ABSTRACT
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.
Subject(s)
Cellulase/chemistry , Cellulase/metabolism , Clostridium thermocellum/enzymology , Receptors, Cell Surface/chemistry , Carboxymethylcellulose Sodium/metabolism , Catalytic Domain , Cloning, Molecular , DNA, Recombinant/genetics , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Escherichia coli/metabolism , Gene Expression , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Mutant Proteins/metabolism , Plasmids/genetics , Polymerase Chain Reaction , Solubility , Structure-Activity Relationship , Substrate Specificity , TemperatureABSTRACT
Clostridium thermocellum encodes a xylanase gene (xynC) which is the major component of its cellulosome. XynC is a multidomain enzyme comprising of a substrate binding domain at the N-terminal followed by the catalytic domain and a dockerin domain. To study the influence of binding domain on activity, stability and expression of the enzyme the protein with the binding domain at C-terminal (XynC-CB), and the one with the binding domain at both N- and C-terminal (XynC-BCB) were expressed in E. coli. Recombinant plasmids, pXynC-CB and pXynC-BCB were constructed by inserting the corresponding gene in pET22b(+). XynC-CB and XynC-BCB were expressed at levels around 30% and 33% of the total E. coli cell proteins, respectively, while losing 40% and 20% of their activities at 70°C for 120 min, respectively. The specific activities of XynC-CB, XynC-BCB were 76 and 98 U mg(-1), while the activities on equimolar basis were 4410 and 7450 U µM(-1) against birchwood xylan, respectively. Their overall activities produced in the culture were 3660 and 5430 U L(-1) OD(600)(-1). Substrate binding studies showed that in case of XynC-C 51% of the activity remained unbound to birchwood xylan, whereas in the cases of XynC-BC, XynC-CB and XynC-BCB the activities left unbound were 33%, 32% and 12%, respectively, under the assay conditions used. Similar binding values were obtained in the case of oat spelt xylan. K(m) values for XynC-CB and XynC-BCB against birchwood xylan were found to be 3.1 and 1.47 mg ml(-1), respectively. Thus addition of a second carbohydrate binding domain at the C-terminal of the catalytic domain enhances activity, substrate affinity as well as thermostability.
