Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii

Thermostable cellulases from glycoside hydrolase family 7 (GH7) are themain components of enzymatic mixtures for industrial saccharification oflignocellulose. Activity improvement of these enzymes via rational design isa promising strategy to alleviate the industrial costs, but it requires detailedstructural knowledge. While substantial biochemical and structural dataare available for GH7 cellobiohydrolases, endoglucanases are more elusiveand only few structures have been solved so far. Here, we report a newcrystal structure and biochemical characterization of a thermostableendoglucanase from the thermophilic ascomycete Rasamsonia emersonii,ReCel7B. The enzyme was compared with the homologous endoglucanasefrom the mesophilic model ascomycete Trichoderma reesei (TrCel7B),which unlike ReCel7B possesses an additional carbohydrate-binding module(CBM). With a temperature optimum of 80 °C, ReCel7B displayed anumber of differences in activity and ability to synergize with cellobiohydrolasescompared to TrCel7B. We improved both binding and kinetics ina chimeric variant of ReCel7B and a CBM, while we observe the oppositeeffect when the CBM was removed in TrCel7B. The crystal structure ofReCel7B was determined at 2.48 A resolution, with Rwork and Rfree factorsof 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7Bhas increased rigidity in a number of peripheral loops compared toTrCel7B and fewer aromatics in the substrate-binding cleft. An increasednumber of glycosylations were identified in ReCel7B, and we propose a stabilizingmechanism for one of the glycans. Global structure–function interpretationsof ReCel7B highlight the differences in temperature stability,turnover, binding, and cellulose accessibility in GH7 endoglucanases

Discovery of hyperstable carbohydrate-active enzymes through metagenomics of extreme environments

International audienceThe enzymes from hyperthermophilic microorganisms populating volcanic sites represent interesting cases of protein adaptation and biotransformations under conditions where conventional enzymes quickly denature. The difficulties in cultivating extremophiles severely limit access to this class of biocatalysts. To circumvent this problem, we embarked on the exploration of the biodiversity of the solfatara Pisciarelli, Agnano (Naples, Italy), to discover hyperthermophilic carbohydrate‐active enzymes (CAZymes) and to characterize the entire set of such enzymes in this environment (CAZome). Here, we report the results of the metagenomic analysis of two mud/water pools that greatly differ in both temperature and pH (T = 85 °C and pH 5.5; T = 92 °C and pH 1.5, for Pool1 and Pool2, respectively). DNA deep sequencing and following in silico analysis led to 14 934 and 17 652 complete ORFs in Pool1 and Pool2, respectively. They exclusively belonged to archaeal cells and viruses with great genera variance within the phylum Crenarchaeota, which reflected the difference in temperature and pH of the two Pools. Surprisingly, 30% and 62% of all of the reads obtained from Pool1 and 2, respectively, had no match in nucleotide databanks. Genes associated with carbohydrate metabolism were 15% and 16% of the total in the two Pools, with 278 and 308 putative CAZymes in Pool1 and 2, corresponding to ~ 2.0% of all ORFs. Biochemical characterization of two CAZymes of a previously unknown archaeon revealed a novel subfamily GH5_19 β‐mannanase/β‐1,3‐glucanase whose hemicellulose specificity correlates with the vegetation surrounding the sampling site, and a novel NAD+‐dependent GH109 with a previously unreported β‐N‐acetylglucosaminide/β‐glucoside specificity