Influence of positioning of carbohydrate binding module on the activity of endoglucanase CelA of Clostridium thermocellum.
This study reports characteristics of different derivatives produced between CelA, a major endoglucanase of Clostridium thermocellum and carbohydrate binding domain of family 3a (CBM3a). In addition to the native form of the endoglucanase containing catalytic and dockerin domains (CelA-CD), its derivatives consisting of catalytic domain without dockerin domain (CelA-C), catalytic domain linked with the binding domain at N-, C- and both termini (CelA-BC, CelA-CB and CelA-BCB, respectively), two catalytic domains cloned in tandem (CelA-CC) and two catalytic domains intervened by a binding domain (CelA-CBC) were expressed in Escherichia coli at levels of 40, 43, 28, 30, 20, 20 and 10%, respectively of the total cell proteins. Specific activities of CelA-CD, CelA-C, CelA-BC, CelA-CB, CelA-CC, CelA-BCB and CelA-CBC against carboxymethyl cellulose (CMC) were 8.1, 7.0, 12.1, 8.5, 11.8, 10.2 and 23.5Umg(-1) enzyme while activities against pre-treated bagasse were 490, 250, 1400, 600, 810, 710 and 2270μmoles reducing sugars released per μmole of the enzyme, respectively, under the assay conditions used. Thus the activities of CelA-BC and CelA-CBC showed nearly 3- and 5-fold increase against pre-treated bagasse as compared to that of the native form of the enzyme, CelA-CD. Molecular modeling studies using MODELLER show that the binding residues of CBM3a and the active site residues of the catalytic domain are more favorably oriented for binding and hydrolysis of the polysaccharide in the case of CelA-BC as compared to those in CelA-CB, which corresponds with higher activity of the former.
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan.
Muhammad
M
Sajjad
M Imran Mahmood
MI
Khan
Rehan
R
Zafar
Sajjad
S
Ahmad
Umar H K
UH
Niazi
Muhammad Waheed
MW
Akhtar
D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D004274:DNA, Recombinant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000737:chemistry*; D012995:Solubility; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
D004274:DNA, Recombinant; D050505:Mutant Proteins; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins; C074775:CelA endoglucanase; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium