Cloning and characterization of a thermostable and halo-tolerant endoglucanase from Thermoanaerobacter tengcongensis MB4 |
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Authors: | Chaoning Liang Yanfen Xue Marco Fioroni Francisco Rodríguez-Ropero Cheng Zhou Ulrich Schwaneberg Yanhe Ma |
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Affiliation: | (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;(2) Graduate School, Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China;(3) Lehrstuhl fuer Biotechnologie, RWTH Aachen University, Worringer weg 1, 52074 Aachen, Germany; |
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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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