Applied Biological Chemistry

, Volume 61, Issue 3, pp 325–336 | Cite as

Cloning, purification, and characterization of the organic solvent tolerant β-glucosidase, OaBGL84, from Olleya aquimaris DAU311

  • Eun-Jung Hwang
  • Yong-Suk Lee
  • Yong-Lark Choi


A marine bacterium, Olleya aquimaris DAU311, was isolated from Goraebul beach in the Republic of Korea. This strain had β-glucosidase activity on Luria–Bertani esculin plates. The β-glucosidase, oabgl84, was isolated, cloned, and sequenced, based on fosmid library. The gene encoded novel β-glucosidase and consisted of an open reading frame of 2304 bp, which encodes 768 amino acids. The deduced amino acid sequence had 99% identity to Olleya sp. VCSM12, 84% identity to Olleya marilimosa, and 78% similarity to Lacinutrix sp. Hel_I_90. OaBGL84 belongs to the glycoside hydrolase family 3, and it was visualized using SDS-PAGE, approximately 84 kDa. The optimal temperature and pH of OaBGL84 were analyzed as 40 °C and 6.0, respectively, using pNPG as substrate. The Km and Vmax values for OaBGL84 were 1.35 mM and 25.3 μM/s, respectively. Furthermore, OaBGL84 activity was completely inhibited by Cu2+ and Hg2+ ions. OaBGL84 demonstrated extraordinary stability until 50% (v/v) benzene, n-hexane, or toluene. These results indicate that OaBGL84 is useful candidate to degrade cellulose or soy isoflavone in the organic solvents for various biotechnological applications.


β-Glucosidase Glycoside hydrolase family 3 Olleya aquimaris Organic solvent tolerance OaBGL84 



This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0008619), the Ministry of Education (2017-0434), and the Ministry of Science and ICT (2017-0182).


  1. 1.
    Bhatia Y, Mishra S, Bisaria VS (2002) Microbial β-glucosidases: cloning, properties, and applications. Crit Rev Biotechnol 22:375–407CrossRefGoogle Scholar
  2. 2.
    Ketudat Carins JR, Esen A (2010) β-glucosidases. Cell Mol Life Sci 67:3389–3405CrossRefGoogle Scholar
  3. 3.
    Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316CrossRefGoogle Scholar
  4. 4.
    Ng IS, Tsai SW, Ju YM, Yu SM, Ho TH (2011) Dynamic synergistic effect on Trichoderma reesei cellulases by novel β-glucosidases from Taiwanese fungi. Bioresour Technol 102:6073–6081CrossRefGoogle Scholar
  5. 5.
    Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A (2013) Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresour Technol 127:500–507CrossRefGoogle Scholar
  6. 6.
    Demain AL, Vaishnav P (2011) Natural products for cancer chemotherapy. Microb Biotechnol 4:687–699CrossRefGoogle Scholar
  7. 7.
    Park DJ, Lee YS, Choi YL (2013) Characterization of a cold-active β-glucosidase from Paenibacillus xylanilyticus KJ-03 capable of hydrolyzing isoflavones daidzin and genistin. Protein J 32:579–584CrossRefGoogle Scholar
  8. 8.
    Joo AR, Jeya M, Lee KM, Sim WI, Kim JS, Kim IW, Kim YS, Oh DK, Gunasekaran P, Lee JK (2009) Purification and characterization of a β-1,4-glucosidase from a newly isolated strain of Fomitopsis pinicola. Appl Microbiol Biotechnol 83:285–294CrossRefGoogle Scholar
  9. 9.
    Ashadi RW, Shimokawa K, Ogawa K (1996) The mechanism of enzymatic cellulose degradation (II). Mode of action of cellulose hydrolyzing enzyme from Aspergillus niger UC. J Gen Appl Microbiol 42:103–108CrossRefGoogle Scholar
  10. 10.
    Li G, Jiang Y, Fan XJ, Liu YH (2012) Molecular cloning and characterization of a novel β-glucosidase with high hydrolyzing ability for soybean isoflavone glycosides and glucose-tolerance from soil metagenomic library. Bioresour Technol 123:15–22CrossRefGoogle Scholar
  11. 11.
    Doukyu N, Ogino H (2010) Organic solvent tolerant enzymes. Biochem Eng J 48:270–282CrossRefGoogle Scholar
  12. 12.
    Grunert O, Reheul D, Van Labeke MC, Permeel M, Hernandez-Sanabria E, Vlaeminck SE, Boon N (2016) Growing media constituents determine the microbial nitrogen conversions in organic growing media for horticulture. Microb Biotechnol 9:389–399CrossRefGoogle Scholar
  13. 13.
    Batra J, Mishra S (2013) Organic solvent tolerance and thermostability of a β-glucosidase co-engineered by random mutagenesis. J Mol Catal B Enzym 96:61–66CrossRefGoogle Scholar
  14. 14.
    Shafiei M, Ziaee AA, Amoozegar MA (2011) Purification and characterization of an organic-solvent-tolerant halophilic α-amylase from the moderately halophilic Nesterenkonia sp. strain F. J Ind Microbiol Biotechnol 38:275–281CrossRefGoogle Scholar
  15. 15.
    Chang J, Park IH, Lee YS, Ahn SC, Zhou Y, Choi YL (2011) Cloning, expression, and characterization of β-glucosidase from Exiguobacterium sp. DAU5 and transglycosylation activity. Biotechnol Bioprocess Eng 16:97–106CrossRefGoogle Scholar
  16. 16.
    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefGoogle Scholar
  17. 17.
    Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar
  18. 18.
    Kelly LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858CrossRefGoogle Scholar
  19. 19.
    Raimondi S, Roncaglia L, Lucia MD, Amaretti A, Leonardi A, Pagnoni UM, Rossi M (2009) Bioconversion of soy isoflavones daidzin and daidzein by Bifidobacterium strains. Appl Microbiol Biotechnol 81:943–950CrossRefGoogle Scholar
  20. 20.
    Lee MH, Jung YT, Park S, Yoon JH (2013) Olleya namhaensis sp. nov., isolated from wood falls, and emended description of the genus Olleya Mancuso Nichols et al. 2005 emend. Lee et al. 2010. Int J Syst Evol Microbiol 63:1610–1615CrossRefGoogle Scholar
  21. 21.
    Lee SY, Park S, Oh TK, Yoon JH (2010) Description of Olleya aquimaris sp. nov., isolated from seawater, and emended description of the genus Olleya Mancuso Nichols et al. 2005. Int J Syst Evol Microbiol 60:887–891CrossRefGoogle Scholar
  22. 22.
    Nichols CM, Bowman JP, Guezennec J (2005) Olleya marilimosa gen. nov., sp. nov., and exopolysaccharide-producing marine bacterium from the family Flavobacteriaceae, isolated from the Southern Ocean. Int J Syst Evol Microbiol 55:1557–1561CrossRefGoogle Scholar
  23. 23.
    Mao X, Hong Y, Shao Z, Zhao Y, Liu Z (2010) A novel cold-active and alkali-stable β-glucosidase gene isolated from the marine bacterium Martelella mediterranea. Appl Biochem Biotechnol 162:2136–2148CrossRefGoogle Scholar
  24. 24.
    Peterson TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786CrossRefGoogle Scholar
  25. 25.
    Chen HL, Chen YC, Lu MJ, Chang JJ, Wang HC, Wang TY, Ruan SK, Li WH (2012) A highly efficient beta-glucosidase from a buffalo rumen fungus Neocallimastix patriciarum W5. Biotechnol Biofuels 5:24CrossRefGoogle Scholar
  26. 26.
    Nakajima M, Yamashita T, Takahashi M, Nakano Y, Takeda T (2012) Identification, cloning, and characterization of β-glucosidase from Ustilago esculenta. Appl Microbiol Biotechnol 93:1989–1998CrossRefGoogle Scholar
  27. 27.
    Liu D, Zhang R, Yang X, Zhang Z, Song S, Miao Y, Shen Q (2012) Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33. Microb Cell Fact 11:25CrossRefGoogle Scholar
  28. 28.
    Rajasree KP, Mathew GM, Pandey A, Sukumaran RK (2013) Highly glucose tolerant β-glucosidase from Aspergillus unguis: NII 08123 for enhanced hydrolysis of biomass. J Ind Microbiol Biotechnol 40:967–975CrossRefGoogle Scholar
  29. 29.
    Saibi W, Amouri B, Gargouri A (2007) Purification and biochemical characterization of a transglucosilating β-glucosidase of Stachybotrys strain. Appl Microbiol Biotechnol 77:293–300CrossRefGoogle Scholar
  30. 30.
    Jiang C, Li SX, Luo FF, Jin K, Wang Q, Hao ZY, Wu LL, Zhao GC, Ma GF, Shen PH, Tang XL, Wu B (2011) Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning. Bioresour Technol 102:3272–3278CrossRefGoogle Scholar
  31. 31.
    Zhou J, Zhang R, Shi P, Huang H, Meng K, Yuan T, Yang P, Yao B (2011) A novel low-temperature-active β-glucosidase from symbiotic Serratia sp. TN49 reveals four essential positions for substrate accommodation. Appl Microbiol Biotechnol 92:305–315CrossRefGoogle Scholar
  32. 32.
    Riou C, Salmon JM, Vallier MJ, Gunata Z, Barre P (1998) Purification, characterization, and substrate specificity of a novel highly glucose-tolerant β-glucosidase from Aspergillus oryzae. Appl Environ Microbiol 64:3607–3614Google Scholar
  33. 33.
    Ng IS, Li CW, Chan SP, Chir JL, Chen PT, Tong CG, Yu SM, Ho TH (2010) High-level production of a thermoacidophilic β-glucosidase from Penicillium citrinum YS40-5 by solid-state fermentation with rice bran. Bioresour Technol 101:1310–1317CrossRefGoogle Scholar
  34. 34.
    Chamoli S, Kumar P, Navani NK, Verma AK (2016) Secretory expression, characterization and docking study of glucose-tolerant β-glucosidase from B. subtilis. Int J Biol Macromol 85:425–433CrossRefGoogle Scholar
  35. 35.
    Ogino H, Watanabe F, Yamada M, Nakagawa S, Hirose T, Noguchi A, Yasuda M, Ishikawa H (1999) Purification and characterization of organic solvent-stable protease from organic solvent-tolerant Pseudomonas aeruginosa PST-01. J Biosci Bioeng 87:61–68CrossRefGoogle Scholar
  36. 36.
    Mallerman J, Papinutti L, Levin L (2015) Characterization of β-glucosidase produced by the white rot fungus Flammulina velutipes. J Microbiol Biotechnol 25:57–65CrossRefGoogle Scholar
  37. 37.
    Gao Z, Hop DV, Yen LTH, Ando K, Hiyamuta S, Kondo R (2012) The production of β-glucosidases by Fusarium proliferatum NBRC109045 isolated from Vietnamese forest. AMB Express 2:49CrossRefGoogle Scholar

Copyright information

© The Korean Society for Applied Biological Chemistry 2018

Authors and Affiliations

  1. 1.Department of BiotechnologyDong-A UniversityBusanRepublic of Korea

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