Correction to: Extraction and bioactive profile of the compounds produced by Rhodococcus sp. VLD-10.

[This corrects the article DOI: 10.1007/s13205-016-0576-6.].

Isolation, identification, optimization, and metabolite profiling of Streptomyces sparsus VSM-30.

Deep sea sediment samples of Bay of Bengal (Visakhapatnam) have been analyzed for actinomycetes as an elite source to screen for the production of bioactive metabolites. The actinomycetes strain VSM-30 has an exciting bioactivity profile and was isolated during our systemic screening of marine actinomycetes. It was identified as Streptomyces sparsus based on morphological, physiological, biochemical, and molecular approaches. Response surface methodology regression analysis was carried out to fit the experimental data of each response by the second-order polynomial. The results have proven right interaction among process variables at optimized values of incubation time at 12 days, pH at 8, temperature at 30 °C, concentrations of starch at 1%, and tryptone at 1% and the data have been adequately fitted into the second-order polynomial models. Under these conditions, the responses (zones of inhibition) of plant pathogenic fungi Aspergillus niger, Aspergillus flavus, Fusarium oxysporum, Fusarium solani, and Penicillium citrinum were also matched with experimental and predicted results. Chemotypic analysis of ethyl acetate extract of the strain was done using LC-Q-TOF-MS revealed the presence of bioactive compounds including tryptophan dehydrobutyrine diketopiperazine, maculosin, 7-o-demethyl albocycline, albocycline M-2, and 7-o-demethoxy-7-oxo albocycline in a negative ion mode. The ethyl acetate extract of actinobacterium has been subjected to gas chromatography and mass spectroscopy (GC-MS) revealed the presence of diverse compounds such as dotriacontane, tetracosane 11-decyl-, diheptyl phthalate, 1-hexadecanesulfonyl chloride, L-alanyl-L-tryptophan, phthalic acid ethyl pentyl ester, 4-trifluoroacetoxyhexadecane, and 1H-imidazole 4,5-dihydro-2,4-dimethyl. Hence, the ethyl acetate extract of Streptomyces sparsus VSM-30 may have antibacterial, antifungal, and antioxidant activities due to the presence of secondary metabolites in ethyl acetate extract. The study also supports marine sediment samples of Bay of Bengal, a promising marine ecosystem remained to be explored for new bioactive compounds.

Isolation and biological evaluation of N-(4-aminocyclooctyl)-3, 5-dinitrobenzamide, a new semisynthetic derivative from the Mangrove-associated actinomycete Pseudonocardia endophytica VUK-10.

The present study was aimed to isolate novel bioactive compounds from actinomycetes species isolated from mangrove habitats. With this connection, Pseudonocardia endophytica (VUK-10) was isolated using dilution plate technique and was examined for its secondary metabolite profiling. After successive purification and spectroscopic characterization viz., FTIR, mass, NMR, DEPT, HMQC, HMBC, and COSY spectroscopy, two compounds were identified including a semi synthetic derivative N-(4-aminocyclooctyl)-3, 5-dinitrobenzamide (1), obtained from the precursor of novel natural product cyclooctane-1,4-diamine (3), along with a known compound 3-((1H-indol-6-yl) methyl) hexahydropyrrolo [1, 2-a] pyrazine-1, 4-dione (2). Anti cancer activities of the characterized compounds against in vitro cancerous cell line models, MDA-MB-231, OAW-42, HeLa, and MCF-7 reveal that HELA cells are most susceptible (IC50-10 nM compound 1 and 2) followed by other studied cells. On the other hand, antibacterial and antifungal activities of the studied compounds against tested pathogens revealed that there is a significant antimicrobial activity with all the tested bacterial and fungal species. Moreover, compound 1 showed the lowest MIC values against Streptococcus mutans as 4  and 16 µg/ml for Candida albicans. In conclusion, the identified novel chemical compounds in the present study may have a potential application in anticancer therapy as well as to mitigate the bacterial and fungal pathogens thus to control the infectious diseases.

Optimization of culture conditions by response surface methodology and unstructured kinetic modeling for bioactive metabolite production by Nocardiopsis litoralis VSM-8.

Response surface methodology-based central composite design on five variables incubation time, pH, temperature, sucrose concentration, and soya peptone concentration was employed for optimization of the production of bioactive compounds by Nocardiopsis litoralis strain VSM 8. The main quadratic effects and interactions of the five variables on the production of bioactive metabolites were investigated. A second-order polynomial model produced a satisfied fit for experimental data with regard to the production of the bioactive metabolites. Regression analysis showed that high R 2 values of all the five responses are significant and adjusted R 2 values showed good agreement with the experimental and predicted values. The present model was used to evaluate the direct interaction and quadratic effects to optimize the physico-chemical parameters for the production of bioactive metabolites that inhibit the pathogenic microorganisms measured in terms of zones of inhibition (responses). Mathematical kinetic model development and estimation of kinetic parameters also showed good approximation in terms of model fitting and regression analysis.

Extraction and bioactive profile of the compounds produced by Rhodococcus sp. VLD-10.

A potent actinobacterial strain isolated from the marine samples of Bheemunipatnam beach, Visakhapatnam, India, was identified as Rhodococcus sp. VLD-10 using the conventional and genomic (16S rRNA) approaches. Bioactive compounds responsible for the antimicrobial activity of the strain were elucidated by cultivating the strain VLD-10 in a modified yeast extract-malt extract-lactose broth followed by subsequent chromatographic and spectroscopic analyses. Extraction, purification, and structural confirmation of five compounds, viz., benzoic acid, 2-nitrobenzaldehyde, 4-chlorobenzaldehyde, nonadeconoic acid, and 3-isopropylhexahydro-1H-pyrido[1,2-a] pyrazine-1,4(6H)-dione, from Rhodococcus sp. VLD-10 were fruitfully described. The bioactivity of the compounds isolated from the strain VLD-10 against Gram-positive as well as Gram-negative bacteria, yeast, and molds was tested and their minimum inhibition concentration was reported. Antibacterial activity of 3-isopropylhexahydro-1H-pyrido[1,2-a] pyrazine-1,4(6H)-dione is more prominent against Bacillus subtilis, B. cereus, B. megaterium, Corynebacterium diphtheriae, and Escherichia coli, whereas its antifungal spectrum showed less potency against yeast and fungi. This is the first report on the natural occurrence and bioactivity of 3-isopropylhexahydro-1H-pyrido[1,2-a] pyrazine-1,4(6H)-dione from Rhodococcus sp. VLD-10.

Improving flavour and quality of tomatoes by expression of synthetic gene encoding sweet protein monellin.

Monellin a sweet-tasting protein exists naturally as a heterodimer of two non-covalently linked subunits chain A and B, which loses its sweetness on denaturation. In this study, we validated the expression of a synthetic monellin gene encoding a single polypeptide chain covalently linking the two subunits under T7 and fruit-ripening-specific promoters in Escherichia coli and tomato fruits, respectively. Purified recombinant monellin protein retained its sweet flavour at 70 °C and pH 2. We developed 15 transgenic T0 tomato plants overexpressing monellin, which were devoid of any growth penalty or phenotypic abnormalities during greenhouse conditions. T-DNA integration and fruit-specific heterologous expression of monellin had occurred in these transgenic tomato lines. ELISA revealed that expression of monellin was 4.5% of the total soluble fruit protein. Functional analyses of transgenic tomatoes of T2-5 and T2-14 lines revealed distinctly strong sweetness compared with wild type. Monellin a potential non-carbohydrate sweetener, if expressed in high amounts in fruits and vegetables, would enhance their flavour and quality.

Bioactive Metabolites Produced by Pseudonocardia endophytica VUK-10 from Mangrove Sediments: Isolation, Chemical Structure Determination and Bioactivity.

Chemical investigation of the actinobacterial isolate Pseudonocardia endophytica VUK-10 has led to the segregation of two known bioactive compounds, namely 4-(2-acetamidoethyl) phenyl acetate and 4-((1, 4-dioxooctahydropyrrolo [1, 2-a] pyrazin-3-yl) methyl) phenyl acetate. The strain was isolated from a sediment sample of the Nizampatnam mangrove ecosystem, south coastal Andhra Pradesh, India. The chemical structure of the active compounds was established on the basis of spectroscopic analysis, including (1)H NMR and (13)C NMR spectroscopies, FTIR, and EIMS. The antimicrobial and cytotoxic activities of the bioactive compounds produced by the strain were tested against opportunistic and pathogenic bacteria and fungi and on MDA-MB-231, OAW, HeLa, and MCF-7 cell lines. The compounds exhibited antimicrobial activities against gram-positive and gram-negative bacteria and fungi and also showed potent cytotoxic activity against MDA-MB-231, OAW, HeLa, and MCF-7 cell lines. This is the first example for this class of bioactive compounds isolated from Pseudonocardia of mangrove origin.

Phloem-specific expression of the lectin gene from Allium sativum confers resistance to the sap-sucker Nilaparvata lugens.

Rice production is severely hampered by insect pests. Garlic lectin gene (ASAL) holds great promise in conferring protection against chewing (lepidopteran) and sap-sucking (homopteran) insect pests. We have developed transgenic rice lines resistant to sap-sucking brown hopper (Nilaparvata lugens) by ectopic expression of ASAL in their phloem tissues. Molecular analyses of T0 lines confirmed stable integration of transgene. T1 lines (NP 1-2, 4-3, 11-6 & 17-7) showed active transcription and translation of ASAL transgene. ELISA revealed ASAL expression was as high as 0.95% of total soluble protein. Insect bioassays on T2 homozygous lines (NP 18 & 32) revealed significant reduction (~74-83%) in survival rate, development and fecundity of brown hoppers in comparison to wild type. Transgenics exhibited enhanced resistance (1-2 score) against brown hoppers, minimal plant damage and no growth penalty or phenotypic abnormalities.

A study on L-asparaginase of Nocardia levis MK-VL_113.

An enzyme-based drug, L-asparaginase, was produced by Nocardia levis MK-VL_113 isolated from laterite soils of Guntur region. Cultural parameters affecting the production of L-asparaginase by the strain were optimized. Maximal yields of L-asparaginase were recorded from 3-day-old culture grown in modified asparagine-glycerol salts broth with initial pH 7.0 at temperature 30°C. Glycerol (2%) and yeast extract (1.5%) served as good carbon and nitrogen sources for L-asparaginase production, respectively. Cell-disrupting agents like EDTA slightly enhanced the productivity of L-asparaginase. Ours is the first paper on the production of L-asparaginase by N. levis.

Isolation, Identification and Molecular Characterization of Rare Actinomycetes from Mangrove Ecosystem of Nizampatnam

Aims: Isolate and characterize the antimicrobial actinomycetes from sediments of Mangrove ecosystems of Nizampatnam located in the south coastal region of Andhra Pradesh, India. Methodology and Results: The Mangrove soil samples were collected, pre-treated and plated on asparagine-glucose 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 potent bioactive metabolite strain was isolated and designated as VUK-10. Further polyphasic studies revealed that the Isolate VUK-10 belongs to the genera Pseudonocardia. Phylogenetic analysis of 16S rRNA sequencing studies revealed that the strain is closely related to Pseudonocardia endophytica and the bioactive metabolites produced by the isolate inhibited Gram positive, Gram negative and Fungi. Conclusion, significance and impact of study: The isolation, characterization of the rare actinomycetes from the mangrove ecosystem will be useful for the discovery of the novel bioactive metabolites that are effective against wide range of 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 30℃, under shaking conditions. This is the first report on the optimization of bioactive metabolites by Pseudonocardia sp. VUK-10.

Optimization and purification of L-asparaginase produced by Streptomyces tendae TK-VL_333.

Cultural factors affecting the production of L-asparaginase by Streptomyces tendae isolated from laterite soil samples of Guntur region were investigated on glycerol-asparagine-salts (modified ISP-5) broth. Optimal yields of L-asparaginase were recorded in the culture medium with the initial pH 7.0 incubated at 30 degrees C for 72 h. The strain utilized sucrose (2%) and yeast (2%) extract as carbon and nitrogen sources for L-asparaginase production. The productivity of L-asparaginase was slightly enhanced when the strain was treated with cell-disrupting agents like EDTA. The crude enzyme was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-100 and CM-Sephadex G-50 gel filtration. By employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the enzyme was recorded as 97.4 kDa. This is the first report on production and purification of L-asparaginase from S. tendae.

Purification and biological evaluation of the metabolites produced by Streptomyces sp. TK-VL_333.

An Actinobacterium strain isolated from laterite soils of the Guntur region was identified as Streptomyces sp. TK-VL_333 by 16S rRNA analysis. Cultural, morphological and physiological characteristics of the strain were recorded. The secondary metabolites produced by the strain cultured on galactose-tyrosine broth were extracted and concentrated followed by defatting of the crude extract with cyclohexane to afford polar and non-polar residues. Purification of the two residues by column chromatography led to isolation of five polar and one non-polar fraction. Bioactivity- guided fractions were rechromatographed on a silica gel column to obtain four compounds, namely 1H-indole-3-carboxylic acid, 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde, 4-(4-hydroxyphenoxy) butan-2-one and acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester from three active polar fractions and 8-methyl decanoic acid from one non-polar fraction. The structure of the compounds was elucidated on the basis of FT-IR, mass and NMR spectroscopy. The antimicrobial activity of the bioactive compounds produced by the strain was tested against the bacteria and fungi and expressed in terms of minimum inhibitory concentration. Antifungal activity of indole-3-carboxylic acid was further evaluated under in vitro and in vivo conditions. This is the first report of 2,3-dihydroxy-5-(hydroxymethyl) benzaldehyde, 4-(4-hydroxyphenoxy) butan-2-one, acetic acid-2-hydroxy-6-(3-oxo-butyl)-phenyl ester and 8-methyl decanoic acid from the genus Streptomyces.

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.

Isolation, characterization and biological evaluation of bioactive metabolites from Nocardia levis MK-VL_113.

An Actinomycete isolate found to be prominent in the laterite soils of Acharya Nagarjuna University (ANU) Campus, Guntur was identified as Nocardia levis MK-VL_113 by 16S rRNA analysis. Cultural, morphological and physiological characteristics of the strain were recorded. Screening of secondary metabolites obtained from 4-day old culture broth of the strain led to the isolation of two fractions active against a wide variety of Gram-positive, Gram-negative bacteria and fungi. The structure of the first active fraction was elucidated using FT-IR, EI-MS, (1)H NMR and (13)C NMR spectra and identified as 1-phenylbut-3-ene-2-ol which is first time reported as a natural product. The compound exhibited good antimicrobial potential against the opportunistic and pathogenic bacteria and fungi. The antifungal activity of the strain and its metabolite were further confirmed with in vitro and in vivo studies. Evidence for the antagonism of the strain against Fusarium oxysporum, causing wilt disease in sorghum was demonstrated by the formation of inhibition zone in in vitro plate assay and reduction in the incidence of wilt of sorghum plants by using a green house trial. Analysis of the rhizosphere soil extracts by high performance liquid chromatography also demonstrated the production of the compound by the strain under in vivo conditions. As compared to the commercial fungicide mancozeb, the bioactive compound, 1-phenylbut-3-ene-2-ol was highly effective in controlling wilt of sorghum. Besides, the partially purified second fraction (PPF) subjected to gas chromatography-mass spectrometry revealed the presence of phenylethyl alcohol, dibutyl phthalate and 1,2-benzenedicarboxylic acid, 3-nitro.

Chitinase production by Streptomyces sp. ANU 6277

Chitinase production by a terrestrial Streptomyces sp. ANU 6277 was studied under sub-merged fermentation. Chitinase production started after 24 h of incubation and reached maximum levels after 60 h of cultivation. A high level of chitinase activity was observed in the culture medium with pH 6 at 35ºC. Culture medium amended with 1 percent chitin was found to be suitable for maximum production of chitinase. An optimum concentration of colloidal chitin for chitinase production was determined. Studies on the influence of additional carbon and nitrogen sources on chitinase production revealed that starch and yeast extract served as good carbon and nitrogen sources to enhance chitinase yield.Chitinase was purified from crude enzyme extract by single step gel filtration by Sephadex G-100. Purified chitinase of the strain exhibited a distinct protein band near 45 kDa by means of SDS-PAGE.

Chitinase Production by Streptomyces sp. ANU 6277.

Chitinase production by a terrestrial Streptomyces sp. ANU 6277 was studied under sub-merged fermentation. Chitinase production started after 24 h of incubation and reached maximum levels after 60 h of cultivation. A high level of chitinase activity was observed in the culture medium with pH 6 at 35°C. Culture medium amended with 1% chitin was found to be suitable for maximum production of chitinase. An optimum concentration of colloidal chitin for chitinase production was determined. Studies on the influence of additional carbon and nitrogen sources on chitinase production revealed that starch and yeast extract served as good carbon and nitrogen sources to enhance chitinase yield. Chitinase was purified from crude enzyme extract by single step gel filtration by Sephadex G-100. Purified chitinase of the strain exhibited a distinct protein band near 45 kDa by means of SDS-PAGE.

Study on bioactive compounds from Streptomyces sp. ANU 6277.

An attempt was made to study the bioactive compounds from a terrestrial Streptomyces sp. ANU 6277 isolated from laterite soil. Four active fractions were recovered from the solvent extracts obtained from the culture broth of five day-old strain. Three bioactive compounds were purified and identified as 3-phenylpropionic acid, anthracene-9,10-quinone and 8-hydroxyquinoline. The components of the partially purified fourth active fraction were analyzed by gas chromatography-mass spectrometry and identified as benzyl alcohol, phenylethyl alcohol and 2H-1, 4-benzoxazin-3 (4H)-one. Four active fractions were screened for antimicrobial activity against Gram-positive and Gram-negative bacteria, and fungi including phytopathogenic, toxigenic and dermatophytic genera. Among these metabolites, 8-hydroxyquinoline exhibited strong antibacterial and antifungal activity as compared to 3-phenylpropionic acid and anthracene-9,10-quinone.

Biological activity of phenylpropionic acid isolated from a terrestrial Streptomycetes.

The strain ANU 6277 was isolated from laterite soil and identified as Streptomyces sp. closely related to Streptomyces albidoflavus cluster by 16S rRNA analysis. The cultural, morphological and physiological characters of the strain were recorded. The strain exhibited resistance to chloramphenicol, penicillin and streptomycin. It had the ability to produce enzymes such as amylase and chitinase. A bioactive compound was isolated from the strain at stationary phase of culture and identified as 3-phenylpropionic acid (3-PPA) by FT-IR, EI-MS, 1H NMR and 13C NMR spectral studies. It exhibited antimicrobial activity against different bacteria like Bacillus cereus, B. subtilis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, P. flourescens, Staphylococcus aureus and some fungi including Aspergillus flavus, A. niger, Candida albicans, Fusarium oxysporum, F. udum and Penicillium citrinum. The antifungal activity of 3-PPA of the strain was evaluated in in vivo and in vitro conditions against Fusarium udum causing wilt disease in pigeon pea. The compound 3-PPA is an effective antifungal agent when compared to tricyclozole (fungicide) to control wilt caused by F. udum, but it exhibited less antifungal activity than carbendazim.