C.S. Vaidyanathan

Evidence for the involvement of multiple pathways in the biodegradation of 1- and 2-methylnaphthalene by Pseudomonas putida CSV86.

Pseudomonas putida CSV86, a soil bacterium, grows on 1- and 2-methylnaphthalene as the sole source of carbon and energy. In order to deduce the pathways for the biodegradation of 1- and 2-methylnaphthalene, metabolites were isolated from the spent medium and purified by thin layer chromatography. Emphasis has been placed on the structural characterisation of isolated intermediates by GC-MS, demonstration of enzyme activities in the cell free extracts and measurement of oxygen uptake by whole cells in the presence of various probable metabolic intermediates. The data obtained from such a study suggest the possibility of occurrence of multiple pathways in the degradation of 1- and 2-methylnaphthalene. We propose that, in one of the pathways, the aromatic ring adjacent to the one bearing the methyl moiety is oxidized leading to the formation of methylsalicylates and methylcatechols. In another pathway the methyl side chain is hydroxylated to-CH2OH which is further converted to-CHO and-COOH resulting in the formation of naphthoic acid as the end product. In addition to this, 2-hydroxymethylnaphthalene formed by the hydroxylation of the methyl group of 2-methylnaphthalene undergoes aromatic ring hydroxylation. The resultant dihydrodiol is further oxidised by a series of enzyme catalysed reactions to form 4-hydroxymethyl catechol as the end product of the pathway.

Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia

2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dichlorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 microM for 2,4-dichlorophenol, and 100 microM for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.

l-asparaginases from Mycobacterium tuberculosis strains H37Rv and H37Ra

M. tuberculosis H37Ra possesses two Image -asparaginases while the H37Rv strain possesses only a single enzyme. These enzymes have been purified and their properties studied. The two Image -asparaginases in H37Ra strain differ from each other in pH optima, heat inactivation, Michaelis constant and effects of inhibitors, while one of them resembles the single Image -asparaginase present in the H37Rv strain. Image -Cysteine inhibits both Image -asparaginases in an allosteric manner probably because it is one of the end-products in Image -asparagine metabolism. This is the first time that a qualitative difference has been reported in the enzyme pattern between the avirulent and virulent strains of M. tuberculosis.

Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae.

The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD+, PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M-1 min-1. A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M-1 min-1. Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.

Purification, properties and induction of a specific benzoate-4-hydroxylase from Aspergillus niger (UBC 814)

An inducible benzoate-4-hydroxylase has been partially purified from crude extracts of the mycelial felts of Aspergillus niger. This enzyme catalyzes the transformation of benzoate to p-hydroxybenzoate with equimolar consumption of NADPH and O2. It requires tetrahydropteridine as a prosthetic group. The optimum activity was found at pH 6.2 with a Km value at 30 degrees C of 1.6-10-minus 4 for NADPH and 1.3-10-minus 4 M for benzoate. Fe-2+ (iron) is required for the enzyme activity. The enzyme is stabilized by the inclusion of benzoate, EDTA and glutathione in the extracting buffer. The enzyme is specific for benzoate as substrate. Sulfhydryl groups(s) are essential for enzyme activity as indicated by p-chloromercuri-benzoate and N-ethylmaleimide inactivation. Benzoate-4-hydroxylase activity is decreased in the mycelial felts of Aspergillus niger grown in the presence of higher concentrations of benzoate. Maximum activity of the enzyme was observed at 36 h after inoculation.

Production of biosurfactant “Biosur-Pm” by Pseudomonas maltophila CSV89: characterization and role in hydrocarbon uptake

Pseudomonas maltophilia CSV89, a soil bacterium, produces an extracellular biosurfactant, “Biosur-Pm”. The partially purified product is nondialyzable and chemically composed of 50% protein and 12–15% sugar, which indicates the complex nature of Biosur-Pm. It reduces the surface tension of water from 73 to 53×10-3 N m-1 and has a critical micellar concentration of 80 mg/l. Compared to aliphatic hydrocarbons, Biosur-Pm shows good activity against aromatic hydrocarbons. The emulsion formed is stable and does not require any metal ions for emulsification. The kinetics of Biosur-Pm production suggest that its synthesis is a growth-associated and pH-dependent process. At pH 7.0, cells produced more Biosur-Pm with less cell surface hydrophobicity. At pH 8.0, however, the cells produced less Biosur-Pm with more cell surface hydrophobicity and showed a twofold higher affinity for aromatic hydrocarbons compared to the cells grown at pH 7.0. The Biosur-Pm showed a pH-dependent release, stimulated growth of the producer strain on mineral salts medium with 1-naphthoic acid when added externally, and facilitated the conversion of salicylate to catechol. All these results suggest that Biosur-Pm is probably a cell-wall component and helps in hydrocarbon assimilation/uptake.

Purification and properties of uridine hydrolase from mung-bean (Phaseolus radiatus) seedlings

The occurrence in plants of an enzyme system catalyzing the cleavage of uridine has been demonstrated. The enzyme from Phaseolus radiatus was purified about 132-fold with 24% recovery by a combination of procedures involving mild acid treatment, ammonium sulphate fractionation, negative adsorption on calcium phosphate gel and DEAE-cellulose chromatography. The enzyme cleaves uridine to uracil and ribose in the absence of phosphate indicating that the mechanism of cleavage was hydrolytic rather than phosphorolytic. The enzyme is specific to uridine and does not act on other purine and pyrimidine compounds. The enzyme shows maximum activity at pH 7.4 and has a temperature optimum of 45 °. It does not require metal ions for activity. Inhibition of the enzyme by p-chloromercuribenzoate as well as N-ethylmaleimide and the reversal of p-chloromercuribenzoate inhibition by sulfhydryl agents indicate the probable involvement of readily oxidizable sulfhydryl groups in enzyme activity.

Substrate-mediated purification and characterization of a 3-hydroxybenzoic acid 6-hydroxylase from Micrococcus

3-Hydroxybenzoic acid-6-hydroxylase from Micrococcus sp. was purified to homogeneity in a single step using the substrate-mediated interaction of the enzyme with blue-Sepharose. The enzyme was bound to the affinity matrix in the presence of 3-hydroxybenzoic acid and was eluted in its absence. The molecular weight of the purified enzyme is 70,000 with no subunit structure. The flavoenzyme required the exogenous addition of FAD for its complete activity and had a strict preference for NADH over NADPH. The activity of the enzyme was drastically inhibited by Cu2+ and Hg2+ and the inhibition was reversed by thiol reagents.

Anthranilic acid oxidase system of Tecoma stans—II. : Studies on the properties of a purified anthranilic acid oxidase system and its separation into different enzymic components

Abstract An enzyme system which catalysed the conversion of anthranilic acid to catechol has been purified 20-fold from a cell-free leaf extract of Tecoma stans . The optimum substrate concentration was 10 −3 M and optimum temperature for the reaction was 45°. The presence of a multi-enzyme system was inferred from inhibition studies. The formation of catechol was inhibited by Mg 2+ , Zn 2+ , and Co 2+ ions, whereas anthranilic acid disappearance was not affected to the same extent. The effect of metal chelating agents like EDTA, cyanide and pyrophosphate showed a similar trend. PCMB inhibited catechol formation but had no effect on anthranilic acid disappearance. The reaction was not inhibited by catalase, nor was it activated by peroxide-donating systems. This ruled out the possibility of peroxidative type of reaction. The overall reaction is markedly activated by NADPH and THFA. This multi-enzyme was separated into three different components, by fractionation with Alumina C γ and calcium phosphate gels. The overall reaction catalysed by these components can be represented as anthranilic acid→3-hydroxy anthranilic acid→ o -aminophenol→catechol.

Ribosome-inactivating proteins and agglutinins from callus and suspension cultures of Ricinus communis L. and Abrus precatorius L.

Abstract Ribosome-inactivating proteins (RIPs) or toxins, as well as agglutinins, were isolated from callus and cell suspension cultures that were established from seed explants of Ricinus communis L. and Abrus precatorius L. The toxins viz. ricin from R. communis and abrin from A. precatorius as well as agglutinins viz., Ricinus communis agglutinin (RCA) and Abrus precatorius agglutinin (APA) were synthesized in these cultures and were secreted into the medium. The lectins (RIPs and agglutinins) were synthesized through several passages of cultured cells at levels which make them attractive as an alternate source of lectins. Biosynthesis of these carbohydrate-binding proteins was regulated by specific exogenous hormones and was positively correlated with the growth of the cultures indicating that toxins and agglutins may have a role to play during cell division. The toxins and agglutinins were secreted into the medium providing a clue that they probably serve as defence molecules. Secretion of lectins into the medium facilitated easy isolation of the lectins. The synthesized lectins were purified from cultures and were partially characterized. They were biologically active, and were found to be comparable with lectins from seeds, in terms of their electrophoretic mobilities.