Insights into the functionality and stability of designer cellulosomes at elevated temperatures

Galanopoulou, Anastasia P.; Moraïs, Sarah; Georgoulis, Anastasios D.; Morag, Ely; Bayer, Edward A.; Hatzinikolaou, Dimitris G.

doi:10.1007/s00253-016-7594-5

Cited by 20 publications

(27 citation statements)

References 52 publications

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“…However, when crystallinity of the remaining cellulose was used as a measure of cellulase efficiency, a negative effect of cellulosomal β-glucosidase on hydrolysis was noted [104]. This is in contrast to a synthetic cellulosome constructed from recombinant enzymes and scaffoldin which incorporated a C. saccharolyticus β-glucosidase [105]. In this case, the β-glucosidase was fused to a dockerin domain similar to native cellulosomal enzymes, and a synergistic effect during corn stover hydrolysis was observed between the β-glucosidase from C. saccharolyticus and endo-cellulase and xylanases from thermophilic Geobacillus strains [105].…”

Section: Heterologous Expression Of Caldicellulosiruptor Enzymes In Omentioning

confidence: 96%

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

Blumer-Schuette

2020

Microorganisms

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Plant polysaccharides continue to serve as a promising feedstock for bioproduct fermentation. However, the recalcitrant nature of plant biomass requires certain key enzymes, including cellobiohydrolases, for efficient solubilization of polysaccharides. Thermostable carbohydrate-active enzymes are sought for their stability and tolerance to other process parameters. Plant biomass degrading microbes found in biotopes like geothermally heated water sources, compost piles, and thermophilic digesters are a common source of thermostable enzymes. While traditional thermophilic enzyme discovery first focused on microbe isolation followed by functional characterization, metagenomic sequences are negating the initial need for species isolation. Here, we summarize the current state of knowledge about the extremely thermophilic genus Caldicellulosiruptor, including genomic and metagenomic analyses in addition to recent breakthroughs in enzymology and genetic manipulation of the genus. Ten years after completing the first Caldicellulosiruptor genome sequence, the tools required for systems biology of this non-model environmental microorganism are in place.

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Section: Heterologous Expression Of Caldicellulosiruptor Enzymes In Omentioning

confidence: 96%

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

Blumer-Schuette

2020

Microorganisms

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“…The combined action of XynDZ5 with the GH52 β-xylosidase of Geobacillus sp. SP24 (XydGH52) (Galanopoulou et al, 2016), resulted in the alleviation of the inhibitory action of xylooligosacchrides on XynDZ5 and an up to 3-fold increase in the total X1 to X4 concentration, with xylose being the main product (80 -95%) ( Figure 6).…”

Section: Overexpression Purification and Biochemical Characterizatiomentioning

confidence: 99%

“…SP24 (Stathopoulou et al, 2012). The latter enzyme has been previously isolated from the gDNA of the strain, overexpressed in E. coli, purified and partially characterized (Galanopoulou et al, 2016). Aliquots were removed from the reaction mixtures at specific time intervals, centrifuged, filtered and analyzed for their xylo-oligosaccharide (X1-X4) concentration in an Agilent 1220 HPLC system equipped with an RI 1260 Infinity detector.…”

Section: Biochemical Characterizationmentioning

confidence: 99%

XynDZ5: A New Thermostable GH10 Xylanase

et al. 2020

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Xylanolytic enzymes have a broad range of applications in industrial biotechnology as biocatalytic components of various processes and products, such as food additives, bakery products, coffee extraction, agricultural silage and functional foods. An increasing market demand has driven the growing interest for the discovery of xylanases with specific industrially relevant characteristics, such as stability at elevated temperatures and in the presence of other denaturing factors, which will facilitate their incorporation into industrial processes. In this work, we report the discovery and biochemical characterization of a new thermostable GH10 xylanase, termed XynDZ5, exhibiting only 26% amino acid sequence identity to the closest characterized xylanolytic enzyme. This new enzyme was discovered in an Icelandic hot spring enrichment culture of a Thermoanaerobacterium species using a recently developed bioinformatic analysis platform. XynDZ5 was produced recombinantly in Escherichia coli, purified and characterized biochemically. This analysis revealed that it acts as an endo-1,4β-xylanase that performs optimally at 65-75 • C and pH 7.5. The enzyme is capable of retaining high levels of catalytic efficiency after several hours of incubation at high temperatures, as well as in the presence of significant concentrations of a range of metal ions and denaturing agents. Interestingly, the XynDZ5 biochemical profile was found to be atypical, as it also exhibits significant exo-activity. Computational modeling of its three-dimensional structure predicted a (β/α) 8 TIM barrel fold, which is very frequently encountered among family GH10 enzymes. This modeled structure has provided clues about structural features that may explain aspects of its catalytic performance. Our results suggest that XynDZ5 represents a promising new candidate biocatalyst appropriate for several high-temperature biotechnological applications in the pulp, paper, baking, animal-feed and biofuel industries.

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“…[5] Cellulosomes are ak ind of metabolon in which scaffoldins contain multiple cohesins that can bind tightly with dockerin-containing hydrolytic enzymes. [8] Second, poor thermostability of miniscaffoldins at elevated temperatures (especially > 50 8C) [9] resultsi nthe disintegration of artificial metabolons. However,t wo inherentp roblemsl imit wide application of the cohesin-dockerin strategy.F irst, addition of ad ockerin to ap rotein with complicated quaternary structure may prevent proper protein folding and thereby greatlyd ecrease its activity.…”

Section: Introductionmentioning

confidence: 99%

“…However,t wo inherentp roblemsl imit wide application of the cohesin-dockerin strategy.F irst, addition of ad ockerin to ap rotein with complicated quaternary structure may prevent proper protein folding and thereby greatlyd ecrease its activity. [8] Second, poor thermostability of miniscaffoldins at elevated temperatures (especially > 50 8C) [9] resultsi nthe disintegration of artificial metabolons. [10] Hydrogen gas, as ac arbon-neutral energy carrier,h as excellent advantages for replacing fossil-fuel-derived liquid fuels.…”

Section: Introductionmentioning

confidence: 99%

Building a Thermostable Metabolon for Facilitating Coenzyme Transport and In Vitro Hydrogen Production at Elevated Temperature

et al. 2018

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To facilitate coenzyme transport and in vitro enzymatic hydrogen production, a multi-enzyme metabolon comprising a miniscaffoldin containing three cohesins, a dockerin-containing mutant dehydrogenase, a dockerin-containing diaphorase, and a Histidine-tagged (His-tagged) NiFe hydrogenase was constructed. As the NiFe hydrogenase has very complicated structure and cannot be fused directly with a dockerin, a bifunctional peptide was designed. The bifunctional peptide, in which one terminus contains a modified dockerin binding the cohesin of the miniscaffoldin and the other, after chemical modification, binds the His-tag of NiFe hydrogenase, enabled His-tagged proteins to be integrated into the cohesin-dockerin-based metabolon. The metabolon exhibited an initial reaction rate 4.5 times that of the enzyme cocktail at the same enzyme loading, which indicated enhanced coenzyme transport of the metabolon. However, this metabolon was unstable owing to the degradation of the miniscaffoldin at elevated temperature. Glutaraldehyde was used to cross-link the metabolon for locking its spatial organization. The cross-linked metabolon not only exhibited 2.5 times the reaction rate of the enzyme cocktail, but also retained its stability at 70 °C. The amount of hydrogen production catalyzed by the cross-linked metabolon was nearly twice that of the metabolon without glutaraldehyde cross-linking and four times that of the enzyme cocktail at 70 °C after 22 h of reaction.

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Insights into the functionality and stability of designer cellulosomes at elevated temperatures

Cited by 20 publications

References 52 publications

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

XynDZ5: A New Thermostable GH10 Xylanase

Building a Thermostable Metabolon for Facilitating Coenzyme Transport and In Vitro Hydrogen Production at Elevated Temperature

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