Cloning and characterization of a thermostable and halo-tolerant endoglucanase from Thermoanaerobacter tengcongensis MB4.
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.
Liang
Chaoning
C
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.
Xue
Yanfen
Y
Fioroni
Marco
M
Rodríguez-Ropero
Francisco
F
Zhou
Cheng
C
Schwaneberg
Ulrich
U
Ma
Yanhe
Y
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000378:metabolism*; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D012965:Sodium Chloride; C074775:CelA endoglucanase; D002480:Cellulase