scholarly journals Two benzaldehyde dehydrogenases in bacterium N.C.I.B. 8250. Distinguishing properties and regulation

1972 ◽  
Vol 130 (4) ◽  
pp. 927-935 ◽  
Author(s):  
A. Livingstone ◽  
C. A. Fewson ◽  
S. I. T. Kennedy ◽  
L. J. Zatman
Keyword(s):  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Gel Filtration ◽  
Wild Type ◽  
Thermal Stabilities ◽  
Molecular Weights ◽  
Ph Values ◽  
Heat Stable ◽  
Benzaldehyde Dehydrogenase ◽  
Wild Type Bacterium

Evidence is presented for the existence in bacterium N.C.I.B. 8250 of two inducible NAD+-linked benzaldehyde dehydrogenases. They may be distinguished in crude extracts by their different thermal stabilities at high pH values, benzaldehyde dehydrogenase I being much more heat-stable than benzaldehyde dehydrogenase II. Only benzaldehyde dehydrogenase I is activated by K+ and certain other univalent cations. Gel-filtration experiments indicate that both enzymes have molecular weights of about 180000. Both enzymes are induced by growth on l-mandelate or phenylglyoxylate; only benzaldehyde dehydrogenase I is gratuitously induced by thiophenoxyacetate and only benzaldehyde dehydrogenase II is induced by benzyl alcohol, by benzaldehyde, and by a number of heterocyclic compounds which do not support growth. Mutants have been isolated that lack either benzaldehyde dehydrogenase II or benzyl alcohol dehydrogenase, or both of the enzymes. Results obtained in induction experiments with the wild-type bacterium N.C.I.B. 8250 and with the mutants show that benzaldehyde dehydrogenase II and benzyl alcohol dehydrogenase are co-ordinately regulated. Overall, the results suggest that benzaldehyde dehydrogenase I is associated with the metabolism of l-mandelate whereas benzaldehyde dehydrogenase II is associated with the metabolism of benzyl alcohol.

scholarly journals Enzymes of the mandelate pathway in bacterium N.C.I.B. 8250

1968 ◽  
Vol 107 (4) ◽  
pp. 497-506 ◽  
Author(s):  
S. I. T. Kennedy ◽  
C A Fewson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Benzoylformate Decarboxylase ◽  
Heat Stable ◽  
Enzyme Systems ◽  
Heat Labile ◽  
Hydroxybenzoate Hydroxylase ◽  
Benzaldehyde Dehydrogenase

1. Bacterium N.C.I.B. 8250 was grown on dl-mandelate, benzyl alcohol, benzoyl-formate, benzaldehyde and benzoate and also on 2-hydroxy, 4-hydroxy, 3,4-dihydroxy and 4-hydroxy-3-methoxy analogues of these compounds. The enzymic complements of the cells were determined and the specificities of some of the enzymes examined. 2. Growth on mandelate or benzoylformate induces l-mandelate dehydrogenase, benzoylformate decarboxylase, benzyl alcohol dehydrogenase and a heat-stable as well as a heat-labile benzaldehyde dehydrogenase. Growth on benzyl alcohol or benzaldehyde induces benzyl alcohol dehydrogenase and the heat-labile benzaldehyde dehydrogenase. 3. The enzymes of the mandelate-to-benzoate pathway are non-specifically active on, and induced by, all the substituted analogues that support growth. 4. Benzoate oxidase is induced by growth on benzoate or on 2-hydroxybenzoate. 2-Hydroxybenzoate hydroxylase, 4-hydroxybenzoate hydroxylase and 4-hydroxy-3-methoxybenzoate O-demethylase are induced only by growth on homologous substrates. 5. The results of the investigation are discussed with regard to the possible regulation of the enzyme systems.

scholarly journals Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Purification and preliminary characterization

1988 ◽  
Vol 250 (3) ◽  
pp. 743-751 ◽  
Author(s):  
R W MacKintosh ◽  
C A Fewson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Reduction Reaction ◽  
Oxidation Reactions ◽  
Benzaldehyde Dehydrogenase ◽  
Blue Sepharose

A quick, reliable, purification procedure was developed for purifying both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from a single batch of Acinetobacter calcoaceticus N.C.I.B. 8250. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernatant, followed by chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose CL-6B and Matrex Gel Red A, and finally gel filtration through a Superose 12 fast-protein-liquid-chromatography column. The enzymes co-purified as far as the Blue Sepharose CL-6B step were separated on the Matrex Gel Red A column. The final preparations of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II gave single bands on electrophoresis under non-denaturing conditions or on SDS/polyacrylamide-gel electrophoresis. The enzymes are tetramers, as judged by comparison of their subunit (benzyl alcohol dehydrogenase, 39,700; benzaldehyde dehydrogenase II, 55,000) and native (benzyl alcohol dehydrogenase, 155,000; benzaldehyde dehydrogenase II, 222,500) Mr values, estimated by SDS/polyacrylamide-gel electrophoresis and gel filtration respectively. The optimum pH values for the oxidation reactions were 9.2 for benzyl alcohol dehydrogenase and 9.5 for benzaldehyde dehydrogenase II. The pH optimum for the reduction reaction for benzyl alcohol dehydrogenase was 8.9. The equilibrium constant for oxidation of benzyl alcohol to benzaldehyde by benzyl alcohol dehydrogenase was determined to be 3.08 x 10(-11) M; the ready reversibility of the reaction catalysed by benzyl alcohol dehydrogenase necessitated the development of an assay procedure in which hydrazine was used to trap the benzaldehyde formed by the NAD+-dependent oxidation of benzyl alcohol. The oxidation reaction catalysed by benzaldehyde dehydrogenase II was essentially irreversible. The maximum velocities for the oxidation reactions catalysed by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were 231 and 76 mumol/min per mg of protein respectively; the maximum velocity of the reduction reaction of benzyl alcohol dehydrogenase was 366 mumol/min per mg of protein. The pI values were 5.0 for benzyl alcohol dehydrogenase and 4.6 for benzaldehyde dehydrogenase II. Neither enzyme activity was affected when assayed in the presence of a range of salts. Absorption spectra of the two enzymes showed no evidence that they contain any cofactors such as cytochrome, flavin, or pyrroloquinoline quinone. The kinetic coefficients of the purified enzymes with benzyl alcohol, benzaldehyde, NAD+ and NADH are also presented.

scholarly journals Characterization of Melanoidins and Color Development in Dulce de Leche, a Confectionary Dairy Product With High Sucrose Content: Evaluation of pH Effect, an Essential Manufacturing Process Parameter

2021 ◽  
Vol 8 ◽  
Author(s):  
Analía Rodríguez ◽  
Patricia Lema ◽  
María Inés Bessio ◽  
Guillermo Moyna ◽  
Cristina Olivaro ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
Ph Effect ◽  
Dairy Product ◽  
Gel Filtration ◽  
Significant Degree ◽  
Product Analysis ◽  
Molecular Weights ◽  
Ph Values ◽  
Initial Ph

The effect on color of the initial pH employed in dulce de leche (DL) production was evaluated through physicochemical and spectroscopical characterization of the melanoidins formed in the process. Melanoidins originated at pH values of 6.5, 7.0, and 7.5, and they were released by the enzymatic hydrolysis of the protein backbone and purified by gel filtration. They showed a significant degree of polydispersity, in general, with molecular weights (MWs) below 1,800 Da. DL produced at a higher pH released melanoidins with higher average MW after the enzymatic hydrolysis. They also presented darker colors (dE*ab, C*), more closely resembling those typical of the commercial product. Analysis of the fractions isolated by gel filtration using HPLC-DAD and multinuclear NMR showed an heterogeneous and complex composition. Even though structurally related, the 1H NMR spectra of melanoidins showed a higher degree of aromaticity at higher pH values. In conclusion, the pH employed in DL production affects the amount and structure of the colored products originated by MR reactions, and thus the color of the final product.

scholarly journals Regulation of the enzymes converting l-mandelate into benzoate in bacterium N.C.I.B. 8250

1972 ◽  
Vol 130 (4) ◽  
pp. 937-946 ◽  
Author(s):  
A. Livingstone ◽  
C. A. Fewson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Single Cell ◽  
Benzyl Alcohol ◽  
Dehydrogenase Activity ◽  
The Other ◽  
Cell Free Extract ◽  
Mutant Strains ◽  
Benzaldehyde Dehydrogenase ◽  
Kinetics Of

l-Mandelate is oxidized to benzoate by the enzymes l-mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I. Conditions have been established for measuring these three enzymes as well as benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase II and catechol 1,2-oxygenase in a single cell-free extract prepared from bacterium N.C.I.B. 8250. The kinetics of induction of all these enzymes have been measured under a variety of conditions. l-Mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I appear to be co-ordinately regulated because (a) their differential rates of synthesis are proportional to one another under various conditions of induction and repression, (b) they are specifically and gratuitously induced by thiophenoxyacetate and a number of other compounds, and (c) mutant strains have been isolated that lack all three enzymes. Phenylglyoxylate is the primary inducer of the regulon as mutant strains lacking phenylglyoxylate carboxy-lyase form the other two enzymes in the presence of l-mandelate or phenylglyoxylate, whereas in mutant strains devoid of l-mandelate dehydrogenase activity only phenylglyoxylate induces phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I.

scholarly journals Purification and properties of α-galactosidases from Vicia faba seeds

1969 ◽  
Vol 113 (1) ◽  
pp. 49-55 ◽  
Author(s):  
P. M. Dey ◽  
J. B. Pridham
Keyword(s):  
Amino Acid ◽  
Vicia Faba ◽  
Aromatic Amino Acid ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Thermal Stabilities ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Enzyme I ◽  
Carbohydrate Contents

Two forms of α-galactosidase, I and II, exist in Vicia faba seeds and these have been purified 3660- and 337-fold respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzymes I and II, as determined by gel filtration, were 209000 and 38000 respectively. The carbohydrate contents of enzymes I and II were 25% and 2·8% respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive subunits in the presence of 6m-urea. α-Galactosidases I and II showed different pH optima and Km and Vmax. values with p-nitrophenyl α-d-galactoside and raffinose as substrates, and also differed in their thermal stabilities.

scholarly journals ntn Genes Determining the Early Steps in the Divergent Catabolism of 4-Nitrotoluene and Toluene in Pseudomonas sp. Strain TW3

1998 ◽  
Vol 180 (8) ◽  
pp. 2043-2049 ◽  
Author(s):  
Keith D. James ◽  
Peter A. Williams
Keyword(s):  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Open Reading Frame ◽  
Pseudomonas Sp ◽  
Tol Plasmid ◽  
Reading Frame ◽  
Toluene Monooxygenase ◽  
Dehydrogenase Gene ◽  
Amino Acid Levels ◽  
Benzaldehyde Dehydrogenase

ABSTRACT Pseudomonas sp. strain TW3 is able to oxidatively metabolize 4-nitrotoluene and toluene via a route analogous to the upper pathway of the TOL plasmids. We report the sequence and organization of five genes, ntnWCMAB*, which are very similar to and in the same order as the xyl operon of TOL plasmid pWW0 and present evidence that they encode enzymes which are expressed during growth on both 4-nitrotoluene and toluene and are responsible for their oxidation to 4-nitrobenzoate and benzoate, respectively. These genes encode an alcohol dehydrogenase homolog (ntnW), an NAD+-linked benzaldehyde dehydrogenase (ntnC), a two-gene toluene monooxygenase (ntnMA), and part of a benzyl alcohol dehydrogenase (ntnB*), which have 84 to 99% identity at the nucleotide and amino acid levels with the corresponding xylWCMABgenes. The xylB homolog on the TW3 genome (ntnB*) appears to be a pseudogene and is interrupted by a piece of DNA which destroys its functional open reading frame, implicating an additional and as-yet-unidentified benzyl alcohol dehydrogenase gene in this pathway. This conforms with the observation that the benzyl alcohol dehydrogenase expressed during growth on 4-nitrotoluene and toluene differs significantly from the XylB protein, requiring assay via dye-linked electron transfer rather than through a nicotinamide cofactor. The further catabolism of 4-nitrobenzoate and benzoate diverges in that the former enters the hydroxylaminobenzoate pathway as previously reported, while the latter is further metabolized via the β-ketoadipate pathway.

scholarly journals Preparation and analysis of peptide fragments produced by pepsin hydrolysis of human plasma albumin and their relationship to its structure

1968 ◽  
Vol 109 (1) ◽  
pp. 107-120 ◽  
Author(s):  
G. Franglen ◽  
G. R. E. Swaniker
Keyword(s):  
Human Plasma ◽  
Gel Filtration ◽  
Tripartite Model ◽  
Peptide Fragments ◽  
Plasma Albumin ◽  
Molecular Weights ◽  
Ph Values ◽  
Cystine Content ◽  
Zone Electrophoresis ◽  
Hydrolysis Of

Human plasma albumin was prepared and subjected to proteolysis by pepsin at pH2·45 at 25° for 10min. with albumin/pepsin ratio 3000:1. Five peptide fragments were detected in the proteolysate by means of zone electrophoresis and gel filtration; these were separated and purified. Molecular weights, amino acid composition and disulphide bond content of the purified fragments were determined. The results show that a high proportion of the polypeptide chain of albumin appears to have a low cystine content, and at low pH values the molecule would be expected to have a considerable degree of freedom in its structure in these regions of the chain. A tripartite model for the structure of plasma albumin is proposed.

scholarly journals Substrate-specificity of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by TOL plasmid pWW0. Metabolic and mechanistic implications

1992 ◽  
Vol 283 (3) ◽  
pp. 789-794 ◽  
Author(s):  
J P Shaw ◽  
F Schwager ◽  
S Harayama
Keyword(s):  
Alcohol Dehydrogenase ◽  
Pseudomonas Putida ◽  
Benzyl Alcohol ◽  
Tol Plasmid ◽  
Benzyl Alcohols ◽  
Substrate Specificities ◽  
Electronic Nature ◽  
Catalysed Oxidation ◽  
Substituted Benzaldehydes ◽  
Benzaldehyde Dehydrogenase

The substrate-specificities of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, encoded by TOL plasmid pWW0 of Pseudomonas putida mt-2, were determined. The rates of benzyl alcohol dehydrogenase-catalysed oxidation of substituted benzyl alcohols and reduction of substituted benzaldehydes were independent of the electronic nature of the substituents at positions 3 and 4. Substitutions at position 2 of benzyl alcohol affected the reactivity of benzyl alcohol dehydrogenase: the velocity of the benzyl alcohol dehydrogenase-catalysed oxidation was lower for compounds possessing electron-withdrawing substitutions. In the reverse reaction of benzyl alcohol dehydrogenase, none of the substitutions tested influenced the apparent kcat. values. The rates of benzaldehyde dehydrogenase-catalysed oxidation of substituted benzaldehydes were influenced by the electronic nature of the substitutions: electron-withdrawing groups at positions 3 and 4 favoured the oxidation of benzaldehydes. Substitution at position 2 of benzaldehyde greatly diminished the benzaldehyde dehydrogenase-catalysed oxidation. Substitution at position 2 with electron-donating groups essentially abolished reactivity, and only substitutions that were strongly electron-withdrawing, such as nitro and fluoro groups, permitted enzyme-catalysed oxidation. The influence of the electronic nature and the position of substitutions on the aromatic ring of the substrate on the velocity of the catalysed reactions provided some indications concerning the transition state during the oxidation of the substrates, and on the rate-limiting steps of the enzymes. Pseudomonas putida mt-2 containing TOL plasmid pWW0 cannot grow on toluene derivatives substituted at position 2, nor can it grow on 2-substituted benzyl alcohols or aldehydes. One of the reasons for this may be the substrate-specificities of the benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase.

scholarly journals Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus

1998 ◽  
Vol 330 (3) ◽  
pp. 1375-1381 ◽  
Author(s):  
J. David GILLOOLY ◽  
G. S. Alan ROBERTSON ◽  
A. Charles FEWSON
Keyword(s):  
Amino Acids ◽  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Catalytic Mechanism ◽  
Acinetobacter Calcoaceticus ◽  
Higher Plant ◽  
Ph Optimum ◽  
Alcohol Dehydrogenases ◽  
Kinetic Coefficients ◽  
Benzaldehyde Dehydrogenase

The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases.

Sign in / Sign up

Export Citation Format

Share Document