Purification of an extracellular cellulose-binding endoglucanase of Cellulomonas sp. ATCC 21399 by affinity chromatography on H3PO4-swollen cellulose

A cellulose-binding endoglucanase (endoglucanase A) of Cellulomonas sp. ATCC 21399 was purified to immunological homogeneity by affinity chromatography ob H(3)PO(4)-swollen cellulose. This method of purification turned out to be an easy and very gentle method for obtaining a high yield of cellulose-binding endoglucanase. The purified enzyme was immunologically homogeneous but appeared heterogeneous when analyzed by denaturing polyacrylamide gel electrophoresis. In addition to the cellulose-binding of endoglucanase A, the enzyme also had a strong affinity for Concanavaline A, indicating that the enzyme was glycosylated. Purified endoglucanase A showed an endo mode of action on carboxymethylcellulose. The enzyme could hydrolyze microcrystalline cellulose when acting alone, and the enzyme had a high specific activity on H(3)PO(4)-swollen cellulose.     

Laccase-based biosensor for determination of polyphenols: determination of catechols in tea.

A new method of amperometric determination of phenolic compounds using an enzyme electrode is proposed. The latter represents the combination of the oxygen electrode and immobilized laccase. Analytical systems of flow injection and batch types were considered. A method of immobilization was developed that provided an increase in the stability of the enzyme. Optimal conditions for biosensor operation were found. The time needed for analysis in the flow injection mode was below 100 s. A column with immobilized enzyme could be used for up to 500 determinations of phenolic compounds without decrease of the enzyme activity. The practical validity of the method was demonstrated by tannin analysis in tea of different brands.     

A Method for the Quantitative Assessment of Wound-induced Chitinase Activity in Potato Tubers

A method for the quantitative assessment of chitinase activity was developed. Dilution series of crude potato tuber chitinase extracts were assayed with a colorimetric microtitre plate assay. using CM-chitin-RBV as enzyme substrate. Linearity between absorbance values mea-sured (540 nm) and enzyme concentration was found to be limited to the low concentration range. where depletion of the substrate was no longer limiting. As as absorbance of 0.1 always fell within the concentration range for which absorbance-concentration linearity was valid. one unit of enzyme activity was defined as the amount of enzyme needed to yield and A of 0.1. A more reliable method for the assessment of chitinase activity was established by basing the difinition of enzyme, activity on a concentration rather than on as absorbance value. as was done previously. Using this method, differences in the rate of chitinase induction upon wounding were demonstrated for six commercial potato cultivars.     

The analysis of low levels of γ-glutamyltransferase activity by high-performance liquid chromatography with electrochemical detection

A method for the analysis of low levels of the enzyme γ-glutamyltransferase in biological samples by high-performance liquid chromatography with electrochemical detection is described. A γ-glutamyl moiety from glutathione is transferred by the enzyme to glycylglycine to produce a tripeptide which is assayed directly after a purification step using octadecylsilica. Confirmation of the method is by use of the inhibitor AT-125. The method is used to measure the level of enzyme activity in rodent tissues and in cultured cells.     

A general method for the rapid measurement of enzyme precursors by the displacement of enzyme from antibody: the measurement of collagen proline hydroxylase precursor in cultured fibroblasts

A method has been developed for measuring inactive forms of an enzyme by displacing active enzyme from its antibody. The unbound enzyme is measured by an enzymatic assay. The method has been applied to the measurement of the precursor of collagen proline hydroxylase in cultured fibroblasts.     

A method of isolation of partially purified alkaline peptide hydrolase from Drosophila melanogaster larvae

A semipreparative method is developed for preparing peptidohydrolase from Drosophila melanogaster larvae which involves the stages of extraction, salting-out, gel-filtration and ion-exchange chromatography. It is established that the maximal (up to 81%) yield of the enzyme is observed with the single extraction in the alkaline medium. The main bulk of the enzyme is salted-out in the low acid 3 M ammonium sulphate solution. Gel-filtration on column with Sephadex-25 provides complete salting-out of the enzyme-containing fraction, and ion exchange chromatography on CM-cellulose--a considerable purification of the enzyme under study. A degree of the obtained purification of the enzyme under study. A degree of the obtained peptidohydrolase preparation purity in acid and alkaline medium is determined by the method of electrophoresis in PAAG. At all stages of the preparation the enzyme possesses the casein-lytic activity and is able of hydrolyzing the ethyl ester and benzoyl arginine p-nitroanilide.     

Identification of denatured enzyme proteins in sodium dodecyl sulfate polyacrylamide gels

A simple modification of the immunological sandwich method of Muilerman et al. for the identification of denatured enzyme proteins in sodium dodecyl sulfate-polyacrylamide gels is described, enabling the method to be used in principle for any enzyme whose activity is not inhibited by binding to antibodies. An immunological sandwich consisting of denatured enzyme, antibodies, and native enzyme is formed on a nitrocellulose filter blot of the gel, the filter is divided into strips, and each strip is tested for enzyme activity. The presence of enzyme activity serves to identify the region in the gel containing denatured enzyme protein. Experiments with human lysosomal alpha-glucosidase as a model system are described. The method was applied to identify a protein of Mr 125,000 as the main component with UDPgalactose pyrophosphatase activity in a partially purified preparation of the enzyme from rat liver.     

Predicting enzyme class from protein structure using Bayesian classification

Predicting enzyme class from protein structure parameters is a challenging problem in protein analysis. We developed a method to predict enzyme class that combines the strengths of statistical and data-mining methods. This method has a strong mathematical foundation and is simple to implement, achieving an accuracy of 45%. A comparison with the methods found in the literature designed to predict enzyme class showed that our method outperforms the existing methods.     

Rate-limiting steps in metabolic pathways.

A method is proposed to detect whether a given enzyme catalyzes a rate-limiting step in a metabolic pathway. With the use of a range of concentrations of specific inhibitors of an enzyme, the finding of a biphasic response with an initial null effect indicates the non-rate-limiting nature of the enzyme. With this method, phosphoenolpyruvate carboxykinase is indicated to catalyze a rate-limiting step in lactate gluconeogenesis in hepatocytes from fasted rats.     

Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae.

A method is shown to be effective over a wide range of enzyme ratios for the simultaneous detection of the two isoenzymes of acetyl coenzyme A synthetase [acetate:coenzyme A ligase (AMP-forming); EC 6.2.1.1] in homogenates and cellular fractions of Saccharomyces cerevisiae. When this method was used, it was found that cells grown under anaerobic conditions contained only one variety of this enzyme, designated the nonaerobic synthetase, whereas cells grown with vigorous aeration contained principally the other, aerobic, synthetase. In cells grown as standing cultures (i.e., semi-aerobically), both enzymes were present and were found mainly in the extramitochondrial material of homogenates. When anaerobic cultures were aerated, the amount of aerobic enzyme increased steadily over a 24-h period, so that at the end of this time, aerated cells contained predominantly aerobic enzyme. During this same period, the amount of nonaerobic enzyme decreased. The percentage of aerobic enzyme that sedimented with the mitochondria increased steadily during this period of aeration, so that, at the end of 24 h of aeration, essentially all of the aerobic enzyme sedimented with the mitochondria. The nonaerobic enzyme was never found in this cellular compartment.     

An automated continuous assay of membrane-bound and solube ATPases and related enzymes.

A simple, rapid, and precise method is described for the continuous automated determination of the activity of membrane-bound enzymes which deliberate inorganic phosphate, e.g., ATPases and phosphatases. The characteristics of this method, which is based on the determination of liberated phosphate in the presence of nucleotides, are: (A) the enzyme reaction can be followed continuously during a certain period, thus providing a higher precision, as compared to other methods in which the enzyme reaction is measured by few distinct determinations; (B) the enzyme protein and other (membrane) proteins of the enzyme preparation have not to be removed during the continuous determination of enzyme activity because they remain solubilized after denaturation; and (C) low or moderate concentration of nonionic detergents do not disturb the reading of the absorbancy.     

The use of 1H-NMR-spectroscopy for the study of peptide degradation by angiotensin-converting enzyme

A new method for continuous registration of enzymatic hydrolysis of peptides involving 1H-NMR spectroscopy was developed. The advantages of the method were demonstrated, using dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg) hydrolysis catalyzed by human kidney angiotensin-converting enzyme as an example. It was shown that the maximal activity of the enzyme towards dalargin is observed at pH 7.8; Km is 0.35 mM. The enzyme is inhibited by the substrate (Kd = 0.55 mM). Cl- do not influence the catalytic activity of the enzyme with respect to dalargin. The stereospecificity of the angiotensin-converting enzyme towards dalargin diasteriomers was studied.     

A new method for the preparation of beta-amylase from sweet potato.

A simple method for the preparation of sweet potato beta-amylase by thymol amylose adsorption is described. The method is far more efficient and gives higher recovery of the enzyme. The crystalline enzyme thus obtained is found to be homogeneous by gel chromatography, polyacrylamide gel electrophoresis.     

Fabrication of butyrylcholinesterase sensor using polyurethane-based ion-selective membranes.

A simple method of enzyme immobilization was investigated which is useful for fabrication of enzyme sensors based on polymeric ion-selective membranes. The enzyme membrane was built by coating a thin hydrophilic polyurethane (HPU) film directly mixed with an enzyme over an underlying polyurethane (PU)-based ion-selective membrane. This highly simple method of enzyme immobilization was applied to the fabrication of a potentiometric butyrylcholinesterase-based biosensor for the determination of organophosphorus pesticides. The enzyme was well entrapped within the HPU film and the intrinsic potentiometric response of the underlying ion-selective PU membrane was not influenced significantly by the outer HPU/enzyme membrane. The enzyme electrode was optimized by changing systematically the composition of the enzyme membrane to evaluate the effect of the changes on sensor response. The sensor was successfully applied to the analysis of paraoxon, an organophosphorus pesticide.     

Assay of aminoglycoside phosphotransferase in situ

A method for the evaluation of the aminoglycoside phosphotransferase activity in bacterial colonies directly is described. The method is based on the ability of the enzyme to modify the substrate immobilized on carboxymethylcellulose paper. The sensitivity and accuracy of the method were tested by comparing the results of the present assay to those obtained with conventional procedures. The method seems to be particularly useful for the detection within a bacterial population producing aminoglycoside phosphotransferase of those cells which do not make the enzyme and for rapid determination of the relative levels of enzyme produced by different clones.     

Wild-type enzyme as a reporter of inhibitor binding by catalytically impaired mutant enzymes.

A method for the determination of inhibition constants for catalytically-debilitated mutant enzymes is described. The inhibitor is partitioned between the mutant and wild-type enzymes. Catalytic rates of the wild-type enzyme are used as the signal of inhibitor binding to the mutant enzyme. The method is validated with scytalone dehydratase, the Y50F mutant, and a potent inhibitor. The K(i) value for Y50F determined by this method is 0.49 +/- 0.10 nM. The K(i) value determined using the Y50F catalytic report for inhibitor binding in the absence of wild-type enzyme is 0.20 +/- 0.030 nM. The wild-type enzyme binds the inhibitor ten-fold less tightly, thus indicating that the hydrogen-bonding interaction between the Y50 hydroxyl group and the inhibitor (suggested by X-ray crystallography) is weak. The method is most useful when the catalytic activity of the wild-type enzyme is the most sensitive report of inhibitor binding and the mutant enzyme is greatly crippled in catalytic activity.     

Silica-immobilized His6-tagged enzyme: alanine racemase in hydrophobic solvent

A new immobilization method for enzymes is presented to facilitate synthetic applications in aqueous as well as organic media. The enzyme Alanine racemase (AlaR) from Geobacillus stearothermophilus was cloned, overexpressed and then immobilized on a silica-coated thin-layer chromatography plate to create an enzyme surface. The enzyme, fused to a His(6)-tag at its N-terminal, was tethered to the chemically modified silica-coated TLC plate through cobalt ions. The immobilized enzyme showed unaltered kinetic parameters in small-scale stirred reactions and retained its activity after rinsing, drying, freezing or immersion in n-hexane. This practical method is a first step towards a general immobilization method for synthesis applications with any enzyme suitable for His6-tagging.     

The Time-Course of Hatching Enzyme Secretion in the Sea Urchin Strongylocentrotus purpuratus

By two independent methods, we have determined approximately the time-course of hatching enzyme secretion in the sea urchin Strongylocentrotus purpuratus . A quick-kill method indicates that a significant fraction of the enzyme is secreted between 90% and 97% of the fertilization-hatching interval. A direct assay method indicates that the remainder of the enzyme is secreted on either side of the 90–97%"window". The entire period of secretion spans from 75% to 100% or more of the fertilization-hatching interval. For embryos raised at 15°C this translates to an interval of 4.8 or more hr.     

Visualization of dihydrouracil dehydrogenase activity after disc gel electrophoresis

A rapid and sensitive method for the location of dihydrouracil dehydrogenase after disc gel electrophoresis based on the reduction of nitroblue tetrazolium to form an insoluble dye has been developed. Semiquantitative evaluation of enzyme activity was achieved by means of densitometer tracings of stained gels. The method permits detection of enzyme activity in partially purified extracts after a minimal number of purification steps. Two enzyme bands, differing mainly in charge, were separated giving the first indication that this enzyme may possibly exist in at least 2 isoenzyme forms in liver.     

New tetrazolium method for the histochemical demonstration of gamma-glutamyl transpeptidase.

A new tetrazolium method for the histochemical demonstration of gamma-glutamyl transpeptidase is proposed. The method is based on a newly synthesized substrate-gamma-L-glutamic acid-1-hydroxy-4-naphthylamide, which upon the enzyme hydrolysis liberates 1,4-aminonaphthol--a powerful reducing agent that reduces tetrazolium salts quickly and quantitatively to deeply colored, water-insoluble formazans, precipitating on the sites of the enzyme activity and marking them accurately. The redox reaction is quick enough and does not need any auxiliary electron-acceptor. The method is very fast and convenient for the histochemical visualization of the enzyme.