Purification of adenosine deaminase from chicken-egg yolk by affinity column chromatography

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.     

An improved purification procedure for sulfite oxidase from bovine liver.

Sulfite oxidase (Sulfite:O2 oxidoreductase, EC 1.8.3.1) has been purified 2,440-fold from bovine liver. The procedure developed was used to isolate the enzyme from 1,000 g of liver and permitted the rapid isolation of enzyme with a very high specific activity (40,405 mU/mg). The enzyme preparations obtained have been characterized by electrophoretic and spectrophotometric analysis and the molecular mass and the Stokes radius of the enzyme have been determined.     

Purification and characterization of an NAD(+)-dependent dehydrogenase that catalyzes the oxidation of thromboxane B2 at C-11 from porcine liver. Development and application of 11-dehydro-thromboxane B2 radioimmunoassay to enzyme assay

11-Dehydro-thromboxane B2 has been identified as a major metabolite of infused as well as endogenous thromboxane B2 in mammalian plasma and urine. This metabolite is derived from thromboxane B2 by enzymatic oxidation at C-11 catalyzed by 11-hydroxythromboxane B2 dehydrogenase. A radioimmunoassay for 11-dehydro-thromboxane B2 has been developed and used for enzyme assay, purification and characterization. Antibodies were generated against 11-dehydro-thromboxane B2 conjugated to bovine thyroglobulin. Labeled marker was prepared by radioiodinating 11-dehydro-thromboxane B2-tyrosine methyl ester conjugate. A sensitive radioimmunoassay capable of detecting 10 pg of 11-dehydro-thromboxane B2 per assay tube was developed. The antibodies showed minimal crossreaction with thromboxane B2 (0.03%), prostaglandin D2 (2.76%) and other eicosanoids (less than 0.03%). The enzyme activity was determined by assaying NAD(+)-dependent formation of immunoreactive 11-dehydro-thromboxane B2 from thromboxane B2. The enzyme was found to be enriched in liver although significant activity was also detected in gastrointestinal tract and kidney in pig. The enzyme was purified from porcine liver cytosol to apparent homogeneity using conventional and affinity chromatography. The purified enzyme exhibited coenzyme specificity for NAD+ and used thromboxane B2 as a substrate. The enzyme also catalyzes NADH-dependent reduction of 11-dehydro-thromboxane B2 to thromboxane B2 indicating the reversibility of the enzyme catalyzed reaction. The apparent Km values for thromboxane B2, 11-dehydro-thromboxane B2 and NAD+ are 8.1, 8.0 and 23 microM, respectively. Subunit Mr was shown to be 55,000, whereas the native enzyme Mr was found to be 110,000 indicating that the enzyme is a dimer. The enzyme is sensitive to sulfhydryl inhibitions suggesting cysteine residues are essential to enzyme activity. The availability of a homogeneous enzyme preparation should allow further studies on the substrate specificity and the structure and function of the enzyme.     

Rapid purification of a restriction endonuclease from Escherichia coli RY13

Summary A two step method for the purification of restriction endonuclease Eco RI was developed. The first step involved the purification of the enzyme on Cibacron Blue-F3GA-agarose column, followed by a hydroxyapatite column. The enzyme was homogeneous on SDS-PAGE and completely free from contaminating nucleases and phosphatases, and can be used for direct DNA hydrolysis.     

Kinetics of chitinase production. II. Relationship between bacterial growth, chitin hydrolysis and enzyme synthesis

A comprehensive model for chitinase production during growth of Serratia marcescens QMB 1466 on chitin was developed taking into account the rate of chitin hydrolysis in order to estimate the rate of bacterial growth. In relating growth with enzyme synthesis the total enzyme concentration was used as the sum of the enzyme present in the bulk of the fermentation broth and the enzyme adsorbed on the chitin particles. The equations constituting the proposed model were fitted to the experimental results from both continuous and batch fermentation to obtain parameters describing substrate yield, metabolic maintenance, and enzyme yields.     

Purification of galactose oxidase from Dactylium dendroides by affinity chromatography on melibiose-polyacrylamide

Galactose oxidase is a fungal enzyme which is known to oxidize the C-6 hydroxymethyl of galactose and galactosamine to an aldehyde group. It has been widely used in glycoconjugate research, for example in the labeling of asialoglycoproteins. We have developed a simple affinity purification for galactose oxidase using melibiose-polyacrylamide. This affinity procedure was used to purify the enzyme from ammonium sulfate precipitates of culture filtrates of Dactylium dendroides. The material containing proteases and other contaminants is eluted in the buffer wash. The galactose oxidase is then specifically eluted from the column with buffer containing 0.1 M D-fucose or D-galactose. Using this procedure, the enzyme was also purified from commercial samples of galactose oxidase which contain high proteolytic activity.     

Pulmonary angiotensin-converting enzyme antienzyme antibody.

A method has been developed for quantitating anticatalytic activity in antibody preparations made in goats against pure solubilized angiotensin-converting enzyme from rabbit pulmonary membranes. Anticatalytic activity was purified about 90-fold from a single batch of serum by a procedure including diethylaminoethylcellulose chromatography and elution from Sepharose columns containing covalently bound pure enzyme. Antiholoenzyme antibody was fractionated with respect to charge and binding affinity; however, these different populations each inhibited enzymatic hydrolysis of hippurylhistidylleucine, angiotensin I, and bradykinin. The inhibition dose-response curves were similar for hydrolysis of hippurylhistidylleucine and angiotensin I despite the difference in molecular weight of these substrates. Evidence is presented suggesting that a single molecule of antibody can bind two molecules of enzyme and that at least 18% of the total antiholoenzyme antibody population is directed against determinants which influence catalytic activity. A competitive immunoassay was developed with radioiodinated pulmonary enzyme as displaceable antigen. The anticatalytic and radioimmune assays were used to examine immunological properties of converting enzymes in various rabbit organs and fluids. Kidney, brain, and serum were found to contain converting enzymes which were immunologically identified with that in rabbit lung. Converting enzyme in seminal plasma was similar to the lung enzyme in the anticatalytic assay, but showed lower immunoreactivity in the radioimmune assay.     

Stabilization of Candida antarctica lipase B in hydrophilic organic solvent by rational design of hydrogen bond

Enzymatic reactions conducted in organic solvents have many advantages. However, organic solvent molecules may replace water molecules at the protein surface and penetrate into the enzyme, which could lead to the denaturation of the enzyme or changes in its reaction kinetics and substrate specificity. Thus, it is important to enhance the stability of enzymes in organic solvents. To date, there has been no efficient rational approach developed to enhance enzyme stability in hydrophilic solvents. We developed a rational approach to enzyme design. The design rules were established by investigating stable mutants from previous studies of directed evolution. Candida antarctica lipase B (CalB) was used as a target enzyme due to its versatile applications in organic solvents. The N97Q, N264Q, and D265E mutants of CalB showed higher organic solvent stability than the wild type.     

Enzyme immunoassay for detection of Salmonellae in foods.

An enzyme immunoassay was developed to detect Salmonella in foods. Indirect test protocols were developed for use with microtitration plates or Gilford microcuvettes. Samples from enrichment cultures were mixed with H-specific immunoglobulin G and allowed to react; unbound antibody was removed by three 5-min centrifugation washes; goat anti-rabbit antibody conjugated to alkaline phosphatase was added and allowed to react; and unbound conjugate was removed by centrifugation washing as before. Salmonella-positive samples were indicated by the production of a chromogenic reaction product after the addition of alkaline phosphatase substrate. The color could be read visually or quantified by absorbance. Ninety-eight food samples were examined to compare the enzyme immunoassay with enrichment serology, immunofluorescence, and the Food and Drug Administration pure culture technique. The enzyme immunoassay was sensitive and specific, and it possessed advantages over methods currently in use. Furthermore, when the enzyme immunoassay was used to screen preenrichment media, the results indicated that it might be decidedly more sensitive than the conventional pure culture technique.     

Diketone cleaving enzyme Dke1 production by Acinetobacter johnsonii--optimization of fermentation conditions

The main objective of this work was the optimization of the production of the novel dioxygenase diketone cleaving enzyme (Dke1) from Acinetobacter johnsonii. Acetylacetone was used as an inducer for enzyme production. In the first step, the growth medium was optimized by using screening designs for finding the optimal carbon and nitrogen source. In the second step, a genetic algorithm was used to optimize the concentrations of all medium components. After six generations the stopping criterion was reached and a growth medium was obtained which produced sixteen times more enzyme than the starting medium. In the next step, an addition profile for the inducer acetylacetone was developed to further increase enzyme production by using a genetic algorithm. In this case, after four generations the stopping criterion was fulfilled. By using the obtained optimal addition profile Dke1 activity was enhanced from 826 to 2584Ul(-1). In comparison to the starting conditions activity could even be increased by a factor of 50.     

Application of capillary zone electrophoresis to study the properties of rhodanese fromAcidithiobacillus ferrooxidans

A new capillary zone electrophoretic method was applied to the assay of enzymic activity of rhodanese from Acidithiobacillus ferroxidans. The enzyme activity determined by capillary zone electrophoresis was compared with that determined by discontinuous spectrophotometry, the values obtained being in good agreement. The method was also used to evaluate Michaelis constants of cyanide and thiocyanate as substrates; a new approach was developed to solve the problem with variable ionic strength of the samples. The pH and temperature optima for the enzyme were also determined.     

Purification and partial amino acid sequences of the enzyme vinorine synthase involved in a crucial step of ajmaline biosynthesis

The acetyl-CoA-dependent enzyme vinorine synthase was isolated from hybrid cell suspension cultures of Rauvolfia serpentina and Rhazya stricta. The sarpagan-type alkaloid gardneral was used as a substrate of the enzyme leading to the ajmalan-type 10-methoxyvinorine. An HPLC-based assay was developed to monitor vinorine synthase activity, which allowed establishing a five step purification procedure combining anion exchange, hydrophobic interaction, hydroxyapatite and gel filtration. Purification resulted in a yield of 0.2% and an approximately 991-fold enrichment of the acetyltransfer activity. SDS-PAGE analysis showed a Mr for the enzyme of approximately 50 kDa. The four peptide fragments generated by proteolysis of the pure enzyme with endoproteinase LysC and the N-terminal part of the enzyme were sequenced. The enzyme preparation (> 875-fold enrichment) delivering the N-terminal sequence was isolated from R. serpentina cell suspensions. Sequence alignment of the five peptides showed highest homologies in a range of 30-71% to acetyltransferases from other higher plants involved in natural plant product biosynthesis. Based on the partial sequences vinorine synthase is probably a novel member of the BAHD enzyme super family.     

Gamma-cystathionase: the non-ideal gel filtration high performance liquid chromatography. Physico-chemical and immunochemical characteristics of the enzyme

The rapid method for gamma-cystathionase purification was developed. It is based on the non-ideal gel filtration HPLC. The isolated homogeneous enzyme was used for immunization and immunosorbent preparation. A monospecific polyclonal antibody was prepared. The substrate specificity of the isolated enzyme was studied.     

Selective preparation and characterization of membranous and soluble forms of alkaline phosphatase from rat tissues. A comparison with the serum enzyme

We developed a method for selective preparation of two forms of alkaline phosphatase from rat tissues. The enzyme was extracted by n-butanol treatment at pH 5.5 and pH 8.5 as soluble and aggregated (membranous) forms, respectively. The soluble form prepared from liver was found to be identical with the serum enzyme. Complete solubilization of the membrane-bound enzyme without detergents had a great advantage in its purification. Rat hepatoma AH-130 cells enriched in alkaline phosphatase were first used for purification of the liver-type enzyme. The hepatoma enzyme, purified by chromatographies on concanavalin-A-Sepharose, Sephacryl S-300 and hydroxyapatite was used for production of antibodies specific for the liver-type isozyme. An immunoaffinity column, prepared with anti-(hepatoma-enzyme) IgG was utilized for the enzyme purification from other tissues including the membranous form. Analyses of amino acid composition of the purified enzymes revealed that all the liver-type enzymes from hepatoma, liver, kidney and serum had the same composition, whereas the intestinal type consisted of the composition distinctly different from that in the liver type. In addition, there was no significant difference in amino acid composition between the soluble and membranous forms, suggesting a possible involvement in the membranous form of a hydrophobic component other than its polypeptide domain. The present method for selective preparation of the soluble and membranous forms of alkaline phosphatase will be useful for a further investigation on the interaction of the enzyme with membranes.     

Method for determination of angiotensin-converting enzyme activity in blood and tissue by high-performance liquid chromatography

A simplified method for an angiotensin-converting enzyme activity assay in biological samples was developed. Samples were incubated with hippurylhistidylleucine, an artificial substrate of angiotensin-converting enzyme. The reaction was terminated by the addition of metaphosphoric acid and liberated hippuric acid in the supernatant was quantitated directly by reversed-phase high-performance liquid chromatography. Tissues were homogenized in the presence of Nonidet-P40, a detergent, and the resulting supernatant was used for the assay of tissue angiotensin-converting enzyme activity by high-performance liquid chromatography. The present procedure made it possible to determine angiotensin-converting enzyme activity in whole blood and the total activity in tissues. A comparative study of angiotensin-converting enzyme activity in plasma, kidney and lung of five experimental animals showed a high degree of variation from species to species.     

Production of α-amylase in a low cost medium by Bacillus licheniformis TCRDC-B13

A low cost synthetic medium producing large quantities of α-amylase has been developed. Bacillus licheniformis TCRDC-B13 isolated from soil was used for α-amylase production. The α-amylase enzyme of this strain showed excellent stability at high temperatures and over a wide pH range. The low cost medium produced 5 times more enzyme than the high cost synthetic medium (using yeast extract and peptone) in shake flasks. In a 2.6-l fermentor, the enzyme production further doubled.     

Fluorometric assay for ADP-ribosylarginine cleavage enzymes

A continuous fluorometric assay for enzyme activities which remove ADP-ribose linked to proteins at arginine was developed. The substrate analog, N alpha-dansyl-N omega-(1,N6-etheno-ADP-ribosyl)arginine methyl ester, was used to assay the catalytic activities of dinitrogenase reductase activating glycohydrolase from Rhodospirillum rubrum and nucleotide pyrophosphatase from Crotalus adamanteus. The assay is based on the increase in fluorescent emission by ethenoadenine accompanying the enzyme-catalyzed hydrolysis of the substrate. The assay has been used to detect activities of 10 fmol substrate cleaved per minute. The substrate anomerizes to give a 40:60 equilibrium of alpha:beta ribosylguanidinium anomers, allowing the determination of enzyme stereospecificity. The substrate was used to determine the kinetic parameters and products of the N-glycohydrolase and the pyrophosphatase.     

Development of a quantitative assay method for 3 beta-hydroxy-delta 5-steroid dehydrogenase in the rat testis

We investigated the first step of the sex steroid hormone biosynthesis pathway by assaying the activities of 3 beta-hydroxy-delta 5-steroid dehydrogenase, the rate-limiting enzyme in this pathway. We have developed a simple and rapid colorimetric assay for 3 beta-hydroxy-delta 5-steroid dehydrogenase in rat testis. The supernatant from rat testis tissue homogenates were used for the enzyme assay. The enzyme activity was determined by measuring the absorbance at 570nm which indicates the rate of conversion of pregnenolone into progesterone in the presence of NAD, using phenazine methosulfate and nitro blue tetrazolium as the color reagent. The activity of this enzyme ranged from 4.57+/-1.34 to 10.56+/-2.13 nmol/mg protein/min with a mean activity of 8.96+/-1.27 nmol/mg protein/min. The K(m) of the enzyme at an optimum pH of 7.25 was about 4.7+/-0.12 nM.     

Purification of Phosphomannanase and Its Action on the Yeast Cell Wall

An improved assay for phosphomannanase (an enzyme required for the preparation of yeast protoplasts) has been developed based on the release of mannan from yeast cell walls. A procedure for the growth of Bacillus circulans on a large scale for maximal production of the enzyme is described. The culture medium containing the secreted enzyme was concentrated, and the enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, gel filtration on P-100, and isoelectric density gradient electrophoresis. Although the enzyme was purified to apparent homogeneity, it still contained laminarinase activity which could not be separated by size or charge. The two enzymatic activities also exhibited two isoelectric points (pH 5.9 and 6.8) on ampholine electrophoresis. The laminarinase was not active on yeast glucan. The enzyme preparation was shown to remove mannan from yeast without removing glucan. Electron microscopic observation supports the idea that this mannan is the outer layer of the yeast wall. Phosphomannanase will produce protoplasts from yeast when supplemented with relatively low amounts of snail enzyme. This activity is present in snail enzyme but appeares to be rate-limiting when snail enzyme alone is used. Phosphomannanase has proven useful for studying the macromolecular organization of polymers in the yeast cell wall.     

Development of an amperometric hypoxanthine biosensor for determination of hypoxanthine in fish and meat tissue

A hypoxanthine (Hx) biosensor based on immobilized xanthine oxidase (XO) as the bio-component was developed and studied for the rapid analysis of fish (sweet water and marine) and goat meat samples. The biosensor was standardized for the determination of Hx in the range of 0.05 to 2 mM. Crosslinking with glutaraldehyde in presence of BSA as a spacer molecule was used for the method of immobilization. One layer of gelatin (10%) was applied over the immobilized enzyme layer to reduce the leaching out of enzyme from the membrane (cellulose acetate) matrix. The optimum pH of the immobilized system was determined to be 8.5 at 25 degrees C instead 7.0-7.2 for free enzyme system. Km and Vmax values were determined for the immobilized system. The developed sensor was applied to determine the amount of Hx present in fish and meat over a period of time. The stability of the enzyme immobilized membrane was also tested over a period of 30 days.