Browsing by Subject "Glycoside hydrolase"
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Item In vitro and in vivo characterization of three Cellvibrio japonicus glycoside hydrolase family 5 members reveals potent xyloglucan backbone-cleaving functions(BioMed Central, 2018) Attia, Mohamed A.; Nelson, Cassandra E.; Offen, Wendy A.; Jain, Namrata; Davies, Gideon J.; Gardner, Jeffrey G.; Brumer, HarryBackground Xyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved. Results Combined bioinformatic and transcriptomic analyses implicated three glycoside hydrolase family 5 subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here. Conclusions Through a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural–functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatic predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications.Item THERMAL DENATURATION AND REFOLDING OF CARBOHYDRATE BINDING MODULES TO IMPROVE ENZYME RECYCLING IN A LIGNOCELLULOSIC BIOREFINERY(2019-08-01) Sanchez Hernandez, Jose; Laufer, Craig S.; Esposito, Dominic; Hirschhorn, Ricky R.; Hood College Biology Department; Hood College Biomedical Science ProgramSecond generation bioethanol using lignocellulosic waste is a promising source of renewable energy. Cellulose, lignin, and other biopolymers make up lignocellulose. Saccharification of lignocellulose requires cellulases, which have a catalytic domain (CD) and a carbohydrate binding module (CBM) that binds the enzyme to substrates. Cellulase activity is known to decrease during saccharification when CBMs irreversibly bind lignin that the CD cannot hydrolyze for release and further reactions. While costly, cellulases must be supplemented after each cycle of saccharification. Here we demonstrate that following denaturation when heated to 5°C above the melting temperature (Tm), CBMs 11 and 44 (CAZy families) from Hungateiclostridium thermocellum (CtCBM11 and CtCBM44) can be released from a bound substrate. Once cooled to a temperature below the Tm, CtCBM11 and CtCBM44, Type B CBMs with a β-sandwich fold, spontaneously refold and regain binding function. Using temperature tunable CBMs could drastically reduce saccharification costs by improving enzyme recycling strategies.