Applied Biochemistry and Biotechnology (v.167, #5)

Preface (929-930).

One-Component Styrene Monooxygenases: An Evolutionary View on a Rare Class of Flavoproteins by Dirk Tischler; Janosch A. D. Gröning; Stefan R. Kaschabek; Michael Schlömann (931-944).
Styrene monooxygenases (SMOs) are catalysts for the enantioselective epoxidation of terminal alkenes. Most representatives comprise a reductase and a monooxygenase which are encoded by separate genes (styA, styB). Only six presumed self-sufficient one-component SMOs (styA2B) have previously been submitted to databases, and one has so far been characterized. StyA2B can be supported by another epoxidase (StyA1) encoded by styA1, a gene in direct neighborhood of styA2B. The present report describes the identification of a further styA1/styA2B-like SMO, which was detected in Rhodococcus opacus MR11. Based on the initially available sequences of styA2B-type SMOs, primers directed at conserved sequences were designed and a 7,012-bp genomic fragment from strain MR11 was obtained after PCRs and subsequent genome walking. Six open reading frames (ORFs) were detected and compared to genomic fragments of strains comprising either two- or one-component SMOs. Among the proteins encoded by the ORFs, the monooxygenase StyA1/StyA2B showed the highest divergence on amino acid level when comparing proteins from different sources. That finding, a rare distribution of styA2B genes among bacteria, and the general observation of evolution from simple to complex systems indicate that one-component SMOs evolved from two-component ancestors. Analysis of gene products from styA/styB- and styA1/styA2B-like SMOs revealed that a fusion of styA/styB to styA2B might have happened at least twice among microorganisms. This points to a convergent evolution of one-component SMOs.
Keywords: One-component styrene monooxygenase; Rhodococcus opacus ; Self-sufficient fusion protein; Covalent evolution; Enantioselective epoxidation; Flavin monooxygenase

Paecilomyces lilacinus (LPS 876) efficiently degraded keratin in chicken feather during submerged cultivation producing extracellular proteases. Characterization of crude protease activity was done including its compatibility in commercial detergents. Optimum pH and temperature were 10.0 and 60 °C, respectively. Protease activity was enhanced by Ca2+ but was strongly inhibited by PMSF and by Hg2+ suggesting the presence of thiol-dependent serine proteases. The crude protease showed extreme stability toward non-ionic (Tween 20, Tween 85, and Triton X-100) and anionic (SDS) surfactants, and relative stability toward oxidizing agent (H2O2 and sodium perborate). In addition, it showed excellent stability and compatibility with various solid and liquid commercial detergents from 30 to 50 °C. The enzyme preparation retained more than 95% of its initial activity with solid detergents (Ariel™ and Drive™) and 97% of its original activity with a liquid detergent (Ace™) after pre-incubation at 40 °C. The protective effect of polyols (propylene glycol, PEG 4000, and glycerol) on the heat inactivation was also examined and the best results were obtained with glycerol from 50 to 60 °C. Considering its promising properties, P. lilacinus enzymatic preparation may be considered as a candidate for use in biotechnological processes (i.e., as detergent additive) and in the processing of keratinous wastes.
Keywords: Paecilomyces lilacinus ; Alkaline serine proteases; Keratinolytic activity; Chicken feather; Detergent stable

Production, Purification, and Characterization of a β-Glucosidase of Penicillium funiculosum NCL1 by Gurusamy Ramani; Balasubramanian Meera; Chinnathambi Vanitha; Mala Rao; Paramasamy Gunasekaran (959-972).
Penicillium funiculosum NCL1, a filamentous fungus, produced significantly higher levels of β-glucosidase. The effect of initial pH, incubation temperature, and different carbon sources on extracellular β-glucosidase production was studied in submerged fermentation. At 30 °C with initial pH 5.0, enzyme production was increased by 48-fold upon induction with paper mill waste, as compared to commercial cellulose powder. In zymogram analysis, four isoforms of β-glucosidases were observed with wheat bran whereas a minimum of one isoform was observed with other carbon sources. A major β-glucosidase (Bgl3A) with the apparent molecular weight of ~120 kDa, induced by paper mill waste, was purified 19-fold to homogeneity, with a specific activity of 1,796 U/mg. Bgl3A was a monomeric glycoprotein with 29% of neutral carbohydrate content. It showed optimum activity at pH 4.0 and 5.0, optimum temperature at 60 °C, and exhibited a half-life of 1 h at 60 °C. K m of Bgl3A was found to be 0.057 mM with p-nitrophenyl β-d-glucoside and V max was 1,920 U/mg. The purified enzyme exhibited glucose tolerance with a K i of 1.5 mM. Bgl3A readily hydrolyzed glucosides with β-linkage. Bgl3A activity was enhanced (156%) by Zn2+ and was not affected by other metal cations and reagents. The supplementation of Bgl3A (5 U/mg) with Trichoderma reesei cellulase complex (5 FPU/mg) resulted in about 70% of enhanced glucose production, which emphasizes the industrial importance of Bgl3A.
Keywords: Penicillium funiculosum NCL1; β-Glucosidase; Paper mill waste; Purification; Thermostability

A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5 °C, was isolated from cow dung; l-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37 °C and in a buffer system of pH 6 to 7. The enzyme exhibited thermostability at 50 °C with a 33% and 28% of activity retention after 45 and 60 min. The kinetic parameters for the enzyme were calculated from Lineweaver–Burk plot, and K m and V max were 0.89 mM and 0.18 U/mg, respectively.
Keywords: Moderately thermotolerant; l-asparaginase II; PelB leader; Nickel affinity; Thermostability

Periplasmic phytase, appA from E. coli has been noticed as a superior feed and food additive owing to its high specific activity, acidic pH optimum and resistance to gastric proteases. E. coli phytase was expressed as a fusion protein with maltose-binding protein, affinity-purified to homogeneity and, subsequently, immobilized in one step using a cost-effective matrix prepared from starch agar bead. Immobilized enzyme revealed an activity optimum at pH 6, while that of free enzyme was observed at pH 4. Both the immobilized and free enzyme showed a temperature optimum at 60 °C. Cleavage of 87 kDa fusion protein using factor Xa released 45 kDa appA. Hydrolysis of soy milk using immobilized enzyme led to 10% increase in release of inorganic phosphate at 50 °C relative to free fusion protein. This study suggests the usability of MBP as an immobilizing linker to other food enzymes for economical use in industry.
Keywords: MBP; E. coli phytase; Starch agar beads; Soy milk; Dephytination

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b(+) expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5 mM) induction for 5 h at 37 °C in LB medium supplemented with 0.3% K2HPO4 and 0.3% KH2PO4. Recombinant His tagged aspartase was purified using Ni–NTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH 8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35 °C. The enzyme showed apparent K m and V max values for L-aspartate as 2.01 mM and 114 U/mg, respectively. The enzyme was stable in pH range of 6.5–9.5 and temperature up to 45 °C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg2+, Mn2+, and Ca2+ ions. The cloned gene (aspA) product showed molecular weight of approximately 51 kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.
Keywords: Aeromonas media ; Aspartase; Amino acid sequence; Purification; Characterization; Thermodynamics

In this study, complete purification and biochemical characterization of protein is presented. The protein was purified by using Sephadex G-75 gel filtration column followed by reverse-phase high-performance liquid chromatography in a C18 column. The molecular weight of the protein was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, mass spectrum matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization tandem mass spectrometry. Protein was fragmented by trypsin based on the m/z values obtained by MALDI-TOF-MS analysis. The peptide fragments sequence showed homology with DEAD-box-ATP-dependent RNA helicase 45, present in a public domain, National Centre for Biotechnology Information. The protein exhibited antibacterial activity against selected Gram +/− bacteria. The analgesic activity was determined by conducting acetic-acid-induced writhing test in mice.
Keywords: Novel protein; Purification; Characterization; Bombyx mori ; MALDI-TOF-MS; LC-ESI-MS/MS

Efficient Microbial Conversion of l-Tyrosine to l-DOPA by Brevundimonas sp. SGJ by Shripad N. Surwase; Sushama A. Patil; Onkar A. Apine; Jyoti P. Jadhav (1015-1028).
l-DOPA (3,4-dihydroxyphenyl-l-alanine), the most widely used drug for the treatment of Parkinson’s disease, was produced in buffer using biomass of Brevundimonas sp. SGJ. The effects of enhancers, such as carrageenan, diatomaceous earth, and activated charcoal, on the l-DOPA production were evaluated to obtain the maximum yield. The optimal process conditions found were pH 8, 2 g l−1 cell mass, 2 g l−1 l-tyrosine, 0.04 g l−1 CuSO4, 0.02 g l−1 l-ascorbic acid, 0.5 g l−1 carrageenan, and 40 °C temperature. In addition, repeated use of cells resulted in the highest yield of 3.81 g l−1 (95.2%) of l-DOPA with utilization of 4 g l−1 l-tyrosine, and the highest tyrosinase activity (9,201 U mg−1) was observed at 18 h of incubation. Furthermore, the produced l-DOPA was confirmed by high-performance thin-layer chromatography, high-performance liquid chromatography, and gas chromatography–mass spectroscopy. Kinetic studies showed significant values of Y p/s, Q s, and q s after optimization of the process. Thus, Brevundimonas sp. SGJ could be an eventual new source for large-scale production of l-DOPA.
Keywords: l-DOPA; Brevundimonas sp. SGJ; Carrageenan; HPTLC; Tyrosinase

Chitosanases produced by microbes and plants are getting attention to explore vastly available marine waste. Chitooligosaccharides and glucosamine can be produced using chitosanase enzyme and have applications in food, pharma and other industries. A potential microbial chitosanase source was found after isolation and screening of chitosan degrading microbes from garden soil. An isolate, designated as C6 produced chitosanase enzyme upon induction by chitosan substrates. Production of 6 U/ml of chitosanase enzyme was achieved from this isolate on chitosan minimal salt broth medium at 32 °C after 3 days of growth. The enzyme was able to hydrolyse both chitosan and cellulosic substrates. Enzymatic production of d-glucosamine and chitooligosaccharides were studied with various chitosan substrates using crude enzyme. The yield of glucosamine was found to be 40% after 2 h of reaction at 40 °C, and chitosan oligomers were produced having two to six polymerizations at 60 °C reaction temperature. The hydrolysates showed 50% antioxidant activity as compared to ascorbic acid.
Keywords: Chitosan; Chitosanase; Chitooligosaccharides; Glucosamine; Antioxidant

Keratinase Production and Biodegradation of Whole Chicken Feather Keratin by a Newly Isolated Bacterium Under Submerged Fermentation by Dipak K. Sahoo; Arpan Das; Hrudayanath Thatoi; Keshab C. Mondal; Pradeep K. Das Mohapatra (1040-1051).
A new feather-degrading bacterium PKD 5 was isolated from feather dumping soil and identified as Bacillus weihenstephanensis based on morphological and physiochemical characteristics as well as 16S rRNA gene analysis. Extracellular keratinase was produced during submerged aerobic cultivation in a medium containing chicken feather as sole carbon and energy source and supplemented with salt solutions (NaCl 5.0, MgSO4 1.0, K2HPO4 1.0, (NH4)2SO4, 2.0 g/l). The optimal conditions for keratinase production include initial pH of 7.0, inoculum size of 2% (v/v), and cultivation at 40 °C. The maximum keratinase production and keratinolytic activity of PKD 5 was achieved after 7 days of fermentation under shaking condition (120 rpm). The enzyme has found application in developing cost-effective feather by-products for feeds and fertilizers. The manuscript first time describes B. weihenstephanensis PKD 5-mediated keratinase production under submerged fermentation and whole chicken feather biodegradation.
Keywords: Keratinolytic bacteria; Submerged fermentation; Keratinase

Regulation of Xylanase Biosynthesis in Bacillus cereus BSA1 by Asish Mandal; Sanjay Kar; Pradeep Kumar Das Mohapatra; Chiranjit Maity; Bikas Ranjan Pati; Keshab Chandra Mondal (1052-1060).
Microbial xylanases have a promising biotechnological potential to be used in industries. In this study, regulation of xylanase production was examined in Bacillus cereus BSA1. Xylanase production was induced by xylan. The enzyme production further increased in the presence of xylose and arabinose in very low concentration with addition of xylan (0.5% up to 6.02 U/ml). Addition of glucose (about 0.1%) to the media supplemented with xylan repressed xylanase production. Even higher concentration (>0.1%) of xylose and arabinose repressed xylanase biosynthesis. Glucose-mediated repression was partially relived by addition of cyclic adenosine monophosphate. Chemical like 2-4-dinitrophenol, which can inhibit adenosine triphosphate synthesis in cell, repressed xylanase synthesis and it suggested xylanase synthesis to be an energy dependent process.
Keywords: Xylan; Xylanase; Regulation of biosynthesis; Bacillus cereus

Repeated batch cultivation (empty-and-fill protocol) using obligate anaerobe Clostridium diolis was attempted in the present study to improve the production of 1,3-propanediol (1,3-PD). In repeated batch operation, 20 % (v/v) culture broth was removed from the bioreactor and supplemented with an equal volume of fresh nutrient medium when the residual glycerol concentration in the bioreactor decreased below 15 g/L. Four cycles of culture broth withdrawal and subsequent replacement resulted in achieving a 1,3-PD concentration of 67.8 g/L with a productivity of 1.04 g/L/h at the end of 65 h. This represented a 1,3-PD concentration and productivity enhancement by 2.6-fold and 1.5-fold, respectively, as compared to batch 1,3-PD fermentation. This is the first report on the use of repeated batch mode of bioreactor operation for enhanced 1,3-PD production.
Keywords: 1,3-Propanediol; Clostridium diolis ; Repeated batch cultivation; Glycerol; Polytrimethylene terephthalate

Graphene-Based Waveguides: Novel Method for Detecting Biological Activity by Jangah Kim; Manasi Kasture; Taihyun Hwang; Atul Kulkarni; Rashid Amin; Sungha Park; Taesung Kim; Suresh Gosavi (1069-1075).
We demonstrate the fabrication of a biosensor based on graphene coupled with polydimethylsiloxane (PDMS) waveguide. Biosensors work on the principle of local evanescent graphene-coupled wave sensor. It is observed that the evanescent field shifts in the presence of chemical or biological species as evanescent waves are extremely sensitive to a change in refractive index. This method helps to monitor the target analyte by attaching the selective receptor molecules to the surface of the PDMS optical waveguide resulting in its optical intensity distribution shift. We monitor the electrical properties of graphene in the dark and under illumination of PDMS waveguide. The changes in photocurrent through the graphene film were monitored for blue, green, and red light. We observed that the fabricated graphene-coupled PDMS optical waveguide sensor is sensitive to visible light for the used bioanalytes.
Keywords: Waveguide; Graphene; PDMS; Local evanescent wave; Biosensor

As a part of a natural biological N-cycle, nitrification is one of the steps included in the conception of artificial ecosystems designed for extraterrestrial life support systems (LSS) such as Micro-Ecological Life Support System Alternative (MELiSSA) project, which is the LSS project of the European Space Agency. Nitrification in aerobic environments is carried out by two groups of bacteria in a two-step process. The ammonia-oxidizing bacteria (Nitrosomonas europaea) realize the oxidation of ammonia to nitrite, and the nitrite-oxidizing bacteria (Nitrobacter winogradskyi), the oxidation of nitrite to nitrate. In both cases, the bacteria achieve these oxidations to obtain an energy and reductant source for their growth and maintenance. Furthermore, both groups also use CO2 predominantly as their carbon source. They are typically found together in ecosystems, and consequently, nitrite accumulation is rare. Due to the necessity of modeling accurately conversion yields and transformation rates to achieve a complete modeling of MELiSSA, the present study focuses on the experimental determination of nitrogen to biomass conversion yields. Kinetic and mass balance studies for axenic cultures of Nitrosomonas europaea and Nitrobacter winogradskyi in autotrophic conditions are performed. The follow-up of these cultures is done using flow cytometry for assessing biomass concentrations and ionic chromatography for ammonium, nitrite, and nitrate concentrations. A linear correlation is observed between cell count and optical density (OD) measurement (within a 10 % accuracy) validating OD measurements for an on-line estimation of biomass quantity even at very low biomass concentrations. The conversion between cell count and biomass concentration has been determined: 7.1 × 1012 cells g dry matter (DM)−1 for Nitrobacter and 6.3 × 1012 cells g DM−1 for Nitrosomonas. Nitrogen substrates and products are assessed redundantly showing excellent agreement for mass balance purposes and conversion yields determination. Although the dominant phenomena are the oxidation of NH 4 + into nitrite (0.95 mol mol N−1 for Nitrosomonas europaea within an accuracy of 3 %) and nitrite into nitrate (0.975 mol mol N−1 for Nitrobacter winogradskyi within an accuracy of 2 %), the Nitrosomonas europaea conversion yield is estimated to be 0.42 g DM mol N−1, and Nitrobacter winogradskyi conversion yield is estimated to be 0.27 g DM mol N−1. The growth rates of both strains appear to be dominated by the oxygen transfer into the experimental setups.
Keywords: Autotrophy; Nitrosomonas europaea ; Nitrobacter winogradskyi ; Ionic chromatography; Flow cytometry

Co-Culture of Microalgae, Cyanobacteria, and Macromycetes for Exopolysaccharides Production: Process Preliminary Optimization and Partial Characterization by S. Angelis; A. C. Novak; E. B. Sydney; V. T. Soccol; J. C. Carvalho; A. Pandey; M. D. Noseda; J. L. Tholozan; J. Lorquin; C. R. Soccol (1092-1106).
In this study, the biomass and exopolysaccharides (EPS) production in co-cultures of microalgae/cyanobacteria and macromycetes was evaluated as a technology for producing new polysaccharides for medical and/or industrial application. Based on biomass and EPS productivity of monocultures, two algae and two fungi were selected and cultured in different co-culture arrangements. The hydrosoluble EPS fractions from mono- and co-cultures were characterized by ¹³C NMR spectroscopy and gas chromatography coupled to mass spectrometry and compared. It was found that co-cultures resulted in the production of an EPS different from those produced by monocultures, showing fungal predominance with microalgal/cyanobacterial traces. Co-cultures conditions were screened (temperature, agitation speed, fungal and microalgae inoculation rate, initial pH, illumination rate, and glucose concentration) in order to achieve maximum biomass and EPS production, resulting in an increase of 33 and 61% in exopolysaccharides and biomass productions, respectively (patent pending).
Keywords: Co-culture; Microalgae; Macromycetes; Exopolysaccharides

Generation of Monoclonal Antibody for 15-deoxy-Δ12,14-Prostaglandin J2 and Development of Enzyme-Linked Immunosorbent Assay for Its Quantification in Culture Medium of Adipocytes by Pinky Karim Syeda; Mohammad Salim Hossain; Abu Asad Chowdhury; Mohammad Sharifur Rahman; Kohji Nishimura; Mitsuo Jisaka; Tsutomu Nagaya; Fumiaki Shono; Kazushige Yokota (1107-1118).
15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2) is a biologically active molecule serving as a pro-adipogenic factor or an anti-inflammatory regulator. This compound is one of naturally occurring derivatives formed by the non-enzymatic dehydration of PGD2. To determine the endogenous synthesis of 15d-PGJ2, a convenient immunological approach is useful. At first, we established a cloned hybridoma cell line to secrete a monoclonal antibody specific for 15d-PGJ2. For the development of a solid-phase enzyme-linked immunosorbent assay (ELISA), the immobilized antigen using a protein conjugate of 15d-PGJ2 was allowed to react competitively with a monoclonal antibody in the presence of free 15d-PGJ2. Under the optimized conditions, a sensitive calibration curve was generated able to determine the amount of 15d-PGJ2 from 0.5 pg to 9.7 ng with 71 pg of 50% displacement in one assay. Our monoclonal antibody did not recognize other related prostanoids except PGJ2 with cross-reaction of 4%. Our ELISA was demonstrated to be reliable for the quantification of 15d-PGJ2 in the maturation medium of cultured adipocytes by confirming the accuracy and specificity of its determination. The application of our assay revealed that the non-enzymatic formation of 15d-PGJ2 became more evident after several hours of incubation with authentic PGD2 at 37 °C. The results indicate the usefulness of our developed solid-phase ELISA with the monoclonal antibody for further studies on the endogenous synthesis of 15d-PGJ2 and its roles in various cells and tissues.
Keywords: Monoclonal antibody; 15-deoxy-Δ12,14-Prostaglandin J2 ; PGD2 ; PGJ2 ; Enzyme-linked immunosorbent assay; Cultured adipocytes; Maturation medium

Production of Microbial Surfactants from Oily Sludge-Contaminated Soil by Bacillus subtilis DSVP23 by Suma C. Pemmaraju; Deepak Sharma; Nivedita Singh; Richa Panwar; Swaranjit S. Cameotra; Vikas Pruthi (1119-1131).
The indigenous microbial community utilizing aliphatic, aromatic, and polar components from the oily sludge as sole source of carbon and energy was selected from the soil samples of Ankleshwar, India for biosurfactant production. Evaluation of biosurfactant production was done using screening assays such as surface tension reduction, hemolytic activity, emulsification activity, drop-collapse assay, and cell surface hydrophobicity studies. Maximum biosurfactant (6.9 g/l) production was achieved after 5 days of growth from Bacillus subtilis DSVP23 which was identified by 16S RNA technique (NCBI GenBank accession no. EU679368). Composition of biosurfactant showed it to be lipopeptide in nature with 15.2% protein content and 18.0% lipid content. Functional group analysis was also done by using Fourier transform infrared spectroscopy which showed it to be a protein-bound lipid thereby imparting them special properties. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric and nuclear magnetic resonance revealed that the major constituents of lipopeptide are leucine and isoleucine. Gas chromatographic analysis data indicated that oily sludge components of chain length C12–C30 and aromatic hydrocarbons were degraded effectively by B. subtilis DSVP23 after 5 days of incubation. These results collectively points toward the importance of B. subtilis DSVP23 as a potential candidate for bioremediation studies.
Keywords: Oily sludge; Bacillus subtilis ; Biosurfactant; Biodegradation

The phenomenon of heat and mass transfer by condensation of water vapour from humid air involves several key concepts in aerobic bioreactors. The high performance of bioreactors results from optimised interactions between biological processes and multiphase heat and mass transfer. Indeed in various processes such as submerged fermenters and solid-state fermenters, gas/liquid transfer need to be well controlled, as it is involved at the microorganism interface and for the control of the global process. For the theoretical prediction of such phenomena, mathematical models require heat and mass transfer coefficients. To date, very few data have been validated concerning mass transfer coefficients from humid air inflows relevant to those bioprocesses. Our study focussed on the condensation process of water vapour and developed an experimental set-up and protocol to study the velocity profiles and the mass flux on a small size horizontal flat plate in controlled environmental conditions. A closed circuit wind tunnel facility was used to control the temperature, hygrometry and hydrodynamics of the flow. The temperature of the active surface was controlled and kept isothermal below the dew point to induce condensation, by the use of thermoelectricity. The experiments were performed at ambient temperature for a relative humidity between 35–65% and for a velocity of 1.0 ms−1. The obtained data are analysed and compared to available theoretical calculations on condensation mass flux.
Keywords: Heat and mass transfer; Bioreactor; Condensation; Humid air; Thermoelectricity

This work aimed to study the production of laccase from Pleurotus ostreatus DSM 1833 and Phoma sp. UHH 5-1-03 using banana peels as alternative carbon source, the subsequent partial purification and characterization of the enzyme, as well the applicability to degrade endocrine disruptors. The laccase stability with pH and temperature, the optimum pH, the K m and V max parameters, and the molar mass were determined. Tests were conducted for assessing the ability of degradation of the endocrine disruptors t-nonylphenol, bisphenol A, and 17α-ethinylestradiol. Laccase production of 752 and 1,117 U L−1 was obtained for Phoma sp. and P. ostreatus, respectively. Phoma sp. laccase showed higher stability with temperature and pH. The laccase from both species showed higher affinity by syringaldazine. The culture broth with banana peels induced the production of two isoforms of P. ostreatus (58.7 and 21 kDa) and one of Phoma sp. laccase (72 kDa). In the first day of incubation, the concentrations of bisphenol A and 17α-ethinylestradiol were reduced to values close to zero and after 3 days the concentration of t-nonylphenol was reduced in 90% by the P. ostreatus laccase, but there was no reduction in its concentration by the Phoma sp. laccase.
Keywords: Laccase; Phoma sp.; Pleurotus ostreatus ; Agro-wastes; Endocrine disruptors; Decontamination of waste effluents

Production of Microbial Cellulose by a Bacterium Isolated from Fruit by Firdaus Jahan; Vinod Kumar; Garima Rawat; R. K. Saxena (1157-1171).
This study presents the production of bacterial cellulose (BC) by a bacterium isolated from a rotten fruit and its process optimization. Here, isolation and screening of potent cellulose producers were carried out from different natural sources, viz., soil, rotten fruits, and vegetables and vinegar. A total of 200 bacterial isolates were obtained, which were screened for cellulose production using Hestrin–Schramm medium. A novel and potent cellulose-producing bacterium was newly isolated from a rotten fruit and identified as Gluconacetobacter sp. F6 through 16S ribosomal DNA sequencing and morphological, cultural, and biochemical characteristics. After optimization of culture conditions, including pH, temperature, agitation, carbon/nitrogen sources, and inducers, the BC production was greatly increased from 0.52 to 4.5 g/l (8.65-fold increase). The optimal culture medium contained 1% (w/v) glucose, 1.5% (w/v) yeast extract, 0.5% (w/v) peptone, 0.27% (w/v) disodium hydrogen phosphate, 0.115% (w/v) citric acid, and 0.4% (w/v) ethanol. BC produced was analyzed for the presence of cellulose fibrils by epiflourescent microscopy using Calcofluor white stain and scanning electron microscopy and confirmed by NMR. There are very scanty reports about the optimization of BC production by bacteria isolated from rotten fruits.
Keywords: Bacterial cellulose; Screening; Gluconacetobacter sp. F6; Production; Process optimization

In this case study, we designed a farnesyl pyrophosphate (FPP) biosynthetic network using hybrid functional Petri net with extension (HFPNe) which is derived from traditional Petri net theory and allows easy modeling with graphical approach of various types of entities in the networks together. Our main objective is to improve the production of FPP in yeast, which is further converted to amorphadiene (AD), a precursor of artemisinin (antimalarial drug). Natively, mevalonate (MEV) pathway is present in yeast. Methyl erythritol phosphate pathways (MEP) are present only in higher plant plastids and eubacteria, but not present in yeast. IPP and DAMP are common isomeric intermediate in these two pathways, which immediately yields FPP. By integrating these two pathways in yeast, we augmented the FPP synthesis approximately two folds higher (431.16 U/pt) than in MEV pathway alone (259.91 U/pt) by using HFPNe technique. Further enhanced FPP levels converted to AD by amorphadiene synthase gene yielding 436.5 U/pt of AD which approximately two folds higher compared to the AD (258.5 U/pt) synthesized by MEV pathway exclusively. Simulation and validation processes performed using these models are reliable with identified biological information and data.
Keywords: Amorpha diene; Artemisinin; Farnesyl pyrophosphate; Hybrid functional Petri net with extensions; Methylerythritol phosphate pathway; Mevalonate Pathway

Volatiles Formation from Grape Must Fermentation Using a Cryophilic and Thermotolerant Yeast by Nikolaos Kopsahelis; Loulouda Bosnea; Maria Kanellaki; Athanasios A. Koutinas (1183-1198).
Grape must fermentation performance and volatiles formation by simultaneously cryophilic and thermotolerant yeast (strain AXAZ-1), isolated from grapes in Greece, was evaluated in a wide temperature range (5–40°C). Yeast strain was immobilized on brewer’s spent grains (BSG) and the formed biocatalyst was introduced into a Multi-Stage Fixed Bed Tower (MFBT) bioreactor. Almost complete sugar utilization from the aforementioned biocatalyst was observed in a wide temperature spectrum, ranging from 5 °C to 37 °C, while at 40 °C residual sugar was up to 29 g/l. Time to complete fermentation with the immobilized yeast ranged from 290 h at 5 °C and 120 h at 40 °C to 25 h at 33 °C. The daily ethanol productivity reached maximum (88.6 g/l) and minimum (5.6 g/l) levels at 33 °C and 5 °C, respectively. The aroma-related compounds’ profiles of immobilized cells at different fermentation temperatures were evaluated by using solid phase microextraction (SPME) gas chromatography/mass spectrometry (GC/MS). Must fermentation resulted in a high-quality fermentation product due to the low concentrations of higher and amyl alcohols at all temperatures tested. AXAZ-1 is a very promising strain for quality wine production, as it is capable of performing fermentations of high ethanol concentration and productivities in both low and high temperatures.
Keywords: Wine production; Immobilization; High temperature; Saccharomyces cerevisiae ; Brewer’s spent grains; Volatiles; GC-MS

Castor (Ricinus communis L.) is an important oil seed crop having its main cultivated area in India, China, and Brazil in dry land farming. Castor husk is generated as waste in castor oil production. Use of castor husk waste as substrate is studied for alkaline protease production by Bacillus altitudinis GVC11 in solid-state fermentation. Various parameters like moisture content, incubation period, particle size, effect of carbon and nitrogen sources are studied and optimized for enzyme production. Highest enzyme production of 419,293 units per gram husk is obtained. Cost of enzyme production can be reduced by using castor husk as substrate.
Keywords: Bacillus altitudinis ; Alkaline protease; Solid state fermentation; Castor husk

Xylanase Isozymes from the Newly Isolated Bacillus sp. CKBx1D and Optimization of Its Deinking Potentiality by Chiranjit Maity; Kuntal Ghosh; Suman K. Halder; Arijit Jana; Atanu Adak; Pradeep K. Das Mohapatra; Bikas R. Pati; Keshab C. Mondal (1208-1219).
Recycling of civic paper waste by enzyme-based technology is nowadays a point of much concern for pollution-less green environment. In this study, the deinking effectiveness of purified xylanase from a newly isolated bacterium was evaluated for recycling of laser jet paper waste. A potent xylanases-producing bacterium from the microbial consortia of termite gut was isolated, which was further identified on the basis of 16S rRNA sequence as Bacillus sp. CKBx1D. In submerged fermentation condition, the isolate produced the highest level of xylanase (480 U/ml) at 36 h of growth. The extracellular xylanase system comprises of three distinct isozymes (est. Mw 35.28, 28.63, 18.94 kDa). The deinking of laser printed paper waste was performed using the purified enzyme mixture. Whole operational parameters were optimized using the Response Surface Methodology; it was found that at pH 6.8 with 47.2 h of continuous shaking at constant temperature of 35 °C, enzymes showed best deinking activity. After enzyme treatment, the physical properties of the pulp like brightness and ERIC (effective residual ink content) values were enhanced, whereas the pulp opacity was more reduced than the control treatment. Hence, the bacterial isolate and its xylanolytic enzyme system could efficiently be used in recycling paper waste as deinking agent.
Keywords: Waste management; Paper wastes; Cellulase-free xylanases; Deinking; ERIC

Production and Characterization of Violacein by Locally Isolated Chromobacterium violaceum Grown in Agricultural Wastes by Wan Azlina Ahmad; Nur Zulaikha Yusof; Nordiana Nordin; Zainul Akmar Zakaria; Mohd Fazlin Rezali (1220-1234).
The present work highlighted the production of violacein by the locally isolated Chromobacterium violaceum (GenBank accession no. HM132057) in various agricultural waste materials (sugarcane bagasse, solid pineapple waste, molasses, brown sugar), as an alternative to the conventional rich medium. The highest yield for pigment production (0.82 g L−1) was obtained using free cells when grown in 3 g of sugarcane bagasse supplemented with 10% (v/v) of l-tryptophan. A much lower yield (0.15 g L−1) was obtained when the cells were grown either in rich medium (nutrient broth) or immobilized onto sugarcane bagasse. Violacein showed similar chemical properties as other natural pigments based on the UV–Vis, Fourier transform infrared spectroscopy, thin-layer chromatography, nuclear magnetic resonance, and mass spectrometry analysis. The pigment is highly soluble in acetone and methanol, insoluble in water or non-polar organic solvents, and showed good stability between pH 5–9, 25–100 °C, in the presence of light metal ions and oxidant such as H2O2. However, violacein would be slowly degraded upon exposure to light. This is the first report on the use of cheap and easily available agricultural wastes as growth medium for violacein-producing C. violaceum.
Keywords: Chromobacterium ; Violacein; Characterization; Stability; Agriculture wastes material; Sugarcane bagasse

Recent Advances on Filamentous Fungal Biofilms for Industrial Uses by Marcel Gutiérrez-Correa; Yvette Ludeña; Gordon Ramage; Gretty K. Villena (1235-1253).
Industrial enzymes are produced by submerged fermentation (SF) and by solid-state fermentation (SSF) to a lesser extent. Although SSF has several advantages, its scale-up is difficult. The role of physiological and genetic properties of microorganisms growing attached to surfaces could explain the advantages of SSF. Filamentous fungi are naturally adapted to growth on surfaces and in these conditions they show a particular physiological behavior which is different from that in SF; thus, they also form biofilms. Fermentation by filamentous fungal biofilms (FFB) is a homogeneous production system within a liquid environment based on the infrastructure of the SF process with the productive efficiency of the SSF. Enzyme production levels of FFB are much higher than those obtained in SF and they are also amenable of mixed fungal cultivation. Transcriptomic and proteomic tools are used to uncover the fundamental biological issues behind FFB. Several genes encoding cellulolytic enzymes are either differentially expressed or overexpressed in FFB. Moreover, our proteomic studies of Aspergillus niger biofilms compared to SF indicate that many intracellular proteins are either differentially expressed or overexpressed. Clinically important fungi like A. fumigatus also form biofilms when they infect lungs and recent studies demonstrate same gene expression features. These results support our hypothesis of cell adhesion and its role in the new schemes for improved fermentative production of industrial enzymes.
Keywords: Aspergillus niger ; Biofilms; Cell adhesion; Cellulase; Gene expression; Fungi

Tannase Production by Penicillium purpurogenum PAF6 in Solid State Fermentation of Tannin-Rich Plant Residues Following OVAT and RSM by Arijit Jana; Chiranjit Maity; Suman Kumar Halder; Keshab Chandra Mondal; Bikash Ranjan Pati; Pradeep Kumar Das Mohapatra (1254-1269).
Tannase production by newly isolated Penicillium purpurogenum PAF6 was investigated by ‘one variable at a time’ (OVAT) approach followed by response surface methodology (RSM). Tannin-rich plant residues were used as supporting solid substrate and sole carbon source and, among them, tamarind seed was found to be the most favorable substrate than haritaki, pomegranate, tea leaf waste and arjun fruit. Physicochemical parameters were initially optimized using OVAT methodology and some important factors like incubation time, incubation temperature, substrate:moisture ratio as well as carbon, nitrogen and phosphate concentrations were verified with Box–Behken design of response surface methodology. Phosphate source, nitrogen source and temperature were found as the most favorable variables in the maximization of production. Tannase production was enhanced from 1.536 U/g to 5.784 U/g using tamarind seed OVAT optimization and further enhancement up to 6.15 U/g following RSM. An overall 3.76- and 4.0-fold increases in tannase production were achieved in OVAT and RSM, respectively.
Keywords: Tannase; Solid state fermentation; Plant residue; OVAT; RSM

Production of Oils from Acetic Acid by the Oleaginous Yeast Cryptococcus curvatus by G. Christophe; J. Lara Deo; V. Kumar; R. Nouaille; P. Fontanille; C. Larroche (1270-1279).
The feasibility of the conversion of acetic acid, a metabolite commonly obtained during anaerobic fermentation processes, into oils using the yeast Cryptococcus curvatus was reported. This microorganism exhibited very slow growth rates on acetate as carbon source, which led to design a two-stage cultivation process. The first consisted of cell growth on glucose as carbon source until its complete exhaustion. The second step involved the use of acetate as carbon source under nitrogen limitation in order to induce lipid accumulation. A typical experiment performed in a bioreactor involved a preliminary yeast growth with a glucose initial concentration of 15 g/L glucose. Further additions of acetate and nitrogen source allowed a final lipid accumulation up to 50% (w/w). These promising results demonstrated the suitability of the technique proposed.
Keywords: Cryptococcus curvatus ; Single cell oils; Acetic acid; Two-staged cultivation; Biodiesel

The bacteriocins of lactic acid bacteria have considerable potential for biopreservation. The Lactococcus lactis strain PSY2 (GenBank account no. JF703669) isolated from the surface of marine perch Perca flavescens produced antibacterial activity against pathogenic and spoilage-causing Gram-positive and Gram-negative bacteria viz. Arthrobacter sp., Acinetobacter sp., Bacillus subtilis, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus and possessed broad inhibitory spectrum. The biopreservative efficacy of the bacteriocin PSY2 was evaluated using fillets of reef cod, Epinephelus diacanthus. The fillets (10 g) were sprayed with 2.0 ml of 1,600 AU/ml bacteriocin, wrapped and kept under different storage temperatures viz., 4, 0 and −18 °C. The biopreservative extended the shelf-life of fillets stored at 4 °C to >21 days as against <14 days observed in the untreated samples. The total count of spoilage bacteria was reduced by 2.5 logarithmic units in the treated sample during the 14th day of storage as against the control. Chemical analysis revealed a significant change (P < 0.05) in the pH value, free fatty acid (as % oleic acid), total volatile base nitrogen and total methyl amine content in the treated samples. The overall acceptability in terms of sensory attributes was significantly higher in the bacteriocin-treated samples stored for 21 days at 4 °C while the untreated samples became unacceptable by the 14th day. The biopreservative gave no significant effect at −18 °C. Thus, the bacteriocin derived from L. lactis PSY2 gave increased protection against spoilage bacteria and offers an alternative for the preservation of high-value sea foods.
Keywords: Lactococcus lactis PSY2; Bacteriocin PSY2; Biopreservative; Storage temperature

Production of Coconut Protein Powder from Coconut Wet Processing Waste and its Characterization by Aduja Naik; S. N. Raghavendra; K. S. M. S. Raghavarao (1290-1302).
Virgin coconut oil (VCO) has been gaining popularity in recent times. During its production, byproducts such as coconut skim milk and insoluble protein are obtained which are underutilized or thrown away to the environment at present. This study deals with utilization of these byproducts to obtain a value-added product, namely, coconut protein powder. When coconut milk was subjected to centrifugation, three phases, namely, fat phase (coconut cream), aqueous phase (coconut skim milk), and solid phase (insoluble protein) were obtained. The coconut skim milk and insoluble protein were mixed and homogenized before spray drying to obtain a dehydrated protein powder. The proximate analysis of the powder showed high protein content (33 % w/w) and low fat content (3 % w/w). Protein solubility was studied as a function of pH and ionic content of solvent. Functional properties such as water hydration capacity, fat absorption capacity, emulsifying properties, wettability, and dispersibility of coconut protein powder were evaluated along with morphological characterization, polyphenol content, and color analysis. Coconut protein powder has shown to have good emulsifying properties and hence has potential to find applications in emulsified foods. Sensory analysis showed high overall quality of the product, indicating that coconut protein powder could be a useful food ingredient.
Keywords: Coconut wet processing; Coconut skim milk; Insoluble protein precipitate; Spray drying; Coconut protein powder

Rheological Behavior and Non-enzymatic Degradation of a Sulfated Galactan from Halymenia durvillei (Halymeniales, Rhodophyta) by Taratra André Fenoradosoa; Céline Laroche; Cedric Delattre; Virginie Dulong; Didier Le Cerf; Luc Picton; Philippe Michaud (1303-1313).
The rheological behavior of a sulfated galactan extracted from Halymenia durvillei, a red seaweed collected in the coastal waters of a small island of Madagascar (Nosy-be in Indian Ocean), was investigated in dilute and semi-dilute solutions. In dilute solution with NaCl at 0.3 M, the polysaccharide adopted a coil conformation whereas, at higher concentrations, the polymer had the behavior of shear-thinning fluid, typical of polymer with high molar mass or semi-rigid conformation. Degradations of this lambda carrageenan-like, using radical depolymerization, and high-pressure homogenization led to several samples of various and controlled molar masses. The measure of their intrinsic viscosities permitted the determination of the relationship of Mark–Houwink–Sakurada.
Keywords: Polysaccharide; Sulfated galactan—Halymenia durvillei ; High-pressure homogenization

Newly Isolated Lactic Acid Bacteria with Probiotic Features for Potential Application in Food Industry by Jayakumar Beena Divya; Kontham Kulangara Varsha; Kesavan Madhavan Nampoothiri (1314-1324).
Five newly isolated lactic acid bacteria were identified as Weissella cibaria, Enterococcus faecium, and three different strains of Lactobacillus plantarum by 16S rRNA sequencing. Essential probiotic requirements of these isolates such as tolerance to phenol, low pH, high sodium chloride, and bile salt concentration were checked. Efficiency in adherence to mucin and hydrophobicity of the bacterial cell were also evaluated by in vitro studies. Antimicrobial activities against some pathogens were tried, and the sensitivity of these strains against 25 different antibiotics was also checked. Further studies revealed Weissella and Enterococcus as substantial producers of folic acid. Folate is involved as a cofactor in many metabolic reactions, and it has to be an essential component in the human diet. The folate level in the fermented samples was determined by microbiological assay using Lactobacillus casei NCIM 2364 as indicator strain. The three strains of L. plantarum showed significant inhibitory activity against various fungi that commonly contaminate food stuffs indicating their potential as a biopreservative of food material.
Keywords: Lactic acid bacteria; Probiotics; Folic acid; Bioavailability; Antifungal; Biopreservative

Bioflavonoids are plant compounds touted for their potential to treat or prevent several diseases including cancer caused by various stress conditions. Galangin (4H-1-Benzopyran-4-one, 3, 5, 7-trihydroxy-2-phenyl-), a flavonoid, is a polyphenolic compound found primarily in medicinal herb, Alpinia galanga. This study aims to demonstrate the galangin as a pharmacological lead compound using in vitro, in vivo, and in silico model targeting specific cancer condition and proteins. The proliferation of MCF-7 and Ehrlich ascites carcinoma (EAC) cells was significantly inhibited with an IC50 of 34.11 and 22.29 μg/ml, respectively. In an animal model system, galangin has inhibited the tumor growth by 73.51% ± 4.742 in EAC-induced Swiss Albino mice with no evidences of mortality as compared to standard drug, 5-fluorouracil. The effectiveness of galangin is proven in an animal system suggesting its pharmacokinetics behavior in an animal model which is also complemented by outcome of in silico analysis with more than 88 % of human intestinal absorption and significant Caco-2 cell, MDCK cell, and skin permeability as predicted by in silico methods. Galangin was docked against 19 different proteins involved in tumorogenesis and apoptosis; the energetic analysis indicates that it exhibits higher predicted binding free energy of −12.7 kcal/mol with Bcl-xL protein.
Keywords: Bioflavonoid; Galangin; Erhlich ascites carcinoma; Apoptosis; Animal model; ADMET; Docking

Leishmaniasis is a group of diseases with a spectrum of clinical manifestations ranging from cutaneous ulcers to visceral leishmaniasis, which results from the bite of an infected sandfly to human. Attempts to develop an effective vaccine have been shown to be feasible but no vaccine is in active clinical use. This study adopts a Reverse Vaccinology approach to identify common vaccine candidates from both highly pathogenic species Leishmania major and Leishmania infantum. Total proteome of both species were compared to identify common proteins, which are further taken for sub-cellular localization and transmembrane helices prediction. Plasma membrane proteins having only one transmembrane helix were first identified and analyzed which are non-homologous in human and mouse in order to avoid molecular mimicry with other proteins. Selected proteins were analyzed for their binding efficiency to both major histocompatibility complex (MHC) class I and class II alleles. As a result, 19 potential epitopes are screened in this study using different approaches, which can be further verified through in vivo experiments in MHC compatible animal models. This study demonstrates that Reverse Vaccinology approach has potential in discovering various immunogenic antigens from in silico analysis of pathogen’s genome or proteome instead of culturing the whole organism by conventional methods.
Keywords: Leishmaniasis; Reverse Vaccinology; Transmembrane helices; Epitope prediction; Major histocompatibility complex (MHC)

Brugia malayi Thioredoxin Peroxidase as a Potential Vaccine Candidate Antigen for Lymphatic Filariasis by Setty Balakrishnan Anand; Vasudevan Rajagopal; Perumal Kaliraj (1351-1364).
Attempts were made to evaluate the protective efficacy of Brugia malayi thioredoxin peroxidase (BmTPX) in a mouse model. Mice immunized with a protein vaccine containing rBmTPX developed higher titres (1:5,000/1:10,000) of anti-BmTPX antibodies, compared with the mice immunized with the alum control. There was a higher level of cellular proliferative response in mice immunized with BmTPX compared with the alum control (p < 0.05), which was associated with a Th2-type of response. In order to compare the prophylactic efficacy of BmTPX in natural infection, we evaluated the human immune responses to these antigens in endemic normals (EN) and infected individuals (microfilaraemic and chronic pathology). Results showed that EN subjects carry BmTPX-specific IgG1 and IgG3 circulating antibodies against natural exposure to filariasis. Peripheral blood mononuclear cells from EN subjects responded strongly to rBmTPX by proliferating, as well as by secreting interferon (IFN)-γ (Th1) and IL-5 (Th2), a mixed type of response to rBmTPX. In the case of infected individuals, there was no IFN-γ or IL-5 response. Thus, there was a clear dichotomy in the cytokine production by infected versus EN individuals. Our findings suggest that BmTPX may be a suitable antigen candidate for lymphatic filariasis, but a further study is still required.
Keywords: Filariasis; Vaccines; Brugia ; Thioredoxin peroxidase; Antigens

Binding and Encapsulation of Doxorubicin on Smart Pectin Hydrogels for Oral Delivery by Valeria E. Bosio; Victoria Machain; Azucena Gómez López; Ignacio O. Pérez De Berti; Sergio G. Marchetti; Magdalena Mechetti; Guillermo R. Castro (1365-1376).
Pectins (Pec) of 33 to 74 % esterification degree were tested with doxorubicin (Dox), a very high toxic drug widely used in cancer therapies. Pec with 35 and 55 % DE were selected because of the Dox binding higher than Pec microspheres of 35 and 55 % obtained by ionotropic gelation with Ca+2 have 88 and 66 % Dox loading capacity. Kinetic Dox release showed more than 80.0 and about 30.0 % free drug from 35 % and 55 % Pec formulations at pH 7.4, and 37 °C after 1-h incubation, respectively. Besides, Dox release decrease to 12 % in 55 % Pec microsphere formulation after 1-year storage at 4 °C. FTIR analysis of Pec–Dox complex showed hipsochromic shifts for the σC=O, δN-H and σC-C vibrational modes compared to Dox spectrum suggesting strong interaction between the drug cargo and the matrix. Rheological studies of Pec and Pec–Dox samples flow behavior exhibited a shear-thinning nature. Fifty-five percent of Pec showed higher viscosity than the viscosity for 35 % Pec in all range of temperatures analyzed, and decreased when the temperature is raised. Besides, Pec–Dox complexes have higher viscosity values than those of the corresponding Pec samples, and viscosity curves as function of shear rate for 35 % Pec–Dox are above the curves of 55 % Pec–Dox. In both cases, the results are confirming significant interaction between the cargo and the matrix, which also was established in viscoelastic dynamic analysis.
Keywords: Drug delivery; Doxorubicin; Pectin; FTIR; Rheology

The prolonged use of the antibiotics over the years has transformed many organisms resistant to multiple drugs. This has made the field of drug discovery of vital importance in curing various infections and diseases. The drugs act by binding to a specific target protein of prime importance for the cell’s survival. Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes are the few gram positive organisms that have developed resistance to drugs. It causes pneumonia, meningitis, pharyngitis, otitis media, sinusitis, bacteremia, pericarditis, and arthritis infections. The present study was carried out to identify potential drug targets and inhibitors for beta subunit of DNA polymerase III in these three Streptococcus species that might facilitate the discovery of novel drugs in near future. Various steps were adopted to find out novel drug targets. And finally 3D structure of DNA polymerase III subunit beta was modeled. The ligand library was generated from various databases to find the most suitable ligands. All the ligands were docked using Molegro Virtual Docker and the lead molecules were investigated for ADME and toxicity.
Keywords: Streptococcus species ; BLAST; DEG; KAAS Server; Homologous proteins; Potential drug targets; Reverse vaccinology; Modeling; Docking; ADMET

Controlled Release of Sulfasalazine Release from “Smart” Pectin Gel Microspheres under Physiological Simulated Fluids by Luciana Costas; Licia M. Pera; Azucena Gómez López; Magdalena Mechetti; Guillermo R. Castro (1396-1407).
Sulfasalazine (SLZ) is a synthetic nonsteroidal anti-inflammatory drug used mainly for the treatment of an inflammatory bowel and other diseases. Two pectins with different methylation degrees were blended to synthesized gel microspheres by ionotropic gelation for SLZ encapsulation. The encapsulation efficiency was found to be around of 99% in all formulations tested. However, different SLZ release profiles related to the methylation degrees of pectin were observed. Mixture of low methylated (LM) and high methylated (HM) pectins in the presence of calcium(II) displayed the best microsphere morphologies among the formulations tested determined by optical and electronic microscopies. The percentage of drug release using a mixture of LM and HM pectins after 255 min in simulated gastric fluid (pH = 1.2), simulated intestinal fluid (pH = 6.8), and phosphate buffer (pH = 7.4) were 15.0%, 47.0%, and 52.2%, respectively.
Keywords: Pectin; Hydrogel; Drug delivery; Sulfasalazine; Biopolymers; Rheological analysis; Physiological simulated fluids

Studies of Ciprofloxacin Encapsulation on Alginate/Pectin Matrixes and Its Relationship with Biodisponibility by Germán A. Islan; Ignacio Pérez de Verti; Sergio G. Marchetti; Guillermo R. Castro (1408-1420).
Screening of ciprofloxacin (Cip) with selected biopolymers brings about 90% antibiotic interactions with a coacervate composed of alginate/high metoxylated pectin in 2:1 ratio. Fourier transform infrared spectroscopy analysis provides information about the nature of this interaction, revealing ionic and hydrophobic patterns among the molecules. Alginate/high methoxylated pectin gel microspheres developed by ionic gelation encapsulates 46.8 ± 5.0% Cip. The gel matrix can release Cip in a sustained manner, releasing 42.7 ± 0.2% in 2 h under simulated stomach pH conditions, and 83.3 ± 1.1% Cip release in 80 mM phosphate at pH = 7.40 (intestinal). The increase of sodium chloride from 50 to 200 mM implies a Cip release from 69.0 ± 1.5% to 95.1 ± 3.6% respectively in 2 h. Scanning electron microscopy revealed the cohesive effect of HM pectin over alginate molecules on the microsphere surface. Those results guarantee all Cip contained in the alginate/HM pectin microspheres could be released in an established kinetic profile along the gastrointestinal tract, avoiding the Cip undesirable side effects during absorption.
Keywords: Pectin; Alginate; Biopolymer blends; Ciprofloxacin; Controlled release

Polyvinyl Alcohol–Pectin Cryogel Films for Controlled Release of Enrofloxacin by Yanina. N. Martinez; Lucrecia. Piñuel; Guillermo. R. Castro; Javier. D. Breccia (1421-1429).
The release of enrofloxacin entrapped in polyvinyl alcohol (PVA) cryogel at pH 5.5 showed a first-order kinetic, releasing 69.7% of the antibiotic after 4.5 h at 37 °C. In order to slow down the fluoroquinolone release rate, high-methoxylated pectin was added into the cryogel (PVA–P). A film containing 1.0% (w/v) HM pectin and 5.0 μg/ml enrofloxacin released only 3.7% of the antibiotic after 4.5 h. Since the FTIR spectrum showed that most of the interactions between PVA–P matrix and enrofloxacin were due to polar groups (carboxylate and amine), a two-layer film system was designed to modulate the releasing rate of the drug. The top film equilibrated with 0.75 or 1.5 M NaCl release up to 41.9% and 89.0% of the enrofloxacin in 4 h, respectively. The release rate of enrofloxacin was found dependent on NaCl concentration in the upper gel layer. The two-layer cryogel system showed attractive features for transcutaneous antibiotic delivery.
Keywords: Transcutaneous delivery; Enrofloxacin; PVA; Pectin; Films

Synthesis, Characterization and Antiproliferative Activity of 1,2-Naphthoquinone and Its Derivatives by S. Shukla; R. S. Srivastava; S. K. Shrivastava; A. Sodhi; Pankaj Kumar (1430-1445).
In the present study substituted 1,2-naphthoquinones were synthesized, purified and characterized by spectroscopic studies (UV, FT-IR, 1H NMR, 13 C NMR and elemental analysis). These compounds were evaluated for cytotoxicity against a panel of human cancer cell lines (Hep-G2 for liver sarcoma, MG-63 for osteosarcoma and MCF-7 for human breast cancer). The cells were dosed with these ortho-naphthoquinone derivatives at varying concentrations, and cell viability was measured by a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay with doxorubicin as positive control. Significant anticancer activities were observed in vitro for some members of the series, and compounds 1,2-naphthoquinone 2-thiosemicarbazone (3), 1,2-naphthoquinone-2-semicarbazone (4), 4-amino-1,2-naphthoquinone 2-thiosemicarbazone (7) and 4-amino-1,2-naphthoquinone-2-semicarbazone (8) are active cytotoxic agents against different cancer cell lines with IC50 values in the range of 5.73–17.67 μM. The obtained data suggested that better anticancer activity was linked with introduction of thiosemicarbazone and semicarbazone moiety in 1,2-naphthoquinone ring system. Outcomes of experimentation also reveal that incorporation of amino group in 1,2-naphthoquinone moiety contributes positively for cytotoxic action of compounds. Docking experiments showed a good correlation between their calculated interaction energies with the topoisomerase-II and the observed IC50 values of all these compounds.
Keywords: Hep-G2 ; MG-63; MCF-7; Cytotoxicity; 1,2-Naphthoquinone

Four hydroxyflavone derivatives have been synthesized with the aim of obtaining a good model of superoxide dismutase. Better to mimic the natural metalloenzyme, copper complexes have been designed. The Cu(II) complexes of general formulae, [CuL] where L = 5-hydroxyflavone-o-phenylenediamine (L1H2)/m-phenylenediamine (L2H2) and 3-hydroxyflavone-o-phenylenediamine (L3H2)/m-phenylenediamine (L4H2) have been synthesized. The structural features have been determined from their analytical and spectral data. All the Cu(II) complexes exhibit square planar geometry. Redox behavior of copper complexes was studied and the present ligand systems stabilize the unusual oxidation state of the copper ion during electrolysis. The in vitro antimicrobial activities of the investigated compounds were tested against the bacterial species Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, and Pseudomonas aeruginosa and fungal species Aspergillus niger, Rhizopus stolonifer, Aspergillus flavus, Rhizoctonia bataicola, and Candida albicans. Superoxide dismutase and anti-inflammatory activities of the copper complexes have also been measured and discussed.
Keywords: Hydroxyflavone; Superoxide; Copper; Metalloenzyme; Anti-inflammatory

Comparison of Bacterial Communities in the Throat Swabs from Healthy Subjects and Pharyngitis Patients by Terminal Restriction Fragment Length Polymorphism by Kannan Balaji; Ramalingam Thenmozhi; Marimuthu Sundaravadivel; Shunmugiah Karutha Pandian (1459-1473).
Terminal restriction fragment length polymorphism (T-RFLP) analysis was applied to characterize bacterial flora present in the throats of healthy subjects and pharyngitis patients. The 16S rRNA genes of bacteria present in throat metagenome were amplified by PCR with 6-carboxy-fluorescein (6-FAM)-labeled universal forward primer (27 F) and a universal reverse primer (1513R). The 16S rDNAs were digested with restriction enzymes with 4-bp recognition sites (MspI or RsaI) and analyzed by using an automated DNA sequencer. T-RFLP patterns were numerically analyzed using computer programs. From analysis of the throat bacterial community, patterns derived from MspI and RsaI digested samples of healthy subjects and pharyngitis patients were grouped into different clusters, though RsaI digested samples showed some uncertainty. Pharyngitis throats generated an average species richness of 9 [±2.1 (SD)] and 10 (±2.9) for MspI and RsaI digests, respectively, whereas healthy throats generated 6.3 (±1.2) and 6.1 (±1.5) in MspI and RsaI digests, respectively. These results suggest that samples from pharyngitis patients contain an unexpected diversity of causative bacteria. The pharyngitis throats were colonized with a rich diversity of bacterial species than that of healthy throats. Using T-RFLP, we are able to detect a model bacterium, Streptococcus pyogenes SF370, and T-RF patterns were consistent with the Streptococcal T-RFLP patterns. Our study indicates that T-RFLP analysis is useful for the assessment of diversity of throat bacterial flora and rapid comparison of the community structure between subjects with and without pharyngitis.
Keywords: Pharyngitis; Normal flora; Metagenome; Restriction enzymes; T-RFLP