Optimization of media components for enhanced carotenoid production by Paracoccus marcusii RSPO1 and assessment of their cytotoxicity against A549 and vero cells.

In this study, we tried to explore the influence of various tricarboxylic acid (TCA) cycle intermediates on carotenoid production and with a focus on enhancing pigment biosynthesis, we conducted two statistical analysis. In case of TCA intermediates influence on pigment production by Paracoccus marcusii RSPO1; fumaric acid, and malic acid were observed as potent enhancers of pigment biosynthesis. Further, to optimize key media components for enhanced carotenoid production, the Plackett-Burman design was employed encompassing carbon, nitrogen sources, TCA cycle intermediates, and metal salts. The selected factors after Plackett Burman were fine-tuned through Response Surface Methodology and the optimal concentrations that have remarkably elevated carotenoid production were starch-2.24 g/l, MgSO4-0.416 g/l, ZnSO4-0.0157 g/l, and fumaric Acid-16 mM. Further, evaluation of pigment cytotoxicity against normal (Vero) and Non-Small Cell Carcinoma (A549) cells was performed. The resultant IC50 values were quantified as 161.3 µg/ml and 7.623 µg/ml for Vero and A549 cells, respectively. Moreover, a reactive oxygen species (ROS) determination study in A549 cells was done which have shown a noteworthy threefold ROS production in A549 cells through fluorescence spectroscopic observation. This implies that the bacterial carotenoids can act as potent pro-oxidants against cancerous cells while being nontoxic toward normal cells.

Characterization of pigment produced by high carotenoid yielding bacteria Paracoccus marcusii RSPO1 and evaluation of its anti-diabetic, anti-microbial and antioxidant properties.

High pigment producing bacteria was isolated and identified as Paracoccus marcusii RSPO1 using biochemical and 16s rRNA identification. Bacterial pigment production was optimised using parameters like inoculum size, nitrogen source, pH, temperature, and agitation speed. Carotenoids production was 7,240 ± 41 µg L-1 after optimization. The silica column purified pigment was characterized using UV-visible spectroscopy, TLC, FTIR, LC-ESI-MS and NMR, which revealed its composition as astaxanthin, zeaxanthin, ζ-carotene and ß-zeacarotene. The inhibition assays against α-amylase and α-glucosidase showed IC50 values as 226 µg ml-1 and 0.7548 µg ml-1 respectively. The MIC of 1000 µg ml-1 of carotenoid was found to be effective against Escherichia coli and Enterobacter aerogenes when tested for antibacterial activity. Moreover, antioxidant activity of carotenoid sample was also determined where antioxidant potential of extracted carotenoid for DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) inhibition were 65 ± 0.06% and 42 ± 07% respectively at 20 µg ml-1 concentration.

Synthesis, characterization and application of lipase-conjugated citric acid-coated magnetic nanoparticles for ester synthesis using waste frying oil.

In the present work, magnetic nanoparticles (MNPs) were prepared by chemical precipitation of trivalent and divalent iron ions which were functionalized using citric acid. The bacterial isolate Staphylococcus epidermidis KX781317 was isolated from oil-contaminated site. The isolate produced lipase, which was purified and immobilized on magnetic nanoparticles (MNPs) for ester synthesis from waste frying oil (WFO). The characterization of MNPs employed conventional TEM, XRD and FTIR techniques. TEM analysis of MNPs showed the particle size in the range of 20-50 nm. FTIR spectra revealed the binding of citric acid to Fe3O4 and lipase on citric acid-coated MNPs. The citric acid-coated MNPs and lipase-conjugated citric acid-coated MNPs had similar XRD patterns which indicate MNPs could preserve their magnetic properties. The maximum immobilization efficiency 98.21% of lipase-containing citric acid-coated MNPs was observed at ratio 10:1 of Cit-MNPs:lipase. The pH and temperature optima for lipase conjugated with Cit-MNPs were 7 and 35 °C, respectively. Isobutanol was found to be an effective solvent for ester synthesis and 1:2 ratio of oil:alcohol observed significant for ester formation. The ester formation was determined using TLC and the % yield of ester conversion was calculated. The rate of ester formation is directly proportional to the enzyme load. Formed esters were identified as isobutyl laurate ester and isobutyl myristate ester through GC-MS analysis.

Biochemical and kinetic study of laccase from Ganoderma cupreum AG-1 in hydrogels.

In the present study, three different types of hydrogels i.e., (poly (-acrylamide)/alginate (P (AAm)/Alg), poly (acrylamide-N-isopropylacrylamide) (P (AAm-NIPA)), and poly (acrylamide-N-isopropylacrylamide)/alginate (P (AAm-NIPA)/Alg)) were synthesized by acrylamide, alginate, and N-isopropylacrylamide for the entrapment of laccase. The hydrogel-entrapped and free laccase showed optimum temperature of 50 °C for the oxidation of ABTS, but the entrapped laccase showed high temperature, pH, and storage stability as compared to the free enzyme. The K m values of free laccase, (P (AAm)/Alg)-L, (P (AAm-NIPA))-L, and (P (AAm-NIPA)/Alg)-L were found to be 0.13, 0.28, 0.33, and 0.50 mM, respectively. The V max values of free laccase, (P (AAm)/Alg)-L, (P (AAm-NIPA))-L, and (P (AAm-NIPA)/Alg)-L were found to be 22.22 × 10(2), 5.55 × 10(2), 5.0 × 10(2), and 4.54 × 10(2) mM/min, respectively. The entrapped laccase hydrogels were used for the decolorization of Reactive Violet 1 dye, with 39 to 45 % decolorization efficiency till the 10th cycle.

Purification and characterization of an extracellular laccase from solid-state culture of Pleurotus ostreatus HP-1.

A native isolate of Pleurotus ostreatus HP-1 (Genbank Accession No. EU420068) was found to have an excellent laccase producing ability. The extracellular laccase was purified to electrophoretic homogeneity from copper sulphate induced solid-state fermentation medium by ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was determined to be monomeric protein with an apparent molecular mass of 68,420 kDa, and an isoelectric point (pI) of 3.5. The inductively coupled plasma spectroscopy showed a presence of iron, zinc and copper in the purified enzyme. The absorption spectrum in the range of 200-700 nm showed the maximum absorption at 610 nm characteristic of fungal laccase and corresponding to the presence of type I copper atom. The laccase was stable at different temperatures up to 70 °C and retained 61 % activity at 50 °C. The enzyme reaction was inhibited by cysteine; sodium azide and EDTA. The enzyme oxidized various known laccase substrates, its lowest Km value being for ortho-dianisidine and highest Kcat and Kcat/Km for ABTS. The purified laccase exhibited different pH optima for different substrates. The N-terminal sequence did not show any similarity with N-terminal sequence of other species of genera Pleurotus.

Optimization of culture condition for enhanced decolorization and degradation of azo dye reactive violet 1 with concomitant production of ligninolytic enzymes by Ganoderma cupreum AG-1.

The strain Ganoderma cupreum AG-1 (Genbank accession no. HQ328947) isolated from the decayed wood was evaluated for its ability to decolorize azo dye reactive violet 1 as well as for the production of ligninolytic enzymes. In the initial decolorization study, the strain was capable of decolorizing 19 different azo dyes. The strain was capable of decolorizing dye over a pH range of 4.5-6 at 30 °C. The optimum pH was found to be 4.5. Various other process parameters like additional carbon and nitrogen source and initial dye concentration were also optimized. The decolorization medium was supplemented with appropriate nitrogen source (yeast extract, 5 g l-1) and carbon source (mannose, 2 g l-1); the decolorization obtained was 98 %. The pattern of enzymes involved in the biodegradation was studied and laccase and MnP were found to be the major enzymes. High laccase activity shown by G. cupreum AG-1 and its ability to decolorize dyes are a good indication of its possible use in the treatment of textile effluents.

Nutrient improvement for simultaneous production of exopolysaccharide and mycelial biomass by submerged cultivation of Schizophyllum commune AGMJ-1 using statistical optimization.

Exopolysaccharides (EPS) of fungal origin have attracted special attention from researchers due to their multifarious applications in the food and pharmaceutical industries. In the present study, optimization of the process parameters for the production of exopolysaccharide by Schizophyllum commune AGMJ-1 was studied using one factor at a time (OFAT) method, Plackett-Burman design (PBD) and response surface methodology (RSM). OFAT method revealed xylose and yeast extract to be the most effective carbon and nitrogen sources and pH 5.3 as an optimum for maximum EPS production. Xylose, yeast extract and KCl were screened as statistically significant variables for EPS production using PBD. RSM based on the central composite design estimated that maximum EPS (4.26 g L-1), mycelial biomass (14 g L-1) and specific yield (0.45 g g-1) were obtained when concentration of xylose, yeast extract and KCl were set at 2.5 g % (w/v), 0.83 g % (w/v) and 6.53 mg % (w/v), respectively, in the production medium.

Biodegradation of fluoranthene by basidiomycetes fungal isolate Pleurotus ostreatus HP-1.

The biodegradation of fluoranthene, a high molecular weight polycyclic aromatic hydrocarbon (PAH), was investigated in submerged culture using the wood decaying fungus isolated from forest locality in Gujarat, India. The basidiomycete fungal isolate was found to have an ability to grow on sabaroud dextrose agar containing 50 mgl(-1) of each naphthalene, anthracene, acenaphthene, benzo (a) anthracene, pyrene, flouranthene, carbazole, and biphenyl. The involvement of extracellular fungal peroxidases such as manganese peroxidase (MnP) and laccase (Phenol oxidase) in the degradation of fluoranthene was studied. On the eighth day of incubation 54.09% of 70 mg l(-1) fluoranthene was removed. There after no PAHs removal was observed till the 20th day of the incubation period. The isolate was identified as Pleurotus ostreatus by 18S rRNA, 5.8S rRNA, and partial 28S rRNA gene sequencing. To the best of our knowledge this is the first time Pleurotus ostreatus have been reported to degrade such a high concentration of fluoranthene within much lower time period of incubation. Depletion in the residual fluoranthene in the culture medium was determined by HPLC. Attempts were made to identify the degradation product in the culture medium with the help of FT-IR, NMR, and HPTLC analysis. In the present study positive correlation between fluoranthene degradation and the ligninolytic enzyme (MnP and laccase) production is observed, thus this isolate can play an effective role for bioremediation of PAHs contaminated sites.