An official website of the United States government
Fervidibacter sacchariis an aerobic hyperthermophile belonging to the phylumArmatimonadotathat degrades a variety of polysaccharides. Its genome encodes 117 enzymes with one or more annotated glycoside hydrolase (GH) domain, but the roles of these putative GHs in polysaccharide catabolism are poorly defined. Here, we describe oneF. saccharienzyme encoding a GH10 domain, Fsa02490Xyn, that was previously shown to be active onMiscanthus, oat β‐glucan, and beech‐wood xylan, with optimal activity at 90–100 °C. We show that Fsa02490Xyn is also active on birch‐wood xylan and gellan gum. The pH range on beech‐wood xylan was 4.5 to 9.5 (pHopt7.0–8.0). Fsa024940Xyn had aKmof 2.375 mm,Vmaxof 1250 μm·min−1, andkcat/Kmof 1.259 × 104 s−1·m−1when using apara‐nitrophenyl‐𝛽‐xylobioside assay. A phylogenetic analysis of GH10 family enzymes revealed a large clade of enzymes from diverse members of the classFervidibacteria, including Fsa02490Xyn and a second enzyme fromF. sacchari, with apparent horizontal gene transfer withinFervidibacteriaand betweenFervidibacteriaand thermophilicBacillota. This study establishes Fsa02490Xyn as a hyperthermophilic GH10 enzyme with endo‐β‐1,4‐xylanase activity and identifies a large clade of homologous GH10 enzymes within the classFervidibacteria. Impact statementThe depolymerization of xylan at high temperatures is important because this process limits the degradation of polysaccharides in nature and the synthesis of biofuels from plant wastes. Our study is also important becauseF. sacchariis one of only a few cultivated members of theArmatimonadota, which are polysaccharide‐degradation specialists.
more »
« less
Hydrolysis of ionic liquid–treated substrate with an Iocasia fonsfrigidae strain SP3-1 endoglucanase
AbstractRecently, we reported the discovery of a novel endoglucanase of the glycoside hydrolase family 12 (GH12), designated IfCelS12A, from the haloalkaliphilic anaerobic bacteriumIocasia fonsfrigidaestrain SP3-1, which was isolated from a hypersaline pond in the Samut Sakhon province of Thailand (ca. 2017). IfCelS12A exhibits high substrate specificity on carboxymethyl cellulose and amorphous cellulose but low substrate specificity on b-1,3;1,4-glucan. Unlike some endoglucanases of the GH12 family, IfCelS12A does not exhibit hydrolytic activity on crystalline cellulose (i.e., Avicel™). High-Pressure Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) analyses of products resulting from IfCelS12-mediated hydrolysis indicate mode of action for this enzyme. Notably, IfCelS12A preferentially hydrolyzes cellotetraoses, cellopentaoses, and cellohexaoses with negligible activity on cellobiose or cellotriose. Kinetic analysis with cellopentaose and barely b-d-glucan as cellulosic substrates were conducted. On cellopentaose, IfCelS12A demonstrates a 16-fold increase in activity (KM = 0.27 mM;kcat = 0.36 s−1;kcat/KM = 1.34 mM−1s−1) compared to the enzymatic hydrolysis of barley b-d-glucan (KM: 0.04 mM,kcat: 0.51 s−1,kcat/KM = 0.08 mM−1s−1). Moreover, IfCelS12A enzymatic efficacy is stable in hypersaline sodium chlorids (NaCl) solutions (up to 10% NaCl). Specifically, IfCel12A retains notable activity after 24 h at 2M NaCl (10% saline solution). IfCelS12A used as a cocktail component with other cellulolytic enzymes and in conjunction with mobile sequestration platform technology offers additional options for deconstruction of ionic liquid–pretreated cellulosic feedstock. Key points•IfCelS12A from an anaerobic alkaliphile Iocasia fronsfrigidae shows salt tolerance•IfCelS12A in cocktails with other enzymes efficiently degrades cellulosic biomass•IfCelS12A used with mobile enzyme sequestration platforms enhances hydrolysis
more »
« less
- PAR ID:
- 10484906
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Applied Microbiology and Biotechnology
- Volume:
- 108
- Issue:
- 1
- ISSN:
- 0175-7598
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Rosenzweig, Amy (Ed.)Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug’s direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition (mfmAandmfmB) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (kcat/KM) of 9.6 × 103M−1s−1and KMfor metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of themfmABgenes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater.more » « less
-
Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silicoAbstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.more » « less
-
The aerobic hyperthermophile“Fervidibacter sacchari”catabolizes diverse polysaccharides and is the only cultivated member of the class“Fervidibacteria”within the phylumArmatimonadota. It encodes 117 putative glycoside hydrolases (GHs), including two from GH family 50 (GH50). In this study, we expressed, purified, and functionally characterized one of these GH50 enzymes, Fsa16295Glu. We show that Fsa16295Glu is a β-1,3-endoglucanase with optimal activity on carboxymethyl curdlan (CM-curdlan) and only weak agarase activity, despite most GH50 enzymes being described as β-agarases. The purified enzyme has a wide temperature range of 4–95°C (optimal 80°C), making it the first characterized hyperthermophilic representative of GH50. The enzyme is also active at a broad pH range of at least 5.5–11 (optimal 6.5–10). Fsa16295Glu possesses a relatively highkcat/KMof 1.82 × 107 s−1M−1with CM-curdlan and degrades CM-curdlan nearly completely to sugar monomers, indicating preferential hydrolysis of glucans containing β-1,3 linkages. Finally, a phylogenetic analysis of Fsa16295Glu and all other GH50 enzymes revealed that Fsa16295Glu is distant from other characterized enzymes but phylogenetically related to enzymes from thermophilic archaea that were likely acquired horizontally from“Fervidibacteria.”Given its functional and phylogenetic novelty, we propose that Fsa16295Glu represents a new enzyme subfamily, GH50_3.more » « less
-
Abstract BackgroundFuture expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Therefore, cellulosic biofuels derived from agricultural waste and dedicated energy crops are necessary. To date, slow and incomplete saccharification as well as high enzyme costs have hindered the economic viability of cellulosic biofuels, and while approaches like simultaneous saccharification and fermentation (SSF) and the use of thermotolerant microorganisms can enhance production, further improvements are needed. Cellulosic emulsions have been shown to enhance saccharification by increasing enzyme contact with cellulose fibers. In this study, we use these emulsions to develop an emulsified SSF (eSSF) process for rapid and efficient cellulosic biofuel production and make a direct three-way comparison of ethanol production betweenS. cerevisiae,O. polymorpha, andK. marxianusin glucose and cellulosic media at different temperatures. ResultsIn this work, we show that cellulosic emulsions hydrolyze rapidly at temperatures tolerable to yeast, reaching up to 40-fold higher conversion in the first hour compared to microcrystalline cellulose (MCC). To evaluate suitable conditions for the eSSF process, we explored the upper temperature limits for the thermotolerant yeastsKluyveromyces marxianusandOgataea polymorpha, as well asSaccharomyces cerevisiae, and observed robust fermentation at up to 46, 50, and 42 °C for each yeast, respectively. We show that the eSSF process reaches high ethanol titers in short processing times, and produces close to theoretical yields at temperatures as low as 30 °C. Finally, we demonstrate the transferability of the eSSF technology to other products by producing the advanced biofuel isobutanol in a light-controlled eSSF using optogenetic regulators, resulting in up to fourfold higher titers relative to MCC SSF. ConclusionsThe eSSF process addresses the main challenges of cellulosic biofuel production by increasing saccharification rate at temperatures tolerable to yeast. The rapid hydrolysis of these emulsions at low temperatures permits fermentation using non-thermotolerant yeasts, short processing times, low enzyme loads, and makes it possible to extend the process to chemicals other than ethanol, such as isobutanol. This transferability establishes the eSSF process as a platform for the sustainable production of biofuels and chemicals as a whole.more » « less
