RESUMEN
Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Uracil-ADN Glicosidasa/metabolismo , Uracilo/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , ADN/química , ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Mutación , Mycobacterium smegmatis/enzimología , Rec A Recombinasas/metabolismo , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Uracil-ADN Glicosidasa/química , Uracil-ADN Glicosidasa/clasificación , Uracil-ADN Glicosidasa/genética , Proteínas Virales/metabolismoRESUMEN
Approximately one third of the world population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. A better understanding of the pathogen biology is crucial to develop new tools/strategies to tackle its spread and treatment. In the host macrophages, the pathogen is exposed to reactive oxygen species, known to damage dGTP and GTP to 8-oxo-dGTP and 8-oxo-GTP, respectively. Incorporation of the damaged nucleotides in nucleic acids is detrimental to organisms. MutT proteins, belonging to a class of Nudix hydrolases, hydrolyze 8-oxo-G nucleoside triphosphates/diphosphates to the corresponding nucleoside monophosphates and sanitize the nucleotide pool. Mycobacteria possess several MutT proteins. However, a functional homolog of Escherichia coli MutT has not been identified. Here, we characterized MtuMutT1 and Rv1700 proteins of M. tuberculosis. Unlike other MutT proteins, MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP, and 8-oxo-GTP to 8-oxo-GDP. Rv1700 then converts them to the corresponding nucleoside monophosphates. This observation suggests the presence of a two-stage mechanism of 8-oxo-dGTP/8-oxo-GTP detoxification in mycobacteria. MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP with a Km of â¼50 µM and Vmax of â¼0.9 pmol/min per ng of protein, and Rv1700 converts 8-oxo-dGDP to 8-oxo-dGMP with a Km of â¼9.5 µM and Vmax of â¼0.04 pmol/min per ng of protein. Together, MtuMutT1 and Rv1700 offer maximal rescue to E. coli for its MutT deficiency by decreasing A to C mutations (a hallmark of MutT deficiency). We suggest that the concerted action of MtuMutT1 and Rv1700 plays a crucial role in survival of bacteria against oxidative stress.
Asunto(s)
Adenosina/metabolismo , Proteínas Bacterianas/metabolismo , Citidina/metabolismo , Desoxiguanosina/análogos & derivados , Mycobacterium tuberculosis/enzimología , Estrés Oxidativo/fisiología , Pirofosfatasas/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Adenosina/genética , Proteínas Bacterianas/genética , Citidina/genética , Desoxiguanosina/genética , Desoxiguanosina/metabolismo , Humanos , Mutación , Mycobacterium tuberculosis/genética , Pirofosfatasas/genética , Homología de Secuencia de Aminoácido , Hidrolasas NudixRESUMEN
A bacterial strain capable of producing extracellular alpha-galactosidase was isolated from sugar cane industrial waste soil sample. Microbiological, physiological, and biochemical studies revealed that isolate belonged to Bacillus sp,. Furthermore, 16S rDNA sequence analysis of new isolates was identified as Bacillus megaterium VHM1. The production of alpha-galactosidase was optimized by various physical culture conditions. Guar gum and yeast extract acted as the best carbon and nitrogen source, respectively for the production of alpha-galactosidase. The enzyme showed an optimum pH at 7.5 and was stable over a pH between 5 and 9. The enzyme was optimally active in 55degreesC and the enzyme was thermostable with half life of 120 minutes at 55 degrees C and lost their 90%, residual activity in 120 minutes at 60 degrees C. alpha-Galactosidase was strongly inhibited by Ag2, Cu2, and Hg2+ at 1mM concentration. The metal ions Fe2, Mn2+, and Mg2+ had no effect on alpha-galactosidase activity, Zn2+,Ni2+, and Ca2+ reduced the enzyme activity slightly. The B megaterium VHM1 enzyme treatment completely hydrolyzed flatulence-causing sugars of soymilk within one and half hour.
Asunto(s)
Antiespumantes/metabolismo , Bacillus megaterium/enzimología , Proteínas Bacterianas/metabolismo , Leche de Soja/química , alfa-Galactosidasa/metabolismo , Antiespumantes/química , Bacillus megaterium/clasificación , Bacillus megaterium/genética , Bacillus megaterium/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Carbohidratos/química , Estabilidad de Enzimas , Flatulencia , Datos de Secuencia Molecular , Filogenia , Saccharum/microbiología , alfa-Galactosidasa/química , alfa-Galactosidasa/genéticaRESUMEN
The aim of this work was to establish optimal conditions for the maximum production of endo-beta-1,4 mannanases using cheaper sources. Eight thermotolerant fungal strains were isolated from garden soil and compost samples collected in and around the Gulbarga University campus, India. Two strains were selected based on their ability to produce considerable endo-beta-1,4 mannanases activity while growing in liquid medium at 37 degrees C with locust bean gum (LBG) as the only carbon source. They were identified as Aspergillus niger gr and Aspergillus flavus gr. The experiment to evaluate the effect of different carbon sources, nitrogen sources, temperatures and initial pH of the medium on maximal enzyme production was studied. Enzyme productivity was influenced by the type of polysaccharide used as the carbon source. Copra meal defatted with n-hexane showed to be a better substrate than LBG and guar gum for endo-beta-1,4 mannanases production by A. niger gr (40.011 U/ml), but for A. flavus gr (33.532 U/ml), the difference was not significant. Endo-beta-1,4 mannanases produced from A. niger gr and A. flavus gr have high optimum temperature (65 and 60 degrees C) and good thermostability in the absence of any stabilizers (maintaining 50% of residual activity for 8 and 6 h, respectively, at 60 degrees C) and are stable over in a wide pH range. These new strains offer an attractive alternative source of enzymes for the food and feed processing industries.
Asunto(s)
Aspergillus flavus/aislamiento & purificación , Aspergillus flavus/metabolismo , Aspergillus niger/aislamiento & purificación , Aspergillus niger/metabolismo , Manosidasas/biosíntesis , Manosidasas/química , Aspergillus flavus/clasificación , Aspergillus niger/clasificación , Inducción Enzimática , Concentración de Iones de Hidrógeno , Manosidasas/metabolismo , Nitrógeno/química , Nitrógeno/farmacología , Estabilidad Proteica , TemperaturaRESUMEN
The treatment of chickpea milk was carried out in batch, repeated batch and continuous reaction by soluble and polyvinyl alcohol (PVA) immobilized Aspergillus oryzae alpha-galactosidase for the removal of raffinose family oligosaccharides (RFOs). In the batch mode of treatment 96 and 92% of RFOs hydrolysis was observed by soluble and immobilized enzyme, respectively. In repeated batch experiments, immobilized enzyme showed 70% RFOs hydrolysis up to sixth cycle. Polyvinyl alcohol immobilized alpha-galactosidase in fluidized bed reactor showed highest reduction of 94% at a flow rate of 30 ml/h. The results obtained from the present study are very interesting for industrial use of PVA-immobilized enzyme.