Investigations of novel molecular features of cellulases from Fibrobacter sp. and their synergistic interactions.

摘要

Fibrobacter succinogenes is a predominant cellulolytic ruminal bacterium that is closely related to the Cytophaga-Flavobacterium-Bacteroides (CFB) group. The objectives of my research were to improve our understanding of the cellulase systems of F. succinogenes and F. intestinalis at the genome level, and to identify important proteins for crystalline cellulose degradation by this group of bacteria.;The genome of Fibrobacter succinogenes strain S85 was sequenced. Annotation of the genome revealed a total of 119 genes coding for plant cell wall polysaccharide degrading enzymes. However, neither exo-acting or processive cellulases required for cellulose solubilization in other eubacteria and fungi were apparent in the genome, nor were cellulosome components identified, suggesting that this bacterium has a unique cellulase system. Suppressive subtractive hybridization was conducted to identify unique genes coding for plant cell wall hydrolytic enzymes and other properties of the gastrointestinal bacterium Fibrobacter intestinalis DR7 not shared by F. succinogenes and vice versa. The majority of plant cell wall degrading enzymes were conserved within the two species. Of genes in F. intestinalis not exhibiting BLASTX similarity to F. succinogenes, 30 encoded putative transposases and six encoded restriction-modification genes. A cluster of genes responsible for cobalamin biosynthesis in F. succinogenes S85 were either missing or diverged greatly in F. intestinalis DR7, which explains the requirement of Vitamin B12 for the growth of the intestinalis species.;The gene encoding a major endoglucanase (endoglucanase 1) of F. succinogenes S85 was identified as cel9B from the genome sequence by reference to internal amino acid sequences of the purified enzyme. In addition, two newly identified cellulases Cel5H and Cel8B produced by S85 were cloned and characterized. Synergistic interactions among Cel9B, Cel5H, Cel8B, and two previously characterized enzymes Cel51A and Cel10A were investigated. Mixtures containing all the enzymes give the highest degree of synergism, which reflected the complimentary nature of their modes of action as well as their substrate specificities.;Cel9D was identified from cellulose-grown S85 because its ability to release reducing sugar from Avicel. cel9D was cloned and characterized. It showed low activity on carboxymethyl cellulose, acid swollen cellulose (ASC) or Avicel. However, the presence of Cel9D enhanced the activities (as measured by reducing sugar produced) with four other glucanases with degrees of synergism ranged from 1.5 to 3. HPLC analysis of the hydrolysis products showed that Cel9D released glucose from ASC as well as cello-oligosaccharides with chain lengths greater than two from the non-reducing end. Cel9D catalyzed the hydrolysis of cellulose by the inverting mechanism, as documented by NMR. Detailed kinetic studies showed Cel9D had increased kcat values on cello-oligosaccharides with a higher degree of polymerization, which indicates that it is a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74).;These data represent the first concerted analysis of synergism among highly expressed cellulases of Fibrobacter. The data are not conclusive that we have identified all major enzymes active in cellulose digestion, but provide a substantial foundation for assembly of the cellulase system of F. succinogenes.