Simple cultural test for relative cellulolytic activity of fungi

A simple method is described for determining the relative cellulolytic activity of fungi. Opaque columns of an agar medium containing a partially crystalline cellulose preparation were inoculated with the fungi. Depth of the clear zone that developed beneath the growing cultures provided a visual measure of cellulolytic activity on a continuous, cumulative basis. Depth of clearing (DC) was determined for 25 species of fungi differing widely in cellulolytic activity, and compared by correlation analysis with results of three other methods for measuring cellulolytic activity. Relatively high coefficients of correlation (greater than 0.6) were obtained between DC and weight loss of cotton sliver, loss in tensile strength of cotton duck, and carboxymethyl cellulase activity in culture filtrates. In comparison with conventional assay procedures, the clearing method offered several advantages: (i) results were at least as well correlated with the capacity to utilize native cellulose as a substrate; (ii) the method measured activity of growing cultures rather than culture filtrates, thus involving less risk of losses due to product inhibition, binding, or denaturation of enzymes; (iii) repeated measurements were made on the same experimental set up, so that errors due to arbitrarily selected times of harvest were avoided conveniently; and (iv) the method required less working time and very simple equipment, making it convenient for large-scale screening tests.     

A simple plate test for screening colicine-inducing substances as a tool for the detection of potential carcinogens.

A simple and rapid plate test is described for screening substances that induce colicine E2. By using chemicals activated with microsomal enzymes and a permeable (rfa) tester bacterium that is also deficient in DNA repair (uvrB), the range of inducing substances that can be detected has been extended. The possible correlation between colicine-inducing substances and carcinogens is discussed.     

A simple agar plate assay for screening siderophore producer yeasts.

Yeasts produce hydroxamate-type siderophores (iron-binding compounds) in response to Fe-stress conditions. Because these siderophores are important to the biocontrol of postharvest diseases of apple and pears, a method for screening siderophore producer yeast was developed.The screening method was carried out in special Petri dishes with eight or nine wells (25-mm diameter). These wells were filled with siderophore production medium and seeded with yeasts isolated from epiphytic apple microflora. After yeasts grew (24-48 h), holes (2-mm diameter) were made in the agar of each well. Holes were filled with an acid solution of ferric perchlorate. After 10-15 min, reddish halos appeared in the bottom of the plate and their intensities were compared with standards. Standards were prepared in the same special dish with rhodotorulic acid solutions (concentrations between 0.05 and 1 g/l) plus 2% agar. When agar solidified into wells, holes were made and filled with ferric perchlorate solution. Color intensities of reddish halos were proportional to siderophore concentration and the detection limit was 0.1 g/l. It was possible to correlate the production of siderophore in solid medium with the results obtained in liquid medium. The methodology was also a useful tool for making a preliminary assessment of the influence of different factors on the siderophore production.     

Sensitive plate assay for screening and detection of bacterial polyurethanase activity

AIMS: A plate assay to screen and detect bacterial polyurethanase in agar medium containing a colloidal polyester-polyurethane and rhodamine B is presented. METHODS AND RESULTS: Substrate hydrolysis causes the formation of orange fluorescent halos visible upon u.v. irradiation. The logarithm of polyurethanase activity from a purified polyurethanase protein is linearly correlated with the diameter of halos, thereby allowing quantification of polyurethanase activities ranging from 0.81 to 7.29 Units. CONCLUSIONS: The potential advantages of this system are in identification and recovery of viable polyurethanolytic bacteria and quantification of polyurethanase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: These advantages are derived largely from the intense fluorescence observed due to the hydrolysis of substrate reacting with rhodamine B allowing for the use of low substrate concentrations and corresponding decrease in time required detecting low levels of enzyme activity.     

Investigating and optimizing the immobilization of levansucrase for increased transfructosylation activity and thermal stability

Levansucrase (LS) represents a key enzyme in glycoside synthesis of novel prebiotics and β-2,6-levan. The study of the effects of immobilization parameters of LS, produced from Bacillus amyloliquefaciens, onto glyoxyl agarose-iminodiacetic acid/Cu (glyoxyl agarose-IDA/Cu) by response surface methodology revealed the significance of their interactive effects. Retention of activity was altered by interactive effects from buffer molarity/time and buffer pH/buffer molarity. The optimized immobilization conditions were identified to be a protein loading of 9.09 mg protein/g support, a buffer concentration of 608 mM at pH 6.8 and an incubation time of 49 h. Normally a reducing agent is applied to the immobilized enzyme in order to promote the formation of covalent bonds. This step was replaced with the addition of the ionic polymer polyethylenimine (PEI), which provided a better compromise between retained activity and thermal stability of the immobilized LS. Indeed, LS immobilized onto glyoxyl agarose-IDA/Cu/PEI had a retention of activity of 70.91% with a protein yield of 44.73% and an activity yield of 54.69%, while exhibiting a half-life 4.7 times higher than that of the free LS at 50 °C.     

A nonradioactive 96-well plate assay for screening of trans-sialidase activity

Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-beta-D-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems.     

A simple screening procedure for selecting fungi with potential for use in the bioremediation of contaminated land

A simple initial screening procedure for selecting strains of white-rot fungi with potential for use in bioremediation of contaminated sites is described. Besides the ability to degrade low molecular weight PAHs, isolates were screened for their growth rate on straw-based agar media, their potential to tolerate high concentrations of phenanthrene and their ability to out-grow the cellulolytic fungus Trichoderma harzianum on straw agar plates. Results from simple in vitro tests were correlated with the ability of the different strains to degrade PAHs in sand microcosms. It was found that fungal growth rate on straw-based agar media in the presence of phenanthrene correlated well with the ability of the different fungi to degrade PAHs in sand microcosms. Whereas growth rate on straw-based agar plates per se was indicative of the ability of white-rot fungi to establish in the presence of a competing fungus, it was a poor indicator of the fungus’ ability to degrade PAHs.     

Synthetic dyes decolourisation by white-rot fungi: Development of original microtitre plate method and screening

A microtitre plate-based method was developed for a fast screening of numerous fungal strains for their ability to decolourise textile dyes. In 3 days, this method allowed to estimate significant fungal decolourisation capability by measuring the absorbance decrease on up to ten dyes. More than 325 white-rot fungi (WRF) strains belonging to 76 fungal genera were compared with regards to their capability to decolourise five azo and two anthraquinone dyes as well as the dyes mixture. The most recalcitrant dyes belonged to the azo group. Several new species unstudied in the bioremediation field were found to be able to efficiently decolourise all the dyes tested.     

Fluorometric assay of erythrocyte protoporphyrin: simple screening test for lead poisoning and iron deficiency.

Erythrocyte protoporphyrin levels are high in lead poisoning, iron deficiency and erythropoietic porphyria. On-site fluorometric assay was used to screen for raised blood levels in three groups of children in one city: 166 who were severely mentally retarded and lived in an institution, 88 who were moderately to severely mentally retarded and attending special schools but lived at home, and 128 who were of normal intelligence and attended a regular school. High erythrocyte protoporphyrin levels (40 micrograms/dl [0.7 mumol/l] or greater) were found in 14 of the children, each of whom was tested for further evidence of lead poisoning and iron deficiency. The two children found to have high blood lead levels (above 30 micrograms/dl [1.5 mumol/l]) were both living in the institution, were ambulatory and had pica. Of the other 12 children 8 had evidence of iron deficiency, though in 4 the probability of a true deficiency was low. The fluorometric assay of erythrocyte protoporphyrin may prove to be a simple method of screening for lead poisoning and iron deficiency.     

Production of fructooligosaccharides by mycelium-bound transfructosylation activity present in Cladosporium cladosporioides and Penicilium sizovae

Different filamentous fungi isolated from molasses and jams (kiwi and fig) were screened for fructooligosaccharides (FOS) producing activity. Two strains, identified as Penicilium sizovae (CK1) and Cladosporium cladosporioides (CF₂15), were selected on the basis of the FOS yield and kestose/nystose ratio. In both strains the activity was mostly mycelium-bound. Starting from 600 g/L of sucrose, maximum FOS yield was 184 and 339 g/L for P. sizovae and C. cladosporioides, respectively. Interestingly, the highest FOS concentration with C. cladosporioides was reached at 93% sucrose conversion, which indicated a notable transglycosylation to hydrolysis ratio. The main FOS in the reaction mixtures were identified by HPAEC–PAD chromatography. C. cladosporioides synthesized mainly 1-kestose (158 g/L), nystose (97 g/L), ¹F-fructosylnystose (19 g/L), 6-kestose (12 g/L), neokestose (10 g/L) and a disaccharide (34 g/L) that after its purification and NMR analysis was identified as blastose [Fru-β(2 → 6)-Glc]. P. sizovae was very selective for the formation of ¹F-FOS (in particular 1-kestose) with minor contribution of neoFOS and negligible of levan-type FOS.     

A simple assay with dyed substrates to quantify cellulase and hemicellulase activity of fungi

Summary A simple assay is described for the quantitative measurement of cellulase and hemicellulase activities. It is based on the enzymatic release of stained cleavage products originating from dyed substrates such as different celluloses, mannan and xylan, diffusing from an upper into a lower agar layer. The amount of cleavage products is assessed by direct absorbance measurement in the agar tubes employing an ordinary filter photometer. The method proved to be applicable to various species of basidiomycete fungi.     

A rapid,sensitive, simple plate assay for detection of microbial alginate lyase activity

Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2–3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also worked well for screening and identification of alginate lyase producers and non-producers from environmental samples on common laboratory media. They did this by clearly showing the presence or absence of clearance zones around the microbial colonies grown. This new method is rapid, efficient, and could easily be performed for screening a large number of microbial cultures. This is the first report on the use of Gram's iodine for the detection of alginate lyase production by microorganisms using plate assay.     

A simple and rapid method for the preparation of a cell-free extract with CCAAT-binding activity from filamentous fungi

A simple and rapid method for the preparation of a cell-free extract with the CCAAT-binding activity was established with Aspergillus nidulans as a model fungus. Proteins were extracted with 6 M guanidine hydrochloride directly from mycelia and renatured by dialysis. This method was found applicable to other filamentous fungi such as Aspergillus oryzae and Trichoderma viride.     

A sensitive diffusion plate assay for screening inhibitors of protease activity in plant cell fractions

Proteolytic activity was detected, using a sensitive radial diffusion plate assay, in the plasma membrane fractions of corn (Zea mays L.) roots and from roots of several other plant species. The proteases could be effectively inhibited in corn with phenylmethane sulfonyl fluoride or chymostatin. Protease activity of oat roots, however, was not significantly reduced by these inhibitors. The results of diffusion plate assay were confirmed with the less sensitive azocasein assay using crude cell homogenates. Chymostatin and phenylmethane sulfonyl fluoride were effective in preventing protease degradation of polypeptides as revealed by electrophoresis. The diffusion plate assay uses a permanent support for a 0.75 millimeter thick agarose slab containing 200 micrograms per milliliter casein. By staining the fixed and dried gel with Coomassie blue R-250, proteolytic activity was visualized as a cleared area around the sample well with a detection limit of about 0.3 nanograms trypsin. The diffusion plate assay should prove useful for screening inhibitors of proteases where limited amounts of material are available, such as with plant cell fractions or highly purified proteins.     

Bacterial test system for the primary screening of substances with potential antitumor activity

A fundamental possibility of antitumor activity testing in the bacterial system represented by Escherichia coli of the wild type and its MS2-induced mutant has been shown. The initial bacterial strain is an indicator of toxic properties of tested substances and the mutant strain is a specific test culture modeling a tumor cell. The comparison of the data described for eukaryotes with the data obtained using the proposed bacterial test system confirms an adequate response of both strains to substances with proved antitumor properties. The data obtained are considered as very promising for the further improvement of this test system towards its application for primary screening of antitumor substances.     

Primary simple assays of cellulose-degrading fungi

Some 25 fungi, including at least 14 basidiomycetes, one ascomycete, and five anamorphic fungi were evaluated for their cellulose-degrading abilities in Difco potato dextrose broth or Difco malt extract broth cultures with cellulosic substrates (e.g., filter paper) in plastic Petri dishes. Among them, Peniophora sp. 06-13 and Phlebia sp. 99-335 reduced the dry weights of the whole cultures with these substrates more than the dry weights of the respective original substrates after 30 days of culture, showing definite cellulose degradation. In the cultures with more than 10 test fungi including Pycnoporus coccineus 84-117, such weight losses did not occur. This assay technique for the primary screening for cellulose degrading fungi is simple, inexpensive, reproducible and accurate.