Thanks to visit codestin.com
Credit goes to link.springer.com

Skip to main content
Springer Nature Link
Log in

Overexpression and characterization of a novel cold-adapted and salt-tolerant GH1 β-glucosidase from the marine bacterium Alteromonas sp. L82

  • Microbial Genetics, Genomics and Molecular Biology
  • Published:
Journal of Microbiology

Abstract

A novel gene (bgl) encoding a cold-adapted β-glucosidase was cloned from the marine bacterium Alteromonas sp. L82. Based on sequence analysis and its putative catalytic conserved region, Bgl belonged to the glycoside hydrolase family 1. Bgl was overexpressed in E. coli and purified by Ni2+ affinity chromatography. The purified recombinant β-glucosidase showed maximum activity at temperatures between 25°C to 45°C and over the pH range 6 to 8. The enzyme lost activity quickly after incubation at 40°C. Therefore, recombinant β-glucosidase appears to be a cold-adapted enzyme. The addition of reducing agent doubled its activity and 2 M NaCl did not influence its activity. Recombinant β-glucosidase was also tolerant of 700 mM glucose and some organic solvents. Bgl had a Km of 0.55 mM, a Vmax of 83.6 U/mg, a kcat of 74.3 s-1 and kcat/Km of 135.1 at 40°C, pH 7 with 4-nitrophenyl-β-D-glucopyranoside as a substrate. These properties indicate Bgl may be an interesting candidate for biotechnological and industrial applications.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+
from £29.99 /Month
  • Starting from 10 chapters or articles per month
  • Access and download chapters and articles from more than 300k books and 2,500 journals
  • Cancel anytime
View plans

Buy Now

Price includes VAT (United Kingdom)

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Explore related subjects

Discover the latest articles, books and news in related subjects, suggested using machine learning.

References

  • Akemi, U.C., Tokuda, G., Watanabe, H., Kitamoto, K., and Arioka, M. 2013. A novel glucose-tolerant β-glucosidase from the salivary gland of the termite Nasutitermes takasagoensis. J. Gen. Appl. Microbiol. 59, 141–145.

    Article  Google Scholar 

  • Bhalla, T.C., Asif, M., and Smita, K. 2017. Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.). Process Biochem. 58, 320–325.

    Article  CAS  Google Scholar 

  • Bhatia, Y., Mishra, S., and Bisaria, V.S. 2002. Microbial β-glucosidases: Cloning, properties, and applications. Crit. Rev. Biotechnol. 22, 375–407.

    Article  PubMed  CAS  Google Scholar 

  • Cairns, J.R.K. and Esen, A. 2010. β-Glucosidases. Cell Mol. Life Sci. 67, 3389–3405.

    Article  CAS  Google Scholar 

  • Chamoli, S., Kumar, P., Navani, N.K., and Verma, A.K. 2016. Secretory expression, characterization and docking study of glucose-tolerant β-glucosidase from B. subtilis. Int. J. Biol. Macromol. 85, 425–433.

    Article  PubMed  CAS  Google Scholar 

  • Cleland, W.W. 1964. Dithiothreitol, a new protective reagent for SH groups. Biochemistry 3, 480–482.

    Article  PubMed  CAS  Google Scholar 

  • Crespim, E., Zanphorlin, L.M., de Souza, F.H.M., Diogo, J.A., Gazolla, A.C., Machado, C.B., Figueiredo, F., Sousa, A.S., Nóbrega, F., Pellizari, V.H., et al. 2016. A novel cold-adapted and glucosetolerant GH1 β-glucosidase from Exiguobacterium antarcticum B7. Int. J. Biol. Macromol. 82, 375–380.

    Article  PubMed  CAS  Google Scholar 

  • Cristóbal, H.A., Breccia, J.D., and Abate, C.M. 2008. Isolation and molecular characterization of Shewanella sp. G5, a producer of cold-active β-D-glucosidases. J. Basic Microbiol. 48, 16–24.

    Article  PubMed  CAS  Google Scholar 

  • Crout, D.H. and Vic, G. 1998. Glycosidases and glycosyl transferases in glycoside and oligosaccharide synthesis. Curr. Opin. Chem. Biol. 2, 98–111.

    Article  PubMed  CAS  Google Scholar 

  • Decker, C.H., Visser, J., and Schreier, P. 2000. β-glucosidases from five black Aspergillus species: study of their physico-chemical and biocatalytic properties. J. Agric. Food Chem. 48, 4929–4936.

    Article  PubMed  CAS  Google Scholar 

  • de Giuseppe, P.O., Souza Tde, A., Souza, F.H., Zanphorlin, L.M., Machado, C.B., Ward, R.J., Jorge, J.A., Furriel Rdos, P., and Murakami, M.T. 2014. Structural basis for glucose tolerance in GH1 β-glucosidases. Acta Crystallogr. D Biol. Crystallogr. 70, 1631–1639.

    Article  PubMed  CAS  Google Scholar 

  • Fan, H.X., Miao, L.L., Liu, Y., Liu, H.C., and Liu, Z.P. 2011. Gene cloning and characterization of a cold-adapted β-glucosidase belonging to glycosyl hydrolase family 1 from a psychrotolerant bacterium Micrococcus antarcticus. Enzyme Microb. Technol. 49, 94–99.

    Article  PubMed  CAS  Google Scholar 

  • Fang, Z., Fang, W., Liu, J., Hong, Y., Peng, H., Zhang, X., Sun, B., and Xiao, Y. 2010. Cloning and characterization of a β-glucosidase from marine microbial metagenome with excellent glucose tolerance. J. Microbiol. Biotechnol. 20, 1351–1358.

    Article  PubMed  CAS  Google Scholar 

  • Feller, G. and Gerday, C. 2003. Psychrophilic enzymes: Hot topics in cold adaptation. Nat. Rev. Microbiol. 1, 200–208.

    Article  PubMed  CAS  Google Scholar 

  • Guadamuro, L., Flórez, A.B., Alegría, Á., Vázquez, L., and Mayo, B. 2017. Characterization of four β-glucosidases acting on isoflavone- glycosides from Bifidobacterium pseudocatenulatum IPLA 36007. Food Res. Int. 100, 522–528.

    Article  PubMed  CAS  Google Scholar 

  • Guo, Y., Yan, Q., Yang, Y., Yang, S., Liu, Y., and Jiang, Z. 2015. Expression and characterization of a novel β-glucosidase, with transglycosylation and exo-β-1,3-glucanase activities, from Rhizomucor miehei. Food Chem. 175, 431–438.

    Article  PubMed  CAS  Google Scholar 

  • Haq, I., Khan, M., Muneer, B., Hussain, Z., Afzal, S., Majeed, S., Rashid, N., Javed, M.M., and Ahmad, I. 2012. Cloning, characterization and molecular docking of a highly thermostable β- 1,4-glucosidase from Thermotoga petrophila. Biotechnol. Lett. 34, 1703–1709.

    Article  PubMed  CAS  Google Scholar 

  • Henrissat, B., Callebaut, I., Fabrega, S., Lehn, P., Mornon, J.P., and Davies, G. 1995. Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc. Natl. Acad. Sci. USA 92, 7090–7094.

    Article  PubMed  CAS  Google Scholar 

  • Hernándezguzmán, A., Floresmartínez, A., Poncenoyola, P., and Villagómezcastro, J.C. 2016. Purification and characterization of an extracellular β-glucosidase from Sporothrix schenckii. FEBS Open Bio 6, 1067–1077.

    Article  CAS  Google Scholar 

  • Jenkins, J., Lo, L.L., Harris, G., and Pickersgill, R. 1995. β-Glucosidase, β-galactosidase, family A cellulases, family F xylanases and two barley glycanases form a superfamily of enzymes with 8-fold β/α architecture and with two conserved glutamates near the carboxy-terminal ends of β-strands four. FEBS Lett. 362, 281–285.

    Article  PubMed  CAS  Google Scholar 

  • Karasová-Lipovová, P., Strnad, H., Spiwok, V., Malá, S., Králová, B., and Russell, N.J. 2003. The cloning, purification and characterization of a cold-active β-galactosidase from the psychrotolerant antarctic bacterium Arthrobacter sp. C2-2. Enzyme Microb. Technol. 33, 836–844.

    Article  CAS  Google Scholar 

  • Li, Y.Y., Jiang, C.J., Wan, X.C., Zhang, Z.Z., and Li, D.X. 2005. Purification and partial characterization of β-glucosidase from fresh leaves of tea plants (Camellia sinensis (L.) O. Kuntze). Acta Biochim. Biophys. Sin. 37, 363–370.

    Article  PubMed  CAS  Google Scholar 

  • Lu, J., Du, L., Wei, Y., Hu, Y., and Huang, R. 2013. Expression and characterization of a novel highly glucose-tolerant β-glucosidase from a soil metagenome. Acta Biochim. Biophys. Sin. 45, 664–673.

    Article  PubMed  CAS  Google Scholar 

  • Mai, Z., Yang, J., Tian, X., Li, J., and Zhang, S. 2013. Gene cloning and characterization of a novel salt-tolerant and glucose-enhanced β-glucosidase from a marine Streptomycete. Appl. Biochem. Biotechnol. 169, 1512–1522.

    Article  PubMed  CAS  Google Scholar 

  • Mao, X., Hong, Y., Shao, Z., Zhao, Y., and Liu, Z. 2010. A novel cold-active and alkali-stable β-glucosidase gene isolated from the marine bacterium Martelella mediterranea. Appl. Biochem. Biotechnol. 162, 2136–2148.

    Article  PubMed  CAS  Google Scholar 

  • Miao, L.L., Hou, Y.J., Fan, H.X., Qu, J., Qi, C., Liu, Y., Li, D.F., and Liu, Z.P. 2016. Molecular structural basis for the cold-adaptedness of psychrophilic β-glucosidase BglU in Micrococcus antarcticus. Appl. Environ. Microbiol. 82, 2021–2030.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Morris, G.M., Goodsell, D., Halliday, R., Huey, R., Hart, W., Belew, R.K., and Olson, A.J. 1998. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 19, 1639–1662.

    Article  CAS  Google Scholar 

  • Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S., and Olson, A.J. 2009. Autodock4 and autodocktools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Pandey, M. and Mishra, S. 1997. Expression and characterization of Pichia etchellsii beta-glucosidase in Escherichia coli. Gene 190, 45–51.

    Article  PubMed  CAS  Google Scholar 

  • Pei, J., Pang, Q., Zhao, L., Fan, S., and Shi, H. 2012. Thermoanaerobacterium thermosaccharolyticum β-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose. Biotechnol. Biofuels 5, 1–10.

    Article  CAS  Google Scholar 

  • Schwede, T., Kopp, J., Guex, N., and Peitsch, M.C. 2003. Swiss-model: An automated protein homology-modeling server. Nucleic Acids Res. 31, 3381–3385.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Shipkowski, S. and Brenchley, J.E. 2005. Characterization of an unusual cold-active β-glucosidase belonging to family 3 of the gly coside hydrolases from the psychrophilic isolate Paenibacillus sp. strain C7. Appl. Environ. Microbiol. 71, 4225–4232.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sinha, S.K. and Datta, S. 2016. β-Glucosidase from the hyperthermophilic archaeon Thermococcus sp. is a salt-tolerant enzyme that is stabilized by its reaction product glucose. Appl. Microbiol. Biotechnol. 100, 8399–8409.

    Article  PubMed  CAS  Google Scholar 

  • Sinnott, M.L. 1990. Catalytic mechanism of enzymic glycosyl transfer. Chem. Rev. 90, 1171–1202.

    Article  CAS  Google Scholar 

  • Sørensen, A., Lübeck, M., Lübeck, P.S., and Ahring, B.K. 2013. Fungal beta-glucosidases: A bottleneck in industrial use of lignocellulosic materials. Biomolecules 3, 612–631.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Tao, Y.L., Yang, D.H., Zhang, Y.T., Zhang, Y., Wang, Z.Q., Wang, Y.S., Cai, S.Q., and Liu, S.L. 2014. Cloning, expression, and characterization of the β-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Bacteroides uniformis ZL1. Appl. Microbiol. Biotechnol. 98, 2519–2531.

    Article  PubMed  CAS  Google Scholar 

  • Uchiyama, T., Miyazaki, K., and Yaoi, K. 2013. Characterization of a novel β-glucosidase from a compost microbial metagenome with strong transglycosylation activity. J. Biol. Chem. 288, 18325–18334.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Ueda, M., Goto, T., Nakazawa, M., Miyatake, K., Sakaguchi, M., and Inouye, K. 2010. A novel cold-adapted cellulase complex from Eisenia foetida: characterization of a multienzyme complex with carboxymethylcellulase, β-glucosidase, β-1,3 glucanase, and β- xylosidase. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 157, 26–32.

    Article  PubMed  CAS  Google Scholar 

  • Wang, Q., Trimbur, D., Graham, R., Warren, R.A., and Withers, S.G. 1995. Identification of the acid/base catalyst in Agrobacterium faecalis β-glucosidase by kinetic analysis of mutants. Biochemistry 34, 14554–14562.

    Article  PubMed  CAS  Google Scholar 

  • Wierzbicka-Woś, A., Bartasun, P., Cieśliński, H., and Kur, J. 2013. Cloning and characterization of a novel cold-active glycoside hydrolase family 1 enzyme with β-glucosidase, β-fucosidase and β-galactosidase activities. BMC Biotechnol. 13, 22.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Woodward, J. and Wiseman, A. 1982. Fungal and other β-D-glucosidases— their properties and applications. Enzyme Microb. Technol. 4, 73–79.

    Article  CAS  Google Scholar 

  • Yang, F., Yang, X., Li, Z., Du, C., Wang, J., and Li, S. 2015. Overexpression and characterization of a glucose-tolerant β-glucosidase from T. aotearoense with high specific activity for cellobiose. Appl. Microbiol. Biotechnol. 99, 8903–8915.

    Article  PubMed  CAS  Google Scholar 

  • Yasumoto, K., Tsuji, H., Iwami, K., and Mitsuda, H. 1977. Isolation from rice bran of a bound form of vitamin B and its identfication as 5′-O-β-D-glucopyranosyl-pyridoxine. Agric. Biol. Chem. 41, 1061–1067.

    CAS  Google Scholar 

  • Zanphorlin, L.M., Giuseppe, P.O.D., Honorato, R.V., Tonoli, C.C.C., Fattori, J., Crespim, E., Oliveira, P.S.L.D., Ruller, R., and Murakami, M.T. 2016. Oligomerization as a strategy for cold adaptation: Structure and dynamics of the GH1 β-glucosidase from Exiguobacterium antarcticum B7. Sci. Rep. 6, 23776.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Zechel, D.L. and Withers, S.G. 2000. Glycosidase mechanisms: anatomy of a finely tuned catalyst. Acc. Chem. Res. 33, 11–18.

    Article  PubMed  CAS  Google Scholar 

  • Zhao, W., Peng, R., Xiong, A., Fu, X., Tian, Y., and Yao, Q. 2012. Expression and characterization of a cold-active and xylosestimulated β-glucosidase from Marinomonas MWYL1 in Escherichia coli. Mol. Biol. Rep. 39, 2937–2943.

    Article  PubMed  CAS  Google Scholar 

  • Zhong, F.L., Ma, R., Jiang, M., Dong, W.W., Jiang, J., Wu, S., Li, D., and Quan, L.H. 2016. Cloning and characterization of ginsenoside-hydrolyzing β-glucosidase from Lactobacillus brevis that transforms ginsenosides Rb1 and F2 into ginsenoside Rd and compund K. J. Microbiol. Biotechnol. 26, 1661–1667.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Key Laboratory of Sustainable Development of Polar Fishery, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, P. R. China

    Jingjing Sun, Wei Wang, Congyu Yao, Fangqun Dai, Xiangjie Zhu, Junzhong Liu & Jianhua Hao

  2. Laboratory for Marine Drugs and Bioproducts, Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, P. R. China

    Jingjing Sun, Wei Wang, Congyu Yao, Fangqun Dai, Xiangjie Zhu, Junzhong Liu & Jianhua Hao

  3. Shanghai Ocean University, Shanghai, 201306, P. R. China

    Congyu Yao & Xiangjie Zhu

  4. Jiangsu Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resource, Lianyungang, 222005, P. R. China

    Jianhua Hao

Authors
  1. Jingjing Sun
    View author publications

    Search author on:PubMed Google Scholar

  2. Wei Wang
    View author publications

    Search author on:PubMed Google Scholar

  3. Congyu Yao
    View author publications

    Search author on:PubMed Google Scholar

  4. Fangqun Dai
    View author publications

    Search author on:PubMed Google Scholar

  5. Xiangjie Zhu
    View author publications

    Search author on:PubMed Google Scholar

  6. Junzhong Liu
    View author publications

    Search author on:PubMed Google Scholar

  7. Jianhua Hao
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Jianhua Hao.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sun, J., Wang, W., Yao, C. et al. Overexpression and characterization of a novel cold-adapted and salt-tolerant GH1 β-glucosidase from the marine bacterium Alteromonas sp. L82. J Microbiol. 56, 656–664 (2018). https://doi.org/10.1007/s12275-018-8018-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1007/s12275-018-8018-2

Keywords