Conversion of crystalline cellulose to solvent by naturally selected and recombinant clostridia

Abstract

Clostridium cellulolyticum, a mesophilic clostridial cellulosomes have a large cluster which is very similar to that of Clostridium acetobutylicum ATCC824. This model mesophilic cellulosome has recently been reported and cellulose gene cluster also characterized. Cel48F, one of the major cellulase of C. cellulolyticum, is active on degraded swallen avicel, carboxymethyl cellulose and even crystalline cellulose. In contrast, C. acetobutylicum produces inactive cellulosome which dues to the mutated amino acid in Cel48A subunit, one of three major cellulases in C. acetobutylicum cellulosome. The cel48A sequences encode polypeptides containing amino acids with calculated molecular mass of 84 000 Da. Protein contain a catalytic domain belonging to the family 48. For this reason, cel48A gene was modified by site-directed mutagenesis method to restore the activity of Cel48A. In parallel, new hybrid molecules of cel48A dockerin domain and cel48F catalytic domain were constructed. The results show that purified hybrid cel48A-cel48F has proficient activity on all cellulolytic substrates inspected. Interestingly, the hybrid protein shows activity on crystalline cellulose as well as efficient native C. cellulolyticum Cel48F (11.882 and 13.4 IU/[micro]mol respectively). Overall results can be concluded that the chimera protein exhibited enhanced action on cellulose more than in native C. acetobutylicum catalytic domain of cellulose. The action of hybrid enzyme over carboxymethylcellulose (CMC) developed for 30.8 folds from native (native Cel48A: 0.011 IU/[micro]M, hybrid: 0.339 IU/[micro]M). Activity over Phosphoric acid swollen cellulose (PASC) was improved for 2771 folds (native Cel48A: 0.002 IU/[micro]M, hybrid: 5.542 IU/[micro]M). Truncate recombinant hybrid protein can be detected in p952-SA-FA C. acetobutylicum strain. In the screening from natural process, Fifteen isolates were rapidly classified as in the class Clostridia by four selected criterias: endospore formation, sulfite reducing ability, metabolic products and 16S rDNA sequence. The selective system in this research was appropriate for the screening of Clostridiaceae in a similarity range between 83-100%. Comparative analysis shows that recombinant also expresses exoglucanase activity as well as naturally selected Clostridia.