4-Nitrophenyl-β-lactoside

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 rapid mass spectrometry method for determining the anomeric configuration of the sugar at the reducing end of an oligosaccharide was demonstrated. The method was employed to identify the nascent anomeric configuration (i.e., before significant mutarotation occurs) of oligosaccharides released by carbohydrate-active enzymes, which enabled determination of the enzyme mechanism. This method was validated by applying it to various enzymes, including α-glucosidase, β-glucosidases, endoglycoceramidase II, β-galactosidase, and β-amylase.

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.