Rapid screening for endo-β-1,4-glucanase and endo-β-1,4-mannanase activities and specific measurement using soluble dye-labelled substrates

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Abstract

Soluble dye-labelled substrates were prepared for the specific and rapid screening and the measurement of endo-β-1,4-glucanase and endo-β-1,4-mannanase activities. Soluble carboxy-methylcellulose and locust bean galactomannan were dyed with Remazol Brilliant Blue R. A specific screening procedure was developed to isolate genes coding for cellulolytic and hemicellulolytic enzymes from a Cellulomonas sp. Ce1B7 library. The assay is advantageous for the simple, rapid and direct detection of cellulase and hemicellulase activities in a large number of clones as found in gene libraries without replica plating or blotting techniques. The prepared dye-labelled substrates can also be used for the quantitative measurement of specific endo activities of cellulolytic and hemicellulolytic enzymes because the undigested substrate can be precipitated and the amount of soluble dye, determined spectrophotometrically, is proportional to enzyme activity.

Introduction

For the conversion of plant cell wall to glucose and other mono- and disaccharides some types of hydrolytic enzymes are needed. The most important enzymes are cellulases and hemicellulases. Endo-β-1,4-glucanase (EC 3.2.1.4), endo-β-1,4-xylanase (EC 3.2.1.8) and endo-β-1,4-mannanase (EC 3.2.1.78) degrade plant cell wall polysaccharides by cleaving internal glucosidic bonds of cellulose, xylan and mannan, respectively.

For the isolation of cellulolytic activities from microorganisms, Congo Red staining of carboxymethylcellulose (CMC) or cellulose supplemented agar plates or polyacrylamide gels [1]is widely used. Conversely, dye-labelled substrates enable the direct selection of appropriate activities by incorporating them into the growth media. The use of 4-O-methyl-d-glucurono-d-xylan–Remazol Brilliant Blue R (RBB–O-xylan) and RBB–carob–galactomannan as selecting media components have been demonstrated in the analysis of bacterial and fungal xylanases and mannanases 2, 3, 4. Dye-labelled substrates, incorporated into culture media are "splitted" into smaller fragments that diffuse rapidly, leaving a bright halo around positive colonies. Direct selection makes the screening of a large number of clones possible, as is found in gene libraries, whether they are made up in λ phage or in plasmids without the need for replica plating or blotting techniques. In order to maintain substrate solubility, for the labelling reaction, partially hydrolyzed polysaccharides are used and this affects the yield of dye-labelled products [5]. Partial hydrolysis of the polysaccharides to be dyed also influences the screening procedure as non-native substrates are used.

Generally, polysaccharide degrading enzymes are assayed by determining the amount of reducing sugars produced during enzymic digestion [6]. Dye-labelled substrates can also be used for the analysis of cellulases, xylanases and mannanases 5, 7, 8. The enzyme kinetics of cellulases and mannanases can be established using this procedure since enzyme activity is proportional to the amount of dye released during enzyme digestion. Although these assay procedures eliminate the reducing sugar background, the precipitation of undigested substrates depends, to a great extent, on the pH, temperature and salt concentration of the reaction mixture [7].

In this paper we report on the production of dye-labelled CMC and locust bean galactomannan for the analysis of plant cell wall degrading enzymes. The procedure enables the dyeing of partially non-digested polysaccharide substrates while their solubility is maintained. An advanced method was developed for the direct screening of a Cellulomonas sp. Ce1B7 gene library for endoglucanase and endomannanase activities. It is also demonstrated that the prepared dye-labelled substrates can be used in enzyme assays to analyze enzyme properties. The modified assay procedure eliminates the need for equilibration as virtually all undigested labelled substrates are precipitated from the reaction mixture [7]. The method determines the endo mode of action of the enzymes more reliably, avoiding artifacts raising when enzymes of unknown purity, found in cell free extracts, are examined.

Section snippets

Reagents and enzymes

Carboxymethylcellulose (CMC, sodium salt, low viscosity), galactomannan (mannan, Gum, Locust Bean; from seeds of Ceratonia siliqua), Remazol Brilliant Blue R (RBB) and chemicals for the dyeing reaction were obtained from Sigma. Media components were purchased from Oxoid, Reanal and Serva. DNA restriction and modification enzymes were from Fermentas.

Preparation and purification of dye-labelled CMC and galactomannan

Dye-labelled CMC and galactomannan were prepared using a modified method of McCleary [5]. One percent (w/v) solution was made of CMC in distilled

Preparation and properties of dyed CMC and galactomannan

For the rapid analysis of Cellulomonas sp. CelB7 gene library and the measurement of cellulase and mannanase activities, RBB–O-CMC and RBB–O-galactomannan were prepared as described above. The dye binding is based on the bimolecular nucleophil substitution reaction which results in covalent binding between the dye and the polysaccharide (Fig. 1).

The optimal conditions for the labelling reactions (see Section 2) were determined by changing polysaccharide and dye concentrations, pH and

Acknowledgements

We thank Professor M. Sajgó and Associate Professor F. Fabian for their help in the preparation of the manuscript. This work was supported by the National Scientific Research Fund (OTKA, Grant No. F-5453).

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