![Sample our Bioscience journals, sign in here to start your access, Latest two full volumes FREE to you for 14 days]({"type":"image" , "src" : "/sda/5925/TOC-Subject-Sample-2021-Bioscience.jpg"})
Abstract
During the course of our studies on the structure–function relationship of cellulosomes, we were interested in converting the free cellulase system of the aerobic bacterium, Thermobifida fusca, to a cellulosomal system. For this purpose, the cellulose-binding modules (CBM) of two T. fusca family-6 cellulases, endoglucanase Cel6A and exoglucanase Cel6B, were replaced by divergent dockerin modules. Thus far, family-6 cellulases have not been shown to be members of natural cellulosome systems. The resultant chimaeric proteins, 6A-c and t-6B, respectively, were purified and found to interact specifically and stoichiometrically with their corresponding cohesin modules, indicating their suitability for use as components in ‘designer cellulosomes’. Both chimaeric enzymes showed somewhat decreased but measurable levels of activity on carboxymethyl cellulose, consistent with the known endo- and exo-glucanase character of the parent enzymes. The activity of 6A-c on phosphoric acid swollen cellulose was also consistent with that of the wild-type endoglucanase Cel6A. The startling finding of the present research was the extent of degradation of this substrate by the chimaeric enzyme t-6B. Wild-type exoglucanase Cel6B exhibited very low activity on this substrate, while the specific activity of t-6B was 14-fold higher than the parent enzyme.
| Abbreviations | ||
| cellulose-binding module | = | CBM |
| T. fusca family-2 CBM | = | CBM2 |
| C. thermocellum family-3a CBM | = | CBM3a |
| glycoside hydrolase | = | GH |
| wild-type T. fusca cellulase containing a CBM2 module downstream of a GH6 catalytic module | = | Cel6A |
| wild-type T. fusca cellulase containing a CBM2 module upstream of a GH6 catalytic module | = | Cel6B |
| chimaeric form of Cel6A with a C. cellulolyticum dockerin substituted for the CBM2 | = | 6A-c |
| chimaeric form of Cel6B with a C. thermocellum dockerin substituted for the CBM2 | = | t-6B |
| bovine serum albumin | = | BSA |
| carboxymethyl cellulose | = | CMC |
| phosphoric acid swollen cellulose (amorphous cellulose) | = | PASC |
| C. cellulolyticum cohesin appended to CBM3a | = | CBM3–Coh-c |
| C. thermocellum cohesin appended to CBM3a | = | CBM3–Coh-t |
| R. flavefaciens cohesin appended to CBM3a | = | CBM3–Coh-f |
| Abbreviations | ||
| cellulose-binding module | = | CBM |
| T. fusca family-2 CBM | = | CBM2 |
| C. thermocellum family-3a CBM | = | CBM3a |
| glycoside hydrolase | = | GH |
| wild-type T. fusca cellulase containing a CBM2 module downstream of a GH6 catalytic module | = | Cel6A |
| wild-type T. fusca cellulase containing a CBM2 module upstream of a GH6 catalytic module | = | Cel6B |
| chimaeric form of Cel6A with a C. cellulolyticum dockerin substituted for the CBM2 | = | 6A-c |
| chimaeric form of Cel6B with a C. thermocellum dockerin substituted for the CBM2 | = | t-6B |
| bovine serum albumin | = | BSA |
| carboxymethyl cellulose | = | CMC |
| phosphoric acid swollen cellulose (amorphous cellulose) | = | PASC |
| C. cellulolyticum cohesin appended to CBM3a | = | CBM3–Coh-c |
| C. thermocellum cohesin appended to CBM3a | = | CBM3–Coh-t |
| R. flavefaciens cohesin appended to CBM3a | = | CBM3–Coh-f |