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Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data are available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far. Here, we report a new crystal structure and biochemical characterization of a thermostable endoglucanase from the thermophilic ascomycete Rasamsonia emersonii, ReCel7B. The enzyme was compared with the homologous endoglucanase from 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 a number of differences in activity and ability to synergize with cellobiohydrolases compared to TrCel7B. We improved both binding and kinetics in a chimeric variant of ReCel7B and a CBM, while we observe the opposite effect when the CBM was removed in TrCel7B. The crystal structure of ReCel7B was determined at 2.48 Å resolution, with Rwork and Rfree factors of 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7B has increased rigidity in a number of peripheral loops compared to TrCel7B and fewer aromatics in the substrate‐binding cleft. An increased number of glycosylations were identified in ReCel7B, and we propose a stabilizing mechanism for one of the glycans. Global structure–function interpretations of ReCel7B highlight the differences in temperature stability, turnover, binding, and cellulose accessibility in GH7 endoglucanases. Database: Structural data are available in RCSB Protein Data Bank database under the accession number 6SU8. Enzymes: ReCel7B, endoglucanase (EC3.2.1.4) from Rasamsonia emersonii; ReCel7A, cellobiohydrolase (EC3.2.1.176) from Rasamsonia emersonii; TrCel7B, endoglucanase (EC3.2.1.4) from Trichoderma reesei; TrCel7A, cellobiohydrolase (EC3.2.1.176) from Trichoderma reesei. [ABSTRACT FROM AUTHOR]

The 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. Databases: The sequencing reads are available in the NCBI Sequence Read Archive (SRA) database under the accession numbers SRR7545549 (Pool1) and SRR7545550 (Pool2). The sequences of GH5_Pool2 and GH109_Pool2 are available in GenBank database under the accession numbers MK869723 and MK86972, respectively. The environmental data relative to Pool1 and Pool2 (NCBI BioProject PRJNA481947) are available in the Biosamples database under the accession numbers SAMN09692669 (Pool1) and SAMN09692670 (Pool2). [ABSTRACT FROM AUTHOR]

Abstract: We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a “high affinity” variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about two‐fold faster around 70 °C. This better thermoactivation of the high‐affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability. [ABSTRACT FROM AUTHOR]