A rapid spectrophotometric method for the determination of esterase activity

We have developed a spectrophotometric assay method which continuously records esterase activity at 510 nm by monitoring absorbance changes due to the formation of a diazo dye complex. In our method, α-naphthyl ester substrates are hydrolyzed by enzymatic action to α-naphthol which couples to Fast Blue RR salt (a diazonium salt) forming a diazo dye complex. Our method is unique in directly monitoring the formation of the diazo dye complex without extracting the color of the complex as in other methods that use naphthyl esters and diazo coupling of reaction products. The method appears to be limited to α-naphthyl ester substrates, however, since β-naphthyl esters did not give a linear change in absorbance in the enzymatic reactions tested. With this assay method, one can use a single substrate both to determine esterase units quantitatively in solution and to detect esterase staining activity on gel electrophoresis.     

A rapid method for determination of ribonucleotide reductase activity

Ribonucleoside diphosphate reductase activity is determined in centrifuged homogenates by following the conversion of cytosine ribonucleotide to cytosine deoxyribonucleotide. The enzymatic reaction is measured by monitoring the radioactivity of the reaction products separated by thin layer chromatography on PEI-cellulose plates. The method is rapid and permits the simultaneous processing of multiple samples.     

A rapid method for determination of viable sulphate-reducing bacteria in human faeces

Adenosine phosphosulphate reductase (APS reductase) (E.C.1.8.99.2), viable counts of sulphate-reducing bacteria and rates of sulphate reduction were determined in 20 human samples of faeces. The activity of APS reductase, in contrast to sulphate reduction rates, correlated well with viable counts ( r = 0.987) and may therefore be used rapidly to quantify sulphate-reducing bacteria in human gut contents.     

Simple and rapid method for the determination of coproporphyrinogen oxidase activity

Coproporphyrinogen oxidase, the sixth enzyme in the biosynthetic heme pathway, catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX. A reversed-phase high pressure liquid chromatography method was developed to measure coproporphyrinogen oxidase enzymatic activity in rat liver. With this method, the separation, identification and quantification of coproporphyrin III (oxidized substrate) and protoporphyrin IX (oxidized product) present in the assays could be carried out with no need of derivatization and in less than 15 min. Rat and human liver coproporphyrinogen oxidase basal activities determined using this method were 0.41+/-0.05 nmol of protoporphyrin IX/h per mg of hepatic protein and 0.87+/-0.06 protoporphyrin IX/h per mg of hepatic protein, respectively. Kinetic studies showed that optimum pH for rat CPGox is 7.3, and that its activity is linear in the range of protein concentrations and incubation times assayed. The present paper describes a sensitive, specific and rapid fluorometric high performance liquid chromatography method to measure coproporphyrinogen oxidase, which could be applied to the diagnosis of human coproporphyria, and which is also suitable for the study of lead and other metal poisoning that produce alterations in this enzymatic activity.     

A modified colorimetric method for the determination of pyrrolidone carboxylyl peptidase activity in bacteria

The direct spectrophotometric estimation of ferrocyanide (1) suggested the possibility of measuring small quantities of reducing sugars. However, as previously described (1), the method is not applicable to the measurement of reducing sugars because of the high temperature required for the required oxidation step.Due to the relative differences in molar extinction coefficients at the wavelengths concerned (1,5), the measurement of ferrocyanide rather than ferricyanide (5) provides a large increase in sensitivity.A comparison of such a method with a modified folin-Wu method (2–4) for estimation of blood glucose should indicate the possibility of its application to biological fluids containing cells and proteins.     

A rapid method for determination of acid phosphatase activity of whole yeast cells

A simple and rapid procedure for determination of intracellular acid phosphatase activity without the need for disruption of cells is described. Candida lipolytica cell suspension was treated with 0.1% Triton X-100 for 30 min at room temperature and with intermittent shaking. The enzyme assay is carried out directly with the permeabilized cell suspension. Permeabilization of the yeast cells to p -nitrophenylphosphate by Triton X-100 provides almost 100% efficiency in determining the total acid phosphatase activity compared to results obtained with disrupted yeast cells.     

A rapid colorimetric method for the determination of mimosine

A rapid colorimetric method has been described for the quantitative determination of mimosine by using activated carbon as a decolorizing agent and measuring the intensity of mimosine-ferric chloride color produced.     

A rapid method for quantitative determination of L-tryptophan

Summary A modified colorimetric technique automatized by an Alpkem micro-continuous flow analyser was described for estimating the concentration of L-tryptophan in fermentation broth. This approach provided a convenient alternative to HPLC for L-tryptophan estimation and may help to avoid the time-consuming and laborious screening work encountered in the strain improvement programme.     

A rapid method for the isolation of eicosapentaenoic acid-producing marine bacteria

Bacterial production of long chain polyunsaturated fatty acids (LC-PUFAs) is a promising biotechnological approach for the mass production of these valuable compounds, but extensive screening is currently needed to select a strain that meets industrial requirements.A method was developed for the rapid screening and isolation of eicosapentaenoic acid (EPA)-producing marine bacteria from mixed cultures using the dye 2,3,5-triphenyltetrazolium chloride (TTC). The method was first validated using two bacteria from the Shewanella genus, S. gelidimarina (known to contain EPA) and S. fidelis (known not to contain EPA), and subsequently applied to a range of bacterial samples collected from seven randomly selected New Zealand fish species.By incorporating TTC in both solid and liquid state fermentation treatments, a clear association between the reduction of TTC to the red-coloured triphenyl formazan (TF) and the presence of EPA within Gram-negative bacteria was confirmed. Incubation in 0.1% w/v TTC was optimal for colour response and cell growth in agar plates and liquid cultures. Bacteria that produce EPA reduced TTC to TF, but a number of non-EPA-producing bacteria also showed this capacity. By conducting a subsequent Gram staining, all EPA-producing strains were revealed to be G (−) rod bacteria while the non-producing ones were all G (+) cocci. The fatty acid methyl esters of the isolated bacteria that reduced TTC to TF were analysed using gas chromatography-mass spectrometry and the content of EPA was confirmed by gas chromatography.From a pool of 2.0 × 10⁸ CFU/ml, this method allowed the rapid isolation of 16 bacteria capable of producing EPA. This new approach significantly reduces the number of samples submitted for GC analysis and therefore the time, effort and cost of screening and isolating strains of EPA-producing marine bacteria.     

A rapid method for the isolation of plasmid DNA from bacteria

Summary A rapid method is described for the isolation of plasmid DNA from Escherichia coli and Pseudomonas putida. The effect of heating the cell preparation during plasmid extraction is discussed in relationship to the final plasmid yield.     

A rapid method for the detection of tryptophanase in anaerobic bacteria

A total of 633 anaerobic bacteria were examined for tryptophanase production using a rapid method which distinguishes within 5 to 180 minutes between anaerobes that contain tryptophanase and those that do not. Of the 196 tryptophanase-positive isolates tested, 99% showed tryptophanase activity within 2 hours as compared with 94.4% in 24 hours by a conventional method. A total of 299 tryptophanase-negative organisms were tested. Ninety three percent of these remained negative after 24 hours as compared with 95.3% when tested with a 24-h conventional method. Additional information was obtained on the sensitivity of this test and the time-dependent production of indole by tryptophanase.     

A rapid colony screening method for the detection of arsenate-reducing bacteria

A rapid and simple method has been developed for the detection of arsenate reducing bacteria based on the presence of arsenite [As (III)], the end product of anaerobic arsenate [As (V)] respiration. Confirmation of As (III) product is made by the reduction of starch-iodine complex. The method can be used over a large pH range (5.5–9.0) and can easily be determined at arsenite concentration as low as 0.025 mM. Major advantages of this technique are that a large number of samples can be analyzed easily at a time.     

A new method for determination of ATPase activity in tissue homogenates]

Authors describe a new method to determine Mg2+ and Na+K+ATPase activity on extremely small samples of tissue homogenates. They use specific inhibitors to discern between the two activities and a sensitive colorimetric method to dose Pi released from ATP, Lower limit of sensitivity is about 0,0100 micromoles of Pi/mg proteins/hour.