An Insoluble Colored Substrate for Dextranase Assay

An assay of dextranase (EC 3.2.1.11) was developed by using Sephadex G-200 coupled with Remazol Brilliant Blue (RBB) as an insoluble substrate. The assay procedure included incubation of the suspension of the colored substrate in a buffer containing the enzyme under study, removal of a residual insoluble substrate, and measurement of the absorbance of supernatant fluid containing colored soluble hydrolysis products at 595 nm. The procedure was examined in the screening of dextranase-forming bacilli from the microbial collection of the Institute of Biology, Ufa Research Center, RAS.     

Development of a plate technique for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble chromogenic substrates

A plate assay based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of different dye-labelled polysaccharides as substrates, provides a specific, reliable and rapid simultaneous detection of corresponding polysaccharide-degrading microorganisms. It has potential for increasing the efficacy of screening of microorganisms, utilizing different polysaccharides, in large numbers of natural samples. Diversely colored insoluble forms of amylose, xylan and hydroxyethyl-cellulose (HE-cellulose) were prepared as chromogenic substrates by using the cross-linking reagent 1,4-butanediol diglycidyl ether and the dyes Brilliant Red 3B-A, Cibacron Blue 3GA and Reactive Orange 14. Using the method, the bacteria with amylase or xylanase or cellulase or a combination of these activities were screened from soil and sludge samples, selected and identified according to 16S rDNA sequencing.     

Revised determination of free and complexed myrosinase activities in plant extracts.

The enzyme myrosinase (thioglucoside glucohydrolase, EC 3.2.1.147, formerly EC 3.2.3.1) catalyzes the hydrolysis of glucosinolates after tissue damage in plants of the order Brassicales. The various myrosinase isoforms occur either as free soluble dimers or as insoluble complexes. We propose a reliable method for determination of both soluble and insoluble myrosinase activity concentrations in partially purified plant extracts. The procedure requires the removal of endogenous glucosinolates through ion-exchange columns previous to enzyme measurements. Myrosinase activity was assayed in continuous mode by photometric quantification of the released glucose using glucose-oxidase with peroxidase and colorimetric indicators. The measurement of the colored product at 492nm has a favorable signal to noise ratio both in clear extract solutions (free dimers) and in turbid pellet suspensions (insoluble complexes). No interferences by ascorbic acid were found in continuous analyses. With the recommended sample preparation methods and assay conditions potential activities in damaged plant tissues can be characterized which are involved in plant defense mechanisms.     

Radial diffusion in gel for micro determination of enzymes. I. Muramidase, alpha-amylase, DNase 1, RNase A, acid phosphatase, and alkaline phosphatase

The principle of radial diffusion in substrate containing agar gel has been applied for the quantitative assessment of several enzymes. Muramidase, alpha-amylase, DNase I, RNase A, acid phosphatase, and alkaline phosphatase have been investigated. Clearing zones in the opalescent agar, staining of the substrate incorporated in the agar, or a colored insoluble hydrolysis product indicate the diffusion zone of the enzyme. A linear relationship was found between the logarithm of the enzyme concentration and the corresponding diameters of the diffusion zones over a wide range. Standard dilutions of the different enzymes are used as reference.     

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 continuous spectrophotometric assay for the determination of cellulase solubilizing activity

A cellulase assay was developed for the continuous measurement of colored cellulose oligosaccharides (total carbohydrates) released during enzymatic hydrolysis of dyed crystal-line cellulose. Several cellulosic substrates were uniformly dyed by Remalzol brilliant blue R salt without altering their physical properties. Dyed Avicel (6.5%, w/w) was selected as the most representative substrate for the assay procedure. The assay was performed continuously in a simple, thermally controlled apparatus designed for filtration of the reaction mixture via a 5-μm-pore-size nylon filter to retain the crystalline dyed cellulose while spectrophotometrically monitoring the absorbance at 595 nm of the reaction filtrate. Crude supernatant cellulase of Trichoderma viride QM9414 was used to test the assay procedure. The activity of cellulase on dyed Avicel as measured by ΔA_595nm correlated directly with the total carbohydrates formed. The initial reaction rate of cellulase solubilizing activity was readily determined with high sensitivity. The continuous assay has utility for the study of cellulase kinetics and for the comparison of activities from different microorganisms.     

Development of a high throughput scintillation proximity assay for hepatitis C virus NS3 protease that reduces the proportion of competitive inhibitors identified

A screening assay has been developed for hepatitis C virus (HCV) NS3 protease using the scintillation proximity assay (SPA) technology. The sequence of the peptide substrate used was taken from the site cleaved by the enzyme in the mature nonstructural protein of HCV. The peptide was biotinylated at the N-terminus and tritiated at the C-terminus so that a decrease in signal was detected as a result of enzyme activity. IC(50) values were calculated for the cleaved product, and it was shown that the value obtained was dependent on the substrate concentration used. The effect of substrate concentration on the inhibition of HCV NS3 protease was further highlighted in a mock screening assay, using colored natural product samples, in which the hit rate was altered by a change in substrate concentration. An increase in substrate concentration reduced the proportion of competitive inhibitors identified. This study highlighted the importance of optimizing the components used in SPA assays in order to obtain an assay format valid for high throughput screening.     

Measurement of saccharifying cellulase

This article sets forth a simple cellulase assay procedure. Cellulose is variable in nature, insoluble and resistant to enzymatic attack. As a result there have been a bevy of bewildering cellulase assays published that yielded irrational results. Certain protocols focused on the rapidity of the assay while ignoring that only the most readily susceptible cellulose regions were being hydrolyzed. Other assays simplified the system by using modified soluble substrates and yielded results that bore no relationship to the real world hydrolysis of insoluble cellulose. In this study Mandels, Andreotti and Roche utilized a common substrate, Whatman filter paper. Hydrolysis of a 50 mg sample of the paper was followed to roughly 4% degradation, which circumvented the problems of attack of only the most susceptible zones. This common hydrolysis target range also resulted in some balance with regard to the interaction of the several cellulase components. The method was subsequently widely adopted.     

New assay for enzymatic phosphate release: application to aspartate transcarbamylase and other enzymes

The colorimetric method for phosphate determination described in the preceding paper is adapted for the assay of orthophosphate liberated in the aspartate transcarbamylase reaction. The method provides for simple, accurate, and sensitive measurement of enzyme activity. The assay uses ammonium molybdate and zinc acetate to form a colored complex with the enzymatically released phosphate; mild conditions which minimize the nonenzymatic background degradation of the substrate, carbamoyl phosphate, are used. Since the assay procedure is relatively rapid, it is especially attractive in situations where results are desired immediately. The method can be used for the assay of any enzyme which releases inorganic phosphate, even in the presence of labile organophosphate compounds.     

13C-n.m.r. study of C hordein.

Insoluble xylan was prepared from ground birch (Betula pubescens) pulp by alkali extraction and precipitation with ethanol. The only sugar detected after acid hydrolysis of the preparation was xylose. The insoluble xylan was used as substrate in a nephelometric assay to determine the xylanase (EC 3.2.1.8, 1,4-beta-D-xylan xylanohydrolase and EC 3.2.1.37, 1,4-beta-D-xylan xylohydrolase) activities of Aspergillus and Trichoderma enzymes. The nephelometric method is reliable in evaluating xylanase hydrolysis of insoluble xylan.     

Properties and repeated use of a reversibly soluble-insoluble yeast lytic enzyme

Summary A yeast lytic enzyme was covalently immobilized on an enteric coating polymer, Eudragit S, that is reversibly soluble and insoluble (S-IS) depending on the pH of the reaction medium. The yeast lytic enzyme immobilized on Eudragit S (Y-E) showed a sharp response of solubility to slight changes in pH without decrease in enzymatic activity. The specific activity per amount of enzyme protein of Y-E for dry yeast cells was about two-thirds that of the native enzyme. In both lysis reactions of dry and pressed baker's yeast cells, changing the pH of the reaction medium from 7.0 to 4.8 at an appropriate interval allows the insoluble Y-E and the reaction products (soluble protein for dry yeast cells and invertase and soluble protein for pressed baker's yeast cells) to be repeatedly separated. The reaction method using a reversible S-IS enzyme is a promising procedure for repeated use of the enzyme in a heterogeneous reaction system containing yeast cells as a substrate.     

Determination of selected xenobiotics with ferrofluid-modified trypsin

Ferrofluid-modified trypsin has been used for the detection and determination of selected xenobiotics that inhibit trypsin activity. The procedure is useful especially when colored samples or samples containing suspended solid impurities are to be assayed. Ferrofluid-modified trypsin was inhibited by Ag⁺ and Pb²⁺, selected dyes (safranin, thionin), bacitracin and 4-aminobenzamidine. Enzymes immobilized on magnetic particles can form a basis of new automated assay procedures for the determination of xenobiotics.     

The use of diaminobenzidine for spectrophotometric and acrylamide gel detection of sulfite oxidase and its applicability to hydrogen peroxide-generating enzymes

DAB, in the presence of HRP, sulfite oxidase and FBS, polymerizes to a product with an absorption difference maximum at 352 nm. This reaction has been used as a sensitive and specific spectrophotometric assay for sulfite oxidase. Incubation of acrylamide gels containing sulfite oxidase with assay mixtures produces an insoluble product which precipitates where the enzyme is localized. This stain is at least as sensitive as the amido black protein stain and its specificity is such that no band is seen in the absence of substrate, and that only one band, migrating identically to purified enzyme is seen in rat liver homogenates. This polymerization reaction has been applied to other H₂O₂-generating enzymes and can be used to demonstrate the presence of these enzymes in the midst of other oxidases. DAB polymerization provides a sensitive spectrophotometric assay which can be used with either ultraviolet or visible optics. The application of this procedure to modified enzymes is discussed.     

An enzymatic cycling procedure for beta-NADP+ generated by 3'-phosphodiesterase, 2':3'-cyclic nucleotide.

An enzymatic cycling procedure for beta-NADP+ generated by the enzyme 3'-phosphodiesterase, 2':3'-cyclic nucleotide (EC 3.1.4.37) from its substrate 2':3'-cyclic NADP+ is described. The enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and diaphorase (EC 1.8.1.4) are used to cycle the cofactor between its oxidized and reduced forms in the presence of glucose-6-phosphate and p-iodonitrotetrazolium violet (INT) with the concomitant production of colored INT-formazan, monitored at 492 nm. The amplification is about 400-fold per hour and is sensitive enough to detect 6 x 10(-13) mol of NADP(H). A simple procedure for the optimization of this cycling assay is also described. Conjugates to 3'-phosphodiesterase, 2':3'-cyclic nucleotide may be used in heterogeneous enzyme immunoassays for the detection of small quantities of haptens or proteins in biological fluids.     

Histochemical method for dipeptidyl aminopeptidase II with a new anthraquinonyl hydrazide substrate.

A new method for the histochemical visualization of lysosomal aminopeptidase dipeptidyl peptidase II activity (DPP II) is developed. The substrate L-Lys-L-Ala-5-chloro-1-anthraquinonylhydrazide-2HBr (Lys-Ala-CAH) is readily hydrolyzed by the enzyme to release 5-Cl-1-anthraquinonylhydrazine (CAH). The last compound is simultaneously coupled to an aromatic aldehyde, e.g. 4-nitrobenzaldehyde (p-NBA) or piperonal (3,4-methylenedioxybenzaldehyde; PPL), to form a highly insoluble deeply colored hydrazone, marking the enzyme locations. Using the new method, DPP II is successfully localyzed in tissue sections from different rat organs.     

A colorimetric procedure for measuring beta-lactamase activity

The enzymatic hydrolysis of benzylpenicillin was measured by a novel colorimetric procedure. The penicilloic acid generated from the hydrolysis of penicillin was reacted with CuSO4 and neocuproine to form a colored complex having a maximal absorption at 454.5 nm. A plot of absorbance versus beta-lactamase activity yielded a straight line from 1 to 5 mU of enzyme. Using TEM-1 as the model beta-lactamase, a Km of 46 microM was observed with benzylpenicillin serving as the substrate. When the assay was used to determine levels of benzylpenicillin, the absorbance was found to be linearly proportional to exogenously added penicillin from 2.8 to 88 microM. This procedure is simple to use and can be employed to measure the hydrolysis of other beta-lactam antibiotics.     

Liposome immunoblotting assay using a substrate-forming precipitate inside immunoliposomes

A new immunoblotting assay which uses antibody-coupled liposomes containing horseradish peroxidase is proposed. A substrate 4-chloro-1-naphthol permeated through the phospholipid membrane of the antibody-coupled liposomes and formed a colored product precipitating inside the liposomes. The precipitates accumulated in the liposomes and could be detected at the positions where the liposomes coupled with a target in blotted samples. Combination of liposomes with average diameter of 350 nm and a PVDF membrane with a pore size of 450 nm, 0.02 ng of IgM was detected, while the conventional immunoblotting using antibody-HRP conjugates detected 2 ng of IgM. The sensitivity increased about two orders of magnitude by the liposome immunoblotting assay. This liposome immunoblotting assay gives a simple detection method of proteins with a high sensitivity, as well as a high sensitivity Western blotting assay.     

A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase

Transfection efficiency of different cell types as well as promoter strength of cloned genes can be easily determined by direct assay of beta-galactosidase activity encoded from recombinant genes containing the E. coli beta-galactosidase gene. A substrate for beta-galactosidase, o-nitrophenyl-beta-D-galactopyranoside (ONPG), can be added to dishes containing the transfected cells, and the intensity of the colored enzyme product released from either the intact cell or cells lysed in the dishes can be determined. The results obtained by this assay are a reliable measure of transfection efficiency as well as promotor strength of the genes introduced into the cells. In addition, cells expressing the transfected gene can be identified and quantitated under a light microscope after incubation with X-gal. Thus, it is more convenient to use the E. coli beta-galactosidase gene than the chloramphenicol acetyltransferase gene as a reporter gene in the evaluation of DNA transfection.     

Direct Image-Based Enumeration of Clostridium phytofermentans Cells on Insoluble Plant Biomass Growth Substrates

A dual-fluorescent-dye protocol to visualize and quantify Clostridium phytofermentans ISDg (ATCC 700394) cells growing on insoluble cellulosic substrates was developed by combining calcofluor white staining of the growth substrate with cell staining using the nucleic acid dye Syto 9. Cell growth, cell substrate attachment, and fermentation product formation were investigated in cultures containing either Whatman no. 1 filter paper, wild-type Sorghum bicolor, or a reduced-lignin S. bicolor double mutant (bmr-6 bmr-12 double mutant) as the growth substrate. After 3 days of growth, cell numbers in cultures grown on filter paper as the substrate were 6.0- and 2.2-fold higher than cell numbers in cultures with wild-type sorghum and double mutant sorghum, respectively. However, cells produced more ethanol per cell when grown with either sorghum substrate than with filter paper as the substrate. Ethanol yields of cultures were significantly higher with double mutant sorghum than with wild-type sorghum or filter paper as the substrate. Moreover, ethanol production correlated with cell attachment in sorghum cultures: 90% of cells were directly attached to the double mutant sorghum substrate, while only 76% of cells were attached to wild-type sorghum substrate. With filter paper as the growth substrate, ethanol production was correlated with cell number; however, with either wild-type or mutant sorghum, ethanol production did not correlate with cell number, suggesting that only a portion of the microbial cell population was active during growth on sorghum. The dual-staining procedure described here may be used to visualize and enumerate cells directly on insoluble cellulosic substrates, enabling in-depth studies of interactions of microbes with plant biomass.     

Two staining methods for selectively detecting isomaltase and maltase activity in electrophoresis gels.

Two methods for specifically detecting maltase, alpha-glucosidase, or isomaltase activity in electrophoresis gels are described. Both systems couple the formation of glucose by enzyme action on maltose or isomaltose to the generation of a colored product. System A uses an agarose overlay which contains substrate, glucose oxidase, peroxidase, 2,4-dichlorophenol, and 4-L-amino-phenazone. A purple color is produced at the site of enzyme activity. No hazardous chemicals are used at any stage. The stain is simple, rapid, sensitive, and inexpensive and does not interfere with subsequent protein staining. However, the stain is not permanent. System B was developed to give a permanent stain. The gel is overlaid with agarose containing substrate, glucose oxidase, phenazine methosulfate, and nitroblue tetrazolium. Glucose production results in the nitroblue tetrazolium being oxidized to an insoluble formazan with a dark blue color. This stain is also sensitive, rapid, and inexpensive but does use hazardous chemicals and if overstaining occurs this can interfere with subsequent protein staining. Neither system inactivates the localized enzymes which can be recovered from the gel if desired.