Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Characterization of Acidic Carboxymethylcellulase Produced by a Marine Microorganism, Psychrobacter aquimaris LBH-10

해양미생물 Psychrobacter aquimaris LBH-10가 생산하는 산성 carboxymethylcellulase의 특성에 대한 연구

  • Kim, Hye-Jin (Department of Medical Bioscience, Graduate School of Donga-A University) ;
  • Gao, Wa (Department of Medical Bioscience, Graduate School of Donga-A University) ;
  • Lee, You-Jung (Department of Medical Bioscience, Graduate School of Donga-A University) ;
  • Chung, Chung-Han (BK21 Bio-Silver Project of Dong-A University) ;
  • Lee, Jin-Woo (BK21 Bio-Silver Project of Dong-A University)
  • 김혜진 (동아대학교 대학원 의생명공학과) ;
  • 고와 (동아대학교 대학원 의생명공학과) ;
  • 이유정 (동아대학교 대학원 의생명공학과) ;
  • 정정한 (동아대학교 BK21 생물자원 실버바이오 산업화 인력양성 사업단) ;
  • 이진우 (동아대학교 BK21 생물자원 실버바이오 산업화 인력양성 사업단)
  • Received : 2010.01.18
  • Accepted : 2010.03.08
  • Published : 2010.04.30
Download PDF
⟨ Previous Next ⟩

Abstract

A microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Psychrobacter aquimaris by analysis of 16S rDNA sequences, and named P. aquimari LBH-10. This strain produced an acidic carboxymethylcellulase (CMCase), which hydrolyzed carboxymethylcellulose (CMC), cellobiose, curdlan, filter paper, p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), pullulan, and xylan, but there was no detectable activity on avicel and cellulose. The optimal temperature for CMCase produced by P. aquimari LBH-10 was $50^{\circ}C$ and more than 90% of its original activity was maintained at broad temperatures ranging from 20 to $50^{\circ}C$ after 24 hr. The optimal pH of the CMCase was 3.5, and more than 70% of its original activity was maintained under acidic conditions between pH 2.5 and 7.0 at $50^{\circ}C$ after 24 hr. The optimal pH of CMCase produced by P. aquimaris LBH-10 seems to be lower than those produced by any other bacterial and fungal strain. $CoCl_2$, EDTA, and $PbCl_2$ at a concentration of 0.1 M enhanced CMCase-produced P. aquimaris LBH-10, whereas $HgCl_2$, KCl, $MnCl_2$, $NiCl_2$, and $SrCl_2$ inhibited it.

Carboxymethylcellulose (CMC)를 분해하는 미생물을 해수에서 분리하였으며, 16S rDNA의 염기서열을 분석하여 동정한 결과, Psychrobacter aquinaris로 학인 되어 P. aquinaris LBH-10로 명명하였다. 이 균주는 CMC, 셀로바이오스, 커드란, 여과지, p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), 풀루란 및 자일란을 분해하였으나, avicel 및 섬유소는 분해하지 못하였다. P. aquinaris LBH-10이 생산하는 carboxymethylcellulase (CMCase)의 최적 반응 온도는 $50^{\circ}C$이었으며, $20^{\circ}C$에서 $50^{\circ}C$의 온도 범위에서 24시간이 경과한 후에도 90% 이상의 활성을 유지하였다. 또한, 이 균주가 생산하는 CMCase의 최적 반응 pH는 3.5이었으며 pH 2.5에서 pH 7.0 사이의 산성 조건하에서 24시간이 경과한 후에도 70% 이상의 활성을 유지하였다. P. aquinaris LBH-10이 생산하는 CMCase의 최적 반응 pH는 지금까지 발견된 섬유소 분해효소 중에서 가장 낮은 pH로 판단된다. 제한된 농도의 $CoCl_2$, EDTA, 및 $PbCl_2$은 P. aquinaris LBH-10가 생산하는 CMCase의 활성을 증가시켰으나, $HgCl_2$, KCl, $MnCl_2$, $NiCl_2$, 및 $SrCl_2$는 이 균주가 생산하는 CMCase의 활성을 감소시켰다.

Keywords

Acknowledgement

Supported by : Korea Science and Engineering Foundation (KOSEF)

References

  1. Akiba, S., Y. Kimura, K. Yamamoto, and H. Kumagai. 1995. Purification and characterization of a-protease-resistant cellulose from Aspergillus niger. J. Ferment. Bioeng. 79, 125-130. https://doi.org/10.1016/0922-338X(95)94078-6
  2. Azevedo, H., D. Bishop, and A. Cavaco-Paulo. 2000. Effects of agitation level on the adsorption, desorption, and activities on cotton fabrics of full length and core domains of EGV (Humicola insolens) and CenA (Cellulonwnas fimi). Enzyme Microb. Technol. 27, 325-329. https://doi.org/10.1016/S0141-0229(00)00205-2
  3. Back, S. C. and Y. J. Kwon. 2007. Optimization of the pretreatment of rice straw hemicellulosic hydrolyzates for microbial production of xylitol. Biotechnol. Bioprocess Eng. 12, 404-409. https://doi.org/10.1007/BF02931063
  4. Ballesteros, M., J. M. Oliva, M. J. Negro, P. Manzanares, and I. Ballesteros. 2009. Ethanol from ligoncelulosic materials by a simultaneous saccharification and fermentation process (SSF) with Kluyveromeces marxianus CECT 10875. Process Biochem. 39, 1843-1848. https://doi.org/10.1016/j.procbio.2003.09.011
  5. Boyer, M. H, J. P. Chambost, M. Magnan, and J. Cattaneo. 1984. Carboxymethyl-cellulase from Erwinia chrysanthermi. II. purification and partial characterization of an endo-B-1,4-glucanase. J. Biotechnol. 1, 241-252. https://doi.org/10.1016/0168-1656(84)90009-9
  6. Cavaco-Paulo, A. 1998. Mechanism of cellulase action in textile processes. Carbohydr. Polymers 37, 273-277. https://doi.org/10.1016/S0144-8617(98)00070-8
  7. Chun, J. 1995. Computer-assisted classification and identification of actinomycestes. Ph. D. Thesis, University of Newcastle, Newcastle upon Tyne, UK.
  8. Delmer, D. P. and C. H Haigler. 2002. The regulation of metabolic flux to cellulose, a major sink for carbon in plants. Metabol. Eng. 4, 22-28. https://doi.org/10.1006/mben.2001.0206
  9. Emtiazi, G. and I. Nahvi. 2000. Multi-enzyme production by Cellulomonas sp. grown on wheat straw. Biomass Bioenergy 19, 31-37. https://doi.org/10.1016/S0961-9534(00)00015-5
  10. Ghose, T. K. 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257-268. https://doi.org/10.1351/pac198759020257
  11. Golias, H., G. J. Dumsday, G. A. Stanley, and N. B. Pamment. 2000. Characteristics of cellulase preparation affecting the simultaneous saccharification and fermentation of cellulose to ethanol. Biotechnol. Lett. 26, 617-621.
  12. Howard, R. L., E. Abotsi, E. L. J. von Rensburg, and S. Howard. 2003. Lignocellulose biotechnology: issues of bioconversion and enzyme production. Afr. J. Biotechnol. 2, 602-619.
  13. Ito, S. 1997. Alkaline cellulases from alkaliphilic Bacillus: enzymatic properties, genetics, and application to detergents. Extremophiles 1, 61-66. https://doi.org/10.1007/s007920050015
  14. Kim, B. K., B. H Lee, Y. J. Lee, I. H Jin, C. H. Chung, and J. W. Lee. 2009. Purification and characterization of carboxymethylcellulase isolated from a marine bacterium, Bacillus subtilis subsp. subtilis A-53. Enzyme Microb. Technol. 44, 411-416. https://doi.org/10.1016/j.enzmictec.2009.02.005
  15. Kulakova, L., A. Galkin, T. Nakayama, T. Nishino, and N. Esaki. 2004. Cold-active estrase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly$\rightarrow$Pro substitution near the active site on its catalytic activity and stability. Biochem. Biophy. Acta 1696, 59-65. https://doi.org/10.1016/j.bbapap.2003.09.008
  16. Kumar, S., K. Tamura, and N. Nei. 1993. MEGA: Molecular evolutionary genetic analysis. Version 1.01. The Pennsylvania State University. University Park, USA.
  17. Kundu, R. K., S. Dube, and D. K. Dube. 1988. Extracellular cellulolytic enyzme system of Aspergillus japnicus: 3. isolation, purification, and characterization of multiple forms of endoglucanase. Enzyme Microb. Technol. 10, 100-109. https://doi.org/10.1016/0141-0229(88)90005-1
  18. Lamed, R., J. Tormo, A. J. hirino, E. Morag, and E. A. Bayer. 1994. Crystallization and preliminary X-ray analysis of the major cellulose-binding domain of the cellulase from Clostridiumthermo eellum. J. Mol. Bioi. 244, 236-237. https://doi.org/10.1006/jmbi.1994.1721
  19. Lee, B. H, B. K. Kim, Y. J. Lee, C. H Chung, and J. W. Lee. 2010. Industrial scale of optimization for the production of carboxymethylcellulase from rice bran by a marine bacterium, Bacillus subtilis subsp. subtilis A-53. Enzyme Microb. Technol. 46, 38-42. https://doi.org/10.1016/j.enzmictec.2009.07.009
  20. Lee, S. M. and Y. M. Koo. 2001. Pilot-scale production of cellulose using Trichoderma reesei Rut C-30 in fed-batch mode. J. Microbial. Biotechnol. 11, 229-233.
  21. Lee, Y. J., B. K. Kim, B. H Lee, K. I. Jo, N. K. Lee, C. H. Chung, Y. C. Lee, and J. W. Lee. 2008. Purification and characterization of cellulase produced by Bacillus amyloliquefaciens DL-3 utilizing rice hull. Bioresource Technol. 99, 378-386. https://doi.org/10.1016/j.biortech.2006.12.013
  22. Liu, J. and W. Xia. 2006. Purification and characterization of a bifunctional enzyme with chotisanase and cellulose activity from commercial cellulose. Biochem. Eng J. 30, 82-87. https://doi.org/10.1016/j.bej.2006.02.005
  23. Mackenzie, L. F., G. Sulzenbacher, C. Divne, T. A. Jones, H. F. Woldike, M. Schulein, S. G. Withers, and G. J. Davies. 1998. Crystal structure of the family 7 endoglucanase I (Cel7B) from Humicola insolens at 2.2 A resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate. Biochem. J. 335, 409-416.
  24. Maeadza, C., R. Hatti-Kaul, R Zvauya, and B. Mattiasson. 2000. Purification and characterization of cellulases produced by two Bacillus strains. J. Biotechnol. 83, 177-187. https://doi.org/10.1016/S0168-1656(00)00305-9
  25. Miller, G .. , L. Blum, R. Glennon, and A. L. Burton. 1960. surement of carboxymethylcellulase activity. Anal. Biochem. 2, 127-132.
  26. Murashima, K., T. Nishimura, Y. Nakamura, J. Koga. T. Moriya, N. Sumida, T. Yaguchi, and T. Kono. 2002. Purification and characterization of new endo-1,4-B-D-glucanases from Rhizopus oryzae. Enzyme Microb. Technol. 30, 319-326. https://doi.org/10.1016/S0141-0229(01)00513-0
  27. Nandakumar, M. P., M. S. Thankur, K. S. M. S. Raghavarao, and N. P. Ghildyal. 1994. Mechanism of solid particle degradation by Aspergillus niger in solid state fermentation. Process Biochem. 29, 545-551. https://doi.org/10.1016/0032-9592(94)80016-2
  28. Okolo, J. C., S. K. C. Obi, and F. J. C. Odibo. 1998. Purification and characterization of two distinct carboxymethylcellulases of Paecilomyces sp. Bioresource Technol. 66, 231-234. https://doi.org/10.1016/S0960-8524(98)00053-4
  29. Parra, L. P., F. Reyes, J. P. Acevedo, O. Salazar, B. A. Andrews, and J. A. Asenjo. 2008. Cloning and fusion expression of a cold-active lipase from marine Antarctic origin. Enzyme Microb. Technol. 42, 371-377. https://doi.org/10.1016/j.enzmictec.2007.11.003
  30. Resmussnen, R S. and M. T. Morrissey. 2007. Marine biotechnology for production of food ingredients. Adv. Food Nut. Res. 52, 237-292. https://doi.org/10.1016/S1043-4526(06)52005-4
  31. Roboson, L. M. and G. H Chambliss. 1989. Celluases of bacterial origin. Enzyme Microb. Technol. 11, 626-644. https://doi.org/10.1016/0141-0229(89)90001-X
  32. Seo, H. P., C. W. Son, C. H Chung, D. I. Jung, S. K. Kim, R. A. Gross, D. L. Kaplan, and J. W. Lee. 2004. Production of high molecular weight pullulan by Aureobasidium pullulans HP-2001 with soybean pomace as a nitrogen source. Bioresource Technol. 95, 293-299. https://doi.org/10.1016/j.biortech.2003.02.001
  33. Takagi, M., S. Abe, S. Suzuki, G. H Emert, and N. Yata. 1977. A method for production of ethanol directly from cellulose using cellulase and yeast, pp. 551-571, In Ghose, T. K. (ed.), Proceedings of Bioconversion Symposium, Delhi, India.
  34. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 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
  35. Thongekkaew, J., H. Ikeda, K. Masaki, and H. Iefuji. 2008. An acidic and fhermostable carboxymethylcellulase from fhe yeast Cryptacaccussp. S-2: purification, characterization and improvement of its recombinant enzyme production by high cell-density fermentation of Pichi a pastaris. Protein Expres. Purif. 60, 140-146. https://doi.org/10.1016/j.pep.2008.03.021
  36. Weisburg. W. G., S. M. Barns, D. A. Pelletire, and D. J. Lane. 1991. 16S ribosomal DNA amplication for phylogenetic study. J. Bacterial. 173, 697-703.
  37. Woo, S. M. and S. D. Kim. 2007. Confirmation of non-side-rophore antifungal substance and cellulase from Bacillus lichenifarmis K11 containing antagonistic ability and plant growth promoting activity. J. Life Sci. 17, 983-989. https://doi.org/10.5352/JLS.2007.17.7.983
  38. Yi, J. C., J. C. Sandra, A. B. John, and T. C. Shu. 1999. Production and distribution of endoglucanase, cellobiohydrolase, and $\beta$-glucosidase components of fhe cellulolytic system of Valvariella valvacea, the edible straw mushroom. Appl. Environ. Microbial. 65, 553-559.
  39. Zhang. Y. H. P. and L. R. Lynd. 2004. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biatechnal. Biaeng. 88, 797-824. https://doi.org/10.1002/bit.20282

Cited by

  1. Enhanced production of cellobiase by marine bacterium Cellulophaga lytica LBH-14 from rice bran under optimized conditions involved in dissolved oxygen vol.20, pp.1, 2015, https://doi.org/10.1007/s12257-014-0486-6
  2. Enhanced Production of carboxymethylcellulase by a marine bacterium, Bacillus velezensis A-68, by using rice hulls in pilot-scale bioreactor under optimized conditions for dissolved oxygen vol.52, pp.9, 2014, https://doi.org/10.1007/s12275-014-4156-3
  3. Optimization of salts in medium for production of carboxymethylcellulase by a psychrophilic marine bacterium, Psychrobacter aquimaris LBH-10 using two statistical methods vol.29, pp.3, 2012, https://doi.org/10.1007/s11814-011-0192-4
  4. Rapid Statistical Optimization of Cultural Conditions for Mass Production of Carboxymethylcellulase by a Newly Isolated Marine Bacterium, Bacillus velezensis A-68 from Rice Hulls vol.23, pp.6, 2013, https://doi.org/10.5352/JLS.2013.23.6.757
  5. Enhanced Production of Carboxymethylcellulase by a Newly Isolated Marine Microorganism Bacillus atrophaeus LBH-18 Using Rice Bran, a Byproduct from the Rice Processing Industry vol.22, pp.10, 2012, https://doi.org/10.5352/JLS.2012.22.10.1295
  6. Comparison of optimal conditions for mass production of carboxymethylcellulase by Escherichia coli JM109/A-68 with other recombinants in pilot-scale bioreactor vol.22, pp.2, 2017, https://doi.org/10.1007/s12257-017-0035-1
  7. Statistical optimization of fermentation conditions and comparison of their influences on production of cellulases by a psychrophilic marine bacterium, Psychrobacter aquimaris LBH-10 using orthogonal array method vol.16, pp.3, 2011, https://doi.org/10.1007/s12257-010-0457-5
  8. Statistical Optimization for Production of Carboxymethylcellulase from Rice Hulls by a Newly Isolated Marine Microorganism Bacillus licheniformis LBH-52 Using Response Surface Method vol.21, pp.8, 2011, https://doi.org/10.5352/JLS.2011.21.8.1083
  9. Enhanced purification of histidine-tagged carboxymethylcellulase produced by Escherichia coli BL21/LBH-10 and comparison of its characteristics with carboxymethylcellulase without histidine-tag pp.1573-4978, 2019, https://doi.org/10.1007/s11033-019-04647-4