Improvement of antimicrobial metabolites by Saccharothrix flava VSM-3 using full factorial design and chemotype analysis

Bioactive metabolite production by marine Saccharothrix flava VSM-3was modeled by response surface methodology(RSM) statistical optimization, and kinetic parameter estimation was executed using unstructured models to depict theimportance of growth-associated metabolite production. RSM-based optimization of the variables and their interactionswas analyzed where the modeled data and experimental data are in concurrence and better responses were yieldedin terms of inhibition zones for active metabolite with good regression coefficients. The regression model developedthe significance of five variables and their influence on the bioactive metabolite production and its effect against theresponses. Logistic, Luedeking–Piret equations were used for batch fermentation to produce bioactive metabolites byS. flava VSM-3, where the anticipated parameter data followed experimental data. Chemotype (using ethyl acetateextract) analysis of actinobacterial isolate S. flava was elucidated for the first time by liquid chromatography quadrupoletime-of-flight mass spectrometry (LC-QTOF-MS) analysis. The main compounds identified in the positive ion modewere 7-Deazaadenosine, 5-Hydroxy-9-Methylstreptimidone, Amiclenomycin, Dihydroabikoviromycin, EpopromycinA, OAP Silane 55 and MKN-003B. In the present study, maritime silt specimen of Bay of Bengal comprising S. flavaVSM-3 recorded prominent broad-spectrum activity against various plant pathogens and LC-QTOF-MS data alsosupported VSM-3 was the most active strain. This study also reveals that under-explored Bay of Bengal of northcoastal Andhra Pradesh should be continuously explored for extracting bioactive compounds from diverse strains.

Production of Antimicrobial Metabolites by Streptomyces lavendulocolor VHB-9 Isolated from Granite Mines

ABSTRACT The actinobacterial strain Streptomyces lavendulocolor VHB-9 was isolated from granite mine soil samples of Khammam district, Telangana state, India. The strain was identified based on detailed microorphological, cultural and phylogenetic analysis. Bioactive guided isolation of the secondary metabolites of the strain was carried out by growing the strain in optimized medium (0.5% lactose, 0.5% peptone, 0.05% K2HPO4, 0.2% CaCO3 with pH adjusted to 7.0). Separation and purification of the active fractions from the crude ethyl acetate extract was carried out by silica gel column chromatography and resulted in the isolation of two active fractions. Structural elucidation of the two (B2 and B3) active compounds was carried out by FT-IR, Mass and NMR spectroscopy and were identified as Bis (7-methyloctyl) phthalate and (Z)-3-aminoacrylic acid. The antimicrobial activity of the bioactive compounds produced by S. lavendulocolor VHB-9 was expressed in terms of minimum inhibitory concentration against opportunistic pathogenic bacteria and fungi. Both fractions exhibited good antimicrobial potential against the bacteria and fungi tested.

Antimicrobial profile of Arthrobacter kerguelensis VL-RK_09 isolated from Mango orchards

Abstract An actinobacterial strain VL-RK_09 having potential antimicrobial activities was isolated from a mango orchard in Krishna District, Andhra Pradesh (India) and was identified as Arthrobacter kerguelensis. The strain A. kerguelensis VL-RK_09 exhibited a broad spectrum of in vitro antimicrobial activity against bacteria and fungi. Production of bioactive metabolites by the strain was the highest in modified yeast extract malt extract dextrose broth, as compared to other media tested. Lactose (1%) and peptone (0.5%) were found to be the most suitable carbon and nitrogen sources, respectively, for the optimum production of the bioactive metabolites. The maximum production of the bioactive metabolites was detected in the culture medium with an initial pH of 7, in which the strain was incubated for five days at 30 °C under shaking conditions. Screening of secondary metabolites obtained from the culture broth led to the isolation of a compound active against a wide variety of Gram-positive and negative bacteria and fungi. The structure of the first active fraction was elucidated using Fourier transform infrared spectroscopy, electrospray ionization mass spectrometry, 1H and 13C nuclear magnetic resonance spectroscopy. The compound was identified as S,S-dipropyl carbonodithioate. This study is the first report of the occurrence of this compound in the genus Arthrobacter.

Pullulan-Stabilized Silver Nanoparticles -Their Synthesis, Characterization and Application as Bactericidal Agents.

A rapid method for Pullulan-stabilized silver nanoparticles (PuAgNPs) synthesis has been developed. Different concentrations of Pullulan and Silver nitrate and effects of reaction time, pH was used to investigate the synthesis of silver nanoparticles. The synthesized Pu-AgNPs were first screened and identified using surface plasmon peaks of UV–VIS spectroscopy. The research results indicated that the surface plasmon resonance peaks were observed between 410–460 nm wavelengths in UV-VIS spectroscopy studies. The morphology of the synthesized AgNPs proved a variation in spherical shape and polydispersed with an average size of 10-55 nm, using TEM. Further, five characteristic peaks confirmed the presence of elemental silver and the crystalline structure of silver nanoparticles from XRD analysis. From FTIR spectra, stretching vibrations of hydroxyl (OH), carbonyl (C=O) and C=C stretches exhibits the reduction and stabilization of AgNPs. Further, clear zones of inhibition (about 10-25 mm) against four bacterial pathogens obtained in the antibacterial studies for the synthesized PuAgNPs. The experimental results demonstrated that pullulan could be used as reducing and stabilizing agent for formation of AgNPs and can be used as redoubtable bactericidal agents.

Production of Amylase by Arthrobacter kerguelensis VL-RK_09 Isolated from Mango Orchards.

Objectives: To optimize the cultural parameters for improved production of amylase by Arthrobacter kerguelensis VL-RK_09 isolated from Mango orchards of Vissannapet, Krishna District, A.P., India. Methods: The strain A. kerguelensis was screened initially for amylase production on Inorganic salts starch agar medium (ISP-4). The enzyme assay was performed as per the procedure described by Bernfield (1955). One amylase unit equals to that amount of enzyme needed to release 1 mg of reducing sugar (maltose as standard) for 15 min at 37°C. Attempts were also made to optimize cultural parameters such as pH, temperature, carbon and nitrogen sources affecting the production of amylase by the strain. Results: Maximal yields of amylase were recorded after 4 days of incubation in Inorganic salts starch medium with initial pH 7.0 and temperature 35°C. ISP-4 broth amended with sorghum flour (2%) and yeast extract (0.5%) with initial pH 7.0 inoculated with Arthrobacter kerguelensis VLRK_ 09 and incubated at 30°C for 96 h resulted in improved production of amylase from initial 4.0 U to 10.4 U/mL. Conclusion: This is the first report on the production and optimization of amylase by A. kerguelensis and further studies on purification and characterization of the enzyme are in progress.

Optimization of Process Parameters for Improved Production of Bioactive Metabolites by Streptomyces tritolerans DAS 165T.

Aims: To optimize the process parameters for enhanced production of bioactive metabolites by Streptomyces tritolerans DAS 165T. Place and Duration of Study: Department of Botany and Microbiology, April 2012 to August 2012. Methodology: Agar well diffusion assay was employed to study the effect of environmental parameters such as incubation period, pH, temperature and salt concentration and influence of various nutrients such as carbon and nitrogen sources and minerals on the bioactive metabolite production by Streptomyces tritolerans DAS 165T.  Results: The production of antimicrobial metabolite was high when the strain was cultured for six days at 35ºC in medium (pH 7.5) with sucrose at the concentration of 2% (carbon source), soya peptone at the concentration of 1% (nitrogen source) and sodium chloride at the concentration of 5%.  Conclusion: This is the first report on the optimization of bioactive metabolite production by Streptomyces tritolerans DAS 165T. As the strain exhibited potent antimicrobial activity, it may be explored for biotechnological purposes.

Optimization of Culture Conditions for Enhanced Antimicrobial Activity of Rhodococcus erythropolis VLK-12 Isolated from South Coast of Andhra Pradesh, India.

Aims: To Isolate and characterize the antimicrobial actinomycetes from the marine habitats of south coast of Andhra Pradesh, India. Place and Duration of the Study: Marine habitats of south coast of Andhra Pradesh, India, between June 2011 and July 2012. Methodology: The soil samples were collected, pre-treated and plated on yeast extractmalt extract dextrose agar medium. Identification of the strain was carried out by employing the polyphasic taxonomical studies including the 16S rRNA sequence based analysis. Phylogenetic tree was constructed using the Molecular Evolutionary Genetic Analysis (MEGA) version 5. The influence of culture conditions and the effect of environmental factors on the biomass and antimicrobial activy\ity of the strain was the focus of this study. Results: A total of 20 actinobacteria were isolated from the marine habitats of south coast of Andhra Pradesh, India, and screened for antimicrobial activity against test bacteria and fungi. The potent bioactive metabolite producing strain was designated as VLK-12. Further polyphasic studies revealed that the Isolate VLK-12 belongs to the genera Rhodococcus. Phylogenetic analysis of 16S rRNA sequencing studies revealed that the strain is closely related to Rhodococcus erythropolis. The crude ethyl acetate extract obtained by culturing the strain on YMD inhibited Gram positive and Gram negative bacteria along with fungi. Conclusion: Rhodococcus erythropolis isolated from the marine habitats of south coast of Andhra Pradesh exhibited antimicrobial activity against pathogens.

Optimization of Culturing Conditions for Improved Production of Bioactive Metabolites by Pseudonocardia sp. VUK-10

The purpose of the present study was to investigate the influence of cultural and environmental parameters affecting the growth and bioactive metabolite production of the rare strain VUK-10 of actinomycete Pseudonocardia, which exhibits a broad spectrum of in vitro antimicrobial activity against bacteria and fungi. Production of bioactive metabolites by the strain was high the in modified yeast extract-malt extract-dextrose (ISP-2) broth, as compared to other tested media. Glucose (1%) and tryptone (0.25%) were found to be the most suitable carbon and nitrogen sources, respectively, for optimum production of growth and bioactive metabolites. Maximum production of bioactive metabolites was found in the culture medium with initial pH 7 incubated with the strain for four days at 30degrees C, under shaking conditions. This is the first report on the optimization of bioactive metabolites by Pseudonocardia sp. VUK-10.

Regulation studies of telomerase gene in cancer cells by lentinan

Lentinan a polysaccharide from medicinal mushroom i.e Lentinus, has been known to have anticancer properties. Telomerase activity is not observed in normal healthy cells, whereas in cancerous cells telomerase expression is high. Telomerase represents a promising cancer therapeutic target. We investigated the inhibitory effect of lentinan on telomerase reverse transcriptase gene [hTERT] which is essential for telomerase activity. To assess the transcriptional effect, DLD -1 cancer cells were cultured in the presence of various concentrations of lentinan. TRAP assay, RT-PCR analysis were performed to find telomerase activity and hTERT gene expression respectively. Since C-myc is known to regulate hTERT, expression of C-myc was also determined. Culturing cells with lentinan resulted in down regulation of hTERT and C-myc expression. These results indicate that lentinan inhibits telomerase activity by down regulating hTERT expression via suppression of C-myc in cancer cells