Evaluation of a high temperature immobilised enzyme reactor for production of non-reducing oligosaccharides

There is interest in the production of non-reducing carbohydrates due to their potential application in various industrial fields, particularly the food industry. In this paper, we describe the development of an immobilised cell bioprocess for the synthesis of non-reducing maltodextrins at high temperatures. The trehalosyl-dextrins-forming enzyme (TDFE) isolated from the thermoacidophilic archaeon Sulfolobus solfataricus (strain MT4), was recently expressed at high yields in Escherichia coli (strain Rb-791). Here, we evaluate different matrices, such as polyacrylamide gel, crude egg white, chitosan and calcium alginate for their effectiveness in immobilising whole recombinant E. coli cells subjected to prior thermal permeabilisation. Calcium-alginate based gels formed a solid biocatalyst with a good activity yield and the best enzymatic stability at the operating temperature (75°C). Therefore, these beads were used to pack a glass column reactor to perform the bioconversion of interest. Optimal operating parameters were defined in relation to the substrate stream flow-rate and the substrate-to-biocatalyst ratio. The production of trehalosylmaltotetraose from maltohexaose reached equilibrium with a constant of about 2.6 at 75°C. The bioreactor was exploited for production of trehalosylmaltodextrins from a commercial mixture of maltodextrins, achieving a productivity of 106.5 mg ml⁻¹ h⁻¹ (g biocatalyst)⁻¹ with ~40% conversion when using a 30% (w/v) solution.     

Assessment of in-line near-infrared spectroscopy for continuous monitoring of fermentation processes

The application of NIR in-line to monitor and control fermentation processes was investigated. Determination of biomass, glucose, and lactic and acetic acids during fermentations of Staphylococcus xylosus ES13 was performed by an interactance fiber optic probe immersed into the culture broth and connected to a NIR instrument. Partial least squares regression (PLSR) calibration models of second derivative NIR spectra in the 700-1800 nm region gave satisfactory predictive models for all parameters of interest: biomass, glucose, and lactic and acetic acids. Batch, repeated batch, and continuous fermentations were monitored and automatically controlled by interfacing the NIR to the bioreactor control unit. The high frequency of data collection permitted an accurate study of the kinetics, supplying lots of data that describe the cultural broth composition and strengthen statistical analysis. Comparison of spectra collected throughout fermentation runs of S. xylosus ES13, Lactobacillus fermentum ES15, and Streptococcus thermophylus ES17 demonstrated the successful extension of a unique calibration model, developed for S. xylosus ES13, to other strains that were differently shaped but growing in the same medium and fermentation conditions. NIR in-line was so versatile as to measure several biochemical parameters of different bacteria by means of slightly adapted models, avoiding a separate calibration for each strain.     

A simple non invasive computerized method for the assessment of bone repair within osteoconductive porous bioceramic grafts

Single energy X-ray imaging, due to its low cost and flexibility, is one of the most used and common technique to assess bone state and bone remodeling over time. Standardized X-ray images are needed to compare sets of radiographs for semi-quantitative analyses of tissue remodeling. However, useful mathematical modeling for the analysis of high level radiographic images are not easily available. In order to propose a useful evaluation tool to a wide clinical scenario, we present an innovative calibration algorithm for a semi-quantitative analysis of non-standardized digitized X-ray images. For calibration on a unique standardization scale, three time invariant regions (ROI) of radiographs were selected and analyzed. The accuracy of the normalization method for X-ray films was successfully validated by using an aluminum step wedge for routine X-ray exposures as tool to standardize serial radiographs (Pearson correlation test: R(2) = 0.96). This method was applied to investigate the progression of the new bone deposition within ceramic scaffolds used as osteoconductive substitute in large bone defects taking advantage of a large animal model. This innovative image-processing algorithm allowed the identification and semi-quantification of the bone matrix deposited within the implant. The osteo-integration at the bone-implant interface was also investigated. A progressively increasing bone tissue deposition within the porous bioceramic implant and a progressive osteo-integration was observed during the 12 months of the trial.     

The role of unstructured extensions in the rotational diffusion properties of a globular protein: the example of the titin i27 module

The possibility of predicting the overall shape of a macromolecule in solution from its diffusional properties has gained increasing importance in the structural genomic era. Here we explore and quantify the influence that unstructured and flexible regions have on the motions of a globular protein, a situation that can occur from the presence of such regions in the natural sequence or from additional tags. I27, an immunoglobulin-like module from the muscle protein titin, whose structure and properties are well characterized, was selected for our studies. The backbone dynamics and the overall tumbling of three different constructs of I27 were investigated using (15)N NMR relaxation collected at two (15)N frequencies (60.8 and 81.1 MHz) and fluorescence depolarization spectroscopy after labeling of a reactive cysteine with an extrinsic fluorophore. Our data show that the presence of disordered tags clearly exerts a frictional drag that increases with the length of the tags, thus affecting the module tumbling in solution. We discuss the use and the limitations of current approaches to hydrodynamic calculations, especially when having to take into account local flexibility.     

A proteomics strategy for the enrichment of receptor-associated complexes

Multimeric protein complexes are important for cell function and are being identified by proteomics approaches. Enrichment strategies, such as those employing affinity matrices, are required for the characterization of such complexes, for example, those containing growth factor receptors. The receptor for the macrophage lineage growth factor, macrophage-colony stimulating factor (M-CSF or CSF-1), is the tyrosine kinase, c-Fms. There is evidence that the CSF-1 receptor (CSF-1R) forms distinct multimeric complexes involving autophosphorylated tyrosines in its cytoplasmic region; however, these complexes are difficult to identify by immunoprecipitation, making enrichment necessary. We report here the use of a tyrosine-phosphorylated, GST-fusion construct of the entire CSF-1R cytoplasmic region to characterize proteins putatively associating with the activated CSF-1R. Besides signalling molecules known to associate with the receptor or be involved in CSF-1R-dependent signalling, mass spectrometry identified a number of other molecules binding to the construct. So far among these candidate proteins, dynein, claudin and silencer of death domains co-immunoprecipitated with the CSF-1R, suggesting association. This affinity matrix method, using an entire cytoplasmic region, may have relevance for other growth factor receptors.     

Aggregation properties of a HPMA-camptothecin copolymer in isotonic solutions

Copolymers of camptothecin (CPT) and [N-(2-hydroxypropyl) methacrylamide] (HPMA) are novel anticancer drugs that show improved pharmacological profile in animal models as compared to the free drug CPT. We investigate here the aggregation properties of a HPMA-glycyl-6-aminohexanoyl-glycyl-CPT copolymer (20,000 Da). The molecular size of HPMA-copolymer CPT is followed over 5 orders of magnitudes of concentration in isotonic buffer by measuring either the time resolved fluorescence anisotropy (FA) of CPT or the autocorrelation function of the light scattered by the copolymer. A detailed analysis of these data suggests the presence of elongated structures with axial ratio 3 in the range 0.1–0.5 μg/ml and aggregates with association number higher than 2 in more concentrated solutions (up to 10 mg/ml). The binding affinity of HPMA-copolymer CPT for serum albumin is inversely dependent on the degree of aggregation of the copolymer. We also show that the copolymer concentration in plasma from mice treated with an active, non-toxic, dose of HPMA-copolymer CPT, decreases from 3 to 0.01 mg/ml in 72 h. In the same range of concentrations in vitro, we do not detect hydrophobic aggregates of polymers with high (>3) association number. Our study indicates that the circulating HPMA-copolymer CPT in mice should not undergo extensive aggregation and should interact with serum albumin more weakly than free CPT.     

Influence of 50-Hz electromagnetic field on anurian (Xenopus laevis) metamorphosis

In this study, we show the effect of a 1-mT magnetic field AC at 50 Hz on Xenopus laevis tadpole populations. In the course of a 65-day exposure to the field, tadpole survival showed a small, but significant, decrease (p < 0.0004), together with a striking parallel 6-day shift in tadpole maturation frequency and a significant impairment of their metamorphosis. Particularly, metamorphosis was successful for 85% of individuals in the unirradiated tadpole population and for 45% of individuals in the irradiated tadpole population, respectively.     

Expression of rolB in tobacco flowers affects the coordinated processes of anther dehiscence and style elongation

The effect of auxin on stamen and pistil development in tobacco flowers was investigated by means of the localized expression of rolB (root loci B), an Agrobacterium oncogene that increases auxin sensitivity in a cell-autonomous fashion. When rolB is driven by the promoter of the meiosis-specific Arabidopsis gene DMC1 (disrupted meiotic cDNA 1), expression occurs earlier in male than in female developing organs, resulting in a delay in anther dehiscence with respect to normal timing of pistil development. As a consequence of this developmental uncoupling, self-pollination is prevented in pDMC1:rolB plants. Histological analysis of pDMC1:GFP plants indicates that in tobacco, this promoter is active not only in meiocytes but also in somatic tissues of the anther. In contrast, simultaneous expression of rolB in anther and pistil somatic tissues, achieved by expressing a construct containing rolB under the control of the promoter of the petunia gene FBP7 (floral binding protein 7), results in a concomitant delay of both anther dehiscence and pistil development without affecting self-pollination of the plants. Analysis of plants harboring the pFBP7:GUS construct shows that in tobacco, this promoter is active not only in the ovules, as described for petunia, but also in pistil and anther somatic tissues involved in the dehiscence program. The delay in anther dehiscence and pistil development could be phenocopied by exogenous application of auxin. Jasmonic acid (JA) could not rescue the delay in anther dehiscence. These results suggest that auxin plays a key role in the timing of anther dehiscence, the dehiscence program is controlled by the somatic tissues of the anther, and auxin also regulates pistil development.