Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.

Influence of package, type of apple juice and temperature on the production of patulin by Byssochlamys nivea and Byssochlamys fulva

Although the production of patulin in apple fruits is mainly by Penicillium expansum, there is no information on the ability of heat resistant moulds that may survive pasteurization to produce this mycotoxin in juice packages during storage and distribution. In this study, the production of patulin by Byssochlamys spp (Byssochlamys nivea FRR 4421, B. nivea ATCC 24008 and Byssochlamys fulva IOC 4518) in cloudy and clarified apple juices packaged in laminated paperboard packages or in polyethylene terephthalate bottles (PET) and stored at both 21 degrees C and 30 degrees C, was investigated. The three Byssochlamys strains were able to produce patulin in both cloudy and clarified apple juices. Overall, the lower the storage temperature, the lower the patulin levels and mycelium dry weight in the apple juices (p<0.05). The greatest variations in pH and degrees Brix were observed in the juices from which the greatest mycelium dry weights were recovered. The maximum levels of patulin recovered from the juices were ca. 150 mu g/kg at 21 degrees C and 220 mu g/kg at 30 degrees C. HPLC-UV, HPCL-DAD and mass spectrometry analyses confirmed the ability of B. fulva IOC 4518 to produce patulin. Due to the heat resistance of B. nivea and B. fulva and their ability to produce patulin either in PET bottles or in laminated paperboard packages, the control of contamination and the incidence of these fungi should be a matter of concern for food safety. Control measures taken by juice industries must also focus on controlling the ascospores of heat resistant moulds. (C) 2010 Elsevier B.V. All rights reserved.

Wettability of Aqueous Rhamnolipids Solutions Produced by Pseudomonas aeruginosa LBI

The wetting behavior of rhamnolipids produced by Pseudomonas aeruginosa LBI strain grown on waste oil substrate and sodium dodecyl sulfate (SDS) on glass, polyethylene terephthalate (PET), poly(vinyl chloride) (PVC), poly(epsilon-caprolactone) (PCL) and polymer blend (PVC-PCL) was investigated by the measuring contact angle of sessile drops, to determine the wetting characteristics of rhamnolipids. The comparison of the wetting profiles showed that at low SDS and rhamnolipid concentrations, the contact angle increased and when the concentration of the surfactant increased further, the contact angle decreased. The blend surface (PVC-PCL) showed better wettability than the homopolymers themselves and the blend changed the surface hydrophobicity of the polymer, making it more hydrophilic. The rhamnolipids produced by the LBI strain exhibited superior wetting abilities than the chemical surfactant SDS one. This is the first work that evaluates the wetting properties of rhamnolipids on polymer blends.

Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352 +/- 21 and 272 +/- 25 mu M, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1 degrees C and pH 8.6. Above 37 degrees C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly. (C) 2010 Elsevier Inc. All rights reserved.

Dynamic Solid Phase DNA Extraction and PCR Amplification in Polyester-Toner Based Microchip

A variety of substrates have been used for fabrication of microchips for DNA extraction, PCR amplification, and DNA fragment separation, including the more conventional glass and silicon as well as alternative polymer-based materials. Polyester represents one such polymer, and the laser-printing of toner onto polyester films has been shown to be effective for generating polyester-toner (PeT) microfluidic devices with channel depths on the order of tens of micrometers. Here, we describe a novel and simple process that allows for the production of multilayer, high aspect-ratio PeT microdevices with substantially larger channel depths. This innovative process utilizes a CO(2) laser to create the microchannel in polyester sheets containing a uniform layer of printed toner, and multilayer devices can easily be constructed by sandwiching the channel layer between uncoated cover sheets of polyester containing precut access holes. The process allows the fabrication of deep channels, with similar to 270 mu m, and we demonstrate the effectiveness of multilayer PeT microchips for dynamic solid phase extraction (dSPE) and PCR amplification. With the former, we found that (i) more than 65% of DNA from 0.6 mu L of blood was recovered, (ii) the resultant DNA was concentrated to greater than 3 ng/mu L., (which was better than other chip-based extraction methods), and (iii) the DNA recovered was compatible with downstream microchip-based PCR amplification. Illustrative of the compatibility of PeT microchips with the PCR process, the successful amplification of a 520 bp fragment of lambda-phage DNA in a conventional thermocycler is shown. The ability to handle the diverse chemistries associated with DNA purification and extraction is a testimony to the potential utility of PeT microchips beyond separations and presents a promising new disposable platform for genetic analysis that is low cost and easy to fabricate.

Studier på nytt fordon för transport av skogsflis

Högskolan Dalarna har i samarbete med Naturbränsle i Mellansverige AB genomfört studier på ett nytt fordon för transport av skogsflis. Eftersom fordonet är utrustat med kran och skopa innebär det att flisskördaren kan tippa flisen direkt på marken, eller på en i förväg utlagd duk för att förhindra att föroreningar (grus, sten etc.) följer med vid lastningen av fordonet. Pro¬jektet har finansierats av EU-strukturfond Mål 2 Norra samt genom naturabidrag från deltag¬ande företag. Studier har även genomförts på lastväxlarfordon med container för att jämförel¬ser skall kunna göras mellan de båda fordonstyperna. Studierna omfattar sammanlagt tjugo lass, varav tio lass med det nya fordonet, fem lass med lastväxlarfordon och två containrar och fem lass med lastväxlarfordon och tre containrar. Den sammanlagda transportvolymen uppgick till 1 940 m3s. Tidsstudier har också genomförts på flisskördarens arbete, eftersom det i vissa delar är direkt beroende på vilket vidaretransportfordon som används. I de fall vidaretransporten sker med lastväxlarfordon vägs flisen innan den tippas i containern (en åtgärd för att fordonet inte skall överskrida tillåten lastvikt). Om lastvikten inte är begränsande genomför flisskördaren vissa åtgärder för att lastvolymen i respektive container skall kunna maximeras (t.ex. utjämning och viss packning av flisen med hjälp av kranen). I det fall vidaretransporten sker med det nya fordonet behöver flisskördaren inte väga flisen eftersom bilen är försedd med kranvåg. Där¬emot tillkommer andra arbetsuppgifter för flisskördaren, t.ex. preparering av avlägget (snö¬packning, sten- och buskröjning) samt hantering och utläggning av dukar.Arbetstiden för vidaretransporten är i huvudsak beroende av transportsträckan och medel¬transporthastigheten. När lastväxlarfordon används är arbetstiden dessutom beroende av last¬volymen (antalet containrar och deras volym) samt om tippmöjligheter endast finns på drag¬fordonet (containrar på släp måste omlastas till dragfordon innan flisen kan tippas) eller om tippmöjligheter finns på såväl dragbil som släp. Om släpet inte kan medföras till uppställ¬ningsplatsen för containrarna (p.g.a. alltför smal och kurvig väg eller utrymmesbrist för vänd¬ning av fordonsekipaget) påverkas även arbetstiden av avståndet mellan containrar och släp samt av medelhastigheten vid ”skotning” av containrar till och från släpet.Medelhastigheten vid transport var i stort sett densamma för de båda fordonstyperna. För last¬växlarfordonet med två containrar tog den totala arbetstiden (inklusive tom- och lasskörning) mellan 1,70 och 2,99 G0-tim per lass, vilket motsvarar en prestation på mellan 32 och 52 m3s/G0-tim. Arbetstiden för lastning och lossning varierade mellan 0,87 och 1,09 G0-tim/lass, eller 0,009-0,011 G0-tim/m3s (medeltal 0,98 G0-tim/lass, eller 0,011 G0-tim/m3s), vilket motsvarar en prestation på mellan 87 och 107 m3s/G0-tim.För fordonet med tre containrar tog den totala arbetstiden (inklusive tom- och lasskörning) mellan 2,40 och 3,53 G0-tim per lass, vilket motsvarar en prestation på mellan 30 och 45 m3s/ G0-tim. Arbetstiden för lastning och lossning varierade mellan 1,07 och 1,62 G0-tim/ lass, eller 0,011-0,016 G0-tim/m3s (medeltal 1,33 G0-tim/lass, eller 0,013 G0-tim/m3s), vilket mot¬svarar en prestation på mellan 64 och 89 m3s/G0-tim.Den totala arbetstiden för det nya fordonet (inklusive tom- och lasskörning) varierade mellan 2,11 och 5,08 G0-tim per lass, vilket motsvarar en prestation på mellan 18 och 45 m3s/G0-tim. Arbetstiden för lastning och lossning varierade mellan 0,96 och 1,46 G0-tim per lass, eller 0,01-0,02 G0-tim/m3s (medeltal 1,22 G0-tim/lass, eller 0,013 G0-tim/m3s), vilket mot¬svarar en prestation på mellan 65 och 94 m3s/G0-tim.