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1.  Psychrotrophic yeast Yarrowia lipolytica NCYC 789 mediates the synthesis of antimicrobial silver nanoparticles via cell-associated melanin 
AMB Express  2013;3:32.
A psychrotrophic marine strain of the ascomycetous yeast Yarrowia lipolytica (NCYC 789) synthesized silver nanoparticles (AgNPs) in a cell-associated manner. These nanostructures were characterized by UV-Visible spectroscopy and scanning electron microscope-energy dispersive spectrometer (SEM-EDS) analysis. The brown pigment (melanin) involved in metal-interactions was obtained from the cells. This extracted pigment also mediated the synthesis of silver nanoparticles that were characterized by a variety of analytical techniques. The melanin-derived nanoparticles displayed antibiofilm activity. This paper thus reports the synthesis of AgNPs by the biotechnologically important yeast Y. lipolytica; proposes a possible mechanism involved in the synthetic process and describes the use of the bio-inspired nanoparticles as antibiofilm agents.
PMCID: PMC3702394  PMID: 23758863
Yarrowia lipolytica; Silver nanoparticles; Melanin; Antibiofilm activity
2.  Disruption of Microbial Biofilms by an Extracellular Protein Isolated from Epibiotic Tropical Marine Strain of Bacillus licheniformis 
PLoS ONE  2013;8(5):e64501.
Marine epibiotic bacteria produce bioactive compounds effective against microbial biofilms. The study examines antibiofilm ability of a protein obtained from a tropical marine strain of Bacillus licheniformis D1.
Methodology/Principal Findings
B. licheniformis strain D1 isolated from the surface of green mussel, Perna viridis showed antimicrobial activity against pathogenic Candida albicans BH, Pseudomonas aeruginosa PAO1 and biofouling Bacillus pumilus TiO1 cultures. The antimicrobial activity was lost after treatment with trypsin and proteinase K. The protein was purified by ultrafiltration and size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75°C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry for a hypothetical protein (BL00275) derived from B. licheniformis ATCC 14580 with the accession number gi52082584. The protein showed minimum inhibitory concentration (MIC) value of 1.6 µg/ml against C. albicans. Against both P. aeruginosa and B. pumilus the MIC was 3.12 µg/ml. The protein inhibited microbial growth, decreased biofilm formation and dispersed pre-formed biofilms of the representative cultures in polystyrene microtiter plates and on glass surfaces.
We isolated a protein from a tropical marine strain of B. licheniformis, assigned a function to the hypothetical protein entry in the NCBI database and described its application as a potential antibiofilm agent.
PMCID: PMC3655075  PMID: 23691235
3.  3, 4-dihydroxy-L-phenylalanine-derived melanin from Yarrowia lipolytica mediates the synthesis of silver and gold nanostructures 
Nanobiotechnology applies the capabilities of biological systems in generating a variety of nano-sized structures. Plants, algae, fungi and bacteria are some systems mediating such reactions. In fungi, the synthesis of melanin is an important strategy for cell-survival under metal-stressed conditions. Yarrowia lipolytica, the biotechnologically significant yeast also produces melanin that sequesters heavy metal ions. The content of this cell-associated melanin is often low and precursors such as L-tyrosine or 3, 4-dihydroxy-L-phenylalanine (L-DOPA) can enhance its production. The induced melanin has not been exploited for the synthesis of nanostructures. In this investigation, we have employed L-DOPA-melanin for the facile synthesis of silver and gold nanostructures. The former have been used for the development of anti-fungal paints.
Yarrowia lipolytica NCIM 3590 cells were incubated with L-DOPA for 18 h and the resultant dark pigment was subjected to physical and chemical analysis. This biopolymer was used as a reducing and stabilizing agent for the synthesis of silver and gold nanostructures. These nanoparticles were characterized by UV-Visible spectra, X-ray diffraction (XRD) studies, and electron microscopy. Silver nanoparticles were evaluated for anti-fungal activity.
The pigment isolated from Y. lipolytica was identified as melanin. The induced pigment reduced silver nitrate and chloroauric acid to silver and gold nanostructures, respectively. The silver nanoparticles were smaller in size (7 nm) and displayed excellent anti-fungal properties towards an Aspergillus sp. isolated from a wall surface. An application of these nanoparticles as effective paint-additives has been demonstrated.
The yeast mediated enhanced production of the metal-ion-reducing pigment, melanin. A simple and rapid method for the extracellular synthesis of nanoparticles with paint-additive-application was developed.
PMCID: PMC3660187  PMID: 23363424
Yarrowia lipolytica; L-DOPA melanin; Nanoparticles; Anti-fungal activity
4.  Disruption of Yarrowia lipolytica biofilms by rhamnolipid biosurfactant 
Aquatic Biosystems  2012;8:17.
Yarrowia lipolytica is an ascomycetous dimorphic fungus that exhibits biofilm mode of growth. Earlier work has shown that biosurfactants such as rhamnolipids are efficient dispersants of bacterial biofilms. However, their effectiveness against fungal biofilms (particularly Y. lipolytica) has not been investigated. The aim of this study was to determine the effect of rhamnolipid on a biofilm forming strain of Y. lipolytica. Two chemical surfactants, cetyl-trimethyl ammonium bromide (CTAB) and sodium dodecyl sulphate (SDS) were used as controls for comparison.
The methylene blue dye exclusion assay indicated an increase in fungal cell permeability after rhamnolipid treatment. Microtiter plate assay showed that the surfactant coating decreased Y. lipolytica biofilm formation by 50%. Rhamnolipid treatment disrupted pre-formed biofilms in a more effective manner than the other two surfactants. Confocal laser scanning microscopic studies showed that biofilm formation onto glass surfaces was decreased by 67% after sub-minimum inhibitory concentration (sub-MIC) treatment with rhamnolipids. The disruption of biofilms after rhamnolipid treatment was significant (P<0.05) when compared to SDS and CTAB.
The results indicate a potential application of the biological surfactant to disrupt Y. lipolytica biofilms.
PMCID: PMC3445841  PMID: 22839701
Biofilm; Biosurfactant; CTAB; Rhamnolipid; SDS; Yarrowia lipolytica
5.  Evaluation of single cell oil (SCO) from a tropical marine yeast Yarrowia lipolytica NCIM 3589 as a potential feedstock for biodiesel 
AMB Express  2012;2:36.
Single cell oils (SCOs) accumulated by oleaginous yeasts have emerged as potential alternative feedstocks for biodiesel production. As lipid accumulation is species and substrate specific, selection of an appropriate strain is critical. Five strains of Y. lipolytica, a known model oleaginous yeast, were investigated to explore their potential for biodiesel production when grown on glucose and inexpensive wastes. All the strains were found to accumulate > 20% (w/w) of their dry cell mass as lipids with neutral lipid as the major fraction when grown on glucose and on wastes such as waste cooking oil (WCO), waste motor oil (WMO). However, amongst them, Y. lipolytica NCIM 3589, a tropical marine yeast, exhibited a maximal lipid/biomass coefficient, YL/X on 30 g L-1 glucose (0.29 g g-1) and on 100 g L-1 WCO (0.43 g g-1) with a high content of saturated and monounsaturated fatty acids similar to conventional vegetable oils used for biodiesel production. The experimentally determined and predicted biodiesel properties of strain 3589 when grown on glucose and WCO, such as density (0.81 and 1.04 g cm-3), viscosity (4.44 and 3.6 mm2 s-1), SN (190.81 and 256), IV (65.7 and 37.8) and CN (56.6 and 50.8) are reported for the first time for Y. lipolytica and correlate well with specified standards. Thus, the SCO of oleaginous tropical marine yeast Y. lipolytica NCIM 3589 could be used as a potential feedstock for biodiesel production.
PMCID: PMC3519684  PMID: 22812483
Y. lipolytica; Single cell oil; Fatty acid methyl ester; Biodiesel; WCO
6.  Single cell oil of oleaginous fungi from the tropical mangrove wetlands as a potential feedstock for biodiesel 
Single cell oils (SCOs) accumulated by oleaginous fungi have emerged as a potential alternative feedstock for biodiesel production. Though fungi from mangrove ecosystem have been reported for production of several lignocellulolytic enzymes, they remain unexplored for their SCO producing ability. Thus, these oleaginous fungi from the mangrove ecosystem could be suitable candidates for production of SCOs from lignocellulosic biomass. The accumulation of lipids being species specific, strain selection is critical and therefore, it is of importance to evaluate the fungal diversity of mangrove wetlands. The whole cells of these fungi were investigated with respect to oleaginicity, cell mass, lipid content, fatty acid methyl ester profiles and physicochemical properties of transesterified SCOs in order to explore their potential for biodiesel production.
In the present study, 14 yeasts and filamentous fungi were isolated from the detritus based mangrove wetlands along the Indian west coast. Nile red staining revealed that lipid bodies were present in 5 of the 14 fungal isolates. Lipid extraction showed that these fungi were able to accumulate > 20% (w/w) of their dry cell mass (4.14 - 6.44 g L-1) as lipids with neutral lipid as the major fraction. The profile of transesterified SCOs revealed a high content of saturated and monounsaturated fatty acids i.e., palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids similar to conventional vegetable oils used for biodiesel production. The experimentally determined and predicted biodiesel properties for 3 fungal isolates correlated well with the specified standards. Isolate IBB M1, with the highest SCO yield and containing high amounts of saturated and monounsaturated fatty acid was identified as Aspergillus terreus using morphotaxonomic study and 18 S rRNA gene sequencing. Batch flask cultures with varying initial glucose concentration revealed that maximal cell biomass and lipid content were obtained at 30gL-1. The strain was able to utilize cheap renewable substrates viz., sugarcane bagasse, grape stalk, groundnut shells and cheese whey for SCO production.
Our study suggests that SCOs of oleaginous fungi from the mangrove wetlands of the Indian west coast could be used as a potential feedstock for biodiesel production with Aspergillus terreus IBB M1 as a promising candidate.
PMCID: PMC3442963  PMID: 22646719
Mangrove wetlands; Oleaginous fungi; Single cell oil; Fatty acid methyl ester; Aspergillus terreus
7.  Antidiabetic Indian Plants: A Good Source of Potent Amylase Inhibitors 
Diabetes is known as a multifactorial disease. The treatment of diabetes (Type II) is complicated due to the inherent patho-physiological factors related to this disease. One of the complications of diabetes is post-prandial hyperglycemia (PPHG). Glucosidase inhibitors, particularly α-amylase inhibitors are a class of compounds that helps in managing PPHG. Six ethno-botanically known plants having antidiabetic property namely, Azadirachta indica Adr. Juss.; Murraya koenigii (L.) Sprengel; Ocimum tenuflorum (L.) (syn: Sanctum); Syzygium cumini (L.) Skeels (syn: Eugenia jambolana); Linum usitatissimum (L.) and Bougainvillea spectabilis were tested for their ability to inhibit glucosidase activity. The chloroform, methanol and aqueous extracts were prepared sequentially from either leaves or seeds of these plants. It was observed that the chloroform extract of O. tenuflorum; B. spectabilis; M. koenigii and S. cumini have significant α-amylase inhibitory property. Plants extracts were further tested against murine pancreatic, liver and small intestinal crude enzyme preparations for glucosidase inhibitory activity. The three extracts of O. tenuflorum and chloroform extract of M. koenigi showed good inhibition of murine pancreatic and intestinal glucosidases as compared with acarbose, a known glucosidase inhibitor.
PMCID: PMC3137644  PMID: 18955350
8.  Potent α-amylase inhibitory activity of Indian Ayurvedic medicinal plants 
Indian medicinal plants used in the Ayurvedic traditional system to treat diabetes are a valuable source of novel anti-diabetic agents. Pancreatic α-amylase inhibitors offer an effective strategy to lower the levels of post-prandial hyperglycemia via control of starch breakdown. In this study, seventeen Indian medicinal plants with known hypoglycemic properties were subjected to sequential solvent extraction and tested for α-amylase inhibition, in order to assess and evaluate their inhibitory potential on PPA (porcine pancreatic α-amylase). Preliminary phytochemical analysis of the lead extracts was performed in order to determine the probable constituents.
Analysis of the 126 extracts, obtained from 17 plants (Aloe vera (L.) Burm.f., Adansonia digitata L., Allium sativum L., Casia fistula L., Catharanthus roseus (L.) G. Don., Cinnamomum verum Persl., Coccinia grandis (L.) Voigt., Linum usitatisumum L., Mangifera indica L., Morus alba L., Nerium oleander L., Ocimum tenuiflorum L., Piper nigrum L., Terminalia chebula Retz., Tinospora cordifolia (Willd.) Miers., Trigonella foenum-graceum L., Zingiber officinale Rosc.) for PPA inhibition was initially performed qualitatively by starch-iodine colour assay. The lead extracts were further quantified with respect to PPA inhibition using the chromogenic DNSA (3, 5-dinitrosalicylic acid) method. Phytochemical constituents of the extracts exhibiting≥ 50% inhibition were analysed qualitatively as well as by GC-MS (Gas chromatography-Mass spectrometry).
Of the 126 extracts obtained from 17 plants, 17 extracts exhibited PPA inhibitory potential to varying degrees (10%-60.5%) while 4 extracts showed low inhibition (< 10%). However, strong porcine pancreatic amylase inhibitory activity (> 50%) was obtained with 3 isopropanol extracts. All these 3 extracts exhibited concentration dependent inhibition with IC50 values, viz., seeds of Linum usitatisumum (540 μgml-1), leaves of Morus alba (1440 μgml-1) and Ocimum tenuiflorum (8.9 μgml-1). Acarbose as the standard inhibitor exhibited an IC50 (half maximal inhibitory concentration)value of 10.2 μgml-1. Phytochemical analysis revealed the presence of alkaloids, tannins, cardiac glycosides, flavonoids, saponins and steroids with the major phytoconstituents being identified by GC-MS.
This study endorses the use of these plants for further studies to determine their potential for type 2 diabetes management. Results suggests that extracts of Linum usitatisumum, Morus alba and Ocimum tenuiflorum act effectively as PPA inhibitors leading to a reduction in starch hydrolysis and hence eventually to lowered glucose levels.
PMCID: PMC3037352  PMID: 21251279
9.  Evaluation of Traditional Indian Antidiabetic Medicinal Plants for Human Pancreatic Amylase Inhibitory Effect In Vitro 
Pancreatic α-amylase inhibitors offer an effective strategy to lower the levels of post prandial hyperglycemia via control of starch breakdown. Eleven Ayurvedic Indian medicinal plants with known hypoglycemic properties were subjected to sequential solvent extraction and tested for α-amylase inhibition, in order to assess and evaluate their inhibitory potential on pancreatic α-amylase. Analysis of 91 extracts, showed that 10 exhibited strong Human Pancreatic Amylase (HPA) inhibitory potential. Of these, 6 extracts showed concentration dependent inhibition with IC50 values, namely, cold and hot water extracts from Ficus bengalensis bark (4.4 and 125 μgmL−1), Syzygium cumini seeds (42.1 and 4.1 μgmL−1), isopropanol extracts of Cinnamomum verum leaves (1.0 μgmL−1) and Curcuma longa rhizome (0.16 μgmL−1). The other 4 extracts exhibited concentration independent inhibition, namely, methanol extract of Bixa orellana leaves (49 μgmL−1), isopropanol extract from Murraya koenigii leaves (127 μgmL−1), acetone extracts from C. longa rhizome (7.4 μgmL−1) and Tribulus terrestris seeds (511 μgmL−1). Thus, the probable mechanism of action of the above fractions is due to their inhibitory action on HPA, thereby reducing the rate of starch hydrolysis leading to lowered glucose levels. Phytochemical analysis revealed the presence of alkaloids, proteins, tannins, cardiac glycosides, flavonoids, saponins and steroids as probable inhibitory compounds.
PMCID: PMC2952308  PMID: 20953430

Results 1-9 (9)