Project description:The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U·mg(-1), respectively. The Km and Vmax of AcCel12B for CMC were 25.47 mg·mL(-1) and 131.75 U·mg(-1), respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future.
Project description:We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene (sharing 48% protein sequence identity to AldO) was identified, cloned and expressed in Escherichia coli. Following 6xHis tag purification, characterization revealed the protein to be a covalent flavoprotein of 47 kDa with a remarkably similar reactivity and substrate specificity to that of AldO. A steady-state kinetic analysis with a number of different polyol substrates revealed lower catalytic rates but slightly altered substrate specificity when compared to AldO. Thermostability measurements revealed that the novel AldO is a highly thermostable enzyme with an unfolding temperature of 84 °C and an activity half-life at 75 °C of 112 min, prompting the name HotAldO. Inspired by earlier studies, we attempted a straightforward, exploratory approach to improve the thermostability of AldO by replacing residues with high B-factors with corresponding residues from HotAldO. None of these mutations resulted in a more thermostable oxidase; a fact that was corroborated by in silico analysis.
Project description:We present here the complete 2.4-Mb genome of the cellulolytic actinobacterial thermophile Acidothermus cellulolyticus 11B. New secreted glycoside hydrolases and carbohydrate esterases were identified in the genome, revealing a diverse biomass-degrading enzyme repertoire far greater than previously characterized and elevating the industrial value of this organism. A sizable fraction of these hydrolytic enzymes break down plant cell walls, and the remaining either degrade components in fungal cell walls or metabolize storage carbohydrates such as glycogen and trehalose, implicating the relative importance of these different carbon sources. Several of the A. cellulolyticus secreted cellulolytic and xylanolytic enzymes are fused to multiple tandemly arranged carbohydrate binding modules (CBM), from families 2 and 3. For the most part, thermophilic patterns in the genome and proteome of A. cellulolyticus were weak, which may be reflective of the recent evolutionary history of A. cellulolyticus since its divergence from its closest phylogenetic neighbor Frankia, a mesophilic plant endosymbiont and soil dweller. However, ribosomal proteins and noncoding RNAs (rRNA and tRNAs) in A. cellulolyticus showed thermophilic traits suggesting the importance of adaptation of cellular translational machinery to environmental temperature. Elevated occurrence of IVYWREL amino acids in A. cellulolyticus orthologs compared to mesophiles and inverse preferences for G and A at the first and third codon positions also point to its ongoing thermoadaptation. Additional interesting features in the genome of this cellulolytic, hot-springs-dwelling prokaryote include a low occurrence of pseudogenes or mobile genetic elements, an unexpected complement of flagellar genes, and the presence of three laterally acquired genomic islands of likely ecophysiological value.
