2-Chloro-4-nitrophenyl-β-cellotrioside

endo-1,4-β-Glucanase (endo-cellulase, EC 3.2.1.4) is one of the most widely used enzymes in industry. Despite its importance, improved methods for the rapid, selective, quantitative assay of this enzyme have been slow to emerge. In 2014, a novel enzyme-coupled assay that addressed many of the limitations of the existing assay methodology was reported. This involved the use of a bifunctional substrate chemically derived from cellotriose. Reported herein is a much improved version of this assay employing a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside.

A specific and sensitive substrate for the assay of endo-1,4-β-glucanase (cellulase) has been prepared. The substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-β-cellotrioside (BzCNPG3) in the presence of thermostable β-glucosidase. Hydrolysis by exo-acting enzymes such as β-glucosidase and exo-β-glucanase is prevented by the presence of the benzylidene group on the non-reducing end D-glucosyl residue. On hydrolysis by cellulase, the 2-chloro-4-nitrophenyl-β-glycoside is immediately hydrolysed to 2-chloro-4-nitrophenol and free D-glucose by the β-glucosidase in the substrate mixture. The reaction is terminated and colour developed by the addition of a weak alkaline solution. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation. This procedure should find widespread applications in biomass enzymology and in the specific assay of endo-1,4-β-glucanase in general.

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

A comparison of the cellulolytic activities found in three commercial Trichoderma reesei preparations is presented. Classical substrates such as native or processed cellulose and substituted (soluble) derivatives (e.g. carboxymethylcellulose) are poorly defined. Chromogenic (fluorogenic) 4-methylumbelliferyl β-glycosides can advantageously be applied to provide clear differentiation of the three main cellulolytic components of the complex from Trichoderma reesei, after separation by isoelectric focusing. Cellobiohydrolase I (CBH I) releases the fluorescent phenol from the lactoside (MULAC) and from the cellobioside (MUC). Endoglucanase I (EG I) liberates the phenol from the same substrates but the enzymes can be differentiated by specific inhibition with cellobiose (suppressing selectively the cellobiohydrolase activity). Endoglucanases III (EG III) is the only enzyme present in the complex capable of catalysing the hydrolysis of the cellotrioside (MUG)₃ at the heterosidic bond, yielding the fluorescent 4-methyl-umbelliferone. The corresponding 2-chloro-4-nitrophenol glycosides offer an attractive alternative to classical reductometric methods. The substrates are sufficiently stable (pH 4–8; 60°C) and the favourable absorption characteristics of the phenol (pK 5•5, absorption coefficient at 405 nm 8750 M^-1 cm^-1, pH 5•6) allow sensitive, continuous and quantitative assays. Using these methods, it was found that Novo (Denmark) cellulase is particularly poor in β-glucosidase activity, an IFP (France) preparation is maximally active on the classical substrates in contrast to the Genencor (USA) cellulase with high activity against the small, chromophoric substrates.