DD268001A1 - QUICK TEST FOR THE DETECTION OF H LOW 2 O LOW 2-FORMAT SUBSTANCE SPECIFIC OXIDASES - Google Patents

Abstract

The invention relates to a rapid test for the detection of H2O2-forming substrate-specific oxidases, which play as new, specific enzymes of increasing importance in the analytical detection of substrates and enzymes in chemical and biotechnological processes, but especially in clinical diagnostics. According to the invention, to culture solutions or cell extracts of various microorganisms, a mixture containing a chromogen, peroxidase and the special substrate of the oxidase is added. When using microtiter plates, the resulting dye is evaluated visually or quantitatively as a rapid test after incubation.

Description

field of use

DS * invention relates to a rapid test for the detection of HjOi-producing, extracellular and intracellular oxidases. These, as new, specific enzymes, are increasingly playing a role in the analytical detection of substrates and enzymes in chemical and biotechnological processes, but above all in clinical diagnostics.

Characteristic of the known state of the art

In the last few years, Η, Ο-producing oxidases are increasingly commercially available for use in analytics. For economic reasons, the isolation of the enzymes takes place increasingly and preferably from microorganisms. The screening for H] O] -forming oxidases turns out to be labor intensive and tedious. By ampe ometric measurement of oxygen consumption or H ₂ O ₂ formation described KAMEI et al. (Chem. Pharm., 31, 3107 (1963)) a screening for an L-glutamate oxidase in which about 500 Actinomycetales strains were examined. For example, analytical methods in the search for HjOj-forming oxides are described in which the quantitative colorimetric test according to EMERSON (J. Org. Chem., 8.41711943) and TRINDER (Ann., Clin., Biochem. 1969)) oxidases can still be demonstrated. However, the disadvantage of the analytical methods lies in their ineffective.

Object of the invention

The object of the invention is to develop a rapid test for the detection of H, O ₂ -blindr oxidases, which is suitable and effective for screening purposes. The test is fast, cheap and suitable for serial screening tests:

Explanation of the essence of the invention

The object of the invention is to develop a rapid analytical test which makes it possible to detect HjOj-producing oxidases, preferably microbial, extracellular or intracellular localized. According to the invention, the object is achieved by giving a chromogen mixture by means of a microtiter test to culture solutions or cell extracts of various microorganisms. When microtiter plates are used, after incubation the dye resulting from the reaction of the oxidase is determined either quantitatively or quantitatively with the aid of an evaluator. The chromogen mixture used contains chromogens, peridase, the special substrate of the oxidase to be detected and a suitable buffer. The relatively simple microtiter test, mainly used for bacteriological and immunological investigations in bacteriological and medical practice and research, allows the analysis of 8 χ 12 samples in 45 s on a single microtiter plate alone. The inventive test for the detection of oxidants is cheap, fast, effective and particularly suitable for serial or screening investigations. The invention is explained below by way of examples.

example 1 Screening for a ml of gross L-glutamate toxase

139 different Streptomyces voice were cultivated in a customary manner in a Submersverfahren, during the course of the

Growth samples were taken for detection of L-glutamate oxidase and centrifuged. During the supernatant

could be used immediately for the detection of extracellular L-glutamate oxidase, which had to be present in the sediment

Biomass to obtain a cell-free extract η I üblicnem procedure be unlocked before. 0.2 ml of a mixture consisting of 1 ml of 0.2% 4-aminoantipyrine, 2 ml of 0.2% was added from culture filtrate and cell-free crude extract. Dimethylaniline, 1 ml of 0.6 mol / L L-glutamate and 5 ml of N / 15 potassium phosphate pH 7.5 with 5 mg peroxidase, 0.1 ml Culture solution or cell extract pipetted with a Multikanalpipetto. After 15-30 min incubation at 28'C shows a violet staining

the presence of an L-Glutama jxldase.

The quantitative evaluation can be carried out in an 8-channel photometer (e.g., Flow Multiscan from FLOW-Laboratories-USA or MTF-10VDE of the Ar! W), whereby 8 parallels can be measured against a Giirid value. It is possible within

45s to measure the 8 x 12 samples of a microtiter plate and print the Absinktionswerte.

Since microorganisms can also otherwise form H! Oi during the fermentation, a control test without Substrate L-glutamate carried. Within a few hours with the help of the microtiter detection system Culture solutions and biomass digests of the 139 microorganisms described above are tested. Example 2 Screening for a microbial alcohol oxydase

46 different methylotrophic yeasts and bacteria were cultured and worked up in usual Weiss submersed.

While the supernatants of the centrifuged samples were immediately subjected to an alcohol oxidase proof test, the Biomass of the sediment subjected to a standard cell disruption and the cell-free extract the alcohol oxidase screening test

subjected. The screening test carried out corresponded to that described in Example 1, except that instead of the substrate L-glutamate used there, 0.5 mol / l of methanol was used. In a single screening step could at a

Sampling all microorganisms to be tested for their ability to form an alcohol oxidase.

Claims (2)

1. rapid test for the detection of H ₂ O ₂ forming, substrate-specific oxidases, characterized in that by a microtiter test to culture solutions or cell extracts of various microorganisms, a chromogen mixture and determines after incubation resulting from the reaction of the oxidase dye visually or quantitatively. 2. rapid test according to claim 1, characterized in that the chromogen mixture contains the special substrate of the detected oxidase, peroxidase, chromogens and a suitable buffer.