A novel Sphingobacterium sp. RB, a rhizosphere isolate degrading para-nitrophenol with substrate specificity towards nitrotoluenes and nitroanilines.

Sphingobacterium sp. RB, a novel bacterial strain isolated from a soil sample, was able to utilize para-nitrophenol (PNP) as sole source of carbon and energy at high concentrations (1.0-5.0 mM). The culture completely degraded 3.0 mM PNP within 36 h with proportionate increase in biomass. With 5.0 mM PNP (700 ppm), 70% degradation was observed within 72 h of incubation. Scanning electron microscope images of the isolate in the presence and absence of PNP showed no significant morphological variations. Liquid chromatography-mass spectrometry analysis indicated that the biodegradation of PNP in this bacterium proceeded via the formation of 1,2,4-benzenetriol. Cells previously exposed to PNP (induced) were 30% more effective in degrading PNP. With catechol and phenol, such induction was not observed. Uninduced cells of Sphingobacterium sp. RB were capable of degrading a variety of other nitroaromatic compounds, including 2-nitroaniline, 2,4-dinitroaniline, 2-nitrotoluene, 3-nitrotoluene and 2,4-dinitrophenol, within 72 h, thus proving its candidacy as a potent bioremediation agent. To the best of our knowledge, this is the first report on a Sphingobacterium species degrading PNP via formation of 1,2,4-benzenetriol.

Comparative structural modeling and docking studies of uricase: possible implication in enzyme supplementation therapy for hyperuricemic disorders.

Uricase (EC 1.7.3.3, UC) catalyzes the oxidation of uric acid (UA) to more soluble allantoin thereby lowering plasma UA levels. In humans, when concentration of UA exceeds >7mg/dl, it leads to hyperuricemia, gout, nephrolithiasis and urolithiasis. A new remedy to cure such metabolic diseases is the enzyme supplementation therapy by UC but with high degree of antigenic independence. Therefore screening of new uricase sources to expand its usefulness and reduced antigenecity is needed. Present study employed cheminformatics approach to construct models of reported UC from different sources viz. Bacillus megaterium, Streptomyces bingchenggensis BCW-1, Paenibacillus sp, Solibacter usitatus Ellin6076, Truepera radiovictrix DSM 17093 and Ktedonobacter racemifer DSM 4496 in order to study their structure-function relationship for enzyme mass production and modification for improved characteristics. BioMed CAChe version 6.1 was further used to study enzyme-substrate interactions of models with uric acid using docking approach. Results indicated that models for UC of Streptomyces bingchenggensis BCW-1 accounted for better regio-specificity towards UA, supporting the interested metabolism and thus may further be implicated in enzyme supplementation therapy for hyperuricemic associated disorders.

Comparative structural modeling and docking studies of oxalate oxidase: Possible implication in enzyme supplementation therapy for urolithiasis.

In humans oxalate is end product of protein metabolism, with no enzyme present to act on it. In conditions of its enhanced endogenous synthesis or increased absorption from the diet, oxalate accumulation leads to hyperoxaluria which can further lead to a number of pathological conditions including urolithiasis. Urolithiasis has been a perplexing problem due to its high incidence and rate of recurrence after treatment like Extracorporeal-shock wave lithotripsy (ESWL). Hence other prophylactic treatment becomes necessary. One of the newer approaches of curing such metabolic disorders is the enzyme supplementation therapy. Oxalate oxidase (OxOx) is a commonly occurring enzyme in plants, bacteria and fungi that catalyses oxidative cleavage of oxalate to CO(2) with reduction of dioxygen to H(2)O(2). Present study, used Hordeum vulgare OxOx crystal structure (PDB ID 2ET1A) as a template for constructing 3D models of OxOx from Triticum aestivum, Arabidopsis thaliana, Sclerotiana sclerotiarum. Similarly Homology models for isoforms Ceriporiopsis subvermispora 336, C. subvermispora 422 were constructed by using template Bacillus subtilis oxalate decarboxylase (Oxdc) (PDB ID 2UY8A) by comparative modeling approach in SWISS MODEL, MODELLER, 3D JIGSAW and GENO 3D program server. Based on overall stereochemical quality (PROCHECK, PROSA, VARIFY 3D), best models were selected, energy minimized, refined and characterized for active site in BioMed CaChe V 6.1 workspace. Selected models were further studied for structure function relationship with substrate (oxalate) and its analogue (glycolate) by using docking approach. Calculated interaction energy between the oxalate and constructed enzyme indicated that homology models for OxOx of T. aestivum, A. thaliana and S. sclerotiarum, can account for better regio-specificity of this enzyme towards oxalate. That supports the interested metabolism and thus may further implement in enzyme supplementation therapy for urolithiasis.

Synthesis and antimicrobial activity of novel pyrazolo[3,4-d]pyrimidin derivatives.

Pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-4-one derivatives have been prepared by cyclocondensation of ethyl 2-cyano-3,3-bis(methylthio)prop-2-enoate with 2-amino-4-(substitutedphenyl)thiazole to give 3-cyano-2-methylthio-4-oxo-4H-6-(substitutedphenyl)thiazolo[3,2-a]pyrimidin (2a-j) and further reacting with hydrazine hydrate to yield the target compounds (3a-j). The chemical structure of the compounds was confirmed by IR and (1)H NMR spectral data. All the compounds of the series have been screened for their antibacterial and antifungal activity studies. The result revealed that all compounds showed significant antimicrobial activity.

Synthesis and biological evaluation of simple methoxylated chalcones as anticancer, anti-inflammatory and antioxidant agents.

Chalcones have been identified as interesting compounds with cytotoxicity, anti-inflammatory and antioxidant properties. In the present study, simple methoxychalcones were synthesized by Claisen-Schmidt condensation reaction and evaluated for above biological activities. The structures of the compounds were established by IR, (1)H NMR and mass spectral analysis. The data revealed that compound 3s (99-100% at 10 microM concentration) completely inhibit the selected five human cancer cell lines as compared to standard flavopiridol and gemcitabine (70-90% at 700 nM and 500 nM concentrations, respectively), followed by 3a, 3n,3o,3p,3q,3r. Among the tested compounds 3l, 3m, 3r, and 3s exhibited promising anti-inflammatory activity against TNF-alpha and IL-6 with 90-100% inhibition at 10 microM concentration. DPPH free radical scavenging activity was given by the compounds 3o, 3n, 3l, 3r, 3m, 3a, 3p, 3c and 3s at 1mM concentration. Overall, 3s was obtained as lead compound with promising anticancer, anti-inflammatory and antioxidant activities. Bioavailability of compounds were checked by in vitro cytotoxicity study and confirmed to be nontoxic. The structure activity relationship (SAR) and in silico drug relevant properties (HBDs, HBAs, PSA, cLogP, ionization potential, molecular weight, E(HOMO) and E(LUMO)) further confirmed that the compounds were potential candidates for future drug discovery study.

Synthesis and biological activity of pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-4-one derivatives: in silico approach.

Xanthine oxidase (XO) is responsible for the pathological condition called gout. Inhibition of XO activity by various pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidine-4-one derivatives was assessed and compared with the standard inhibitor allopurinol. Out of 10 synthesized compounds, two compounds, viz. 3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-4-one (3b) and 3-amino-6-(4-chloro-2-hydroxy-5-methylphenyl)-1H-pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-4-one (3g) were found to have promising XO inhibitory activity of the same order as allopurinol. Both compounds and allopurinol inhibited competitively with comparable Ki (3b: 3.56 microg, 3g: 2.337 microg, allopurinol: 1.816 microg) and IC(50) (3b: 4.228 microg, 3g: 3.1 microg, allopurinol: 2.9 microg) values. The enzyme-ligand interaction was studied by molecular docking using Autodock in BioMed Cache V. 6.1 software. The results revealed a significant dock score for 3b (-84.976 kcal/mol) and 3g (-90.921 kcal/mol) compared with allopurinol (-55.01 kcal/mol). The physiochemical properties and toxicity of the compounds were determined in silico using online computational tools. Overall, in vitro and in silico study revealed 3-amino-6-(4-chloro-2-hydroxy-5-methylphenyl)-1H-pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-4-one (3g) as a potential lead compound for the design and development of XO inhibitors.

Synthesis and biological evaluation of a novel series of pyrazole chalcones as anti-inflammatory, antioxidant and antimicrobial agents.

A novel series of 1-(2,4-dimethoxy-phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-propenone (3) have been prepared by the Claisen-Schmidt condensation of 1-(2,4-dimethoxy-phenyl)-ethanone (1) and substituted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes (2). Substituted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes (2) were prepared by Vilsmeir-Haack reaction on acetophenonephenylhydrazones to offer the target compounds. The structures of the compounds were established by IR, (1)H NMR and mass spectral analysis. All the compounds were evaluated for their anti-inflammatory (TNF-alpha and IL-6 inhibitory assays), antioxidant (DPPH free radical scavenging assay) and antimicrobial activities (agar diffusion method) against some pathogenic bacteria and fungi. Of 10 compounds screened, compounds 3a, 3c and 3g exhibited promising IL-6 inhibitory (35-70% inhibition, 10 microM), free radical scavenging (25-35% DPPH activity) and antimicrobial activities (MIC 100 microg/mL and 250 microg/mL) at varied concentrations. The structure-activity relationship (SAR) and in silico drug relevant properties (HBD, HBA, PSA, cLogP, molecular weight, E(HOMO) and E(LUMO)) further confirmed that the compounds are potential lead compounds for future drug discovery study. Toxicity of the compounds was evaluated theoretically and experimentally and revealed to be nontoxic except 3d and 3j.

Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9 / Purificação e caracterização de uma lipase extracelular produzida por uma nova cepa: Pseudomonas aeruginosa SRT9

An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16) fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg2+, Zn2+, Cu2+, Ag2+ and Fe2+ decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/minrespectively.

Uma lipase extracelular foi isolada e purificada a partir de um caldo de cultura de Pseudomonas aeruginosa SRT9 até homogeneidade visível empregando-se precipitação com sulfato de amônia, seguida de técnicas cromatográficas em colunas de fenil sefarose CL-4B e Mono Q HR 5/5, obtendo-se um fator de purificação de 98 vezes, e atividade especifica de 12307,8 U/mg. Por SDS_PAGE, estimou-se que o peso molecular da lipase purificada é 29kDa, com um ponto isoelétrico de 4,5. A lipase apresentou atividade máxima em uma ampla faixa de temperatura e pH, com ótimos a 55ºC e pH 6,9. A lípase foi mais ativa sobre triacilglicerois de cadeia longa (C14-C16). A lipase foi fortemente inibida por EDTA, o que sugere que a enzima pode ser uma metaloproteína. SDS e íons metálicos, como Hg2+, Zn2+,Cu2+, Ag2+ e Fe2+, diminuíram marcadamente a atividade da lipase. Sua grande estabilidade e atividade em solventes organicos sugerem que esta lípase pode ser uma excelente ferramenta tecnológica com várias aplicações como reações organosintéticas e preparação de produtos farmacêuticos enantiomericamente puros. Os valores de Km e Vmax para a enzima purificada na hidrólise de trioleina foram 1,11 mmol/L e 0,05 mmol/L/min, respectivamente.

Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9.

An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16) fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg(2+), Zn(2+), Cu(2+), Ag(2+) and Fe(2+) decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/min respectively.

Microbial and xanthine dehydrogenase inhibitory activity of some flavones.

Xanthine dehydrogenase (XDH) is responsible for the pathological condition called Gout. In the present study different flavones synthesized from chalcone were evaluated in vitro for their inhibitory activity. Inhibitory activity of flavones on XDH was determined in terms of inhibition of uric acid synthesis from Xanthine. The enzymatic activity was found maximum at pH 7.5 and temperature 40 degrees C. The flavones 6-chloro-2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(1)) and 6-chloro-7methyl-2-[3-(4-chloro-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one(F(2)),were noncompetitive and competitive inhibitor with Ki values 1.1 and 0.22 respectively. The flavones (F(1)), (F(2)), 6-chloro-2-[3-(4-chloro-phenyl)-1phenyl-1-H-pyrazol-4-yl]-chromen-4-one(F(3)), 8-bromo-6-chloro-2-[3-(4-chloro-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(4)), 2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(5)) and 6-methyl-2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(6)) were also screened for their antimicrobial activity, measured in terms of zone of inhibition. A broad spectrum antifungal activity was obtained against Trichoderma viridae, Candida albicans, Microsporum cannis, Penicillium chrysogenum and Fusarium moniliformae. In case of Aspergillus niger and Aspergillus flavous only spore formation was affected, while antibacterial activity was observed against Staphylococcus aureus, Bacillus subtilis and Serratia marsecens only. The flavones were further analyzed for quantitative structural activity relationship study (QSAR) by using PASS, online software to determine their Pa value. Toxicity and drug relevant properties were revealed by PALLAS software in terms of their molecular weight. Log P values were also studied. The result showed both the F(1) and F(2) flavones as antigout and therefore supports the development of novel drugs for the treatment of gout.