Journal of Microbiology and Biotechnology

  • 제28권12호
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  • Pages.2036-2045
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  • 2018
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Roles of Carbohydrate-Binding Module (CBM) of an Endo-β-1,4-Glucanase (Cel5L) from Bacillus sp. KD1014 in Thermostability and Small-Substrate Hydrolyzing Activity

  • Lee, Jae Pil (Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University) ;
  • Shin, Eun-Sun (Department of Agricultural Chemistry, Sunchon National University) ;
  • Cho, Min Yeol (Department of Agricultural Chemistry, Sunchon National University) ;
  • Lee, Kyung-Dong (Department of Oriental Medicine Materials, Dongshin University) ;
  • Kim, Hoon (Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University)
  • 투고 : 2018.10.02
  • 심사 : 2018.10.19
  • 발행 : 2018.12.28
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초록

An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.

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참고문헌

  1. Lynd LR, Liang X, Biddy MJ, Allee A, Cai H, Foust T, et al. 2017. Cellulosic ethanol: status and innovation. Curr. Opin. Biotechnol. 45: 202211.
  2. Zheng F, Tu T, Wang X, Wang Y, Ma R, Su X, et al. 2018. Enhancing the catalytic activity of a novel GH5 cellulase GtCel5 from Gloeophyllum trabeum CBS 900.73 by sitedirected mutagenesis on loop 6. Biotechnol. Biofuels 11: 76. https://doi.org/10.1186/s13068-018-1080-5
  3. Lima AO, Quecine MC, Fungaro MH, Andreote FD, Maccheroni W Jr, Araujo WL, et al. 2005. Molecular characterization of a ${\beta}$-1,4-endoglucanase from an endophytic Bacillus pumilus strain. Appl. Microbiol. Biotechnol. 68: 5765.
  4. Berlemont R, Martiny AC. 2016. Glycoside hydrolases across environmental microbial communities. PLoS Comput. Biol. 12: e1005300. https://doi.org/10.1371/journal.pcbi.1005300
  5. Berlemont R, Martiny AC. 2013. Phylogenetic distribution of potential cellulases in bacteria. Appl. Environ. Microbiol. 79: 1545-1554. https://doi.org/10.1128/AEM.03305-12
  6. Talamantes D, Biabini N, Dang H, Abdoun K, Berlemont R. 2016. Natural diversity of cellulases, xylanases, and chitinases in bacteria. Biotechnol. Biofuels 9: 133. https://doi.org/10.1186/s13068-016-0538-6
  7. Guo H, Chang Y, Lee DJ. 2018. Enzymatic saccharification of lignocellulosic biorefinery: research focuses. Bioresour. Technol. 252: 198-215. https://doi.org/10.1016/j.biortech.2017.12.062
  8. Kim IJ, Lee HJ, Choi IG, Kim KH. 2014. Synergistic proteins for the enhanced enzymatic hydrolysis of cellulose by cellulase. Appl. Microbiol. Biotechnol. 98: 8469-8480. https://doi.org/10.1007/s00253-014-6001-3
  9. Park SH, Kim HK, Pack MY. 1991. Characterization and structure of the cellulase gene of Bacillus subtilis BSE616. Agric. Biol. Chem. 55: 441448.
  10. Lee YJ, Kim BK, Lee BH, Jo KI, Lee NK, Chung CH, et al. 2008. Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull. Bioresour. Technol. 99: 378-386. https://doi.org/10.1016/j.biortech.2006.12.013
  11. Santos C R, Paiva J H, Sforca ML, Neves JL, Navarro RZ, Cota J, et al. 2012. Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168. Biochem. J. 441: 95-104. https://doi.org/10.1042/BJ20110869
  12. Venditto I, Santos H, Sandy J, Sanchez-Weatherby J, Ferreira LM, Sakka K, et al. 2014. Crystallization and preliminary Xray diffraction analysis of a trimodular endo-${\beta}$-1,4-glucanase (Cel5B) from Bacillus halodurans. Acta Crystallogr. F Struct. Biol. Commun. 70: 1628-1630. https://doi.org/10.1107/S2053230X1402319X
  13. Guan X, Chen P, Xu Q, Qian L, Huang J, Lin B. 2017. Expression, purification and molecular characterization of a novel endoglucanase protein from Bacillus subtilis SB13. Protein Expr. Purif. 134: 125-131. https://doi.org/10.1016/j.pep.2017.04.009
  14. Lee KD, Kim J, Kim H. 1996. Isolation and characterization of Bacillus sp. KD1014 producing carboxymethyl-cellulase. J. Microbiol. 34: 305-310.
  15. Lee JP, Kim YA, Kim SK, Kim H. 2018. Characterization of a multimodular endo-${\beta}$-1,4-glucanase (Cel9K) from Paenibacillus sp. X4 with a potential additive for saccharification. J. Microbiol. Biotechnol. 28: 588-596.
  16. Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786. https://doi.org/10.1038/nmeth.1701
  17. Bradford MM. 1976. Arapid 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
  18. 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
  19. Yang MJ, Lee HW, Kim H. Enhancement of thermostability of Bacillus subtilis endoglucanase by error-prone PCR and DNA shuffling. Appl. Biol. Chem. 60: 73-78.
  20. Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination reducing sugar. Anal. Chem. 31: 426428.
  21. Jeong YS, Na HB, Kim SK, Kim YH, Kwon EJ, Kim J, et al. 2012. Characterization of xyn10J, a novel family 10 xylanase from a compost metagenomic library. Appl. Biochem. Biotechnol. 166: 1328-1339. https://doi.org/10.1007/s12010-011-9520-8
  22. Shin ES, Yang MJ, Jung KH, Kwon EJ, Jung JS, Park SK, et al. 2002. Influence of the transposition of the thermostabilizing domain of Clostridium thermocellum xylanase (XynX) on xylan binding and thermostabilization. Appl. Environ. Microbiol. 68: 3496-3501. https://doi.org/10.1128/AEM.68.7.3496-3501.2002
  23. Lin L, Meng X, Liu P, Hong Y, Wu G, Huang X, et al. 2009. Improved catalytic efficiency of endo-beta-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution. Appl. Microbiol. Biotechnol. 82: 671679.
  24. Robson LM, Chambliss GH. 1987. Endo-beta-1,4-glucanase gene of Bacillus subtilis DLG. J. Bacteriol. 169: 20172025.
  25. Endo K, Hakamada Y, Takizawa S, Kubota H, Sumitomo N, Kobayashi T, et al. 2001. A novel alkaline endoglucanase from an alkaliphilic Bacillus isolate: enzymatic properties, and nucleotide and deduced amino acid sequences. Appl. Microbiol. Biotechnol. 57: 109116.
  26. Henrissat B, Claeyssens M, Tomme P, Lemesle L, Mornon JP. 1989. Cellulase families revealed by hydrophobic cluster analysis. Gene. 81: 8395.
  27. Ducros V, Czjzek M, Belaich A, Gaudin C, Fierobe HP, Belaich JP, et al. 1995. Crystal structure of the catalytic domain of a bacterial cellulase belonging to family 5. Structure 9: 939949.
  28. Rattu G, Joshi S, Satyanarayana T. 2016. Bifunctional recombinant cellulase-xylanase (rBhcell-xyl) from the polyextremophilic bacterium Bacillus halodurans TSLV1 and its utility in valorization of renewable agro-residues. Extremophiles 6: 831-842.
  29. Ruiz DM, Turowski VR, Murakami MT. 2016. Effects of the linker region on the structure and function of modular GH5 cellulases. Sci. Rep. 6: 28504. https://doi.org/10.1038/srep28504
  30. Boraston AB, Kwan E, Chiu P, Warren RA, Kilburn DG. 2003. Recognition and hydrolysis of noncrystalline cellulose. J. Biol. Chem. 8: 61206127.
  31. Jung KH, Lee KM, Kim H, Yoon KH, Park SH, Pack MY. 1998. Cloning and expression of a Clostridium thermocellum xylanase gene in Escherichia coli. Biochem. Mol. Biol. Int. 44: 283-292.
  32. Feng JX, Karita S, Fujino E, Fujino T, Kimura T, Sakka K, et al. 2000. Cloning, sequencing, and expression of the gene encoding a cell-bound multidomain xylanase from Clostridium josui, and characterization of the translated product. Biosci. Biotechnol. Biochem. 64: 614-2624.
  33. Pastor FI, Pujol X, Blanco A, Vidal T, Torres AL, Diaz P. 2001. Molecular cloning and characterization of a multidomain endoglucanase from Paenibacillus sp BP-23: evaluation of its performance in pulp refining. Appl. Microbiol. Biotechnol. 55: 61-68. https://doi.org/10.1007/s002530000470
  34. Lee JP, Lee HW, Na HB, Lee JH, Hong YJ, Jeon JM, et al. 2018. Characterization of truncated endo-${\beta}$-1,4-glucanases from a compost metagenomic library and their saccharification potentials. Int. J. Biol. Macromol. 115: 554-562. https://doi.org/10.1016/j.ijbiomac.2018.04.102
  35. Kim H, Kim SF, Ahn DH, Lee JH, Pack MY. 1995. Internal cleavage of Bacillus subtilis BSE616 endo- ${\beta}$-1,4-glucanase expressed in Escherichia coli. J. Microbiol. Biotechnol. 5: 26-30.
  36. Aspeborg H, Coutinho PM, Wang Y, Brumer 3rd. H, Henrissat B. 2012. Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). BMC Evol. Biol. 12: 186. https://doi.org/10.1186/1471-2148-12-186
  37. Liberato MV, Silveira RL, Prates ET, de Araujo EA, Pellegrini VO, Camilo CM, et al. 2016. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism. Sci. Rep. 6: 23473. https://doi.org/10.1038/srep23473
  38. Posta K, Béki E, Wilson DB, Kukolya J, Hornok I. 2005. Cloning, characterization and phylogenetic relationships of cel5B, a new endoglucanase encoding gene from Thermobifida fusca. J. Basic Microbiol. 44: 383399.
  39. Akita M, Kayatama K, Hatada Y, Ito S, Horikoshi K. 2005. A novel ${\beta}$-glucanase gene from Bacillus halodurans C-125. FEMS Microbiol. Lett. 248: 9-15. https://doi.org/10.1016/j.femsle.2005.05.009
  40. Dadheech T, Shah R, Pandit R, Hinsu A, Chauhan PS, Jakhesara S, et al. 2018. Cloning, molecular modeling and characterization of acidic cellulase from buffalo rumen and its applicability in saccharification of lignocellulosic biomass. Int. J. Biol. Macromol. 113: 7381.
  41. Wang Y, Yuan H, Wang J, Yu Z. 2009. Truncation of the cellulose binding domain improved thermal stability of endo-beta-1,4-glucanase from Bacillus subtilis JA18. Bioresour. Technol. 100: 345349.
  42. Dhar H, Kasana RC, Dutt S, Gulati A. 2015. Cloning and expression of low temperature active endoglucanase EG5C from Paenibacillus sp. IHB B 3084. Int. J. Biol. Macromol. 81: 259-266. https://doi.org/10.1016/j.ijbiomac.2015.07.060
  43. Kim DU, Kim HJ, Jeong YS, Na HB, Cha YL, Koo BC, et al. 2015. Enhanced saccharification of reed and rice straws by the addition of ${\beta}$-1,3-1,4-glucanase with broad substrate specificity and calcium ion. J. Korean Soc. Appl. Biol. Chem. 58: 29-33. https://doi.org/10.1007/s13765-015-0013-2
  44. Singh A, Bishnoi NR. 2012. Optimization of enzymatic hydrolysis of pretreated rice straw and ethanol production. Appl. Microbiol. Biotechnol. 93: 1785-1793. https://doi.org/10.1007/s00253-012-3870-1
  45. Park JI, Steen EJ, Burd H, Evans SS, Redding-Johnson AM, Batth T, et al. 2012. A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels. PLoS One 7: e37010. https://doi.org/10.1371/journal.pone.0037010