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Rapid Statistical Optimization of Cultural Conditions for Mass Production of Carboxymethylcellulase by a Newly Isolated Marine Bacterium, Bacillus velezensis A-68 from Rice Hulls

통계학적 방법을 사용한 해양미생물 Bacillus velezensis A-68균주의 섬유소 분해효소 생산 조건 최적화

  • Received : 2012.03.15
  • Accepted : 2013.05.28
  • Published : 2013.06.30

Abstract

A microorganism producing carboxymethylcellulase (CMCase) was isolated from seawater, identified as Bacillus velezensis by analyses of 16S rDNA and partial sequences of the gyrA, and designated as B. velezensis A-68. The optimal conditions for production of CMCase by B. velezensis A-68 were established using response surface methodology (RSM). The optimal concentrations of rice hulls and yeast extract, and initial pH of the medium for cell growth were 60.2 g/l, 7.38 g/l, and 7.18, respectively, whereas those for production of CMCase were 50.0 g/l, 5.00 g/l, and 7.30. The analysis of variance (ANOVA) implied that the most significant factor for cell growth as well as production of CMCase was yeast extract. The optimal concentrations of $K_2HPO_4$, NaCl, $MgSO_4{\cdot}7H_2O$, and $(NH_4)_2SO_4$ in the medium for cell growth were 7.50, 1.00, 0.10, and 0.80 g/l, respectively, which were the same as those for production of CMCase. The optimal temperatures for cell growth and production of CMCase were 30 and $35^{\circ}C$, respectively. The maximal production of CMCase under optimized conditions was 83.8 U/ml, which was 3.3 times higher than that before optimization. In this study, rice hulls, agro-byproduct, were developed as a substrate for production of CMCase and time for production of CMCase was reduced to 3 days using a newly isolated marine bacterium.

섬유소 분해효소(carbxymethylcellulase)를 생산하는 미생물을 해수에서 분리하여 16S rDNA 및 gyrase 유전자의 염기서열을 분석하여 동정한 결과, Bcaillus velezensis로 확인되었으며 B. velezensis A-68로 명명하였다. 이 균주가 생산하는 섬유소 분해효소의 생산 조건을 최적화하기 위하여 통계학적 방법인 response surface method (RSM)를 사용하였다. 이 균주의 생육에 최적인 왕겨, 효모 추출물 및 배지의 초기 pH는 60.2 g/l, 7.38 g/l 및 7.18이었으나, 섬유소 분해효소 생산에 최적인 왕겨, 효모 추출물 및 배지의 초기 pH는 50.0 g/l, 5.99 g/l 및 7.30이었다. 통계학적인 분석 결과, 균주의 생육 및 균주의 섬유소 분해효소 생산에 가장 큰 영향을 미치는 것은 효모 추출물이었다. 이 균주의 생육과 섬유소 분해 효소의 생산에 최적인 $K_2HPO_4$, NaCl, $MgSO_4{\cdot}7H_2O$$(NH_4)_2SO_4$의 농도는 각각 7.50, 1.00, 0.10, and 0.80 g/l이었다. 이 균주의 생육 및 섬유소 분해효소 생산에 최적인 온도는 각각 $30^{\circ}C$$35^{\circ}C$로 생육에 최적인 조건과 섬유소 분해효소 생산에 최적인 조건이 다름을 알 수 있었다. 이 균주가 생산하는 섬유소 분해효소의 생산성은 83.8 m/l이며, 이는 최적화하기 전의 생산성에 비하여 3.3배 증가한 것이다. 이 연구를 통하여 농업 부산물인 왕겨를 섬유소 분해효소 생산을 위한 기질로 개발하였으며, 해수에서 분리한 미생물을 사용함으로써 섬유소 분해효소의 생산기간을 3일로 단축할 수 있었다.

Keywords

References

  1. Blumer-Schuette, S. E., Kataeva, I., Westpheling, J., Adams, M. W. W. and Kelly, R. M. 2008. Extremely thermophilic microorganisms for biomass conversion: status and prospects. Curr Opin Biotechnol 19, 210-217. https://doi.org/10.1016/j.copbio.2008.04.007
  2. Chun, J. 1995 Computer-assisted classification and identification of actinomycestes. Ph D Thesis, University of Newcastle, Newcastle upon Tyne, UK.
  3. Chun, J. and Bae, K. S. 2000. Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequence. Antonie van Leeuwenhoek 78, 123-127. https://doi.org/10.1023/A:1026555830014
  4. Gao, W., Lee, E. J., Lee, S. U., Li, J., Chung, C. H. and Lee, J. W. 2012. Enhanced carboxymethylcellulase production by a newly isolated marine bacterium, Cellulophaga lytica LBH-14, using rice bran. J Microbiol Biotechnol 22, 1415-1425.
  5. Gao, W., Lee, S. U., Li, J. and Lee, J. W. 2013. Enhanced production of carboxymethylcellulase by Cellulophaga lytica LBH-14 in pilot-scale bioreactor under optimized conditions involved in dissolved oxygen. Korean J Chem Eng 30, 1105-1110. https://doi.org/10.1007/s11814-012-0219-5
  6. Irwin, J. A., Alfredsson, G. A., Lanzetti, A. J., Gudmundsson, H. M. and Engel, P. C. 2001. Purification and characterization of a serine peptidase from the marine psychrophile strain PA-43. FEMS Microbiol 201, 285-290. https://doi.org/10.1111/j.1574-6968.2001.tb10770.x
  7. Jecu, L. 2000. Solid state fermentation of agricultural wastes for endogulcanse production. Ind Crop Prod 11, 1-5. https://doi.org/10.1016/S0926-6690(99)00022-9
  8. Jin, I. H., Jung, D. Y., Son, C. W., Kim, S. K., Gao, W., Chung, C. H. and Lee, J. W. 2011. Enhanced production of hetero-polysaccharide-7 by Beijerinkia indica HS-2001 in repeated batch culture with optimized substitution of culture medium. Biotechnol Bioproces Eng 16, 245-255. https://doi.org/10.1007/s12257-010-0120-1
  9. Jo, K. I., Lee, Y. J., Kim, B. K., Lee, B. H., Chung, C. H., Nam, S. W., Kim, S. K. and Lee, J. W. 2008. Pilot-scale production of carboxymethylcellulase from rice hull by Bacillus amyloliquefaciens DL-3. Biotechnol Bioprocess Eng 13, 182-188. https://doi.org/10.1007/s12257-007-0149-y
  10. Kalogeris, E., Christakopoulos, P., Katapodis, P., Alexious, A., Vlachou, S., Kekos, D. and Macris, B. J. 2003. Production and characterization of cellulolytic enzymes from the thermophilic fungus Thermoascus aurantiacus under solid state cultivation of agricultural waste. Process Biochem 38, 1099-1104. https://doi.org/10.1016/S0032-9592(02)00242-X
  11. Kang, S. W., Park, Y. S., Lee, J. S., Hong, S. I. and Kim, S. W. 2004. Production of cellulase and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. Bioresour Technol 91, 153-156. https://doi.org/10.1016/S0960-8524(03)00172-X
  12. Kim, B. K., Lee, B. H., Lee, Y. J., Jin, I. H., Chung, C. H. and Lee, J. W. 2009. Purification and characterization of carboxymethylcellulase isolated from a marine bacterium, Bacillulus subsp. subtilis A-53. Enzyme Microb Technol 44, 411-416. https://doi.org/10.1016/j.enzmictec.2009.02.005
  13. Kim, H. J., Gao, W., Chung, C. H. and Lee, J. W. 2011. Statistical optimization for production of carboxymethylcellulase from rice hulls by a new isolated marine microorganism Bacillus licheniformis LBH-52 using response surface method. J Life Sci 21, 1083-1093. https://doi.org/10.5352/JLS.2011.21.8.1083
  14. Kim, H. J., Gao, W., Lee, Y. J., Chung, C. H. and Lee, J. W. 2010. Characterization of acidic carboxymethylcellulase produced by a marine microorganism, Psychrobacter aquimaris LBH-10. J Life Sci 20, 487-495. https://doi.org/10.5352/JLS.2010.20.4.487
  15. Kim, H. J., Lee, Y. J. Gao, W., Chung, C. H., and Lee, J. W. 2012. Optimization of salts in medium for production of carboxymethylcellulase by a psychrophilic marine bacterium, Psychrobacter aquimaris LBH-10 using two statistical methods. Korean J Chem Eng 29, 384-391. https://doi.org/10.1007/s11814-011-0192-4
  16. Kim, H. J., Lee, Y. J., Gao, W., Chung, C. H., Son, C. W. and Lee, J. W. 2011. Statistical optimization of fermentation conditions and comparison of their influences on production of cellulases by Psychrophilic marine bacterium, Psychrobacter aquimaris LBH-10 using orthogonal array method. Biotechnol Bioprocess Eng 16, 542-548. https://doi.org/10.1007/s12257-010-0457-5
  17. Kim, Y. J., Gao, W., Lee, S. U., and Lee, J. W. 2012. Enhanced production of carboxymethylcellulase by a newly isolated marine microorganism Bacillus atrophaeus LBH-18 using rice bran, a byproduct from the rice processing industry. J Life Sci 22, 1295-1306. https://doi.org/10.5352/JLS.2012.22.10.1295
  18. Kumar, S., Tamura, K. and Nei, N. 1993. MEGA N. Molecular evolutionary genetic analysis, version 1.01, The Pennsylvania State University, University Park. USA.
  19. Lee, B. H., Kim, B. K., Lee, Y. J., Chung, C. H. and Lee, J. W. 2010. Industrial scale of optimization for the production of carboxymethylcellulase from rice bran by a marine bacterium, Bacillulus subsp. subtilis A-53. Enzyme Microb Technol 46, 38-42. https://doi.org/10.1016/j.enzmictec.2009.07.009
  20. Lee, E. J., Lee, B. H., Kim, B. K. and Lee, J. W. 2013. Enhanced production of carboxymethylcellulase of a marine microorganism, Bacillus subtilis subsp. subtilis A-53 in a pilot-scaled bioreactor by a recombinant Escherichia coli JM109/A-53 from rice bran. Mol Biol Rep 40, 3609-3621. https://doi.org/10.1007/s11033-012-2435-9
  21. Lee, N. K., Jo, Y. B., Jin, I. H., Son, C. W. and Lee, J. W. 2009. The effect of potassium phosphate as a pH stabilizer on the production of gellan by Sphingomonas paucibilis Nk-2000. J Life Sci 19, 1033-1038. https://doi.org/10.5352/JLS.2009.19.8.1033
  22. Lee, Y. J., Kim, H. J., Gao, W., Chung, C. H., and Lee, J. W. 2012. Statistical optimimization for production of carboxymethylcellulase of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 from rice bran using response surface method. Biotechnol Bioprocess Eng 17, 227-235. https://doi.org/10.1007/s12257-011-0258-5
  23. Liu, J., Zhang, Z., Liu, Z., Zhu, H., Dang, H., Lu, J. and Cui, Z. 2011. Production of cold-adapted amylase by marine bacterium Wangia sp. 53: optimization, modeling, and partial characterization. Mar Biotechnol 13, 837-844. https://doi.org/10.1007/s10126-010-9360-5
  24. MercoPress 2011. Global rice production reaches 476 million tons in 2011; strong Mercosur recovery. South Atlantic News Agency, June 27th, USA.
  25. Park, E. Y., Ikeda, Y. and Okuda, N. 2002. Empirical evaluation of cellulose on enzymatic hydrolysis of waste office paper. Biotechnol Bioprocess Eng 7, 268-274. https://doi.org/10.1007/BF02932835
  26. Roboson, L. M. and Chambliss, G. H. 1989. Cellulases of bacterial origin. Enzyme Microb Technol 11, 626-644. https://doi.org/10.1016/0141-0229(89)90001-X
  27. Saha, B. C. and Cotta, M. A. 2008. Lime pretreatment, enzymatic saccharification and fermentation of rice hulls to ethanol. Biomass Bioenergy 32, 971-977. https://doi.org/10.1016/j.biombioe.2008.01.014
  28. Senthikumar, S. R., Ashokkumar, A., Raj, K. C. and Cunasekraran, P. 2005. Optimization of medium composition for alkali-stable xylanase production by Aspergillus fischeri Fxn 1 in solid-state fermentation using central composite rotary desing. Bioresour Technol 96, 1380-1386. https://doi.org/10.1016/j.biortech.2004.11.005
  29. Sukumaran, R. K., Singhania, R. R., Mathew, G. M. and Pandey, A. 2009. Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production. Renew Energy 34, 421-424. https://doi.org/10.1016/j.renene.2008.05.008
  30. Suzuki, T., Yamamoto, K., Tada, H. and Uda, K. 2012. Cold-adapted features of arginine kinase from the deep-sea clam Calyptogena kaikoi. Mar Biotechnol 13, 294-303.
  31. Takagi, M., Abe, S., Suzuki, S., Emert, G. H. and Yata, N. 1977. A method for production of ethanol directly from cellulose using cellulase and yeast. Ghose, T. K. (ed), Proceedings of Bioconversion Symposium, Dheli, India.
  32. Thompson, J. D., Higgins, D. G. and Gibson, T. J. 1994. CLUSTAL W. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice Nucleic Acids Res 22, 4673-4680. https://doi.org/10.1093/nar/22.22.4673
  33. Wei, G. Y., Lee, Y. J., Kim, Y. J., Jin, I. H., Lee, J. H., Chung, C. H. and Lee, J. W. 2010. Kinetic study on the pretreatment and enzymatic saccharification of rice hull for the production of fermentable sugars. Appl Biochem Biotechnol 162, 1471-1482. https://doi.org/10.1007/s12010-010-8926-z

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