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1.
Appl Microbiol Biotechnol ; 74(3): 609-16, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17139509

ABSTRACT

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man-Rogosa-Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.


Subject(s)
Bacterial Proteins/biosynthesis , Gene Expression , Lacticaseibacillus casei/genetics , Lactococcus lactis/enzymology , Organic Anion Transporters/biosynthesis , Acetoin/metabolism , Acetoin Dehydrogenase/analysis , Bacterial Proteins/genetics , Citric Acid/metabolism , Cloning, Molecular , Culture Media , DNA Helicases/genetics , Diacetyl/metabolism , Genetic Vectors , Hydrogen-Ion Concentration , Lacticaseibacillus casei/metabolism , Lactococcus lactis/genetics , Methyltransferases/genetics , Organic Anion Transporters/genetics , Plasmids/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
2.
Anal Biochem ; 293(2): 157-68, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11399028

ABSTRACT

Several forms of diacetyl-reducing enzyme were found to exist in the human liver cytosol. Three (DAR-2, DAR-5, and DAR-7) of them were purified as a single band on SDS-PAGE by a combination of a few kinds of column chromatographies. The in-gel tryptic digests of the purified enzymes were analyzed by nano-liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS), which provided peptide masses at a ppm-level accuracy. The enzymes, DAR-2, DAR-5, and DAR-7, were identified as alcohol dehydrogenase beta subunit (ADH2), carbonyl reductase (CBR1), and aldehyde reductase (AKR1A1), respectively, by peptide mass fingerprinting. In addition, an alternating-scan acquisition of nano-LC/FT ICR mass spectra, i.e., switching of normal acquisition conditions and in-source fragmentation conditions scan by scan, provided sets of parent and fragment ion masses of many of the tryptic peptides in a single LC/MS run. The peptide sequence-tag information at the ppm-level accuracy was used to further confirm the protein identities. It was demonstrated that nano-LC/FT ICR MS can be used for rigorous protein identification at a subpicomole level as an alternative technique to nano-LC/MS/MS.


Subject(s)
Acetoin Dehydrogenase/analysis , Acetoin Dehydrogenase/chemistry , Fourier Analysis , Liver/enzymology , Mass Spectrometry/methods , Acetoin Dehydrogenase/isolation & purification , Amino Acid Sequence , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Databases as Topic , Humans , Mass Spectrometry/instrumentation , Molecular Sequence Data , Peptide Mapping , Spectrometry, Mass, Electrospray Ionization
3.
J Biochem ; 98(5): 1349-57, 1985 Nov.
Article in English | MEDLINE | ID: mdl-3910648

ABSTRACT

Kinetic and physicochemical properties of hamster liver diacetyl reductase have been examined. The results of kinetic studies on the reduction of diacetyl and NADPH to acetoin and NADP+ suggest that the reaction follows an Ordered Bi Bi mechanism in which NADPH binds first before diacetyl. The enzyme is a tetrameric glycoprotein of single subunits of a molecular weight of 23,500 with a sedimentation coefficient of 6.0S. The enzyme does not contain Zn, Cu, or Fe. The amino acid composition revealed an unusually low proportion of proline residues (0.9%). p-Chloromercuriphenylsulfonate and phenylglyoxal inactivated the enzyme, but the presence of NADPH prevented the loss of activity due to thiol and arginine modification. The enzyme transferred the pro 4S hydrogen atom of NADPH to the substrate and the binding of the enzyme to NADPH resulted in a red shift of the ultraviolet absorption spectrum of the cofactor.


Subject(s)
Acetoin Dehydrogenase/analysis , Alcohol Oxidoreductases/analysis , Liver/enzymology , Acetoin Dehydrogenase/antagonists & inhibitors , Acetoin Dehydrogenase/metabolism , Amino Acids/analysis , Animals , Centrifugation, Density Gradient , Cricetinae , Electrophoresis, Disc , Enzyme Reactivators/pharmacology , Kinetics , Male , Metals/analysis , NADP/analysis , Protein Denaturation , Proteins/analysis , Spectrophotometry, Ultraviolet
5.
Biol Bull Acad Sci USSR ; 6(3): 356-61, 1979.
Article in English | MEDLINE | ID: mdl-45079

ABSTRACT

A highly active preparation of diacetyl(acetoin) reductase was isolated from cell-free extracts of the yeast Saccharomyces vini. Since the activity ratio of 2,3-butanediol dehydrogenase and diacetyl(acetoin) reductase was practically unchanged in the process of 65-fold purification, it can be assumed that the yeast cells contain one enzyme, which catalyzes both the reversible oxidation of 2,3-butanediol to acetoin by NAD and the practically irreversible reduction of diacetyl to acetoin by NAD-H2. Some properties of this enzyme were studied.


Subject(s)
Acetoin Dehydrogenase/analysis , Alcohol Oxidoreductases/analysis , Saccharomyces/enzymology , Acetoin Dehydrogenase/isolation & purification , Butylene Glycols/metabolism , Chromatography, Ion Exchange , Hydrogen-Ion Concentration , Isoelectric Focusing , Protein Conformation , Temperature
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