[Show abstract][Hide abstract] ABSTRACT: Chitobiase (CHB) is an important enzyme for the production of N-acetyl-D-glucosamine from the chitin biopolymer in the series of chitinolytic enzymes. Majority of over-expressed CHB (58 %) in E. coli expression system led to formation of inclusion bodies. The production and soluble yield of active CHB was enhanced by co-expression with GroEL/ES chaperonin, optimizing culture conditions and solubilization followed by refolding of remaining inactive chitobiase present in the form of inclusion bodies. The growth of recombinant E. coli produced 42 % CHB in soluble form and the rest (~58 %) as inclusion bodies. The percentage of active CHB was enhanced to 71 % by co-expression with GroEL/ES chaperonin system and optimizing culture conditions (37 °C, 200 rpm, IPTG-0.5 mM, L-arabinose-13.2 mM). Of the remaining inactive CHB present in inclusion bodies, 37 % could be recovered in active form using pulsatile dilution method involving denaturants (2 M urea, pH 12.5) and protein refolding studies (1.0 M L-arginine, 5 % glycerol). Using combinatorial approach, 80 % of the total CHB expressed, could be recovered from cells grown in one litre of LB medium is a step forward in replacing hazardous chemical technology by biotechnological process for the production of NAG from chitinous waste.
Full-text · Article · Jan 2016 · The Protein Journal
[Show abstract][Hide abstract] ABSTRACT: A series of chalconyl blended triazole allied silatranes (7a-g/8a-g/9a-g) were synthesized in good yields using a simple, economical and biocompatible synthetic route. The blend of three different pharmacologically active moieties into a single scaffold resulted into synergistic effect in their bio-activity. Various substitutions were tried to study the structure activity relationship (SAR) of the synthesized compounds on the basis of biological results. All the newly synthesized compounds were well characterized by IR, (1)H and (13)C NMR, low resolution mass spectroscopy and elemental analysis. The structures of 7a and 7c were authenticated by single crystal X-ray crystallography. These compounds were screened by using Molinspiration software for their physicochemical properties and all the compounds showed good oral bioavailability. The antiparasitic activity of the newly synthesized compounds was evaluated against unicellular parasites (Giardia lamblia and Trichomonas vaginalis) in comparison to standard drug (metronidazole) by 3-(4,5-dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) assay. All the compounds displayed significant activity against G. lamblia and T. vaginalis with IC50 values ranging from 19.58-131.2 μM to 18.24-101.26 μM respectively. The entire library of compounds was found to be more active than metronidazole except 9a, 9f and 9g. Notably, 9e and 7e were found to be most significant against G. lamblia and T. vaginalis respectively.
No preview · Article · Dec 2015 · European Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: To control the infection caused by a multi-drug resistant bacterial pathogen, Acinetobacter baumannii, antimicrobial peptides (AMPs) are being considered as a viable option because of their broad range of antimicrobial activity and non-specific mode of action. However, high cost of synthesis of AMPs has led to the development of several computational tools to predict the biological and physicochemical properties of AMPs. In the present study, a comparative analysis has been done between in vitro activity of seven anti-Acinetobacter α-helical peptides with in silico prediction tools for studying antimicrobial related properties of AMPs. Database of Antimicrobial Activity and Structure of Peptides (DBAASP) and two algorithms, Support Vector Machine (SVM) and Random Forest (RF) of another server, Collection of Antimicrobial Peptides (CAMP) were compared with in vitro results of AMP/non-AMP nature of anti-Acinetobacter peptides. The results of the influence of biophysical properties of peptides on their antimicrobial activity suggested that amphipathicity is more important than spatial arrangement and charge of α-helical peptides. No correlation was observed between the MIC50 values and the hydropathy of AMPs. Based on the present study, it is suggested that with further refinements in the available servers/algorithms for correctly predicting the antimicrobial nature of the peptides, in silico tools can be used to assist the development of new antimicrobial agents.
No preview · Article · Oct 2015 · Journal of Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: In view of the emergence of multidrug-resistant cancer cells, there is a need for therapeutic alternatives. Keeping this in mind, the present study was aimed at evaluating the synergism between nisin (an antimicrobial peptide) and doxorubicin (DOX) against DMBA-induced skin carcinogenesis. The possible tumoricidal activity of the combination was evaluated in terms of animal bioassay observations, changes in hisotological architecture of skin tissues, in situ apoptosis assay (TUNEL assay) and in terms of oxidant and antioxidant status of the skin tissues. In vivo additive effect of the combination was evidenced by larger decreases in mean tumour burden and tumour volume in mice treated with the combination than those treated with the drugs alone. Histological observations indicated that nisin-DOX therapy causes chromatin condensation and marginalisation of nuclear material in skin tissues of treated mice which correlated well with the results of TUNEL assay wherein a marked increase in the rate of apoptosis was revealed in tissues treated with the combination. A slightly increased oxidative stress in response to the adjunct therapy as compared to dox-alone-treated group was revealed by levels of lipid peroxidation (LPO) and nitrite generation in skin tissue-treated mice. An almost similar marginal enhancement in superoxide dismutase levels corresponding with a decrease in catalase activity could also be observed in nisin + DOX-treated groups as compared to nisin and dox-alone-treated groups. These results point towards the possible use of nisin as an adjunct to doxorubicin may help in developing alternate strategies to combat currently developing drug resistance in cancer cells.
[Show abstract][Hide abstract] ABSTRACT: Stenotrophomonas maltophilia is an emerging nosocomial pathogen responsible for serious human infections. This study was carried out to determine antibiotic susceptibility, resistance mechanisms (integrons, sul1 and sul2), and genetic relatedness (Enterobacterial Repetitive Intergenic Consensus [ERIC]-PCR) among 106 clinical isolates of S. maltophilia from India. Twenty-four (22.6%) of S. maltophilia isolates exhibited resistance to mainstay antibiotic trimethoprim-sulfamethoxazole (TMP-SMX). Except for 2 isolates which contained both TMP-SMX resistance determinants sul1 and sul2 genes, all other 22 TMP-SMX-resistant isolates carried either sul1 (10 isolates) or sul2 (12 isolates) genes. Class 1 integrons were present in 8.5% (9 out of 106) of S. maltophilia isolates, and only 5 out of these isolates were TMP-SMX resistant and positive for sul1 gene. The same isolates also carried resistance cassettes containing qac/smr gene. Minocycline and levofloxacin exhibited the maximum in vitro activity against S. maltophilia. ERIC-PCR revealed high diversity among S. maltophilia isolates. The present study demonstrated high (22.4%) TMP-SMX resistance in clinical isolates of S. maltophilia from India. TMP-SMX-resistant isolates carried relatively higher percentage of sul2 gene than sul1 gene as against the reported literature. Majority (58.3%) of sul1 gene positive were not associated with class 1 integrase gene.
No preview · Article · Mar 2015 · Microbial drug resistance (Larchmont, N.Y.)
[Show abstract][Hide abstract] ABSTRACT: An efficient method for C-N bond formation via aza-Michael addition of γ-aminopropylsilatranes to substituted N-phenylmaleimides under mild conditions is described. The five receptors enclosing the maleimide moiety have been synthesized by the aza-Michael addition to strong nucleophile γ-aminopropylsilatrane producing silatranes possessing substituted N-phenylsuccinimide at the axial position without any catalyst. All the compounds have been characterized by elemental analysis, spectroscopic techniques and thermo-gravimetric analysis. The synthesized compounds have been screened for possible antimicrobial properties and results have shown modest activity.
[Show abstract][Hide abstract] ABSTRACT: Intracellular lipase producer screened from the library available in the laboratory, identified through 16S rRNA as Pseudomonas stutzeri, was studied for maximum enzyme production in shake flask. The work was intended to evaluate the effect of different physicochemical factors like carbon, nitrogen, metal ions, surfactant, inoculum, pH, temperature, agitation, and aeration on lipase production. Optimized media showed 1.62-fold increase in lipase production when compared to basal media. Scale-up of lipase in in situ bioreactor showed reduction in fermentation time in both basal and optimized media, giving 41 and 99 U/mg of lipase activity after 48 h of fermentation.
[Show abstract][Hide abstract] ABSTRACT: A nitrile-hydrolysing bacterium, identified as Isoptericola variabilis RGT01, was isolated from industrial effluent through enrichment culture technique using acrylonitrile as the carbon source. Whole cells of this microorganism exhibited a broad range of nitrile-hydrolysing activity as they hydrolysed five aliphatic nitriles (acetonitrile, acrylonitrile, propionitrile, butyronitrile and valeronitrile), two aromatic nitriles (benzonitrile and m-Tolunitrile) and two arylacetonitriles (4-Methoxyphenyl acetonitrile and phenoxyacetonitrile). The nitrile-hydrolysing activity was inducible in nature and acetonitrile proved to be the most efficient inducer. Minimal salt medium supplemented with 50 mM acetonitrile, an incubation temperature of 30 °C with 2 % v/v inoculum, at 200 rpm and incubation of 48 h were found to be the optimal conditions for maximum production (2.64 ± 0.12 U/mg) of nitrile-hydrolysing activity. This activity was stable at 30 °C as it retained around 86 % activity after 4 h at this temperature, but was thermolabile with a half-life of 120 min and 45 min at 40 °C and 50 °C respectively.
No preview · Article · Jun 2014 · Indian Journal of Microbiology
[Show abstract][Hide abstract] ABSTRACT: Optimization of cultural conditions for enhanced cellulase production by Aspergillus niger NS-2 were studied under solid-state fermentation. Significant increase in yields (CMCase 463.9 ± 20.1 U/g, FPase 101.1 ± 3.5 U/g and β-glucosidase 99 ± 4.0 U/g) were obtained under optimized conditions. Effect of different nutritional parameters was studied to induce the maximum production of cellulase complex. Scale-up studies for enzyme production process were carried out. Characterization studies showed that enzymes produced by A. niger NS-2 were highly temperature- and pH stable. At 50 °C, the half life for CMCase, FPase, β-glucosidase were approximately 240 h. Cellulases from A. niger NS-2 were stable at 35 °C for 24 h over a broader pH range of 3.0-9.0. We examined the feasibility of using steam pretreatment to increase the saccharification yields from various lignocellulosic residues for sugar release which can potentially be used in bioethanol production. Saccharification of pretreated dry potato peels, carrot peels, composite waste mixture, orange peels, onion peels, banana peels, pineapple peels by crude enzyme extract from A. niger NS-2, resulted in very high cellulose conversion efficiencies of 92-98 %.
No preview · Article · Sep 2013 · Applied biochemistry and biotechnology
[Show abstract][Hide abstract] ABSTRACT: The emergence of multi-drug-resistant bacterial
pathogens along with labor-intensive and time-consuming
drug discovery methods has put an emphasis on alternative
approaches which can overcome these drawbacks. One such
approach is in silico identification of novel bacterial inhibitors
using structure-based drug design methodology. In the
present study, novel inhibitors have been proposed against
two essential enzymes (MurA and MurZ) involved in the
biosynthesis of cell wall of Enterococcus faecalis, a serious
nosocomial bacterial pathogen. Homology models of MurA
and MurZ of E. faecalis were constructed using template
MurA of Enterobacter cloacae and Haemophilus influenza,
respectively. The validation of the refined models, using
Ramachandran, Errat and ProSA energy plots, showed that
constructed models were reliable and of good quality. The
active site amino acid residues, as predicted by CASTp
server, were in accordance with the residues identified from
MurA crystal structures available for other bacteria. The
present study revealed for the first time the importance of two
additional amino acid residues (Thr305 and Tyr329) in
MurA and MurZ enzymes, which might be crucial for the
binding of ligands/inhibitors with the target. Based on the
GLIDE score and interaction profile, 5-sulfonoxyanthranilic acid derivatives, T6361 and T6362 were found to
be the potential inhibitors of MurA and MurZ enzymes.
Full-text · Article · Sep 2013 · Medicinal Chemistry Research
[Show abstract][Hide abstract] ABSTRACT: Emergence of the multidrug-resistant pathogens has rendered the current therapies ineffective thereby, resulting in the need for new drugs and drug targets. The accumulating protein sequence data has initiated a drift from classical drug discovery protocols to structure-based drug designing. In the present study, in silico subtractive genomics approach was implemented to find a set of potential drug targets present in an opportunist bacterial pathogen, Acinetobacter baumannii (A. baumannii). Out of the 43 targets identified, further studies for protein model building and lead-inhibitor identification were carried out on two cell-essential targets, MurA and MurB enzymes (of A. baumannii designated as MurAAb and MurBAb) involved in the peptidoglycan biosynthesis pathway of bacteria. The homology model built for each of them was further refined and validated using various available programs like PROCHECK, Errat, ProSA energy plots, etc. Compounds showing activity against MurA and MurB enzymes of other organisms were collected from the literature and were docked into the active site of MurAAb and MurBAb enzymes. Three inhibitors namely, T6361, carbidopa, and aesculin, showed maximum Glide score, hydrogen bonding interactions with the key amino acid residues of both the enzymes and acceptable ADME properties. Furthermore, molecular dynamics simulation studies on MurAAb-T6361 and MurBAb-T6361 complexes suggested that the ligand has a high binding affinity with both the enzymes and the hydrogen bonding with the key residues were stable in the dynamic condition also. Therefore, these ligands have been propsed as dual inhibitors and promising lead compounds for the drug design against MurAAb and MurBAb enzymes.
[Show abstract][Hide abstract] ABSTRACT: Loop-mediated isothermal amplification (LAMP) assay has come forward as a rapid, cost-effective molecular technique for diagnosis of tuberculosis (TB) in developing countries. This study evaluated Mycobacterium tuberculosis-specific in-house LAMP assay targeting 16s rRNA and compared it with other conventional tests and nucleic acid amplification assay (IS6110 PCR).
A total of 133 sputum specimens (103 from suspected pulmonary TB cases and 30 from non-TB controls) were subjected to conventional tests, IS6110 PCR and 16s rRNA LAMP assay.
Of the 103 patients, the maximum number of cases were found to be positive by LAMP assay, that is, in 87 (84.5%) patients, followed by culture positive in 78 (75.7%), IS6110 PCR in 74 (71.8%), and smear positive in 70 (67.9%) patients. Of the 83 smear positive and/or culture positive cases, LAMP detected 77 (92.77%) cases, and was found to be superior to IS6110 PCR, which could detect 69 (83.1%) cases; a concordance of 0.6 was obtained between the two tests using kappa statistics.
Overall, LAMP was simple and efficacious for early diagnosis of smear positive, culture positive cases as well as for confirmation of smear negative, culture negative cases, and was found to be superior to IS6110 PCR.
No preview · Article · Jul 2013 · Journal of Clinical Laboratory Analysis
[Show abstract][Hide abstract] ABSTRACT: Phosphate solubilizing bacteria (PSB) are known to convert the insoluble forms of phosphate to soluble one and make them available for plant uptake. The present study aimed to isolate PSB from the rhizosphere of chickpea (Cicer arietinum L. cv. GPF2) and examine their effect on the growth and seed number. The isolated PSB were analyzed for phosphate solubilization, indole acetic acid and siderophore production. PSB were characterized for phenotypic and biochemical properties, BIOLOG and whole-cell fatty acid methyl ester profile and found to be closely related to Pantoea cypripedii and Enterobacter aerogenes based on 16s rRNA gene sequencing. A high increase in growth of C. arietinum was observed when innoculated with PSB in tricalcium phosphate amended soils. A higher uptake in total P (53 %) of plants was observed when inoculated with mixture of P. cypripedii and E. aerogenes along with Rhizobium ciceri as compared to respective control plants which significantly increased the seed number (98.3 %) and seed weight (46.1 %). This study demonstrated the ability of novel PSB P. cypripedii along with E. aerogenes and R. ciceri to promote chickpea growth.
No preview · Article · May 2013 · Plant Growth Regulation
[Show abstract][Hide abstract] ABSTRACT: Background
Mycobacterium tuberculosis (M.tb) is the causative agent of tuberculosis, killing ~1.7 million people annually. The remarkable capacity of this pathogen to escape the host immune system for decades and then to cause active tuberculosis disease, makes M.tb a successful pathogen. Currently available anti-mycobacterial therapy has poor compliance due to requirement of prolonged treatment resulting in accelerated emergence of drug resistant strains. Hence, there is an urgent need to identify new chemical entities with novel mechanism of action and potent activity against the drug resistant strains.
This study describes novel computational models developed for predicting inhibitors against both replicative and non-replicative phase of drug-tolerant M.tb under carbon starvation stage. These models were trained on highly diverse dataset of 2135 compounds using four classes of binary fingerprint namely PubChem, MACCS, EState, SubStructure. We achieved the best performance Matthews correlation coefficient (MCC) of 0.45 using the model based on MACCS fingerprints for replicative phase inhibitor dataset. In case of non-replicative phase, Hybrid model based on PubChem, MACCS, EState, SubStructure fingerprints performed better with maximum MCC value of 0.28. In this study, we have shown that molecular weight, polar surface area and rotatable bond count of inhibitors (replicating and non-replicating phase) are significantly different from non-inhibitors. The fragment analysis suggests that substructures like hetero_N_nonbasic, heterocyclic, carboxylic_ester, and hetero_N_basic_no_H are predominant in replicating phase inhibitors while hetero_O, ketone, secondary_mixed_amine are preferred in the non-replicative phase inhibitors. It was observed that nitro, alkyne, and enamine are important for the molecules inhibiting bacilli residing in both the phases. In this study, we introduced a new algorithm based on Matthews correlation coefficient called MCCA for feature selection and found that this algorithm is better or comparable to frequency based approach.
In this study, we have developed computational models to predict phase specific inhibitors against drug resistant strains of M.tb grown under carbon starvation. Based on simple molecular properties, we have derived some rules, which would be useful in robust identification of tuberculosis inhibitors. Based on these observations, we have developed a webserver for predicting inhibitors against drug tolerant M.tb H37Rv available at http://crdd.osdd.net/oscadd/mdri/.
Full-text · Article · Mar 2013 · Chemistry Central Journal
[Show abstract][Hide abstract] ABSTRACT: Aims:
In view of the changing sensitivity pattern of Salmonella, this study was carried out to investigate the current antibiogram and clonal relatedness among the drug-resistant Salmonella enterica serovar Typhi in Northern India.
Re-emergence in the sensitivity to chloramphenicol and cotrimoxazole with increase in the resistance to various quinolones has been observed. The efficacy of nalidixic acid resistance to be used as a marker for decrease in sensitivity to ciprofloxacin may vary. Clonal differences on the basis of amplification of repetitive elements (rep-PCR) were found in the two different areas within the same region.
The present study creates a paramount baseline for rationalizing the judicious use of quinolones and re-examine the use of chloramphenicol and cotrimoxazole. Molecular analysis of clinical Salmonella isolates depicts the clonal expansion of the isolates that may serve as a reference to which newer outbreak strains can be compared. rep-PCR techniques may be useful in molecular discrimination of isolates for better understanding of the Salmonella epidemiology and as a basis for development of rational control strategies.
No preview · Article · Jan 2013 · Microbial drug resistance (Larchmont, N.Y.)
[Show abstract][Hide abstract] ABSTRACT: Like any other enteric pathogen, Salmonella also encounters acidic stress in the stomach as well as within the host macrophage milieu. However, the pathogen is reported to combat this stress through acid tolerance response (ATR), expressing a number of genes and eventually the proteins. Recently, an acid induced outer membrane phenotype encoded by fliC gene in Salmonella enterica serovar Typhi has been identified. In the present study, fliC gene was cloned to study its biological implications. The recombinant FliC (rFliC) protein was observed to stimulate the production of antibodies. These antibodies could also recognize the FliC protein (antigen) in the clinical samples i.e. blood samples from typhoid patents as well as healthy blood samples spiked with serovar Typhi. Moreover, the rFliC also reacted with the sera from patients suffering with typhoid fever indicating its in-vivo immunogenicity. Ex-vivo study revealed that rFliC has the potential to stimulate the macrophages to generate higher levels of inflammatory mediators such as malondialdehyde (MDA) and nitrite. The inflammatory potential of FliC was also confirmed in-vivo, by the paw oedema test as well as by flicking response of the inflamed paw indicating hyperalgesia occurring during inflammatory response. The findings of the present study indicate that acid induced FliC might be one of the factors enhancing the virulence of serovar Typhi under the host acidic conditions and may prove to be helpful in designing the prophylactic measures.
[Show abstract][Hide abstract] ABSTRACT: Restriction fragment length polymorphism (RFLP) based on IS6110 is considered the gold standard for Mycobacterium tuberculosis molecular typing. It is useful to discriminate among M. tuberculosis strains, investigate outbreaks and distinguish between reactivation and re-infection. We studied polymorphisms among M. tuberculosis isolates from northern India using RFLP to determine the presence of a correlation between IS6110 based fingerprints and drug resistance and to look for relapse and transmission among patients and their contacts. RFLP patterns of PvuII digested genomic DNA of 100 M. tuberculosis isolates were analyzed using southern blotting with a 245 bp IS6110 probe. Drug sensitivity testing (DST) was conducted for rifampicin (40 microg/ml), isoniazid (1 microg/ml), ethambutol (2 microg/ml) and streptomycin (4 microg/ml) using the proportion method. A high degree of polymorphism was seen among the M. tuberculosis isolates and the number of IS6110 copies varied from 0 to 14, with a predominance of isolates with 11 bands. Seventy-five isolates had a high number of bands, 9 had an intermediate number, 6 isolates had a low number and 10 isolates had no bands. No correlation between IS6110 band numbers and RFLP banding patterns was found with drug resistance or for any particular geographical area, although clustering was seen amongst MDR-TB cases. No cases of relapses or transmissions were seen.
No preview · Article · Sep 2012 · The Southeast Asian journal of tropical medicine and public health
[Show abstract][Hide abstract] ABSTRACT: Background:
The diagnosis of pulmonary tuberculosis is still a major challenge. Using a polymerase chain reaction (PCR), one can detect Mycobacterium tuberculosis in clinical samples within a few hours. However, single gene targets may result in false negativity due to the absence of target DNA in some M. tuberculosis isolates. The objective of this study was to develop and evaluate a multiplex PCR (M-PCR) using IS6110 and devR primers for the detection of M. tuberculosis in sputum samples.
Sputum samples were collected from: (1) 200 confirmed cases of tuberculosis; (2) 100 suspected cases of tuberculosis diagnosed on the basis of clinical and radiological findings; (3) 200 non-tubercular patients suffering from respiratory diseases other than tuberculosis, in whom tuberculosis had been excluded. All 500 sputum samples were subjected to PCR using IS6110 primers, and M-PCR using IS6110 and devR primers; results were compared with conventional techniques.
It was found that M-PCR was 97.5% successful in detecting the presence of tuberculosis in the confirmed tuberculosis group as compared to 84.5% by IS6110-based PCR. In the suspected tuberculosis group, M-PCR could detect 45% of cases as compared to 40% by IS6110-based PCR. Overall, the specificities of both the PCR and M-PCR were found to be 96.5%.
This study demonstrated that the M-PCR assay is more sensitive than the IS6110-based PCR for the detection of M. tuberculosis in sputum specimens and could be applied in situations of highly suspected tuberculosis when all others tests including IS6110 PCR are negative.
No preview · Article · Jun 2012 · Scandinavian Journal of Infectious Diseases