Gelatin hydrogels find broad medical application. The current materials, however, are from animal sources, and their molecular structure and thermal properties cannot be controlled. This study describes recombinant gelatin-like polymers with a general design that inherently offers independent tuning of the cross-link density, melting temperature, and biocompatibility of the gel. The polymers contain small blocks with thermoreversible trimerization capacity and defined melting temperature, separated by hydrophilic nontrimerizing blocks defining the distance between the knot-forming domains. As an example, we report the secreted production in yeast at several g/L of two nonhydroxylated approximately 42 kDa triblock copolymers with terminal trimerizing blocks. Because only the end blocks formed cross-links, the molecular architecture of the gels is much more defined than that of traditional gelatins. The novel hydrogels had a approximately 37 degrees C melting temperature, and the dynamic elasticity was independent of the thermal history. The concept allows to produce custom-made precision gels for biomedical applications.

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An amphiphilic silk-like protein polymer was efficiently produced in the yeast Pichia pastoris. The secreted product was fully intact and was purified by solubilization in formic acid and subsequent precipitation of denatured host proteins upon dilution with water. In aqueous alkaline solution, the negatively charged acidic polymer assumed extended helical (silk III-like) and unordered conformations. Upon subsequent drying, it assumed a conformation rich in beta-turns. In water at low pH, the uncharged polymer aggregated and the solution became turbid. Concentrated solutions in 70% (v/v) formic acid slowly formed gels. Replacement of the formic acid-water mixture with methanol and subsequent drying resulted in beta-sheets, which stacked into fibril-like structures. The novel polymer instantaneously lowered the air-water interfacial tension under neutral to alkaline conditions and reversed the polarity of hydrophobic and hydrophilic solid surfaces upon adsorption.

The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay, NALFIA). Prior to the detection with NALFIA, a rapid amplification method with tagged primers was applied. In the evaluation of the optimised NALFIA strips, no cross-reactivity was found for any of the antibodies used. The limit of detection was higher than for quantitative PCR (q-PCR), in most cases between 104 and 105 colony forming units/mL or 0.1–0.9 ng/μL DNA. NALFIA strips were applied to 48 isolates from cattle faeces, and results were compared to those achieved by q-PCR. E. coli virulence factors identified by NALFIA were in very good agreement with those observed in q-PCR, showing in most cases sensitivity and specificity values of 1.0 and an almost perfect agreement between both methods (kappa coefficient larger than 0.9). The results demonstrate that the screening method developed is reliable, cost-effective and user-friendly, and that the procedure is fast as the total time required is <1 h, which includes amplification.FigureResults achieved with multi-analyte NALFIA for E. coli virulence factors. First strip: blank; second to sixth strip: each of the STEC factors; seventh strip: all factorsElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-010-4334-z) contains supplementary material, which is available to authorized users.

To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisindependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenasedeficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.Mannitol is a reduced form of fructose and is produced by a variety of microorganisms including bacteria, yeasts, and fungi. Besides the ability of several organisms to maintain their redox balance by the production of mannitol (9, 21, 22), mannitol has a physiological function in microorganisms as an osmolyte (16) and can serve as a protecting agent. It has been reported that mannitol enhances survival of Lactococcus lactis cells during drying processes (10). The viability of starter cultures of L. lactis, a lactic acid bacterium (LAB) extensively used in dairy industry, may thus be enhanced by mannitol production. In addition, the use of a mannitol-producing L. lactis may result in fermented products with extra value, since mannitol is assumed to have several beneficial effects as a food additive. It can serve as an antioxidant (4,5,25,26) and as a low-calorie sweetener that can replace sucrose (6,8).In heterofermentative LABs such as Leuconostoc mesenteroides, mannitol is formed from fructose in a single conversion by mannitol dehydrogenase, and fructose-to-mannitol conversion rates of Ͼ90% are common (13,24,27). In contrast, most homofermentative LABs, such as Lactococcus lactis, do not normally produce mannitol. Mannitol formation in homofermentative LABs is limited to strains whose ability to regenerate NAD to fulfill the redox balance is severely hampered. In these strains, mannitol 1-phosphate dehydrogenase (M1PDH) and mannitol 1-phosphatase (M1Pase) are the enzymes involved in the mannitol biosynthesis route (Fig. 1). Transient formation of high concentrations of intracellular mannitol (90 mM) and mannitol 1-phosphate (76 mM) were detected in high-density nongrowing cell suspensions of a lactate dehydrogenase (LDH)-deficient L. lactis strain (22). During growth, only small amounts of mannitol (Ͻ0.4 mM) were transiently produced extracellularly (23). Recently, inactivation of the mannitol transport system in an LDH-def...