Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
1.
Prikl Biokhim Mikrobiol ; 50(1): 39-43, 2014.
Article in English | MEDLINE | ID: mdl-25272750

ABSTRACT

The nicosulfuron-degrading enzymes from Bacillus subtilis strain YB1 were purified and their genes were cloned. The proteins of bacterial culture filtrate were precipitated with ammonium sulfate or acetone. The extracellular proteins concentrated by acetone were purified from DEAE-Sepharose Fast Flow chromatography. The four protein peaks eluted from DEAE-column were separated and purified by native PAGE. Three components (P1-1, P3-2, P4-3) had nicosulfuron-degrading activity, and component P4-3 degradated 57.5% of this compound. The molecular weights of the components were 33.5, 54.8 and 37.0 kDa, respectively. The amino acid sequences of nicosulfuron-degrading enzymes from B. subtilis YB1 were determined by MALDI-TOF-MS, indicating these enzymes as manganese ABC transporter, vegetative catalase 1 and acetoin dehydrogenase E1, respectively. Using PCR amplification, genes 918, 1428, 1026 bp in size were detected for the enzymes studied.


Subject(s)
ATP-Binding Cassette Transporters/isolation & purification , Acetoin Dehydrogenase/isolation & purification , Bacillus subtilis/chemistry , Bacterial Proteins/isolation & purification , Catalase/isolation & purification , Pyridines/chemistry , Sulfonylurea Compounds/chemistry , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , Acetoin Dehydrogenase/chemistry , Acetoin Dehydrogenase/genetics , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biodegradation, Environmental , Catalase/chemistry , Catalase/genetics , Chromatography, Ion Exchange , Cloning, Molecular , Environmental Pollutants/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Herbicides/chemistry , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
2.
Appl Microbiol Biotechnol ; 98(2): 641-50, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23568047

ABSTRACT

Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K(m) values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD⁺, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.


Subject(s)
Acetoin Dehydrogenase/metabolism , Butylene Glycols/metabolism , Rhodococcus/enzymology , Acetoin Dehydrogenase/chemistry , Acetoin Dehydrogenase/genetics , Acetoin Dehydrogenase/isolation & purification , Chromatography, Affinity , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enzyme Stability , Escherichia coli/genetics , Gene Expression , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Molecular Weight , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Rhodococcus/genetics , Sequence Analysis, DNA , Stereoisomerism , Substrate Specificity , Temperature
3.
Biochim Biophys Acta ; 1760(11): 1636-45, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17030441

ABSTRACT

An alpha,beta-dicarbonyl reductase activity was purified from Saccharomyces cerevisiae and identified as the cytosolic enzyme D-Arabinose dehydrogenase (ARA1) by MALDI-TOF/TOF. Size exclusion chromatography analysis of recombinant Ara1p revealed that this protein formed a homodimer. Ara1p catalyzed the reduction of the reactive alpha,beta-dicarbonyl compounds methylglyoxal, diacetyl, and pentanedione in a NADPH dependant manner. Ara1p had apparent Km values of approximately 14 mM, 7 mM and 4 mM for methylglyoxal, diacetyl and pentanedione respectively, with corresponding turnover rates of 4.4, 6.9 and 5.9 s(-1) at pH 7.0. pH profiling showed that Ara1p had a pH optimum of 4.5 for the diacetyl reduction reaction. Ara1p also catalyzed the NADP+ dependant oxidation of acetoin; however this back reaction only occurred at alkaline pH values. That Ara1p was important for degradation of alpha,beta-dicarbonyl substrates was further supported by the observation that ara1-Delta knockout yeast mutants exhibited a decreased growth rate phenotype in media containing diacetyl.


Subject(s)
Oxidoreductases Acting on Aldehyde or Oxo Group Donors/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Sugar Alcohol Dehydrogenases/chemistry , Acetoin Dehydrogenase/chemistry , Acetoin Dehydrogenase/isolation & purification , Amino Acid Sequence , Diacetyl/chemistry , Diacetyl/metabolism , Kinetics , Mass Spectrometry , Molecular Sequence Data , Oxidation-Reduction , Oxidoreductases Acting on Aldehyde or Oxo Group Donors/genetics , Oxidoreductases Acting on Aldehyde or Oxo Group Donors/metabolism , Pyruvaldehyde/chemistry , Pyruvaldehyde/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Sugar Alcohol Dehydrogenases/genetics , Sugar Alcohol Dehydrogenases/metabolism
4.
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
5.
FEBS Lett ; 457(3): 298-301, 1999 Sep 03.
Article in English | MEDLINE | ID: mdl-10471796

ABSTRACT

With the publication of the three-dimensional structures of several thiamin diphosphate-dependent enzymes, the chemical mechanism of their non-oxidative and oxidative decarboxylation reactions is better understood. Chemical models for these reactions serve a useful purpose to help evaluate the additional catalytic rate acceleration provided by the protein component. The ability to generate, and spectroscopically observe, the two key zwitterionic intermediates invoked in such reactions allowed progress to be made in elucidating the rates and mechanisms of the elementary steps leading to and from these intermediates. The need remains to develop chemical models, which accurately reflect the enzyme-bound conformation of this coenzyme.


Subject(s)
Acetoin Dehydrogenase/metabolism , Coenzymes/chemistry , Flavins/metabolism , Thiamine Pyrophosphate/metabolism , Thioctic Acid/metabolism , Acetoin Dehydrogenase/chemistry , Acetyl Coenzyme A/chemistry , Acetyl Coenzyme A/metabolism , Amines/chemistry , Amines/metabolism , Coenzymes/metabolism , Decarboxylation , Oxidation-Reduction , Protons , Pyruvate Decarboxylase/chemistry , Pyruvate Decarboxylase/metabolism , Pyruvate Oxidase/chemistry , Pyruvate Oxidase/metabolism , Thiamine Pyrophosphate/chemistry
6.
Antonie Van Leeuwenhoek ; 64(1): 9-15, 1993.
Article in English | MEDLINE | ID: mdl-8274006

ABSTRACT

In Clostridium magnum strain Wo Bd P1 the formation of the enzyme components of the acetoin dehydrogenase enzyme system E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase Ao:DCPIP OR), E2 (dihydrolipoamide acetyltransferase DHLTA) and E3 (dihydrolipoamide dehydrogenase DHLDH) were induced during growth on acetoin. Ao:DCPIP OR was purified from acetoin-grown cells in two steps by chromatography on DEAE-Sephacel and on Mono Q HR. Native Ao:DCPIP OR exhibited a M(r) of 138,000; it consisted of two different subunits of M(r) alpha 38,500 and M(r) beta 34,000, and it occurred most probably in a tetrameric alpha 2 beta 2 structure. The N-terminal amino acid sequences of the alpha- and beta-subunits revealed homologies to the N-termini of the corresponding subunits of Ao:DCPIP OR from Pelobacter carbinolicus and from Alcaligenes eutrophus; furthermore, the N-terminus of the beta-subunit exhibited homologies to the N-termini of beta-subunits from different 2-oxo acid dehydrogenases.


Subject(s)
Acetoin Dehydrogenase/isolation & purification , Clostridium/enzymology , 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) , Acetoin/metabolism , Acetoin Dehydrogenase/chemistry , Acetoin Dehydrogenase/metabolism , Acyltransferases/isolation & purification , Alcaligenes/enzymology , Amino Acid Sequence , Chromatography, Ion Exchange , Culture Media , Dihydrolipoamide Dehydrogenase/isolation & purification , Electrophoresis, Polyacrylamide Gel , Glucose/metabolism , Gram-Negative Anaerobic Bacteria/enzymology , Ketone Oxidoreductases , Molecular Sequence Data , Molecular Weight , Multienzyme Complexes/chemistry , Oxidoreductases/chemistry , Sequence Alignment
SELECTION OF CITATIONS
SEARCH DETAIL
...