Сarbohydrate binding module CBM28 of endoglucanase Cel5D from Caldicellulosiruptor bescii recognizes crystalline cellulose

Dvortsov, Igor A.; Lunina, N. A.; Chekanovskaya, L. A.; Gromov, A.V.; Schwarz, Wolfgang H.; Zverlov, Vladimir V.; Velikodvorskaya, G. A.; Demidyuk, Ilya V.; Kostrov, Sergey V.

doi:10.1016/j.ijbiomac.2017.08.165

Cited by 4 publications

(2 citation statements)

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“…Cel5D was therefore selected to be integrated into our hyperthermophilic designer cellulosome. The Cel5D endoglucanase possesses a glycoside hydrolase 5 module and a CBM28 that has been found to bind to amorphous cellulose [68]. The CBM28 was removed and replaced by a dockerin, in order to convert the enzyme into the cellulosomal mode.…”

Section: Resultsmentioning

confidence: 99%

Creation of a functional hyperthermostable designer cellulosome

Kahn

Moraïs

Galanopoulou

et al. 2019

Biotechnol Biofuels

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Background Renewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a particularly high potential as an alternative source of energy. Industrial deconstruction of biomass, however, is an onerous, exothermic process, the cost of which could be decreased significantly by use of hyperthermophilic enzymes. An efficient way of breaking down cellulosic substrates can also be achieved by highly efficient enzymatic complexes called cellulosomes. The modular architecture of these multi-enzyme complexes results in substrate targeting and proximity-based synergy among the resident enzymes. However, cellulosomes have not been observed in hyperthermophilic bacteria. Results Here, we report the design and function of a novel hyperthermostable “designer cellulosome” system, which is stable and active at 75 °C. Enzymes from Caldicellulosiruptor bescii , a highly cellulolytic hyperthermophilic anaerobic bacterium, were selected and successfully converted to the cellulosomal mode by grafting onto them divergent dockerin modules that can be inserted in a precise manner into a thermostable chimaeric scaffoldin by virtue of their matching cohesins. Three pairs of cohesins and dockerins, selected from thermophilic microbes, were examined for their stability at extreme temperatures and were determined stable at 75 °C for at least 72 h. The resultant hyperthermostable cellulosome complex exhibited the highest levels of enzymatic activity on microcrystalline cellulose at 75 °C, compared to those of previously reported designer cellulosome systems and the native cellulosome from Clostridium thermocellum . Conclusion The functional hyperthermophilic platform fulfills the appropriate physico-chemical properties required for exothermic processes. This system can thus be adapted for other types of thermostable enzyme systems and could serve as a basis for a variety of cellulolytic and non-cellulolytic industrial objectives at high temperatures. Electronic supplementary material The online version of this article (10.1186/s13068-019-1386-y) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Creation of a functional hyperthermostable designer cellulosome

Kahn

Moraïs

Galanopoulou

et al. 2019

Biotechnol Biofuels

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“…To generate CL-containing enzymes for the CIQ system, four hyperthermophilic enzymes involved in the hydrolysis of cellulose were chosen, namely exoglucanase Cel48S m3 and endoglucanases Cel8A* from C. thermocellum (proteins engineered ,, ), GH5D , from Caldicellulosiruptor bescii, and β-glucosidase CoGH1A from Caldicellulosiruptor owensensis . The native CBM and dockerins of these modular enzymes were removed prior to fusing with CL2, 7, 8, and 9, respectively.…”

Section: Resultsmentioning

confidence: 99%

Development of a Hyperthermostable Artificial Scaffold Based on Ultrahigh-Affinity Protein Pairs and Its Application in Cellulose Degradation

Yang

Wang

Yang

et al. 2022

ACS Sustainable Chem. Eng.

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The binding affinities between E-group colicin (CE) and cognate immunity proteins (Im) are among the strongest interactions in nature. Aiming at mining protein pairs with ultrahigh affinity for artificial multienzyme complexes, DNase domains of CE2, 8, and 9 were engineered to generate CL2, 8, and 9 variants without DNA binding and catalytic activity but retaining Im binding activity, respectively. Further study discovered that all these variants, in addition to a CE7 variant (CL7) from the previous report, and their cognate Im proteins are highly thermostable. Taking advantage of the specific and ultrahigh affinity of these hyperthermostable affinity pairs, a hyperthermostable artificial scaffold was established and applied for cellulose degradation. Four hyperthermostable cellulolytic enzymes were integrated into the system through specific pairing of CL and cognate Im proteins. This complex with four enzymes assembled sequentially exhibited an obvious synergistic effect in hydrolyzing cellulosic substrates at elevated temperature. In comparison with free enzymes, the catalytic activity of the complex enhanced more than half-fold at 70 °C with phosphoric acid-swollen cellulose as the substrate, while it increased almost onefold at 75 °C with Avicel as the substrate. The present study provided four affinity pairs with ultrahigh affinity, hyperthermostability, high specificity, a small and unified structure, and high assembly efficiency. More importantly, it provided an unconventional idea of mining hyperthermostable affinity pairs from mesophilic microorganisms instead of the less-abundant thermophiles, which are the main source pool of the thermostable affinity pairs for the time being.

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