scholarly journals Conversion of a Thiol Precursor into Aroma Compound 4-mercapto-4-methyl-2-pentanone Using Microbial Cell Extracts

Fermentation ◽  
2021 ◽  
Vol 7 (3) ◽  
pp. 129
Author(s):  
Hao-Kai Li ◽  
Chi-Fong Chang ◽  
Hsuan-Ju Lin ◽  
Jung-Lee Lin ◽  
Yu-Ting Lee ◽  
...  
Keyword(s):  
Colorimetric Method ◽  
Cell Extract ◽  
Microbial Cell ◽  
Aroma Compound ◽  
Resonance Spectroscopy ◽  
Black Currant ◽  
Crude Cell Extract ◽  
Cell Extracts ◽  
Lyase Activity ◽  
Environmentally Friendly Approach

4-Mercapto-4-methyl-2-pentanone (4MMP), a high-impact aroma compound with the box tree and black currant flavors was first identified in wines and could be released by microbial cysteine-S-conjugate β-lyases from its precursors. In this study, various yeasts and bacteria encoding β-lyases were selected to examine their β-lyase activities. A thiol precursor of 4MMP, cysteine-conjugate of 4MMP (cys-4MMP), was synthesized with a purity of >95% in a relatively environmentally friendly approach, and its chemical structure was confirmed by nuclear magnetic resonance spectroscopy. The β-lyase activities of the crude cell extract from the bacteria and yeast strains for different substrates were examined using a colorimetric method. Shewanella putrefaciens cell extract exhibited the highest β-lyase activity for all tested substrates. Additionally, the optimum pH and temperature for their β-lyase activities were determined. To monitor the conversion efficiency of precursor cys-4MMP to 4MMP, liquid chromatography-mass spectrometry was used. Our data indicate that selected bacteria and yeasts could convert cys-4MMP into 4MMP, and S. putrefaciens exhibited the best conversion yield. This study demonstrated the potential use of microbial cell extracts to produce sulfur-containing aroma compounds such as 4MMP.

scholarly journals An enzyme degrading reduced nicotinamide-adenine dinucleotide in Proteus vulgaris

1978 ◽  
Vol 175 (2) ◽  
pp. 669-674 ◽  
Author(s):  
R Davies ◽  
H K King
Keyword(s):  
Rapid Heating ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Cell Extract ◽  
Maximum Activity ◽  
Proteus Vulgaris ◽  
Crude Cell Extract ◽  
Major Band ◽  
Cell Extracts ◽  
Reduced Nicotinamide Adenine Dinucleotide

Cell-free extracts of a strain of Proteus vulgaris degrade NADH to reduced nicotinamide riboside, adenosine and two molecules of phosphate. The system is weakly active in fresh cell extracts, but activity is increased about 10-fold on rapid heating to 70-100 degrees C. On returning to room temperature, the activity returns rapidly to its initial low value but can be re-activated by again heating to 70-100 degrees C. Reversible activation can also be effected by extremes of pH or by teatment with 8M-urea. Activation appears to be due to reversible changes in conformation of the protein of the enzyme rather than to combination of the enzyme with a heat-labile inhibitor. The active form can be stabilized by addition of PPi. The system, which also possesses 5′-nucleotidase activity not separable from the NADH pyrophosphatase, requires Co2+ (0.4mM) for maximum activity. Although activated at relatively high temperatures, it is not enzymically active until cooled to 50-60 degrees C. It may be purified by affinity chromatography (with NAD+ as ligand) to an activity over 400 times that of the crude cell extract, and yields only one major band on polyacrylamide-gel electrophoresis.

Disulfiram Determination by Proton Magnetic Resonance Spectroscopy and by Colorimetry

1973 ◽  
Vol 56 (1) ◽  
pp. 124-127 ◽  
Author(s):  
Eric B Sheinin ◽  
Walter R Benson ◽  
Myron M Smith
Keyword(s):  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
Proton Magnetic Resonance ◽  
Proton Magnetic Resonance Spectroscopy ◽  
Colorimetric Method ◽  
Drug Substance ◽  
Resonance Spectroscopy ◽  
Pmr Spectroscopy ◽  
Label Claim ◽  
Tablet Excipients

Abstract Disulfiram was determined in disulfiram drug substance and tablets by proton magnetic resonance (PMR) spectroscopy at the 100–480 mg level and by a colorimetric technique involving cuprous iodide at the 50 mg level. The tablet excipients do not interfere in the analysis. The average result for disulfiram in a tablet composite was 100.8±1.4% of label claim by PMR and 100.7±0.4% by the colorimetric method.

scholarly journals Effect of Polyphenols on 3-Hydroxy-3-methylglutaryl-Coenzyme A Lyase Activity in Human Hepatoma HepG2 Cell Extracts

2013 ◽  
Vol 36 (12) ◽  
pp. 1902-1906 ◽  
Author(s):  
Saori Nakagawa ◽  
Yuko Kojima ◽  
Koichi Sekino ◽  
Susumu Yamato
Keyword(s):  
Hepg2 Cell ◽  
Coenzyme A ◽  
Human Hepatoma ◽  
Cell Extracts ◽  
Lyase Activity ◽  
Hepatoma Hepg2

scholarly journals Rapid, selective and stable HaloTag-LbADH immobilization directly from crude cell extract for the continuous biocatalytic production of chiral alcohols and epoxides

2018 ◽  
Vol 3 (1) ◽  
pp. 8-12 ◽  
Author(s):  
J. Döbber ◽  
M. Pohl ◽  
S. V. Ley ◽  
B. Musio
Keyword(s):  
Cell Extract ◽  
Chiral Alcohols ◽  
Crude Cell Extract ◽  
Biocatalytic Production

A strategy for biocatalyst immobilization in flow directly from the crude cell extract is described.

scholarly journals The Saccharomyces cerevisiae ICL2 Gene Encodes a Mitochondrial 2-Methylisocitrate Lyase Involved in Propionyl-Coenzyme A Metabolism

2000 ◽  
Vol 182 (24) ◽  
pp. 7007-7013 ◽  
Author(s):  
Marijke A. H. Luttik ◽  
Peter Kötter ◽  
Florian A. Salomons ◽  
Ida J. van der Klei ◽  
Johannes P. van Dijken ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Source ◽  
Coenzyme A ◽  
Isocitrate Lyase ◽  
Significant Activity ◽  
Mrna Levels ◽  
Wild Type ◽  
Cell Extracts ◽  
Lyase Activity ◽  
Null Mutants

ABSTRACT The Saccharomyces cerevisiae ICL1 gene encodes isocitrate lyase, an essential enzyme for growth on ethanol and acetate. Previous studies have demonstrated that the highly homologousICL2 gene (YPR006c) is transcribed during the growth of wild-type cells on ethanol. However, even when multiple copies are introduced, ICL2 cannot complement the growth defect oficl1 null mutants. It has therefore been suggested thatICL2 encodes a nonsense mRNA or nonfunctional protein. In the methylcitrate cycle of propionyl-coenzyme A metabolism, 2-methylisocitrate is converted to succinate and pyruvate, a reaction similar to that catalyzed by isocitrate lyase. To investigate whetherICL2 encodes a specific 2-methylisocitrate lyase, isocitrate lyase and 2-methylisocitrate lyase activities were assayed in cell extracts of wild-type S. cerevisiae and of isogenicicl1, icl2, and icl1 icl2 null mutants. Isocitrate lyase activity was absent in icl1 andicl1 icl2 null mutants, whereas in contrast, 2-methylisocitrate lyase activity was detected in the wild type and single icl mutants but not in the icl1 icl2mutant. This demonstrated that ICL2 encodes a specific 2-methylisocitrate lyase and that the ICL1-encoded isocitrate lyase exhibits a low but significant activity with 2-methylisocitrate. Subcellular fractionation studies and experiments with an ICL2-green fluorescent protein fusion demonstrated that theICL2-encoded 2-methylisocitrate lyase is located in the mitochondrial matrix. Similar to that of ICL1, transcription of ICL2 is subject to glucose catabolite repression. In glucose-limited cultures, growth with threonine as a nitrogen source resulted in a ca. threefold induction ofICL2 mRNA levels and of 2-methylisocitrate lyase activity in cell extracts relative to cultures grown with ammonia as the nitrogen source. This is consistent with an involvement of the 2-methylcitrate cycle in threonine catabolism.

scholarly journals Accumulation of α-Keto Acids as Essential Components in Cyanide Assimilation by Pseudomonas fluorescens NCIMB 11764

1998 ◽  
Vol 64 (11) ◽  
pp. 4452-4459 ◽  
Author(s):  
Daniel A. Kunz ◽  
Jui-Lin Chen ◽  
Guangliang Pan
Keyword(s):  
Pseudomonas Fluorescens ◽  
Culture Fluid ◽  
Maximal Activity ◽  
Resonance Spectroscopy ◽  
Keto Acid ◽  
Growth Substrate ◽  
Reaction Products ◽  
Cell Extracts ◽  
Keto Acids ◽  
Essential Components

ABSTRACT Pyruvate (Pyr) and α-ketoglutarate (αKg) accumulated when cells of Pseudomonas fluorescens NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L. Chen and D. A. Kunz, FEMS Microbiol. Lett. 156:61–67, 1997). The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 μmol of cyanide removed min−1 ml of culture fluid−1) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM. The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by 13C nuclear magnetic resonance spectroscopy. Both the Pyr and α-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates. Radiotracer experiments showed that CO2 (and NH3) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct. Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate. Pyr and αKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate. The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role.

scholarly journals Detection of Antinuclear Antibodies by Use of an Enzyme Immunoassay with Nuclear HEp-2 Cell Extract and Recombinant Antigens: Comparison with Immunofluorescence Assay in 307 Patients

2001 ◽  
Vol 47 (9) ◽  
pp. 1649-1659 ◽  
Author(s):  
Nobuhide Hayashi ◽  
Tomoko Kawamoto ◽  
Masahiko Mukai ◽  
Akio Morinobu ◽  
Masahiro Koshiba ◽  
...  
Keyword(s):  
Connective Tissue ◽  
Enzyme Immunoassay ◽  
Sensitivity And Specificity ◽  
Lupus Erythematosus ◽  
Antinuclear Antibodies ◽  
Connective Tissue Diseases ◽  
Cell Extract ◽  
Healthy Individuals ◽  
Cell Extracts ◽  
Disease Specific

Abstract Background: A new enzyme immunoassay (EIA) for automated detection of antinuclear antibodies (ANAs) uses a mixture of HEp-2 cell extracts and multiple recombinant nuclear antigens immobilized on beads. We compared this EIA and an immunofluorescence (IF) assay in a large group of patients and controls. Methods: We studied 492 healthy individuals and 307 patients with connective tissue diseases (CTDs). Sera were tested by an automated EIA (COBAS® Core HEp2 ANA EIA; Roche Diagnostics) and IF. Samples were also tested for eight disease-specific antibodies, including antibodies against U1RNP, Sm, SSA/Ro, SSB/La, Scl-70, Jo-1, dsDNA, and centromere. Results: Areas under ROC curves for the EIA were greater than (P = 0.008–0.012) or numerically identical to areas for the IF method for each of six CTDs studied. ROC areas for EIA were 0.98 (95% confidence interval, 0.95–0.99), 0.99 (0.96–1.00), and 0.99 (0.98–1.00) in systemic lupus erythematosus (n = 111), systemic sclerosis (n = 39), and mixed connective tissue disease (n = 33), respectively. For all 258 CTD patients with conditions other than rheumatoid arthritis (RA), the sensitivity and specificity of the IF method at a cutoff dilution of 1:40 were 92% and 65%, respectively, vs 93% and 79% for the EIA at a cutoff of 0.6. For the IF method at a cutoff dilution of 1:160, sensitivity and specificity were 81% and 87%, respectively, vs 84% and 94%, respectively, for the EIA at a cutoff of 0.9. For 207 sera containing at least one of eight disease-specific ANAs, positivities for the EIA and the IF method were 97.1% and 97.6%, respectively, at cutoffs of 0.6 and 1:40 (P = 0.76). Conclusions: An EIA that can be performed by a fully automated instrument distinguishes CTDs (except RA) from healthy individuals with both higher sensitivity and specificity than the IF method when the cutoff index was set at 0.9. Moreover, it can be used to exclude the presence of disease-specific ANAs by setting the cutoff index at 0.6 with almost the same efficacy as the IF method.

scholarly journals STUDIES ON OXIDATION AND REDUCTION BY PNEUMOCOCCUS

1924 ◽  
Vol 39 (5) ◽  
pp. 745-755 ◽  
Author(s):  
Oswald T. Avery ◽  
James M. Neill
Keyword(s):  
Hydrogen Peroxide ◽  
Molecular Oxygen ◽  
Hemolytic Activity ◽  
Active Agent ◽  
Organic Peroxide ◽  
Cell Extract ◽  
The Other ◽  
Cell Extracts ◽  
Other Hand ◽  
The Stability

In the present work on oxidation and reduction by sterile extracts of pneumococcus, the preparations employed contain among other constituents, a hemolytic substance the properties of which have been described by Cole (1, 2) in his studies on pneumococcus hemotoxin. Pneumococcus extracts prepared by the methods described are actively hemolytic, 0.005 cc. of extract causing complete lysis of 2.5 cc. of a 1 per cent suspension of red cells from rabbit blood. This hemolytic property of pneumococcus extracts is destroyed by 10 minutes exposure to 55°C. When pneumotoxin-containing extracts are protected from the action of molecular oxygen, their hemolytic activity remains unimpaired for considerable periods of time. In the presence of air, on the other hand, the stability of the hemolytic substance depends upon whether the particular type of extract contains a "complete" or "incomplete" oxidation-reduction system. Sterile broth extracts of unwashed pneumococci are reactive with molecular oxygen, and as a result of this union peroxide is formed whenever these extracts are exposed to air. The hemolytic activity of "complete" extracts of this type is rapidly decreased and finally destroyed in the presence of molecular oxygen. On the other hand, the "incomplete" type of extract prepared by saline extraction of washed pneumococci may be exposed to air with little or no loss of hemolytic power. This "incomplete" washed cell extract, unless reactivated, does not undergo autoxidation in the presence of air; under these circumstances peroxide is not formed and the hemolytic activity of this type of extract is not impaired by exposure to air. The stability of the hemolytic agent in the "incomplete" type of extract is evidence that this substance is itself not reactive with or affected by molecular oxygen, even in the presence of the cell enzymes. The destruction of the same hemolytic substance in extracts capable of undergoing autoxidation may be ascribed to the action of some peroxide formed by the union of molecular oxygen with easily oxidized or autoxidizable substances of the extract. It is now known that a peroxide, having the reactions of hydrogen peroxide, accumulates in sterile pneumococcus extracts during oxidation. It has been shown in the present study that the addition of preformed hydrogen peroxide destroys the hemolytic activity of pneumococcus extracts, although higher concentrations were required than were detected in oxidized extracts themselves. These facts and the known action of superoxides in analogous types of reaction make it seem not unlikely that the active agent in the destruction of pneumotoxin in oxidized cell extracts may be a peroxide; either hydrogen peroxide or some higher organic peroxide formed during autoxidation of the extract.

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