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Cloning and characterization of a thermostable and halo-tolerant endoglucanase from Thermoanaerobacter tengcongensis MB4

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Abstract

A β-1,4-endoglucanase (Cel5A) was cloned from the genomic DNA of saccharolytic thermophilic eubacterium Thermoanaerobacter tengcongensis MB4 and functionally expressed in Escherichia coli. Substrate specificity analysis revealed that Cel5A cleaves specifically the β-1,4-glycosidic linkage in cellulose with high activity (294 U mg−1; carboxymethyl cellulose sodium (CMC)). On CMC, kinetics of Cel5A was determined (K m 1.39 ± 0.12 g l−1; k cat/K m 1.41 ± 0.13 g−1 s−1). Cel5A displays an activity optimum between 75 and 80 °C. Residues Glu187 and Glu289 were identified as key catalytic amino acids by sequence alignment. Interestingly, derived from a non-halophilic bacterium, Cel5A exhibits high residual activities in molar concentration of NaCl (3 M, 49.3%) and KCl (4 M, 48.6%). In 1 M NaCl, 82% of Cel5A activity is retained after 24 h incubation. Molecular Dynamics studies performed at 0 and 3 M NaCl, correlate the Cel5A stability to the formation of R-COO−···Na+ ···−OOC-R salt bridges within the Cel5A tertiary structure, while activity possibly relates to the number of Na+ ions trapped into the negatively charged active site, involving a competition mechanism between substrate and Na+. Additionally, Cel5A is remarkably resistant in ionic liquids 1-butyl-3-methyllimidazolium chloride (1 M, 54.4%) and 1-allyl-3-methylimidazolium chloride (1 M, 65.1%) which are promising solvents for cellulose degradation and making Cel5A an attractive candidate for industrial applications.

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Acknowledgement

This research was supported by the grant of Natural Science Foundation of China (No.30621005) and Ministry of Sciences and Technology of China (973 Program: 2007CB707801 and 2009CB724705, 863 Programs: 2006AA020201 and 2007AA021306). The authors would like to express our sincere gratitude to Deutscher Akademischer Austausch Dienst (DAAD) for its valuable support, the Cluster of Excellence Tailor Made Fuels from Biomass (TMFB, RWTH Aachen University), BioNoCo (Biocatalysis using non-conventional media, Deutsche Forschungsgemeinschaft) and the computational resources offered by the RZ (Rechen Zentrum, Super Computer Center RWTH Aachen). We would like to thank Mr. Wolfgang Glory and Mr. Caesar Garfield for their assistance in experiments.

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Authors and Affiliations

  1. State Key Laboratory of Microbial Resource, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Beijing, Chaoyang District, 100101, People’s Republic of China

    Chaoning Liang, Yanfen Xue, Cheng Zhou & Yanhe Ma

  2. Graduate School, Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

    Chaoning Liang

  3. Lehrstuhl fuer Biotechnologie, RWTH Aachen University, Worringer weg 1, 52074, Aachen, Germany

    Marco Fioroni, Francisco Rodríguez-Ropero & Ulrich Schwaneberg

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  1. Chaoning Liang
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  2. Yanfen Xue
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  5. Cheng Zhou
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Correspondence to Yanhe Ma.

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Liang, C., Xue, Y., Fioroni, M. et al. Cloning and characterization of a thermostable and halo-tolerant endoglucanase from Thermoanaerobacter tengcongensis MB4. Appl Microbiol Biotechnol 89, 315–326 (2011). https://doi.org/10.1007/s00253-010-2842-6

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