Sensitive high-throughput screening for the detection of reducing sugars

The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2.     

An o-toluidine method for detection of carbohydrates in protein hydrolysates

The o-toluidine high-performance thin-layer chromatography (HPTLC) method for detection of reducing sugars has been demonstrated to be a facile method for composition analysis of protein hydrolysates with a maximum sensitivity range of 50-100 pmol. The solution phase reaction of o-toluidine with reducing sugars has been previously used for spectrophotometric detection of glucose at 480-630 nm. In contrast, the heterogeneous reaction of o-toluidine with reducing sugars resolved by thin-layer chromatography produces chromophoric derivatives which have a broad absorbance at 295 nm. Detection of these chromophoric derivatives is achieved by uv diffuse reflectance scanning densitometry. It is demonstrated that detection limits of less than 10 ng can be achieved by using HPTLC plates and is therefore equal or more sensitive for some sugars than recently reported high-pressure liquid chromatography methods using amperometric or fluorescence detection.     

Assessment of biochemical methods to detect enzymatic depolymerization of polysaccharides

Biochemical methods for detection of depolymerisation were compared. Currently used methods such as reducing sugars assays, double bond monitoring or molecular weight determination were tested to follow the kinetic of depolymerization with different enzyme/polysaccharide combinations. The range of concentrations of different enzymes allowed us to identify the most sensitive and appropriate method to detect polysaccharide degradation. Reducing sugars assays are quantitative, sensitive and usable with all kind of polysaccharide but some compounds may interfere with them. When the polysaccharide is charged, agarose gel electrophoresis, although being a qualitative assay is as sensitive as high performance size exclusion chromatography analysis, easy to handle, high-throughput and this is preferred.     

A sensitive fluorometric assay for reducing sugars.

A simple and rapid fluorometric assay for reducing sugars that is sensitive to the nanomolar range has been developed. The assay involves the derivatization of a given sugar with hydrazine at pH 3 to form a hydrazone, which is reacted with fluorescamine following adjustment of pH to first 9.4 and then 7.4. The amount of sugar in a sample is quantitated by measuring the fluorescence intensity at an excitation wavelength of 400 nm and an emission wavelength of 490 nm. The assay is precise and reproducible, as indicated by intra- and inter-run variations of at most 3% and 4%, respectively. In addition to reducing sugars, the assay can also be used to measure aliphatic and aromatic aldehydes, but not acetone. Compared with an existing fluorometric sugar assay, the assay reported here does not require chromatographic separation of the fluorescent derivative from unreacted fluorescamine. The assay can, however, be potentially adapted for postcolumn detection of aldehydes, reducing sugars, and hydrazones in HPLC.     

An ultraviolet-spectrophotometric method with 2-cyanoacetamide for the determination of the enzymatic degradation of reducing polysaccharides.

A rapid, facile, and sensitive uv-spectrophotometric assay has been developed for the determination of the enzymatic degradation of polysaccharides that generates reducing sugars. The assay was carried out with 2-cyanoacetamide in a single test tube. The solution was left at pH 9 by the addition of borate buffer within 5 min. Measurement of the reaction mixture at 274 nm allows a simple determination up to 600 mumol/liter of reducing sugars. The coefficient of variation was less than 2% on all measurements. The assay was developed with pectin and polygalacturonic acid from apples and has been compared with the Somogyi-Nelson method. The new assay was then exemplarily used for the determination of the enzymatic hydrolysis products of pectin from cotton.     

New reducing sugar assay for the study of cellulases

A simple assay for reducing sugars based on the production of a coloured formazan and solvent extraction has been developed and used to study the cellulase complex of a Gram-negative bacterium. This assay is more sensitive than others previously described and allows direct study of unconcentrated culture supernatants. The levels of enzyme activity in subcellular fractions were measured after growth on different carbohydrate sources.     

A manual method for reducing sugar determinations with 2,2′-bicinchoninate reagent

A method was developed to analyze reducing sugars manually by use of a 2,2′-bicinchoninate reagent. Potassium phosphate was used to buffer the reagent. Ethylene glycol increased the sensitivity of the assay for several sugars. Sugar determinations can be performed in the presence of borate ion. In comparative assays of a number of monosaccharides and disaccharides, the bicinchoninate reagent was about as sensitive as the Nelson reagent but more convenient to use. The effects of ethylene glycol concentration, reagent pH, heating time, and borate ion on color development were evaluated.     

Application of high performance anion exchange-pulsed amperometric detection to measure the activity of key sucrose metabolising enzymes in sugarcane

A novel method using an HPAE-PAD system, which is routinely applied to detect carbohydrates at low levels (ng per sample injection), has been applied to the measurement of key sucrose metabolising enzyme activities in partially purified extracts of sugarcane tissues. Extraction and assay procedures tailored for the HPAE-PAD system enabled the accurate measurement of enzyme activities in more mature internodes than had previously been possible using enzyme coupled assay methodology. A major advantage of the HPAE-PAD method is the capability to monitor a broad range of sugars in each assay and provides an overarching perspective of the mix of competing enzymes that may be operating simultaneously in crude extracts. The technique has been successfully applied to measuring the activity of key sucrose metabolising enzymes in sugarcane stem tissue that is generally low in protein and high in endogenous sugars, primarily sucrose.     

Fluorometric assay of O-linked glycoproteins by reaction with 2-cyanoacetamide

A simple assay for O-glycosylated glycoproteins involving the liberation of oligosaccharides by beta-elimination with dilute alkali and the subsequent derivatization of the reducing end with 2-cyanoacetamide is reported. The method can be used to quantitate microgram amounts of mucin within 30 min. The assay is 30 times less sensitive to protein or N-linked glycoproteins and 100 times less sensitive to DNA than to the corresponding weight of canine tracheal mucin. Of the substances tested, only cesium chloride and potassium thiocyanate caused substantial interference, but in neither case was this sufficiently serious to prevent the method being used for monitoring mucin purification schemes utilizing these reagents. The coefficients of variation for replicate analyses of canine tracheal mucin (14.0, 5.0, and 2.0 micrograms) were 3.6, 6.5, and 12.3%, respectively.     

Simple and rapid measurement of acetylcholine and choline by HPLC and enzymatic-electrochemical detection

A simple, rapid and sensitive method for the detection of acetylcholine and choline in tissue extracts is reported. Acetylcholine and choline are first separated by HPLC then react in a mini-column with acetylcholinesterase and choline oxidase immobilized on Sepharose. The resulting H₂O₂ produced by choline oxidase is then detected electrochemically. The assay is more sensitive than existing methods. We believe that the principle involved in this method namely the combination of immobilized enzymes and the high sensitivity of electrochemical detection may be applied to other substances that can be converted by immobilized enzymes into an electrochemically detectable compound.     

Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate

Cellulose-binding domain (CBD) enriches cellulolytic enzymes on cellulosic surfaces and contributes to the catalytic efficiency by increasing enzyme-substrate complex formations. Thus, high affinity CBDs are essential for the development of efficient cellulose-degrading enzymes. Here, we present a microtiter plate-based assay system to measure the binding affinity of CBDs to cellulose. The assay uses a periplasmic alkaline phosphatase (AP) as a fusion reporter and its activity is detected using a fluorogenic substrate, 4-methylumbelliferyl phosphate. Lignocellulose discs of 6 mm in diameter were used as substrates in 96-well plate. As a result, the enzyme-linked assay detected the binding of CBDs on the cellulosic discs in a highly sensitive manner, detecting from 0.05 to 1.0 μg/mL of APCBD proteins, which is several hundred times more sensitive than conventional protein measurements. The proposed method was applied to compare the binding affinity of different CBDs from Cellulomonas fimi to lignocellulose discs.     

A sensitive radiometric assay for enkephalin convertase and other carboxypeptidase B-like enzymes

A sensitive radiometric assay for carboxypeptidase B-like enzymes has been developed using enkephalin convertase, an enkephalin synthesizing carboxypeptidase. The assay is based on the differential solubility of 3H-labeled substrate and product in chloroform. The substrates 3H-benzoyl-Phe-Ala-Arg or 3H-benzoyl-Phe-Leu-Arg are poorly soluble in chloroform due to the charged arginine. The products of carboxypeptidase B-like activity on these substrates, 3H-benzoyl-Phe-Ala or 3H-benzoyl Phe-Leu partition quantitatively into chloroform, allowing rapid separation of product from substrate. This assay is approximately 100 times more sensitive than a similar fluorometric assay utilizing dansyl-Phe-Ala-Arg as a substrate.     

A simple modification of the Salmonella liquid-incubation assay. Increased sensitivity for detecting mutagens in human urine

A simple modification of the Salmonella/microsome liquid-incubation procedure improves the sensitivity of the assay for detecting mutagens in human urine. Extracts from cigarette smokers' urine were used as a model complex mutagenic mixture for validation of the assay. The modification consists of adding increased numbers of bacterial cells (approximately 10(9] in a concentrated suspension to liver homogenate mix and urine extract, all in 0.2-ml volume. After 90 min incubation at 37 degrees C, the mixture is processed according to the standard Ames test protocol. This procedure is 20 times more sensitive than the standard plate-incorporation test and 13 times more sensitive than a previously reported liquid-incubation protocol. The number of spontaneous revertants did not increase under these conditions and, compared to the plate-incorporation test, 10-fold less liver homogenate and 5-fold less enzymatic cofactors were needed per plate. The procedure was approximately 14 times more sensitive in detecting the mutagenic activity of benzo[ a ]pyrene. We also used the modification to determine mutagenic activity in urine from a group of nonsmokers. The method may be generally useful for investigations of mutagenic activity in human urine samples.     

Two time-resolved fluorometric high-throughput assays for quantitation of GDP-L-fucose

Two rapid and simple procedures for the quantitative analysis of GDP-l-fucose (GDP-Fuc) are described. The methods are based on time-resolved fluorescence and microplate assay technology. The first assay relies on measuring the enzyme activity of alpha1, 3-fucosyltransferase. In this assay, transfer of fucose from GDP-Fuc converts sialyllactosamine to sialyl Lewis x tetrasaccharide, which is detected and quantified by relevant antibodies on a microplate. The formation of the reaction product is directly dependent on the presence of GDP-Fuc in the concentration range of 10-10,000 nM. In the second method GDP-Fuc inhibits the binding of fucose-specific Aleuria aurantia lectin to fucosylated glycan on a microwell. The lectin-based assay is less sensitive than the enzyme assay, but it is cheaper and faster. We used these assays in monitoring the amount of GDP-Fuc in crude lysates of transgenic yeast, which expresses the enzymes producing GDP-Fuc. The newly developed assays are versatile and applicable to measure also other nucleotide sugars or glycosyltransferase activities in a high-throughput manner.     

Comparison of a spectrophotometric, a fluorometric, and a novel radiometric assay for carboxypeptidase E (EC 3.4.17.10) and other carboxypeptidase B-like enzymes

Carboxypeptidase E (CPE) is a carboxypeptidase B-like enzyme involved in the biosynthesis of numerous peptide hormones and neurotransmitters. A sensitive assay for CPE and other carboxypeptidase B-like enzymes has been developed using 125I-acetyl-Tyr-Ala-Arg (125I-AcYAR) as the substrate. This peptide is poorly soluble in ethyl acetate whereas the product of carboxypeptidase B-like enzymatic activity (125I-AcYA) can be quantitatively extracted with this solvent, allowing the rapid separation of product from substrate. This radiometric assay can detect less than 1 pg of either CPE or carboxypeptidase B. For CPE, the assay with 125I-AcYAR is approximately 1000 times more sensitive than a fluorescent assay using dansyl-Phe-Ala-Arg (dans-FAR), and 6000 times more sensitive than a spectrophotometric assay using hippuryl-Arg (hipp-R). CPE hydrolyzes the three substrates with Kcat values of 16 s-1 for AcYAR, 13 s-1 for dans-FAR, and 8.5 s-1 for hipp-R. The Km values for CPE with AcYAR (28 microM) and dans-FAR (34 microM) are similar, and are much lower than the Km with hipp-R (400 microM). Thus, the primary reason for the increased sensitivity of the 125I-AcYAR assay over the fluorescent assay is not a result of kinetic differences but is due to the detection limit of iodinated product (10(-15) mol), compared to the fluorescent product (5 x 10(-11) mol). Applications of this rapid and sensitive radiometric assay to detect CPE in cultured cells and in subcellular fractions of the pituitary are described.     

The use of Tween 20 in a sensitive turbidimetric assay of lipolytic enzymes

A turbidimetric esterase assay was developed using a Tween 20 solution in the presence of CaCl2 and Lysobacter enzymogenes esterase (EC 3.1.1.1) as the enzyme source. The reaction was followed by measuring the increase in the optical density at 500 nm (OD500) due to the hydrolytic release of the fatty acids from Tween 20 and their precipitation as the calcium salts. Concentrations of 1.8% Tween and 3 mM CaCl2 were found to be optimal for the assay of 0.036 to 0.15 esterase units in a 4-mL reaction mixture over a 30-min period. The esterase reactions were linear with time at least up to 1.2 OD500 and the rate of increase in the OD500 was proportional to the enzyme concentration. Low initial reaction rates were seen with low esterase activity, presumably because of the limited solubility of the fatty acid - calcium salt in a 1.8% Tween solution. This turbidimetric method is much simpler and at least 36 times more sensitive than the titrimetric assay with Tween 20, and at least four times more sensitive than a spectrophotometric assay with p-nitrophenyl palmitate. This assay has been used to determine the activities of cell-associated and excreted esterases produced by Lysobacter enzymogenes and Pseudomonas aeruginosa, and of lipolytic enzymes from porcine liver, Chromobacterium viscosum, Candida cylindracea, and wheat germ.     

Non-specificity of a colorimetric method for the estimation of N-acetyl-D-glucosamine.

The colorimetric method of Reissig et al. for the estimation of N-acetylamino sugars, is often used as a specific method for the quantification of the N-acetyl-D-glucosamine. Although this assay is more sensitive to the monomer, it recognizes all soluble N-acetyl-D-glucosamine oligomers. This result is very important because this method is extensively used in biology for the estimation of chitinolytic activity.     

A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper

This study describes a procedure for the selective determination of endo- (EG) and exo- (ExG) cellulase activities using filter paper as the sole substrate. The procedure is based on the enzymes mode of action whereby EG activity predominantly forms insoluble reducing sugars and ExG activity soluble reducing sugars. The procedure was developed using filter paper as substrate for hydrolysis with three cellulase preparations of Hypocrea jecorina containing either endoglucanase (EG), predominantly exoglucanase (ExG) or both endo- and exoglucanase activities. Hydrolysis experiments, which were followed assessing the formation of total, soluble and insoluble reducing sugars (RS), showed that up to 30min of hydrolysis predominantly insoluble reducing sugars were formed, while after this initial hydrolysis stage soluble reducing sugar formation increased significantly, making it thus possible to measure separately EG and ExG activity. FPA activities obtained from the reaction products at different reaction times suggest that EG-activity (FPA(insol)) should be measured between 10 and 20min of hydrolysis. The proposed procedure allows to evaluate the EG and ExG activity contribution to total cellulase activity and to calculate the endo/exo activity ratio of any cellulase preparation.     

A radioisotope method for assays of amylomaltase and D-enzyme

A method has been developed for the assay of amylomaltase based on the incorporation of a [14C]glucose moiety of uniformly 14C-labeled maltose into a maltodextrin fraction insoluble in aqueous ethanol. The presence of dextrin at a high concentration greatly enhances [14C]glucose incorporation and serves to minimize interference with the assay by contaminating enzymes that hydrolyze substrates and products in the assay mixture. Since a number of other enzymes are capable of forming glucose in the assay mixture, the 14C incorporation is a more specific method of enzyme assay than are previously reported assays based on glucose release.