[Show abstract][Hide abstract] ABSTRACT: Background
Enzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes.
We have developed a new generation of multi-coloured chromogenic polysaccharide and protein substrates that can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates of biomass materials in which the complexity of plant cell walls is partially maintained.
We show that these substrates can be used to screen the activities of glycosyl hydrolases, lytic polysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. We envisage that the assays we have developed will be used primarily for first-level screening of large numbers of putative carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic enzyme screening systems.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-015-0250-y) contains supplementary material, which is available to authorized users.
Biotechnology for Biofuels 12/2015; 8(1). DOI:10.1186/s13068-015-0250-y · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lignin-carbohydrate complexes (LCCs) are believed to influence the recalcitrance of lignocellulosic plant material preventing optimal utilization of biomass in e.g. forestry, feed and biofuel applications. The recently emerged carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) has been proposed to degrade ester LCC bonds between glucuronic acids in xylans and lignin alcohols thereby potentially improving delignification of lignocellulosic biomass when applied in conjunction with other cellulases, hemicellulases and oxidoreductases. Herein, we report the synthesis of four new GE model substrates comprising α- and ɣ-arylalkyl esters representative of the lignin part of naturally occurring ester LCCs as well as the cloning and purification of a novel GE from Cerrena unicolor (CuGE). Together with a known GE from Schizophyllum commune (ScGE), CuGE was biochemically characterized by means of Michaelis-Menten kinetics with respect to substrate specificity using the synthesized compounds. For both enzymes, a strong preference for 4-O-methyl glucuronoyl esters rather than unsubstituted glucuronoyl esters was observed. Moreover, we found that α-arylalkyl esters of methyl α-D-glucuronic acid are more easily cleaved by GEs than their corresponding ɣ-arylalkyl esters. Furthermore, our results suggest a preference of CuGE for glucuronoyl esters of bulky alcohols supporting the suggested biological action of GEs on LCCs. The synthesis of relevant GE model substrates presented here may provide a valuable tool for the screening, selection and development of industrially relevant GEs for delignification of biomass. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Biotechnology and Bioengineering 05/2015; 112(5). DOI:10.1002/bit.25508 · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome
sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and
-modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of
methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays
with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme
screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of
single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples.
Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide
lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by
identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal
[Show abstract][Hide abstract] ABSTRACT: Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs on the market. Whilst they are considered safe, several NSAIDs have been withdrawn from the market as a result of adverse drug reactions. NSAIDs are extensively metabolised to their 1-β-O-acyl glucuronides (AGs), and the risk of NSAID AGs covalently modifying biomacromolecules such as proteins or DNA, leading to immune responses and cellular dysfunction constitutes a major concern in drug discovery and development. The assessment of the degree of protein modification and potential toxicity of individual NSAID AGs is therefore of importance in both drug monitoring and development. Herein, we report the covalent reaction of 1-β-O-acyl glucuronides of ibuprofen and several NSAID analogues with human serum albumin (HSA) protein in vitro under concentrations encountered in therapy. Stable transacylation and glycosylation adducts are formed; the observed protein product ratios can be rationalised by the degree of α-substitution in the acyl group. Structure-based protein reactivity correlations of AGs, such as these, may prove a useful tool in distinguishing between carboxylic acid-containing drugs of similar structure that ultimately prove beneficial (e.g., ibuprofen) from those that prove toxic (e.g., ibufenac).
Chemical Science 07/2014; 5(10). DOI:10.1039/C4SC01329H · 9.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent advances in the cleavage and formation of C–C bonds at the anomeric center of carbohydrates are reviewed. Both chemical and enzymatic transformations are covered with particular emphasis on aldol condensations, radical reactions, and organometallic transformations. The report contains 230 references. Figure optionsView in workspace
[Show abstract][Hide abstract] ABSTRACT: A straightforward synthesis of substituted quinolines is described by cyclocondensation of anilines with 1,3-diols. The reaction proceeds in mesitylene solution with catalytic amounts of RuCl(3)·xH(2)O, PBu(3) and MgBr(2)·OEt(2). The transformation does not require any stoichiometric additives and only produces water and dihydrogen as byproducts. Anilines containing methyl, methoxy and chloro substituents as well as naphthylamines were shown to participate in the heterocyclisation. In the 1,3-diol a substituent was allowed in the 1- or the 2-position giving rise to 2- and 3-substituted quinolines, respectively. The best results were obtained with 2-alkyl substituted 1,3-diols to afford 3-alkylquinolines. The mechanism is believed to involve dehydrogenation of the 1,3-diol to the 3-hydroxyaldehyde which eliminates water to the corresponding α,β-unsaturated aldehyde. The latter then reacts with anilines in a similar fashion as observed in the Doebner-von Miller quinoline synthesis.
[Show abstract][Hide abstract] ABSTRACT: A catalytic procedure is described for decarbonylation of unprotected aldoses to afford alditols with one less carbon atom. The reaction is performed with the rhodium complex Rh(dppp)2Cl in a refluxing diglyme-DMA solution. A slightly improved catalyst turnover is observed when a catalytic amount of pyridine is added. Under these conditions most hexoses and pentoses undergo decarbonylation into the corresponding pentitols and tetrols in isolated yields around 70%. The reaction has been applied as the key transformation in a five-step synthesis of L-threose from D-glucose.
The Journal of Organic Chemistry 01/2008; 72(25):9782-5. DOI:10.1021/jo7017729 · 4.72 Impact Factor