Comparative analysis of three hyperthermophilic GH1 and GH3 family members with industrial potential

Cota, Júnio; Corrêa, Thamy Lívia Ribeiro; Damásio, André R.L.; Diogo, José; Hoffmam, Zaira B.; Garcia, Wânius; Oliveira, Leandro C.; Prade, Rolf A.; Squina, Fábio M.

doi:10.1016/j.nbt.2014.07.009

Cited by 44 publications

(41 citation statements)

References 48 publications

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“…The lines are just a guide for the eye (color figure online) in the supernatant after lysis. The recombinant enzymes were purified to apparent homogeneity by a two-step protocol using both affinity and size-exclusion chromatographies (Cota et al 2015). Upon 15 % SDS-PAGE under reducing conditions the purified enzymes migrated as large homogenous bands, consistent with the expected molecular mass for TpBGL1 (54 kDa) and TpBGL3 (84 kDa) monomers (inset of Fig.…”

Section: Resultsmentioning

confidence: 61%

“…Cloning, expression and purification of the TpBGL1 (GenBank: ABQ46970.1) and TpBGL3 (GenBank: ABQ46916.1) were carried out as described previously (Cota et al 2015;Squina et al 2009). The purity of the final products, TpBGL1 and TpBGL3, were checked by Coomassie-stained 15 % SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

“…The standard enzymatic assay for β-glucosidase activities was performed according to previously described method (Cota et al 2015). 100 ng of enzyme, 40 μL of buffer solution adjusted at different pH values (pH range between 2 and 10) were used and 50 μL of 1 mM 4-nitrophenyl-β-dglucopyranoside (pNPG) was used as substrate.…”

Section: Enzymatic Activity Measurementsmentioning

confidence: 99%

“…In a recent study, we have provided a biochemical characterization of two hyperthermostable β-glucosidases from T. petrophila belonging to the families GH1 (TpBGL1) and GH3 (TpBGL3) (Cota et al 2015). Here, as part of a continuing investigation, the oligomeric state, the net charge, and the structural stability, at acidic pH, of the TpBGL1 and TpBGL3 were characterized and compared.…”

Section: Introductionmentioning

confidence: 99%

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Oligomeric state and structural stability of two hyperthermophilic β-glucosidases from Thermotoga petrophila

et al. 2015

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The β-glucosidases are enzymes essential for several industrial applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts. In a recent study, we have provided a biochemical characterization of two hyperthermostable β-glucosidases from Thermotoga petrophila belonging to the families GH1 (TpBGL1) and GH3 (TpBGL3). Here, as part of a continuing investigation, the oligomeric state, the net charge, and the structural stability, at acidic pH, of the TpBGL1 and TpBGL3 were characterized and compared. Enzymatic activity is directly related to the balance between protonation and conformational changes. Interestingly, our results indicated that there were no significant changes in the secondary, tertiary and quaternary structures of the β-glucosidases at temperatures below 80 °C. Furthermore, the results indicated that both the enzymes are stable homodimers in solution. Therefore, the observed changes in the enzymatic activities are due to variations in pH that modify protonation of the enzymes residues and the net charge, directly affecting the interactions with ligands. Finally, the results showed that the two β-glucosidases displayed different pH dependence of thermostability at temperatures above 80 °C. TpBGL1 showed higher stability at pH 6 than at pH 4, while TpBGL3 showed similar stability at both pH values. This study provides a useful comparison of the structural stability, at acidic pH, of two different hyperthermostable β-glucosidases and how it correlates with the activity of the enzymes. The information described here can be useful for biotechnological applications in the biofuel and food industries.

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Section: Resultsmentioning

confidence: 61%

Section: Methodsmentioning

confidence: 99%

Section: Enzymatic Activity Measurementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Oligomeric state and structural stability of two hyperthermophilic β-glucosidases from Thermotoga petrophila

et al. 2015

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“…A simple solution is simultaneous saccharification and fermentation wherein glucose is rapidly removed; however, the low temperatures at which current commercial fermentative organisms operate do not take full advantage of the higher temperature optimum of the hydrolytic enzymes and the trade-off is typically not in favour of this strategy, particularly with more recent commercial enzyme preparations (Ask et al, 2012;Wirawan et al, 2012;Cannella & Jørgensen, 2014;Agrawal et al, 2015). Another possible solution is the use of certain GH1 family -d-glucosidases with a much higher apparent K i for glucose; however, many if not most of these have poor activity on cellobiose, although exceptions have been reported (Pei et al, 2012;Cota et al, 2015). A partial solution is to use enzymes with high catalytic efficiency such as AfG, which at 50 C has a k cat more than twice that of AoG and 37% higher than that of the A. niger -d-glucosidase found in the commonly used -dglucosidase source Novozyme 188 (Bohlin et al, 2010).…”

Section: The Importance Of B-d-glucosidases In Industrial Biotechnologymentioning

confidence: 99%

Three-dimensional structures of two heavily N-glycosylatedAspergillussp. family GH3 β-D-glucosidases

Agirre

Ariza

Offen

et al. 2016

Acta Crystallogr D Struct Biol

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The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo-and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant -d-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. -d-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3 -d-glucosidases from Aspergillus fumigatus and A. oryzae, solved by molecular replacement and refined at 1.95 Å resolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley -d-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as highmannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the d-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy 4 C 1 chair conformation and should be of general application to refinements of other crystal structures containing O-or N-glycosylation. The Aspergillus GH3 structures, in light of other recent three-dimensional structures, provide insight into fungal -d-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.

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AA9 and AA10: from enigmatic to essential enzymes

Corrêa

Santos

Pereira

2015

Appl Microbiol Biotechnol

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The lignocellulosic biomass, comprised mainly of cellulose, hemicellulose, and lignin, is a strong competitor for petroleum to obtain fuels and other products because of its renewable nature, low cost, and non-competitiveness with food production when obtained from agricultural waste. Due to its recalcitrance, lignocellulosic material requires an arsenal of enzymes for its deconstruction and the consequent release of fermentable sugars. In this context, enzymes currently classified as auxiliary activity 9 (AA9/formerly GH61) and 10 (AA10/formerly CBM 33) or lytic polysaccharide monooxygenases (LPMO) have emerged as cellulase boosting enzymes. AA9 and AA10 are the new paradigm for deconstruction of lignocellulosic biomass by enhancing the activity and decreasing the loading of classical enzymes to the reaction and, consequently, reducing costs of the hydrolysis step in the second-generation ethanol production chain. In view of that disclosed above, the goal of this work is to review experimental data that supports the relevance of AA9 and AA10 for the biomass deconstruction field.

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Comparative analysis of three hyperthermophilic GH1 and GH3 family members with industrial potential

Cited by 44 publications

References 48 publications

Oligomeric state and structural stability of two hyperthermophilic β-glucosidases from Thermotoga petrophila

Oligomeric state and structural stability of two hyperthermophilic β-glucosidases from Thermotoga petrophila

Three-dimensional structures of two heavily N-glycosylatedAspergillussp. family GH3 β-D-glucosidases

AA9 and AA10: from enigmatic to essential enzymes

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