Chemoenzymatic Total Synthesis of (R)-4-Dodecanolide and (R)-4-Octanolide.

A simple and efficient chemoenzymatic total synthesis of the naturally occurring insect pheromones, (4R)-dodecanolide and (4R)-octanolide is described.

Evaluation of antibody response in mice against avian influenza A (H5N1) strain neuraminidase expressed in yeast Pichia pastoris.

Avian influenza has raised many apprehension in the recent years because of its potential transmitability to humans. With the increasing emergence of drug-resistant avian influenza strains, development of potential vaccines are imperative to manage this disease. Two structural antigens, haemagglutinin and neuraminidase, have been the target candidates for the development of subunit vaccine against influenza. In an effort to develop a faster and economically beneficial vaccine, the neuraminidase gene of a highly pathogenic avian influenza isolate was cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant neuraminidase (rNA) antigen was purified, and its bioactivity was analysed. The rNA was found to be functional, as determined by the neuraminidase assay. Four groups of mice were immunized with different concentrations of purified rNA antigen, which were adjuvanted with aluminium hydroxide. The immune response against rNA was analysed by enzyme-linked immunosorbent assay (ELISA) and neuraminidase inhibition assay. The mice groups immunized with 25 (mu) g and 10 (mu) g of antigen had a significant immune response against rNA. This method can be utilized for faster and cost-effective development of vaccines for a circulating and newer strain of avian influenza, and would aid in combating the disease in a pandemic situation, in which production time matters greatly.

Recovery and purification of rapamycin from the culture broth of Mtcc 5681.

In this study of the recovery and purification of rapamycin from the culture broth of an actinomycetes strain MTCC 5681, we investigated various factors such as biomass separation, suitable solvents for extraction, normal phase and flash chromatographic conditions and selective precipitation to obtain rapamycin in substantially pure form of the product. Adsorption chromatography particularly with normal phase and flash chromatography, in combination with centrifugal decantation is found to be the most suitable for separation as well as purification of rapamycin. Centrifugal decantation technique is likely to emerge as an efficient, industrially scalable, high yielding and economical process for biomass separation. The purity of rapamycin obtained through the method described was 99.4% which has not been reported so far.

Improvement of microbial strain and fermentation process of rapamycin biosynthesis.

The purpose of this investigation is to enhance the production of the immunosuppressant drug rapamycin by subjecting the strain CBS 773.23 to ultraviolet (UV) and N-methyl-N'-nitro-N-nitroso guanidine (NTG) mutations. Among all the mutants tested, MTCC 5681 (NRC-CM03/SH) obtained by NTG mutagenesis of strain CBS 773.72 showed the highest activity, 210 mg/L. The effect of different factors including medium composition, pH, temperature, and intensity of mixing on rapamycin production was studied. Based on the study, the optimal concentrations of soluble starch and dry yeast granules were found to be 50 g/L and 1.5 g/L, respectively. Furthermore, optimal values for pH, temperature, and shaking speed were found to be 6.0, 28°C, and 220 rpm, respectively. The production of rapamycin increased 1.6-fold, to 360 mg/L, in shake-flask culture using the optimal combination of factors observed compared with basal cultivation medium using MTCC 5681 mutant strain.

Molecular characterization of α-amylase from Staphylococcus aureus.

Staphylococcus aureus is one of the prominent Gram positive human pathogen secretes many surface and secretary proteins including various enzymes and pathogenic factors that favour the successful colonization and infection of host tissue. α-amylase is one of the enzymes secreted by S. aureus which catalyses the breakdown of complex sugars to monosaccharides, which are required for colonization and survival of this pathogen in any anatomical locales. In the present study we have cloned, sequenced, expressed and characterized α-amylase gene from S. aureus ATCC12600. The recombinant enzyme has a molecular weight of 58kDa and the kinetics showed Vmax 0.0208±0.033 (mg/ml)/mg/min and Km 10.633±0.737mg/ml. The multiple sequence analysis showed α- amylase of S. aureus exhibited large differences with Bacillus subtilis and Streptococcus bovis. As the crystal structure of S. aureus α- amylase was unavailable, we used homology modelling method to build the structure. The built structure was validated by Ramachandran plot which showed 90% of the residues in the allowed region while no residue was found in the disallowed region and the built structure was close to the crystal structure with Z-Score: -6.85. The structural superimposition studies with α- amylases of Bacillus subtilis and Streptococcus bovis showed distinct differences with RMSD values of 18.158Åand 7.091Å respectively which correlated with enzyme kinetics, indicating α-amylase is different among these bacteria.

Cloning, expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus.

Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound role in the pathogenesis.

Pearl millet [Pennisetum glaucum (L.) R. Br.] consensus linkage map constructed using four RIL mapping populations and newly developed EST-SSRs.

BACKGROUND: Pearl millet [Pennisetum glaucum (L.) R. Br.] is a widely cultivated drought- and high-temperature tolerant C4 cereal grown under dryland, rainfed and irrigated conditions in drought-prone regions of the tropics and sub-tropics of Africa, South Asia and the Americas. It is considered an orphan crop with relatively few genomic and genetic resources. This study was undertaken to increase the EST-based microsatellite marker and genetic resources for this crop to facilitate marker-assisted breeding. RESULTS: Newly developed EST-SSR markers (99), along with previously mapped EST-SSR (17), genomic SSR (53) and STS (2) markers, were used to construct linkage maps of four F7 recombinant inbred populations (RIP) based on crosses ICMB 841-P3 × 863B-P2 (RIP A), H 77/833-2 × PRLT 2/89-33 (RIP B), 81B-P6 × ICMP 451-P8 (RIP C) and PT 732B-P2 × P1449-2-P1 (RIP D). Mapped loci numbers were greatest for RIP A (104), followed by RIP B (78), RIP C (64) and RIP D (59). Total map lengths (Haldane) were 615 cM, 690 cM, 428 cM and 276 cM, respectively. A total of 176 loci detected by 171 primer pairs were mapped among the four crosses. A consensus map of 174 loci (899 cM) detected by 169 primer pairs was constructed using MergeMap to integrate the individual linkage maps. Locus order in the consensus map was well conserved for nearly all linkage groups. Eighty-nine EST-SSR marker loci from this consensus map had significant BLAST hits (top hits with e-value ≤ 1E-10) on the genome sequences of rice, foxtail millet, sorghum, maize and Brachypodium with 35, 88, 58, 48 and 38 loci, respectively. CONCLUSION: The consensus map developed in the present study contains the largest set of mapped SSRs reported to date for pearl millet, and represents a major consolidation of existing pearl millet genetic mapping information. This study increased numbers of mapped pearl millet SSR markers by >50%, filling important gaps in previously published SSR-based linkage maps for this species and will greatly facilitate SSR-based QTL mapping and applied marker-assisted selection programs.

Polyphenolics profile, antioxidant and radical scavenging activity of leaves and stem of Raphanus sativus L.

Aerial parts (leaves and stem) of Raphanus sativus, which are usually discarded were found to possess potent antioxidant and radical scavenging activity, as measured by standard antioxidant assays. Methanolic and acetone extracts of R. sativus leaves had total polyphenolic content of 86.16 and 78.77 mg/g dry extract, which were comparable to the traditional rich sources such as green tea and black tea. HPLC identification of polyphenolics indicated the presence of catechin, protocatechuic acid, syringic acid, vanillic acid, ferulic acid, sinapic acid, o-coumaric acid, myricetin, and quercetin in leaves and stem. Among the different extraction solvents, methanolic extract of leaves and stem showed potent reductive capacity, significantly inhibited linoleic acid peroxidation and displayed metal chelating activity. Further, they scavenged free radicals effectively with IC50 (half maximal inhibitory concentration) of 31 and 42 microg/ml for DPPH radical, 23 and 52 microg/ml for superoxide radical, 67 and 197 microg/ml for hydrogen peroxide,and 56 and 62 microg/ml for nitric oxide, respectively. Leaves showed most potent antioxidant and radical scavenging activity as compared to stem, which may be accounted for the high polyphenolic content. Leaves and stem of R. sativus,often under-utilized part of this vegetable, thus possessed considerable amount of polyphenolics. Hence, it should be egarded as a potential source of natural antioxidants and could be effectively employed as an ingredient in health or in functional food.

Effect of FMD vaccine antigen payload on protection, sub-clinical infection and persistence following needle challenge in sheep.

The relationship of Foot-and-Mouth Disease (FMD) virus antigen payload and single and double vaccinations in conferring protection against virus challenge in sheep was studied. Sheep vaccinated with half the cattle dose (1 ml) containing 15 and 3.75 µg of FMDV antigen with or without booster resisted virulent challenge on 21 days post vaccination or 7 days post booster. FMDV RNA could be detected in nasal secretions in 26% of vaccinated sheep (10(3.12) to 10(3.82) viral RNA copies) on day 35 post challenge. No live virus could be isolated after 5 days post challenge indicating that the risk of transmission of disease was probably very low. The finding showed that vaccines containing antigen payload of 1.88 µg may prevent or reduce the local virus replication at the oropharynx and shedding of virus from nasal secretions and thereby reduce the amount of virus released into the environment subsequent to exposure to live virus. Sheep with no vaccination or with poor sero conversion to vaccination can be infected without overt clinical signs and became carriers.

Cloning, high expression and purification of recombinant human intereferon-beta-1b in Escherichia coli.

Sequential evaluation and process control strategy were employed for impurity profile and high recovery with quality of rhIFN-beta-1b expressed in Escherichia coli. The high-level expression was achieved by using codon substitution (AT content of 52.6% at N-terminal region) and optimization of culture conditions. The addition of rifampicin at a concentration of 200 microg/ml has increased the specific product yield of 66 mg optical density(-1) l(-1) (43.5% of total cellular protein). Eighty-three percent of lipopolysaccharides, 32% of host deoxyribonucleic acid (DNA), and 78% of host cell proteins were removed by 0.75% Triton X-100 and 2 M urea wash. Eleven percent of lipopolysaccharides, 39% of host DNA, and 12% of host cell proteins were removed at the solubilization step. Ninety-two percent of protein refolding was achieved by high-pressure diafiltration method. Refolding by high-pressure diafiltration, bed height, and height equivalent to the theoretical plate value in chromatography column were identified as key parameters for high recovery with purity. Finally, the established process yielded 34% of purified protein with greater than 99% purity and is acceptable for preclinical toxicological studies. The purified rhIFN-beta-1b obtained in this study is the highest that has been reported so far.

Hepatoprotective activity of Sapindus mukorossi and Rheum emodi extracts: in vitro and in vivo studies.

AIM: To study the hepatoprotective capacity of Sapindus mukorossi (S. mukorossi) and Rheum emodi (R. emodi) extracts in CCl(4) treated male rats. METHODS: The dried powder of S. mukorossi and R. emodi was extracted successively with petroleum ether, benzene, chloroform, and ethanol and concentrated in vacuum. Primary rat hepatocyte monolayer cultures were used for in vitro studies. In vivo, the hepatoprotective capacity of the extract of the fruit pericarp of S. mukorossi and the rhizomes of R. emodi was analyzed in liver injured CCl(4)-treated male rats. RESULTS: In vitro: primary hepatocytes monolayer cultures were treated with CCl(4) and extracts of S. mukorossi & R. emodi. A protective activity could be demonstrated in the CCl(4) damaged primary monolayer culture. In vivo: extracts of the fruit pericarp of S. mukorossi (2.5 mg/mL) and rhizomes of R. emodi (3.0 mg/mL) were found to have protective properties in rats with CCl(4) induced liver damage as judged from serum marker enzyme activities. CONCLUSION: The extracts of S. mukorossi and R. emodi do have a protective capacity both in vitro on primary hepatocytes cultures and in in vivo in a rat model of CCl(4) mediated liver injury.

A purification method for improving the process yield and quality of recombinant human granulocyte colony-stimulating factor expressed in Escherichia coli and its characterization.

A purification method employing a process-control strategy was developed for improving the yield of rhG-CSF (recombinant human granulocyte colony-stimulating factor). A purity of >/=99% with an overall yield of 2.18 g/l was achieved in the present study. Analysis of the product during purification indicated that detergents removed 72% of LPS (lipopolysaccharides) and 98% of HCPs (host cell proteins) without removing nucleic acid. Cysteine concentration was a key parameter in protein refolding. The bed height and HETP (height equivalent theoretical plates) value in the SEC (size-exclusion chromatography) column was evaluated and its impact on the resolution was studied. Formulation during SEC was found to be crucial for increasing the product yields with saving of time and process costs. The yield obtained in the present study is nearly four times higher than that reported in the literature. The product obtained was found to be acceptable for toxicological studies.