Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1991-10-16
1994-03-15
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 37, 435 711, 435189, 435814, 435921, 435930, C12Q 126, C12Q 112, C12N 902, C12R 172
Patent
active
052945391
DESCRIPTION:
BRIEF SUMMARY
The invention relates to NAD(P)H-dependent nitrate reductase from yeasts, to a process for the preparation thereof and to the use thereof in a reagent for determining nitrate.
The quantitative determination of nitrate in a wide variety of samples, for example in water (mineral water, tap water, wastewater), foodstuffs (fruit, vegetables, beer, wine, juices, meat and meat products, milk and dairy products, baby food) and soil samples is becoming increasingly important. Nitrate determinations are extremely important, especially when there is suspicion of excessive concentrations injurious to health, in order to be able to prevent, for example, a potential carcinogenic nitrosamine formation.
Both physical (HPLC, gel chromatography) and chemical methods for determining the nitrate concentration are known. In the customary chemical methods, the nitrate is reduced to nitrite and the latter is detected by a diazotization reaction. These methods are very elaborate and cannot be used without problems for determinations in many organic materials.
EP 244 771 discloses a stabilized nitrate reductase and a reagent for determining nitrate using this nitrate reductase. The reagent contains, besides the enzyme and NAD(P)H, a zwitterionic buffer and a nitrogen heterocycle buffer. However, the enzyme has the disadvantage that its preparation is very elaborate, and that it shows an interfering nitrite reductase activity, which makes the described completely enzymatic nitrate determination difficult. In addition, it has emerged that the nitrate reductase described in EP 244 771 satisfactorily converts nitrate to nitrite only with the relatively costly NADPH, but not with NADH.
The object of the invention was to find and isolate a nitrate reductase which is stable enough, and with which it is possible, to determine nitrate accurately even in low concentrations with a simple, low-cost and rapid test.
It has emerged, surprisingly, that yeasts which are able to grow with nitrate as the sole nitrogen source in a completely synthetic nutrient medium under aerobic conditions have a nitrate reductase which can be isolated in a simple process and which can be stabilized. The nitrate reductase prepared in this way can be used in a nitrate determination which avoids the disadvantages of the known methods.
The invention relates to a NAD(P)H-dependent nitrate reductase which is characterized by a molecular weight in the native state of about 350 000 D and which can be obtained by yeast cells which have been cultivated in a completely synthetic nutrient medium with nitrate as the sole nitrogen source and which contain nitrate reductase of the EC 1.6.6.2 type being disrupted in phosphate buffer, pH 6.5-8.5, the crude extract being chromatographed on an anion exchanger, the fractions containing nitrate reductase being mixed with 10-50 % protein, concentrated by ultrafiltration and dried by fluidized bed granulation.
The invention also relates to a process for the preparation of a NAD(P)H-dependent nitrate reductase, which is characterized in that yeast cells which have been cultivated in a completely synthetic nutrient medium with nitrate as the sole nitrogen source are disrupted in phosphate buffer, pH 6.5-8.5, in the presence of a complexing agent, mercaptoethanol, FAD, potassium hexacyanoferrate and a protease inhibitor, after centrifugation the crude extract is directly chromatographed on an anion exchanger, the fractions containing nitrate reductase are mixed with 10-50 % protein, concentrated by ultrafiltration and dried by fluidized bed granulation, and to a reagent for determining nitrate which is characterized by a content of the nitrate reductase prepared in this way, NAD(P)H and a color reagent for determining nitrite.
Examples of yeasts suitable for the preparation of the NAD(P)H-dependent nitrate reductase according to the invention are those of the genera Candida, Hansenula and Williopsis, preferably the genera Candida and Hansenula and the organisms Candida boidinii (DSM 70 026), Hansenula ciferrii (DSM 70 780), Hansenula satu
REFERENCES:
patent: 5169758 (1992-12-01), Fischer et al.
Chem Abs 105 No. 25 221446p (Dec. 1986) Choudary et al. Abs of Microbiol 1986, 47 (192-193) 135-47.
Chem Abs 85 No. 19 139035p (Nov. 8, 1976) Choudary et al. Abs "Indian Acad. Sci" Sec B 1976 (83) 5 (185-95).
Gromes Reiner
Heinrich Martin
Johannssen Walther
Schwartz Harry
Lilling Herbert J.
Merck Patent Gesellschaft mit beschrankter Haftung
LandOfFree
Nitrate reductase from yeasts, the preparation and use thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nitrate reductase from yeasts, the preparation and use thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nitrate reductase from yeasts, the preparation and use thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1535436