Rhamnolipid from Pseudomonas desmolyticum NCIM-2112 and its role in the degradation of Brown 3REL.

The biosurfactant produced by Pseudomonas desmolyticum NCIM 2112 (Pd 2112) was confirmed as rhamnolipid based on the formation of dark blue halos around the colonies in CTAB-methylene blue agar plates and the content of rhamnose sugar. The average yield of rhamnolipid was 0.398 g/l/day when grown on hexadecane as sole carbon source. Pd 2112 emulsification potential associated with cell free culture broth was stable for 72 h using various hydrocarbons and vegetable oils. Chemical structure of the biosurfactant was identified as mono-rhamnolipid (Rha-C(6) -C(8) ) using HPTLC, fourier transform infrared spectroscopy, (1) H and (13) C NMR and gas chromatography-mass spectroscopy analysis. Pd 2112 mono-rhamnolipid (1 mg/ml) had increased permeabilization of Bacillus sp VUS NCIM 5342 and increased decolorization rate of textile dye Brown 3REL by 50%. Extracellular activities of lignin peroxidase and veratryl alcohol oxidase, enzymes involved in dye degradation, were significantly increased in the presence of mono-rhamnolipid by 324.52% and 100% respectively. Scanning electron micro-scopy observations revealed that rhamnolipid did not exert any disruptive action on Bacillus cells as compared to Tween 80. The mono-rhamnolipid of Pd 2112 has potential for its application in biodegradation of textile dyes.

Evaluation of substituted triazol-1-yl-pyrimidines as inhibitors of Bacillus anthracis acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS), a potential target for antimicrobial agents, catalyzes the first common step in the biosynthesis of the branched-chain amino acids. The genes of both catalytic and regulatory subunits of AHAS from Bacillus anthracis (Bantx), a causative agent of anthrax, were cloned, overexpressed in Escherichia coli, and purified to homogeneity. To develop novel anti-anthracis drugs that inhibit AHAS, a chemical library was screened, and four chemicals, AVS2087, AVS2093, AVS2387, and AVS2236, were identified as potent inhibitors of catalytic subunit with IC(50) values of 1.0 +/- 0.02, 1.0 +/- 0.04, 2.1 +/- 0.12, and 2.0 +/- 0.08 microM, respectively. Further, these four chemicals also showed strong inhibition against reconstituted AHAS with IC(50) values of 0.05 +/- 0.002, 0.153 +/- 0.004, 1.30 +/- 0.10, and 1.29 +/- 0.40 microM, respectively. The basic scaffold of the AVS group consists of 1-pyrimidine-2-yl-1H-[1,2,4]triazole-3-sulfonamide. The potent inhibitor, AVS2093 showed the lowest binding energy, -8.52 kcal/mol and formed a single hydrogen bond with a distance of 1.973 A. As the need for novel antibiotic classes to combat bacterial drug resistance increases, the screening of new compounds that act against Bantx-AHAS shows that AHAS is a good target for new anti-anthracis drugs.

Characterization of Capsicum annuum recombinant alpha- and beta-tubulin.

There are several conditions which might modulate polymerization to produce polymers having normal lattice structure. In the absence of 1 mM MgCl(2) the assembly was reduced by 36% in Capsicum annuum tubulin (CAnm tubulin). There was no significant difference in the final assembly formation in the presence of 5% to 10% glycerol. However, nucleation rate was slow and apparent study state was achieved lately in the presence of 10% glycerol. Taxol at 100 microM concentration increased 23% tubulin assembly. One millimolar CaCl(2), >or=1% dimethyl sulfoxide (DMSO) and physiologically low temperature reduced CAnm tubulin assembly. A value of 0.089 mg/ml was obtained as critical concentration for polymerization. Benomyl significantly reduced the number of cysteine residues accessible to 5,5'-dithiobis-(2-nitrobenzoic acid); there were 4.77 +/- 0.21 and 3.49 +/- 0.35 residues accessible per tubulin dimer in the presence of 50 and 100 microM benomyl respectively.

Molecular cloning and biochemical characterization of alpha- and beta-tubulin from potato plants (Solanum tuberosum L.).

Few studies have investigated microtubules from plants that host pathogenic fungi. Considerable efforts are underway to find an antimitotic agent against plant pathogens like Phytophthora infestans. However, screening the effects of antifungal agents on plant tubulin in vivo or using purified native microtubule in vitro is a time consuming process. A recombinant, correctly folded, microtubule-like structure forming tubulin could accelerate research in this area. In this study, we cloned full length cDNAs isolated from potato leaves using reverse-transcribed polymerase chain reaction (RT-PCR). Solanum tuberosum (Stub) alpha-tubulin and beta-tubulin were predicted to encode 449 and 451 amino acid long proteins with molecular masses of 57 kDa and 60 kDa, respectively. Average yields of alpha- and beta-tubulin were 2.0-3.5 mg l(-1) and 1.3-3.0 mg l(-1) of culture, respectively. The amino acids, His6, Glu198, and Phe170 involved in benomyl sensitivity were conserved in Stub tubulin. The dimerization of tubulin monomers was confirmed by western blot analysis. When combined under appropriate conditions, these recombinant alpha- and beta-tubulins were capable of polymerizing into microtubules. Accessibility of cysteine residues of tubulin revealed that important ligand binding sites were folded correctly. This recombinant tubulin could serve as a control of phytotoxicity of selected antimitotic fungicide compounds during in vitro screening experiments.

Protective antigen detection using horizontally stacked hexagonal ZnO platelets.

Anthrax toxin detection before bacteremia, when toxin concentration is low, improves the chances of efficient treatment and cure. We present a novel technique for ultrasensitive detection of a protective antigen (PA(83)) of anthrax using an array of zinc oxide nanorods in conjunction with a FITC-labeled PA affinity peptide. The nanorods are composed of horizontally stacked hexagonal platelets which are uniformly spaced and grown unidirectionally upon a glass substrate via a new and simple technique. Images taken under UV emission demonstrate fluorescence sensitivity to PA as a function of antigen concentration, and a negative control using bovine serum albumin produced no fluorescence signal. The fluorescence signal of the PA-peptide complex is also significantly reduced in the absence of the nanorods, suggesting that the presence of ZnO nanorods inhibits the self-quenching properties of the fluorophore. A lower limit of detection for the assay system for PA is estimated at 150 aM, which demonstrates the possibility of using ZnO nanorods in biological sensor systems.

Purification and partial characterization of lignin peroxidase from Acinetobacter calcoaceticus NCIM 2890 and its application in decolorization of textile dyes.

Lignin peroxidase was purified (72-fold) from Acinetobacter calcoaceticus NCIM 2890. The purified lignin peroxidase (55-65 kDa) showed dimeric nature. The maximum enzyme activity was observed at pH 1.0, between a broad temperature range of 50 and 70 degrees C, at H2O2 concentration (40 mM) and the substrate concentration (n-propanol, 100 mM). Purified lignin peroxidase was able to oxidize a variety of substrates including Mn2+, tryptophan, mimosine, L-Dopa, hydroquinone, xylidine, n-propanol, veratryl alcohol, and ten textile dyes of various groups indicating as a versatile peroxidase. Most of the dyes decolorized up to 90%. Tryptophan stabilizes the lignin peroxidase activity during decolorization of dyes.

Alpha- and beta-tubulin from Phytophthora capsici KACC 40483: molecular cloning, biochemical characterization, and antimicrotubule screening.

Internal fragments of alpha- and beta-tubulin genes were generated using reverse transcription polymerase chain reaction (RT-PCR), and the termini were isolated using 5'- and 3'-rapid amplification of cDNA ends. Phytophthora capsici alpha- and beta-tubulin specific primers were then used to generate full-length cDNA by RT-PCR. The recombinant alpha- and beta-tubulin genes were expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, and average yields were 3.38-4.5 mg of alpha-tubulin and 2.89-4.0 mg of beta-tubulin, each from 1-l culture. Optimum conditions were obtained for formation of microtubule-like structures. A value of 0.12 mg/ml was obtained as the critical concentration of polymerization of P. capsici tubulin. Benomyl inhibited polymerization with half-maximal inhibition (IC(50)) = 468 +/- 20 microM. Approximately 18.66 +/- 0.13 cysteine residues per tubulin dimer were accessible to 5,5'-dithiobis-(2-nitrobenzoic acid), a quantification reagent of sulfhydryl and 12.43 +/- 0.12 residues were accessible in the presence of 200 microM benomyl. The order of preference for accessibility to cysteines was benomyl > colchicine > GTP > taxol, and cysteine accessibility changes conformed that binding sites of these ligands in tubulin were folding correctly. Fluorescence resonance energy transfer technique was used for high throughput screening of chemical library in search of antimitotic agent. There was significant difference in relative fluorescence by 210-O-2 and 210-O-14 as compared to colchicine.

Cloning, purification, and polymerization of Capsicum annuum recombinant alpha and beta tubulin.

Alpha and beta tubulin genes were cloned from the Capsicum annuum leaves using rapid amplification of cDNA ends (RACE)-PCR. Nucleotide sequence analysis revealed that 1,353 bp Capsicum annuum alpha/beta-tubulin (CAnm alpha/beta-TUB) encodes a protein of 450 amino acids (aa) each. The recombinant alpha/beta tubulin was overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with 0.2 mM isopropyl-beta-D-thiogalactopyranoside (IPTG), and its content was as high as 50% of the total protein content. Effective fusion protein purification and refolding are described. The average yields of alpha and beta tubulin were 2.0 and 1.3 mg/l of culture respectively. The apparent molecular weight of each tubulin was estimated to be 55 kDa by SDS-polyacrylamide gel electrophoresis (PAGE). The tubulin monomers were found to be assembly competent using a standard dimerization assay, and also retained antigenicity with anti-His/T7 antibodies. The purified tubulins were polymerized to microtubule-like structures in the presence of 2 mM guanosine 5'-triphosphate (GTP).

Diesel and kerosene degradation by Pseudomonas desmolyticum NCIM 2112 and Nocardia hydrocarbonoxydans NCIM 2386.

Pseudomonas desmolyticum NCIM 2112 (Pd 2112) and Nocardia hydrocarbonoxydans NCIM 2386 (Nh 2386) demonstrated an ability to degrade diesel and kerosene. Triton X-100 had enhanced the diesel degradation process by reducing the time required for the maximum utilization of total petroleum hydrocarbon. Fourier transform infrared spectroscopy spectrum of degraded diesel indicates the presence of aliphatic and aromatic aldehydes, C=C aromatic nuclei, and substituted benzenes. Surface tension reduction and stable emulsification was increased using consortium when compared to individual strains. Triton X-100 showed increase in microbial attachment to hydrocarbon among the various chemical surfactants tested. For generating a rapid assay to screen microorganisms capable of degrading kerosene, the acetaldehyde produced in the degradation process could be used as an indicator of degradation. These results indicate diesel and kerosene degradation ability of both of the strains.

Biodegradation of kerosene by Aspergillus ochraceus NCIM-1146.

The filamentous fungus Aspergillus ochraceus NCIM-1146 was found to degrade kerosene, when previously grown mycelium (96 h) was incubated in the broth containing kerosene. Higher levels of NADPH-DCIP reductase, aminopyrine N-demethylase and kerosene biodegradation activities were found to be present after the growth in potato dextrose broth for 96 h, when compared with the activities at different time intervals during the growth phase. NADPH was the preferred cofactor for enzyme activity, which was inhibited by CO, indicating cytochrome P450 mediated reactions. A significant increase in all the enzyme activities was observed when mycelium incubated for 18 h in mineral salts medium, containing cholesterol, camphor, naphthalene, 1,2-dimethoxybenzene, phenobarbital, n-hexane, kerosene or saffola oil as inducers. Acetaldehyde produced by alcohol dehydrogenase could be used as an indicator for the kerosene biodegradation.