonas MF, Jonas R. Optimization of pH, temperature, and inoculum ratio for the production of $\beta$-D-galactosidase by Kluyveromyces marxianus using a lactose free medium. Acta Biotechnol. 21: 57-64 (2001) https://doi.org/10.1002/1521-3846(200102)21:1<57::AID-ABIO57>3.0.CO;2-Q
  38. Inchaurrondo VA, Yantorno OM, Voget CE. Yeast growth and $\beta$-galactosidase production during aerobic batch cultures in lactoselimited synthetic medium. Process Biochem. 29: 47-54 (1994) https://doi.org/10.1016/0032-9592(94)80058-8
  39. Fujimura Y, Rokushika S, Ohnishi M. Purification and molecular characterization of $\beta$-galactosidase from yeast Kluyveromyces lactis. J. Biol. Macromol. 3: 97-103 (2003) https://doi.org/10.1016/0141-8130(81)90074-X
  40. Cavaille D, Combes D. Characterization of $\beta$-galactosidase from Kluyveromyces lactis. Biotechnol. Appl. Bioc. 22: 55-64 (1995)
  41. Fleming M, Barron N, McHale L, Marchant R, McHale AP. Studies on the growth of a thermotolerant yeast strain, Kluyveromyces marxianus IMB3, on sucrose containing media. Biotechnol. Lett. 15: 1195-1198 (1993) https://doi.org/10.1007/BF00130296
  42. Takahashi T, Sugahara T, Yamaya S. Purification and characterization of a $\beta$-galactosidase from Treponema phagedenis (Reiter strain). Curr. Microbiol. 8: 341-345 (1983) https://doi.org/10.1007/BF01573706
  43. Li L, Zhang M, Jiang Z, Tang L, Cong Q. Characterization of a thermostable family 42 $\beta$-galactosidase from Thermotoga maritime. Food Chem. 112: 844-850 (2009) https://doi.org/10.1016/j.foodchem.2008.06.058
  44. Chen W, Chen H, Xia Y, Zhao J, Tian F, Zhang H. Production, purification, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus. J. Dairy Sci. 91: 1751-1758 (2008) https://doi.org/10.3168/jds.2007-617
  45. Samoshina NM, Samoshin VV. The Michaelis constants ratio for two substrates with a series of fungal (mould and yeast) $\beta$-galactosidases. Enzyme Microb. Tech. 36: 239-251 (2005) https://doi.org/10.1016/j.enzmictec.2004.07.011