One-step magnetic modification of yeast cells by microwave-synthesized iron oxide microparticles.

UNLABELLED: Baker's yeast (Saccharomyces cerevisiae) cells were magnetically modified with magnetic iron oxide particles prepared by microwave irradiation of iron(II) sulfate at high pH. The modification procedure was very simple and fast. Both non-cross-linked and glutaraldehyde cross-linked magnetic cells enabled efficient sucrose conversion into glucose and fructose, due to the presence of active intracellular invertase. The prepared magnetic whole-cell biocatalyst was stable; almost the same catalytic activity was observed after 1-month storage at 4°C. Simple magnetic separation and stability of the developed biocatalyst enabled its reusability without significant loss of enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Magnetic whole yeast cell biocatalyst containing intracellular invertase in its natural environment has been prepared. Magnetic properties enable its easy separation from reaction mixture. Magnetically modified Saccharomyces cerevisiae cells have been used for invert sugar production, hydrolysing sucrose into glucose and fructose. The described magnetization procedure employing microwave-synthesized iron oxide microparticles is a low-cost and easy-to-perform alternative to already existing magnetization techniques.

Biosorption of water-soluble dyes on magnetically modified Saccharomyces cerevisiae subsp. uvarum cells.

Brewer's yeast (bottom yeast, Saccharomyces cerevisiae subsp. uvarum) cells were magnetically modified using water based magnetic fluid stabilized with perchloric acid. Magnetically modified yeast cells efficiently adsorbed various water soluble dyes. The dyes adsorption can be described by the Langmuir adsorption model. The maximum adsorption capacity of the magnetic cells differed substantially for individual dyes; the highest value was found for aniline blue (approx. 220 mg per g of dried magnetic adsorbent).

Magnetic alginate microparticles for purification of alpha-amylases.

Spherical magnetic alginate microparticles (25-60 microm in diameter) were prepared using the microemulsion system, with water-saturated 1-pentanol as the organic phase. The limited solubility of 1-pentanol in water enabled simple removal of the organic solvent from the prepared beads with water solution. The prepared alginate microparticles were used as magnetic affinity adsorbents for specific purification of alpha-amylases. Enzyme activity was eluted by 1.0 M maltose. alpha-Amylases from Bacillus amyloliquefaciens and porcine pancreatic acetone powder were purified 9- and 12-fold with 88 and 96% activity recovery, respectively.

Detection of low concentrations of malachite green and crystal violet in water.

A simple procedure for the detection of low concentrations of malachite green and crystal violet in water is presented. The dyes were preconcentrated from 1,000 ml of water samples with magnetic solid phase extraction using magnetic affinity adsorbent (magnetite with immobilized copper phthalocyanine dye). Due to the magnetic properties of the adsorbent the preconcentration process can also be performed in water samples containing suspended solids. After elution of the captured dyes, their presence in eluates was detected spectrophotometrically. Concentrations of both dyes in the range 0.5-1.0 microgl(-1) of water could be reproducibly detected. The dyes can be detected not only in potable water, but also in river ones.

Immunomagnetic separation of Escherichia coli O26, O111 and O157 from vegetables.

AIMS: Raw fruits and vegetables have been increasingly associated with human infections caused by Shiga toxin-producing Escherichia coli. This study evaluates the isolation and detection of E. coli O26, O111 and O157 from vegetable samples using immunomagnetic particles. METHODS AND RESULTS: Standard cultivation and immunomagnetic separation (IMS) procedures were compared. It was found that immunomagnetic particles could efficiently concentrate E. coli cells, detecting significantly more bacteria than with standard cultivation procedures. CONCLUSION: Bacteria were detected in 93-100% of the inoculated samples using the IMS procedure, but only 36-93% samples tested by standard cultivation procedures were found to be positive. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that E. coli O26, O111 and O157 immunomagnetic particles can be a very useful and efficient tool for the detection of E. coli strains in raw vegetables, and could probably be used with samples of animal origin.

Purification and characterization of the lysozyme from the gut of the soft tick Ornithodoros moubata.

The gut of the adult soft ticks Ornithodoros moubata displays high lytic activity against the bacteria Micrococcus luteus. The activity differed in the range of two orders of magnitude among individual animals and increased on average 4 fold during the first week following ingestion. In homogenates of first instar nymphs the activity was much lower increasing exponentially as nymphs neared the first molt. The protein responsible for this activity was purified out of gut contents of adult ticks by means of affinity adsorption on magnetic-chitin followed by two chromatography steps on cation exchange FPLC column MonoS. The homogeneous active protein has a mass of 14006 +/- 20 Daltons as determined by MALDI-TOF mass spectrometry. The N-terminal amino-acid sequence of this protein is K-V-Y-D-R-C-S-L-A-S-E-L-R with the highest similarity to the lysozyme from liver of rainbow trout and to lysozymes from digestive tracts of several mammals. The motif DRCSLA is specific for the digestive lysozymes of several dipteran insects. Based on this evidence, we have identified the protein as the tick gut lysozyme. The tick gut lysozyme has a pI near 9.7 and retains its full activity after treatment at 60 degrees C for 30 minutes. The pH optimum of the tick lysozyme was in the range from pH 5-7. Only marginal activity could be detected at pH > 8 which raises the question about the function of lysozyme in anti-bacterial defense in the environment of the tick gut.

Use of magnetic techniques for the isolation of cells.

Magnetic separation is an emerging technology using magnetism, sometimes in combination with conventional separation or identification methods, to purify cells, cell organelles and biologically active compounds (nucleic acids, proteins, xenobiotics) directly from crude samples. Several magnetic separation procedures have been developed to isolate target cells specifically. The purpose of this short review is to summarize various methodologies, strategies and materials which can be employed for the selection and separation of target cells with the help of magnetic field and thus to help the novices in this field to be able to orient themselves in vast amount of literature available. Immunomagnetic separations employing specific antibodies to label the target cells represent the most often used approach and are discussed in detail.

Improved properties of bovine erythrocyte acetylcholinesterase, isolated by papain cleavage.

A simple and rapid procedure involving papain cleavage of the membrane anchor was used to isolate membrane-bound acetylcholinesterase from bovine erythrocytes. The solubilized enzyme was purified 930-fold by ion exchange chromatography and gel filtration. The properties of the papain-cleaved acetylcholinesterase were compared with those of a commercial acetylcholinesterase, solubilized from the erythrocyte membranes by detergents. Cleavage of the membrane anchor eliminated dimer aggregation, caused a pH shift in thermal stability and resulted in increased stability in organic solvents. Bovine serum albumin, used as stabilizer of the commercial enzyme preparation, increased the thermal stability but concomitantly decreased the activity of acetylcholinesterase at pH 6-8. The improved stability of the cleaved acetylcholinesterase, especially in organic solvents, may enhance the biosensor performance of the enzyme.

Spectrophotometric determination of effective proteolytic activity in biodetergents.

A new procedure for the determination of effective proteolytic activity in biodetergents has been developed. Effective enzyme activity is defined as the activity exhibited by the tested enzyme under real washing conditions, i.e., in the water suspensions of the biodetergent tested at the working temperature (usually 40 degrees C). Two insoluble chromolytic substrates, namely black gelatin (gelatin cross-linked with glutaraldehyde in the presence of black drawing ink) and the protease substrate based on the immobilization of dyed casein in the structure of polyacrylamide gel (blue casein-PAAG) were successfully used. It was found that there is a great difference in effective proteolytic activity among various biodetergents. The proposed procedure is suitable both for the manufacturers for a quick control of their products and for the quality control laboratories.

Batch isolation of hen egg white lysozyme with magnetic chitin.

Biospecific magnetic sorbent for lysozyme isolation (magnetic chitin) has been prepared from magnetic chitosan after acetylation with acetic anhydride. The capacity of magnetic chitin was 2.5 mg of lysozyme per 1 ml of sorbent.

Black substrate for spectrophotometric determination of cellulase activity in coloured solutions.

A simple procedure for spectrophotometric determination of cellulase activity in coloured solutions is described. CM-cellulose, cross-linked with epichlorhydrin in the presence of black drawing ink, is used as an insoluble chromolytic substrate. The absorbance of reaction mixture filtrates are read in the near infra-red region (at 800-900 nm) where the absorbances of contaminating coloured substances are substantially lowered; by contrast, black drawing ink released from the substrate during the action of cellulases absorbs strongly at these wavelengths.

A spectrophotometric assay for lipase activity utilizing immobilized triacylglycerols.

New substrates for the determination of lipase activity have been developed. Triacylglycerols were immobilized by adsorption on an appropriate carrier or adsorbent yielding a lipase substrate in a powder form. The adsorbed triacylglycerols were easily hydrolyzed by lipases present in a reaction mixture. The released fatty acids were extracted with benzene and converted to the corresponding Cu (II) salts (copper soaps) which were measured spectrophotometrically.

Spectrophotometric determination of amylase activity in coloured solutions.

A simple procedure for spectrophotometric determination of amylase activity in coloured solutions is described. Starch cross-linked with a bifunctional reagent in the presence of black drawing ink is used as an insoluble chromolytic substrate. The absorbances of reaction mixture filtrates are read in the near-infrared region (at 800-900 nm) where the majority of coloured substances does not absorb; on the contrary, black drawing ink released from the substrate during the action of amylase absorbs strongly even at these wavelengths.

Dye-stained gelatin microcarriers as insoluble chromolytic substrates for the determination of proteolytic activity.

Dye-stained gelatin microcarriers were used as insoluble chromolytic substrates for the determination of proteolytic activity. Trypsin at concentration 100 ng/ml could be easily detected. The spontaneous leakage of dyed fragments into water solutions was negligible.

Rapid detection of dextranases in liquid samples.

A rapid procedure for detecting dextranases in fractions after liquid chromatography and other liquid samples has been developed. The detection is based on the hydrolysis of a very thin layer of cross-linked dextran (Sephadex). Dextranase positive and negative fractions can be distinguished within a short time.

Spectrophotometric determination of proteolytic activity in coloured solutions.

A simple procedure for spectrophotometric determination of proteolytic activity in coloured solutions is described. Gelatin cross-linked with glutaric dialdehyde or formaldehyde in the presence of black drawing ink is used as an insoluble chromolytic substrate. The absorbances of reaction mixture filtrates are read in the near infra-red region (at 800-900 nm) where the majority of coloured substances does not absorb; on the contrary black drawing ink released from the substrate during the proteolysis absorbs strongly even at these wavelengths.

Detection of proteinase inhibitors in fractions after liquid chromatography.

A technique for rapid detection of proteinase inhibitors in fractions after liquid chromatography is described. Aliquots of the tested solutions are placed onto a thin layer of gelatin, crosslinked with glutardialdehyde in the presence of water soluble nigrosin, to form small drops. A proteinase solution is added to the above-mentioned drops and the plate is incubated at ambient temperature for approximately 15-25 min. After the gelatin layer is washed with water the inhibitor negative fractions are visualized as colorless zones on a blue background. With inhibitor positive fractions no change of gelatin layer occurs.