Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester

Sunner, Hampus; Charavgi, Maria-Despoina; Olsson, Lisbeth; Christakopoulos, Paul

doi:10.3390/molecules201017807

Cited by 21 publications

(16 citation statements)

References 18 publications

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“…GE activity was detected by using the K-URONIC kit (Megazyme, Ireland), as described previously (Sunner et al 2015). Briefly, GE assays were performed in 200 μL reactions containing 73 mM potassium phosphate buffer pH 6.0, 2 mM substrate and 2% DMSO, and incubated for 1–30 min at 35 °C.…”

Section: Methodsmentioning

confidence: 99%

Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds

Hüttner

Klaubauf

Vries

et al. 2017

Appl Microbiol Biotechnol

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The glucuronoyl esterases (GEs) that have been identified so far belong to family 15 of the carbohydrate esterases in the CAZy classification system and are presumed to target ester bonds between lignin alcohols and (4-O-methyl-)d-glucuronic acid residues of xylan. Few GEs have been cloned, expressed and characterised to date. Characterisation has been done on a variety of synthetic substrates; however, the number of commercially available substrates is very limited. We identified novel putative GEs from a wide taxonomic range of fungi and expressed the enzymes originating from Acremonium alcalophilum and Wolfiporia cocos as well as the previously described PcGE1 from Phanerochaete chrysosporium. All three fungal GEs were active on the commercially available compounds benzyl glucuronic acid (BnGlcA), allyl glucuronic acid (allylGlcA) and to a lower degree on methyl glucuronic acid (MeGlcA). The enzymes showed pH stability over a wide pH range and tolerated 6-h incubations of up to 50 °C. Kinetic parameters were determined for BnGlcA. This study shows the suitability of the commercially available model compounds BnGlcA, MeGlcA and allylGlcA in GE activity screening and characterisation experiments. We enriched the spectrum of characterised GEs with two new members of a relatively young enzyme family. Due to its biotechnological significance, this family deserves to be more extensively studied. The presented enzymes are promising candidates as auxiliary enzymes to improve saccharification of plant biomass.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-017-8266-9) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds

Hüttner

Klaubauf

Vries

et al. 2017

Appl Microbiol Biotechnol

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“…The reaction of the CkGE15A domain with the BnzGlcA substrate at 40 °C showed an approximately twofold reduction in K m and a threefold increase in k cat . It was not possible to conduct reactions at higher temperatures due to rapid autohydrolysis of the substrates that resulted in extremely high background, which shows that the model substrates are not thermostable, as reported previously [58]. Therefore, an optimal temperature for this domain could not be determined.…”

Section: Determination Of Glucuronoyl Esterase Activitymentioning

confidence: 94%

“…Activity was measured using a spectrophotometric assay described previously [58]. Briefly, the synthetic substrate analogs benzyl-, allyl-, or methyl-d-glucuronoate or methyl-d-galacturonoate (Carbosynth) were dissolved in dimethylsulfoxide (DMSO).…”

Section: Determination Of Glucuronoyl Esterase Activitymentioning

confidence: 99%

Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules

Krska

Larsbrink

2020

Biotechnol Biofuels

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Background: Efficient degradation of lignocellulosic biomass has become a major bottleneck in industrial processes which attempt to use biomass as a carbon source for the production of biofuels and materials. To make the most effective use of the source material, both the hemicellulosic as well as cellulosic parts of the biomass should be targeted, and as such both hemicellulases and cellulases are important enzymes in biorefinery processes. Using thermostable versions of these enzymes can also prove beneficial in biomass degradation, as they can be expected to act faster than mesophilic enzymes and the process can also be improved by lower viscosities at higher temperatures, as well as prevent the introduction of microbial contamination. Results: This study presents the investigation of the thermostable, dual-function xylanase-glucuronoyl esterase enzyme CkXyn10C-GE15A from the hyperthermophilic bacterium Caldicellulosiruptor kristjanssonii. Biochemical characterization of the enzyme was performed, including assays for establishing the melting points for the different protein domains, activity assays for the two catalytic domains, as well as binding assays for the multiple carbohydratebinding domains present in CkXyn10C-GE15A. Although the enzyme domains are naturally linked together, when added separately to biomass, the expected boosting of the xylanase action was not seen. This lack of intramolecular synergy might suggest, together with previous data, that increased xylose release is not the main beneficial trait given by glucuronoyl esterases. Conclusions: Due to its thermostability, CkXyn10C-GE15A is a promising candidate for industrial processes, with both catalytic domains exhibiting melting temperatures over 70 °C. Of particular interest is the glucuronoyl esterase domain, as it represents the first studied thermostable enzyme displaying this activity.

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“…GE activity was confirmed on the model substrate Benzyl‐ d ‐glucuronate (BenzGlcA) (Carbosynth, Compton, UK) and GlcA was detected using the Megazyme K‐URONIC kit, according to the assay described by Sunner et al . . One unit of GE activity is defined as the amount of enzyme required to release 1 μmol of GlcA per minute at pH 6.0 and 35 °C.…”

Section: Methodsmentioning

confidence: 99%

A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin‐carbohydrate ester bonds

et al. 2016

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Edited by Ulf-Ingo Fl€ uggeThe Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and 31 P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood.Keywords: 31 P NMR; carbohydrate esterase; lignin-carbohydrate complexes; size exclusion chromatography; spruce and birch An obstacle to intact and efficient extraction of the wood polymers cellulose, hemicelluloses and lignin may be the proposed chemical linkages connecting lignin with hemicelluloses, the so-called lignin-carbohydrate complexes (LCCs). In a lignocellulose-based biorefinery context it is therefore of interest to find mild methods, for example, enzymatic treatment, that specifically would break these lignin-carbohydrate (LC) linkages. Three types of existing covalent LC bonds have been suggested, namely benzyl ether-, ester-and phenyl glycosidic linkages [1]. The first described and most studied LC ester bond is the benzyl a-ester bond [2]. However, several more recent studies have also shown the presence of c-esters [3,4], suggested to arise due to uronosyl migrations of the linkage from the a-to the c-position [5,6].It has been proposed that glucuronoyl esterases (GE), members of the recently discovered carbohydrate esterase 15 family (CE15), could be used to target the LC ester bond between aliphatic alcohols in lignin and the 4-O-methyl-D-glucuronic acid residues of glucuronoxylans ( Fig. 1) [7,8].The first characterized GE was produced by the wood-rotting fungus Schizophyllum commune [7] and at the present time nine GEs have been characterized for their activity and kinetics on different model substrates [7][8][9][10][11][12][13][14][15][16][17][18][19][20].Cleavage of model substrates mimicking LC ester bonds has been investigated for a wide range of esters. Studies have covered simple glucuronic acid (GlcA) esters with the 'lignin moiety' being single arylalkyl units at the c-position [8,10] or at the a-position [14].

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Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester

Cited by 21 publications

References 18 publications

Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds

Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds

Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules

A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin‐carbohydrate ester bonds

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