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Biomedical subjects

W Hummel

Publications and source records attributed to W Hummel.

At least 19 recordsLinked to original sources
PubMed1997

Screening and characterization of new enzymes for biosensing and analytics.

The development of new or improved analytical methods requires new enzymes. Screening techniques utilizing enrichment cultures and rapid assay methods supported by automated or miniaturized methods are useful tools to detect new enzyme producers. Notably, oxidoreductases are well suited for analytical purposes. The NAD(P)- and oxygen-independent quinoprotein dehydrogenases with a covalently bound redox cofactor can be used advantageously for the development of biosensors. Examples are given of selective enrichment methods used in screening for useful enzyme-producing microorganisms. Enrichment under chemostatic conditions proved to be successful because enzymes with a remarkably high affinity against the analyte could be obtained. This is demonstrated by the screening of a trimethylamine-converting enzyme. The frequently observed high selectivity of these enzymes against the substrate is demonstrated in a few examples. In exploitation of these new oxidoreductases, new analytical methods were developed which are useful for the detection and during monitoring of phenylketonuria (PKU) or maple syrup urine disease (MSUD).

Alcohols↗
PubMed1997

New alcohol dehydrogenases for the synthesis of chiral compounds.

The enantioselective reduction of carbonyl groups is of interest for the production of various chiral compounds such as hydroxy acids, amino acids, hydroxy esters, or alcohols. Such products have high economic value and are most interesting as additives for food and feed or as building blocks for organic synthesis. Enzymatic reactions or biotransformations with whole cells (growing or resting) for this purpose are described. Although conversions with whole cells are advantageous with respect to saving expensive isolation of the desired enzymes, the products often lack high enantiomeric excess and the process results in low time-space-yield. For the synthesis of chiral alcohols, only lab-scale syntheses with commercially available alcohol dehydrogenases have been described yet. However, most of these enzymes are of limited use for technical applications because they lack substrate specificity, stability (yeast ADH) or enantioselectivity (Thermoanaerobium brockii ADH). Furthermore, all enzymes so far described are forming (S)-alcohols. Quite recently, we found and characterized several new bacterial alcohol dehydrogenases, which are suited for the preparation of chiral alcohols as well as for hydroxy esters in technical scale. Remarkably, of all these novel ADHs the (R)-specific enzymes were found in strains of the genus Lactobacillus. Meanwhile, these new enzymes were characterized extensively. Protein data (amino acid sequence, bound cations) confirm that these catalysts are novel enzymes. (R)-specific as well as (S)-specific ADHs accept a broad variety of ketones and ketoesters as substrates. The applicability of alcohol dehydrogenases for chiral syntheses as an example for the technical use of coenzyme-dependent enzymes is demonstrated and discussed in this contribution. In particular NAD-dependent enzymes coupled with the coenzyme regeneration by formate dehydrogenase proved to be economically feasible for the production of fine chemicals.

Alcohol Dehydrogenase↗
PubMed1995

Enzymatic method for determination of branched-chain amino acid aminotransferase activity.

A spectrophotometric assay for the determination of branched-chain L-amino acid aminotransferase activity is described. It is based on the transamination of L-leucine in the presence of 2-oxoglutarate yielding 4-methyl-2-oxopentanoate. The rate of formation of the branched-chain 2-oxo acid is specifically monitored in a coupled enzymatic reaction using NAD(+)-dependent D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei ssp. pseudoplantarum as coupling enzyme by measuring the decrease in NADH absorbance at 334 nm. Optimized assay conditions are provided as evaluated for the (iso)enzyme from rat heart.

Animals↗
PubMed1993

Neonatal screening for maple syrup urine disease by an enzyme-mediated colorimetric method.

A microplate-based, enzyme-mediated, colorimetric method using L-leucine dehydrogenase (EC 1.4.1.9) has been developed for the determination of the combined branched-chain amino acids in plasma and blood-spot specimens. The test exhibits acceptable precision and fits into a new concept according to which the different parameters of neonatal screening programs for metabolic disorders, such as phenylalanine (phenylketonuria), galactose/galactose-1-phosphate (galactosemia) and branched-chain amino acids (maple syrup urine disease) can be measured in the same blood-spot eluate by use of different specific NAD(H)-dependent enzymes.

Amino Acid Oxidoreductases↗
PubMed1992

Determination of (S)- and (R)-2-oxo-3-methylvaleric acid in plasma of patients with maple syrup urine disease.

An enzymatic method for the separate measurement of both chiral 2-oxo-3-methylvaleric acid (OMV) compounds, (S)- and (R)-OMV, by NADH-dependent enantioselective amination using leucine dehydrogenase in the presence of a NADH regenerating system is described. This method allows the quantitative determination of all branched-chain 2-oxo acids, simultaneously. In plasma samples from classical maple syrup urine disease patients under therapy the average (R)-OMV/(S)-OMV ratio was 0.35 and great differences in the transamination equilibria of the diastereomeric branched-chain amino acids L-isoleucine and L-alloisoleucine were demonstrated.

Amino Acid Oxidoreductases↗
PubMed1991

[Collection and processing of ophthalmologic findings with a personal computer].

In a recently presented paper we proved that it is easy possible to store and analyse data form patients in a strabological department by using purchable data base programs and personal computers. The new program, we now use, has the opportunity to show all inputs in a special data field (i.e. the diagnosis field) in alphabetical order on the screen. For standardization in the data file we have created a special record. In all fields all possible and allowed inputs are entered. When entering new data of a patient, the user only has to select and to transfer the data from one field into the other. This has three advantages: 1. Time is saved when entering data, because it is not necessary to encode the data in an alphanumerical code. 2. The presentation of the data on the monitor is easy understandable. 3. It is not possible to enter no standardized data.

Electronic Data Processing↗
PubMed1990

A new approach to the newborn screening for hyperphenylalaninemias: use of L-phenylalanine dehydrogenase and microtiter plates.

We adapted the recently described colorimetric method for the specific determination of phenylalanine to a microplate assay using a NAD(H)-dependent L-phenylalanine dehydrogenase. With respect to sensitivity, analytical recovery and interrun imprecision this method for measuring phenylalanine in eluates of paper-dried blood spots is suitable for routine newborn screening for hyperphenylalaninemias. In contrast to the microbiological Guthrie assay, with the enzymatic method quantitative data may be obtained on the same day, also in the blood of newborns on antibiotic treatment.

Amino Acid Oxidoreductases↗
PubMed1990

PEANUT: computer graphics program to represent atomic displacement parameters.

PEANUT is an easy to use computer graphics program for the visualization and real-time manipulation of the atomic displacement parameters of small molecules. A flexible, dynamic data structure allows the user to compute complicated, nonspherical atomic surfaces and to handle the point group symmetry of the molecules automatically. Pictures with hidden-line removal may be plotted in publication quality on appropriate output devices.

Azepines↗
PubMed1989

A convenient enzymatic method for the determination of 4-methyl-2-oxopentanoate in plasma: comparison with high performance liquid chromatographic analysis.

A simple and rapid spectrophotometric method for the estimation of 4-methyl-2-oxopentanoate in plasma samples by use of NAD+-dependent D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei ssp. pseudoplantarum is described. It is based on the kinetic measurement of the decrease of NADH absorbance at 334 nm. Applicability is demonstrated by comparative measurement of 4-methyl-2-oxopentanoate content in plasma of patients with maple syrup urine disease by the enzymatic and a reversed phase high performance liquid chromatographic method.

Alcohol Oxidoreductases↗
PubMed1989

Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase.

A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis.

Amino Acid Oxidoreductases↗
PubMed1988

Enzymatic determination of L-phenylalanine and phenylpyruvate with L-phenylalanine dehydrogenase.

An enzymatic method is described for the determination of L-phenylalanine or phenylpyruvate using L-phenylalanine dehydrogenase. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine or the reductive amination of the 2-oxoacid, respectively. The stoichiometric coupling of the coenzyme allows a direct spectrophotometric assay of the substrate concentration. The equilibrium of the reaction favors L-phenylalanine formation; however, by measuring initial reaction velocities, the enzyme can be used for L-phenylalanine determination, too. Standard solutions of L-phenylalanine in the range of 10-300 microM and of phenylpyruvate (5-100 microM) show a linearity between the value for dENADH/min and the substrate concentration. Besides phenylalanine, the enzyme can convert tyrosine and methionine, and their oxoacids, respectively. The Km values of these substrates are higher. The influence of tyrosine on the determination of phenylalanine was studied and appeared tolerable for certain applications.

Amino Acid Oxidoreductases↗
PubMed1988

[A computer program for registering all daily accumulated data at a school for vision disorders].

Despite increasing medical use of the computer, many clinics have experienced only limited success (or none at all) in computerized registration of patient data. One important reason for this lies in the complicated user interfaces of most programs. The clinician has no time to familiarize himself with the complicated command sequences involved, while necessary acceptance of the new technology by administrative staff is often lacking. This paper shows that computerized registration and interpretation of patient data are well within the bounds of possibility, given the use of newly-developed user-friendly operating systems and suitable personal computers. A concrete example of computerized registration of data in a remedial center is given: a case with squint symptoms for which 176 parameters can be entered.

Documentation↗
PubMed1986

Characterization of hydantoinase from Pseudomonas fluorescens strain DSM 84.

The hydantoinase (EC 3.5.2.2) from Pseudomonas fluorescens strain DSM 84 was purified either by hydrophobic interaction chromatography on phenyl-Sepharose or by salting out chromatography on Sepharose 4B, gel filtration on Sephacryl S-400, and preparative electrophoresis. Molecular weight values of 230,000 and 60,000 for the native enzyme and each of the four subunits were estimated for the hydantoin hydrolysing activity. The hydantoinase was stable at temperatures up to 40 degrees C but showed an optimal activity at 55 degrees C. The enzyme was markedly inhibited by copper, para-hydroxymercuribenzoate, 8-hydroxyquinoline, and 2,2'-dipyridyl but not by zinc, and poorly by EDTA and o-phenanthroline. The hydantoin-hydrolyzing activity could be reactivated by ferrous ions. Dihydrouracil was the most readily hydrolyzed substrate. The dihydropyrimidinase produced by strain DSM 84 could also hydrolyze 5-substituted hydantions such as isopropylhydantoin (valine derivative) continuously for 10 days in a membrane reactor at a conversion rate of 30%. The only identified end product was N-carbamyl-D-valine.

Amidohydrolases↗
PubMed1985

Improved enzyme screening by automated fast protein liquid chromatography.

The assay for NADH-dependent dehydrogenases in crude extracts is often interfered with non-specific reactions. Therefore a screening for such enzymes is hampered by high blank values. To overcome such problems we chromatographed crude extracts on a fast protein liquid chromatography system during part of an enzyme screening for 2-hydroxyisocaproate dehydrogenases and lactate dehydrogenases. The automated chromatography procedure presented consists of a combination of gel filtration and ion-exchange chromatography. The total time needed to perform one cycle of the two-column purification, including the equilibration and regeneration steps, is about 35 min. The procedure described separates the desired enzyme, 2-hydroxyisocaproate dehydrogenase, totally from any interfering activity such as NADH-oxidase and also from the second enzyme of interest, the lactate dehydrogenase. Besides the elimination of the side reactions the desired enzymes are purified up to 20-fold.

Alcohol Oxidoreductases↗