Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor

<p>Abstract</p> <p>Background</p> <p>Staphylococcal (or micrococcal) nuclease or thermonuclease (SNase or Nuc) is a naturally-secreted nucleic acid degrading enzyme that participates in <it>Staphylococcus aureus </it>spread in the infected host. Purified Nuc protein can be used as an exogenous reagent to clear cellular extracts and improve protein purification. Here, a recombinant form of Nuc was produced and secreted in a Gram-positive host, <it>Lactococcus lactis</it>, and purified from the culture medium.</p> <p>Results</p> <p>The gene segment corresponding to the <it>S. aureus </it>nuclease without its signal peptide was cloned in an expression-secretion vector. It was then fused to a lactococcal sequence encoding a signal peptide, and expressed under the control of a lactococcal promoter that is inducible by zinc starvation. An <it>L. lactis </it>subsp <it>cremoris </it>model strain (MG1363) transformed with the resulting plasmid was grown in either of two media (GM17v and CDM) that are free of animal compounds, allowing GMP (Good Manufacturing Practice) production. Induction conditions (concentration of the metal chelator EDTA and timing of addition) in small-scale pH-regulated fermentors were optimized using LacMF (Lactis Multi-Fermentor), a home-made parallel fermentation control system able to monitor 12 reactors simultaneously. Large amounts of recombinant Nuc (rNuc) were produced and secreted in both media, and rNuc was purified from GM17v medium in a single-step procedure.</p> <p>Conclusions</p> <p>In <it>L. lactis</it>, rNuc production and secretion were optimal after induction by 0.5 mM EDTA in small scale (200 mL) GM17v exponential phase cultures (at an OD₆₀₀of 2), leading to a maximal protein yield of 210 mg per L of culture medium. Purified rNuc was highly active, displaying a specific activity of 2000 U/mg.</p

Application of the final flotation waste for obtaining the glass-ceramic materials

This work describes the investigation of the final flotation waste (FFW), originating from the RTB Bor Company (Serbia), as the main component for the production of glass-ceramic materials. The glass-ceramics was synthesized by the sintering of FFW, mixtures of FFW with basalt (10%, 20%, and 40%), and mixtures of FFW with tuff (20% and 40%). The sintering was conducted at the different temperatures and with the different time duration in order to find the optimal composition and conditions for crystallization. The increase of temperature, from 1100 to 1480°C, and sintering time, from 4 to 6h resulted in a higher content of hematite crystal in the obtained glass-ceramic (up to 44%). The glass-ceramics sintered from pure FFW (1080°C/36h) has good mechanical properties, such as high propagation speed (4500 m/s) and hardness (10800 MPa), as well as very good thermal stability. The glass-ceramics obtained from mixtures shows weaker mechanical properties compared to that obtained from pure FFW. The mixtures of FFW with tuff have a significantly lower bulk density compared to other obtained glass-ceramics. Our results indicate that FFW can be applied as a basis for obtaining the construction materials

Detection of Anatoxin-a(s) in Environmental Samples of Cyanobacteria by Using a Biosensor with Engineered Acetylcholinesterases

Bioassays are little used to detect individual toxins in the environment because, compared to analytical methods, these assays are still limited by several problems, such as the sensitivity and specificity of detection. We tentatively solved these two drawbacks for detection of anatoxin-a(s) by engineering an acetylcholinesterase to increase its sensitivity and by using a combination of mutants to obtain increased analyte specificity. Anatoxin-a(s), a neurotoxin produced by some freshwater cyanobacteria, was detected by measuring the inhibition of acetylcholinesterase activity. By using mutated enzyme, the sensitivity of detection was brought to below the nanomole-per-liter level. However, anatoxin-a(s) is an organophosphorous compound, as are several synthetic molecules which are widely used as insecticides. The mode of action of these compounds is via inhibition of acetylcholinesterase, which makes the biotest nonspecific. The use of a four-mutant set of acetylcholinesterase variants, two mutants that are sensitive to anatoxin-a(s) and two mutants that are sensitive to the insecticides, allows specific detection of the cyanobacterial neurotoxin

Conjugation of genetically-engineered protein phosphatases to magnetic particles for okadaic acid detection

This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3μg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1μg/L)

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An improved biosensor for acetaldehyde determination using a bienzymatic strategy at poly(neutral red) modified carbon film electrodes

Improved biosensors for acetaldehyde determination have been developed using a bienzymatic strategy, based on a mediator-modified carbon film electrode and co-immobilisation of NADH oxidase and aldehyde dehydrogenase. Modification of the carbon film electrode with poly(neutral red) mediator resulted in a sensitive, low-cost and reliable NADH detector. Immobilisation of the enzymes was performed using encapsulation in a sol-gel matrix or cross-linking with glutaraldehyde. The bienzymatic biosensors were characterized by studying the influence of pH, applied potential and co-factors. The sol-gel and glutaraldehyde biosensors showed a linear response up to 60 [mu]M and 100 [mu]M, respectively, with detection limits of 2.6 [mu]M and 3.3 [mu]M and sensitivities were 1.7 [mu]A mM-1 and 5.6 [mu]A mM-1. The optimised biosensors showed good stability and good selectivity and have been tested for application for the determination of acetaldehyde in natural samples such as wine.http://www.sciencedirect.com/science/article/B6TF4-4NBH223-4/1/df3a7ae65fbb5f9739654441b2c733e