Chromatographic Methods

1. HPLC Method

Separation of substrates and products by HPLC can be used to analyze product distribution of individual cel-lulases for classification and to determine specificity of cellulases when using cello-oligosaccharides. Several different procedures have been used successfully with a variety of different separation procedures. Labeled and unlabeled substrates and products have also been used.

a. Unlabeled Sugar Substrates (15). Separation of unlabeled sugars using Aminex columns with detection by refractive index is one of the more common procedures used. These columns have the distinct advantage that they use water, or in some applications, dilute sulfuric acid as the eluent. In application to cel-lulases analysis, the HPX-87P (Pb form) and the HPX-42A (Ag form) for monosaccharides and oligosacchar-ides analyses, respectively, are used most frequently.

An HPLC system using an Aminex column (ideally with a deashing guard column), a column heater, and a refractive index detector is required. Samples are prepared using an appropriate hydrolysis system followed by inactivation of the enzyme by heating (5 min in boiling water). If the final pH is not between 5 and 9 for the HPX-87P or between 6 and 8 for the HPX-42A, the pH should be adjusted to prevent loss of column performance. Samples are then filtered through 0.45-^m filters and injected (10-20 ^L) into the HPLC system. Flow rates of 0.4-0.8 mL/min are used with column temperatures of 60-85°C. Quantification of results is accomplished by running external standards and constructing response factors for each sugar.

b. MeUmb-Labeled Substrates (13). MeUmb-labeled cello-oligosaccharides are prepared according to Tilbeurgh et al. (14), or they are available commercially. Enzymes are mixed with substrates in 0.05 M acetate buffer, pH 5.0, with substrate concentration from 300-500 ^M and incubated at 25° C. Samples are taken from the reaction mixture (at 5 to 10-min intervals for 30-60 min) and diluted 1:3 in acetonitrile to stop the reaction. Inactivated samples (20 ^L) are injected into an HPLC system consisting of a 25 x 0.46 cm Rsil-Polyol 5-^m particle-size column from Alltech and a UV detector monitoring at 313 nm. The eluent is a mixture of acetonitrile and water (39:11, v/v) at a flow rate of 1.5 mL/min. Quantification of results is accomplished by running external standards and constructing response factors for each sugar using integrated peak areas. Using the same column and eluent, the unmodified cello-oligosaccharides can be analyzed using a refractive index detector.

2. Enzyme Detection in Polyacrylamide Gels (16)

The detection of cellulases in polyacrylamide gels is based on the hydrolysis of 5-bromoindoxyl-^-D-cello-bioside by endo-1,4-^-D-glucanases to produce 5-bro-moindoxyl. The product 5-bromoindoxyl is then oxidized with nitroblue monotetrazolium (NBMT) producing an insoluble blue precipitate.

Standard gel electrophoresis is carried out using nondenaturing PAGE or isoelectric focusing systems. The incubation solution is prepared by first dissolving 5 mg of 5-bromoindoxyl-^-D-cellobioside and 20 mg NBMT in 0.5 mL dimethylformamide followed by mixing with 20 mL of 0.1 M sodium acetate buffer, pH 5.0. The gel is added to the solution and incubated in the dark at 40°C until dark blue bands appear. The development time is 2-15 h and is dependent on the concentration of active enzyme in the gel. Gels can be scanned or photographed for preservation. The staining solution can be used many times before discarding.

3. Gel Electrophoresis of Products (17-19)

a. ANTS Reagents. A 0.2-M solution is prepared by dissolving 0.086 g of 8-aminonaphthyl-1,3-6-trisul-fonate (ANTS) in 5 mL of a solution of glacial acetic acid/water (3:17; v/v).

b. Reducing Agent. A 1.0-M solution of sodium cyanoborohydride (NaCNBH4) is prepared in HPLC-grade dimethylsulfoxide (DMSO) by the addition of 2.5 mL DMSO to 0.25 g NaCNBH4.

c. Procedure. Enzymatic hydrolysis reactions are carried out using 0.05 M ammonium acetate buffer, pH 5.0, and inactivated by a change in pH using ammonium hydroxide. The use of this volatile buffer system prevents high salt concentrations and allows samples to be dried before labeling with the ANTS reagent. Following hydrolysis, the inactivated reaction is centrifuged and the necessary volume (10-50 nmoles reducing sugar) is transferred to a microcentrifuge tube and dried using a vacuum centrifuge.

The dried sample containing 10-50 nmoles of reducing sugar is labeled by adding 5-10 ^L of the ANTS reagent and 5-10 ^L of the reducing agent. This mixture is vortexed for 10 sec and centrifuged briefly to bring reagents together. The reaction is incubated at 40° C for 15 h.

Electrophoresis is done using a 5-36% gradient acrylamide gel with a Tris-glycine buffer system, pH 8.3, and a 5% stacking gel, pH 6.8 (no SDS is incorporated). [The acrylamide stock solution used to prepare gels is acrylamide/bis-acrylamide (39:2 w/w).] The labeled sample is diluted with four times loading buffer (40% glycerol in 0.2 M Tris-HCL buffer, pH 6.8) and applied to sample wells. Current is applied and the migration monitored by use of a hand-held UV moni tor. The gel is removed when the fluorescent dye front reaches the bottom of the gel. Gels are documented by photography on a UV gel box. A Polaroid camera is used with a 4 x 5 film holder model 545, type 55 film, which gives a negative and a positive image, and a No. 8 filter.

4. Capillary Electrophoresis of Products (17, 19)

a. Saturated Trisodium Phosphate (TSP). An excess of TSP is added to water and mixed. Solution is kept at room temperature.

b. Phosphoric Acid. Commercial 85% phosphoric acid (8.67 M) is diluted to 1 M with deionized water.

c. Running Buffer. Phosphoric acid 100 mM is titrated to pH 2.5 using triethylamine (TEA). The final solution is ~ 100 mM phosphate, 36 mM TEA.

d. Procedure. Hydrolysis samples are labeled as above for gel electrophoresis but diluted with water (1:10) rather than loading buffer. A capillary electro-phoresis system with an inactivated capillary column (60 cm x 50 ^m) and phosphate-TEA buffer, pH 2.5, is used. Injection is done using pressure at 20 psi * sec. Separation is monitored at 235 nm and samples are run at 20 kV with the polarity (—) to (+). Capillary and sample chamber temperatures are set at 35°C. Run times are ~ 15 min. The column is flushed with fresh buffer between runs.

The capillary is conditioned and cleaned before separation by sequentially flushing the column with saturated TSP, deionized water, 1 M phosphoric acid, deionized water, and running buffer for 5 min each. When the column performance decreases, as indicated by a change in elution times, the column is again cleaned.

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