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41.
An indigenously isolated fungal strain Aspergillus flavus MTCC 10938 was subjected to pectin lyase (PNL) production under submerged fermentation conditions. The enzyme was purified to homogeneity from the culture filtrate of the fungus involving concentration by ultrafiltration, anion exchange chromatography on DEAE cellulose and gel filtration chromatography on Sephadex G-100. The purified PNL gave a single protein band in SDS-PAGE analysis with a relative molecular mass corresponding to 50 kDa. Using citrus pectin as the substrate the K m and k cat values of the enzyme were obtained as 1.7 mg/ml and 66 s?1, respectively. The optimum pH of the purified PNL from A. flavus MTCC 10938 was 8.0 and up to 90% of its activity retained in the pH range from 3.0 to 11.0 after 24 h incubation. The optimum temperature of the purified enzyme was revealed at 55°C and it was completely stable up to 40°C when exposed for 30 min. The purified A. flavus MTCC 10938 PNL showed efficient retting of Crotalaria juncea fibres. 相似文献
42.
Arun P. Chandrasekharappa Sangamesh E. Badiger Pramod K. Dubey Sunil K. Panigrahi Sekhar Reddy V.V.V. Manukonda 《Bioorganic & medicinal chemistry letters》2013,23(9):2579-2584
A series of benzoxazole compounds containing oxamic acid were synthesized and screened for the PTP1B inhibition. Compound 31d showed best biochemical potency (Ki) of 6.7 μM. Structure–activity relationship were explained with the help of molecular modeling approach. 相似文献
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Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimensional structure of active procerain B was modeled by homology modeling using X-ray crystal structure of actinidin (PDB ID: 3P5U), a cysteine protease from the fruits of Actinidia arguta. The structural aspect of the enzyme is also discussed. 相似文献
46.
We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology. 相似文献
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Dharmendra Singh Chandan Kumar Singh Ram Sewak Singh Tomar Jyoti Taunk Ranjeet Singh Sadhana Maurya Ashish Kumar Chaturvedi Pal Madan Singh Rajendra Dubey Sarawan Kumar 《PloS one》2016,11(1)
The success of drought tolerance breeding programs can be enhanced through molecular assortment of germplasm. This study was designed to characterize molecular diversity within and between Lens species with different adaptations to drought stress conditions using SSR markers. Drought stress was applied at seedling stage to study the effects on morpho-physiological traits under controlled condition, where tolerant cultivars and wilds showed 12.8–27.6% and 9.5–23.2% reduction in seed yield per plant respectively. When juxtaposed to field conditions, the tolerant cultivars (PDL-1 and PDL-2) and wild (ILWL-314 and ILWL-436) accessions showed 10.5–26.5% and 7.5%–15.6% reduction in seed yield per plant, respectively under rain-fed conditions. The reductions in seed yield in the two tolerant cultivars and wilds under severe drought condition were 48–49% and 30.5–45.3% respectively. A set of 258 alleles were identified among 278 genotypes using 35 SSR markers. Genetic diversity and polymorphism information contents varied between 0.321–0.854 and 0.299–0.836, with mean value of 0.682 and 0.643, respectively. All the genotypes were clustered into 11 groups based on SSR markers. Tolerant genotypes were grouped in cluster 6 while sensitive ones were mainly grouped into cluster 7. Wild accessions were separated from cultivars on the basis of both population structure and cluster analysis. Cluster analysis has further grouped the wild accessions on the basis of species and sub-species into 5 clusters. Physiological and morphological characters under drought stress were significantly (P = 0.05) different among microsatellite clusters. These findings suggest that drought adaptation is variable among wild and cultivated genotypes. Also, genotypes from contrasting clusters can be selected for hybridization which could help in evolution of better segregants for improving drought tolerance in lentil. 相似文献
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Sirakova TD Thirumala AK Dubey VS Sprecher H Kolattukudy PE 《The Journal of biological chemistry》2001,276(20):16833-16839
Multidrug-resistant tuberculosis is a major global health emergency. Cell wall lipids of Mycobacterium tuberculosis can play crucial roles in the pathogenesis. The enzymes involved in their synthesis can be ideal new drug targets against tuberculosis, because many such lipids are unique to this pathogen. A variety of multiple methyl-branched fatty acids are among such unique lipids. We have identified seven genes highly homologous to the mas gene, which is known to be involved in the production of one class of such multiple methyl-branched fatty acids. One of these mas-like genes, pks2, was disrupted using a phage-mediated delivery of the disruption construct. Gene disruption by homologous recombination was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Thin-layer and radio gas-chromatographic analyses of lipids derived from [1-14C]propionic acid and gas chromatography/mass spectrometry analysis of the fatty acids and hydroxy fatty acids showed that the pks2 mutant was incapable of producing hepta- and octamethyl phthioceranic acids and hydroxyphthioceranic acids that are the major acyl constituents of sulfolipids. Consequently, pks2 mutant does not produce sulfolipids. Sulfolipid deficiency in pks2 mutant was confirmed by two-dimensional thin-layer chromatographic analysis of lipids derived from [1-14C]propionic acid and 35SO4(-2). With this sulfolipid-deficient mutant, it should be possible to test for the postulated important roles for sulfolipids in the pathogenesis of M. tuberculosis. 相似文献