Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-08-03
2002-04-16
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007100, C435S007400, C435S007720, C435S007900, C435S034000
Reexamination Certificate
active
06372446
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the field of determining fungal biomass in a variety of samples including environmental samples, food products, plant materials, building construction materials, human and animal body samples and industrial fungal cultures.
TECHNICAL BACKGROUND AND PRIOR ART
Fungal species are used in the fermentation of foods and for the production of desired gene products like enzymes and antibiotics. Fungi may also cause spoilage of foods and agricultural products, cause rot in building materials, and may be pathogenic to mammals including humans.
Currently used methods for detecting fungal biomass include extracting fungus specific cell components such as ergosterol (Grant and West, 1986) and phospholipid fatty acids (PLFA) (Frosteg{dot over (a)}rd et al., 1993), selectively staining cellular organelles (Kropp, 1990; U.S. Pat. No. 5,445,946), fluorescently detecting fungal metabolites present in cellular extracts (SU 1,744,114; JP 7,095,898), and immunological detection of structural components of the fungal cell wall (U.S. Pat. No. 5,004,699).
However, all of these methods involve several drawbacks, in particular due to complicated sample preparations including disruption of cells and hyphae and isolation of cell components. In addition, the methods generally involve extended assay periods and besides, they are time consuming, laborious and require specialised technical skills.
There is therefore a need for an improved, simple and rapid method of selectively determining fungal biomass which can be performed without the above drawbacks.
It has now been found that a reproducible correlation exists between certain enzymatic activities which is present in substantially all fungal species, and the amount of fungal biomass and/or the amount of fungal cell components. This discovery has led to the development of a novel method of determining fungal biomass that fulfills the above need for an improved method.
SUMMARY OF THE INVENTION
Accordingly, the present invention pertains in one aspect to a method of selectively detecting a fungal biomass in a sample, comprising detecting the amount, presence or activity of at least one enzyme that is present in substantially all fungal species, the amount or activity of which enzyme is correlated with the amount of fungal biomass present in the sample, the detection being made under conditions where enzymes of non-fungal origin, if present, cannot be detected. In particular, there is provided a method comprising the steps of (i) contacting a sample with a substrate molecule comprising a detectable moiety releasable from said substrate molecule in the presence of a selectively detectable fungal enzymatic activity, and (ii) detecting the released moiety.
In a further aspect, the invention pertains to a product comprising (i) an agent that reacts with an enzyme that is present in substantially all fungal species and the amount or activity of which enzyme is correlated with the amount of fungal biomass present in a sample, the reaction between the enzyme and the agent resulting in a detectable signal and (ii) means for detecting said signal, as a combined system for the detection of a fungal biomass present in the sample.
DETAILED DISCLOSURE OF THE INVENTION
The invention provides, as it mentioned above, a method of selectively detecting a fungal biomass in a sample, comprising detecting the amount, presence or activity of at least one enzyme being present in substantially all fungal species and the amount or activity of which is correlated with the amount of fungal biomass present in the sample.
As used herein, the expression “selectively detecting” indicates that, when a sample is tested in accordance with the invention, the test conditions are selected so as to exclude any detection of non-fungal enzymes or enzymatic activities which could otherwise interfere with a selective assay for fungal enzymes.
In the present context, the expression “fungal biomass” refers to any cellular components of fungal species as they are defined in Henderson's Dictionary of Biological Terms, 10th edition, Longman Scientific & Technical, 1990. Thus, as used herein fungal biomass includes single cells, mycelia, thalli, hyphae and spores of the fungal species mentioned in the above reference book. Fungal species as defined herein include species belonging to the subdivisions Zygomycotina, Ascomycotina, Basidiomycotina and Deuteromycotina.
In one embodiment, the method of the invention is based on the detection in a sample to be tested of the activity of at least one fungal enzyme by contacting the sample with a substrate molecule comprising a detectable moiety releasable from said substrate molecule in the presence of the fungal enzyme followed by detecting the released moiety.
Thus, an assay for detecting a fungal enzymatic activity can be based upon specific cleavage of a substrate molecule into one or more readily detectable moieties. Fluorogenic moieties can be detected with a high sensitivity and the use of substrate molecules comprising fluorescently detectable moieties in conventional assays of enzymatic activities is well characterised and can be used in accordance with the invention. As an example, the high sensitivity of detection of fluorogenic moieties has facilitated the development of assays for the detection of e.g. chitinase produced by bacterial species (McCreath and Gooday, 1992) and &bgr;-glucanase activities in fungal species, including yeast (Claeyssens et al., 1989)
Thus, the present method can include the detection of an enzymatic activity that is associated with the metabolism of cell structural components. As used in this context, the expression “structural components” includes naturally occurring substances which confer rigidity and mechanical strength to fungal cell walls including septae such as e.g. chitin, chitosan, cellulose, glycogen, glucan, polygalactosamine and polypeptides.
Fungal enzymatic activities which can be used in the present method include those naturally produced in a fungal biomass to be detected. Alternatively, a detectable fungal enzymatic activity in a given fungal biomass can be expressed from a gene inserted by genetic recombination.
In accordance with the invention, detectable enzymatic activities are preferably activities that are expressed constitutively, expressed in all growth phases of the fungal biomass and/or expressed independently of the physiological state of the fungal biomass. The enzymatic activities can be cell associated and/or extracellular.
In other embodiments, the method is based on a detectable enzymatic activity which is expressed in both the presence and absence of biologically cleavable polymers present in a fungal cell wall, such as e.g. chitin, glucans and polypeptides. In yet another embodiment, the detectable enzymatic activity is expressed in both the presence and absence of biologically cleavable polymers such as polysaccharides e.g. including cellulose, hemicellulose, amylose, amylopectin, mannan, xanthan, xylan, arabinan and galactan or is a fungal enzymatic activity that degrades any of such polysaccharides or polypeptides. A presently preferred embodiment of the method according to the invention is based on the detection in a sample of an enzymatic activity selected from an enzyme hydrolysing &bgr;-(1-4) bonds between N-acetyl-hexosaminide groups and an enzyme synthesising such bonds. In this connection, &bgr;-(1-4) bond hydrolysing enzymes include any fungal enzymatic activity that hydrolyses such bonds in polymers and glycosylated proteins containing N-acetylglucosaminide groups, such as the polysaccharides chitin and/or chitosan which are present in substantially all fungal species.
Accordingly, the present method can be based upon detection of a fungal enzymatic activity associated with chitin metabolism, in particular a chitinase (E.C. 3.2.1.14), a &bgr;-N-acetylhexosaminidase (E.C. 3.2.1.52) and/or a chitin synthase (E.C. 2.4.1.16). Alternatively, the method is based on an enzymatic activity that is associated with the metabolism of
Miller Morten
Reeslev Morten
Graser Jennifer E.
Hunton & Williams
Mycometer ApS
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