Catalytic properties of endoxylanase fusion proteins from Neocallimastix frontalis and effect of immobilization onto metal-chelate matrix

The production of hybrid enzymes with novel properties and the research for new methods for enzyme immobilization in bioreactors are of major interest in biotechnology. We report here the second part of a study concerning the improvement of the properties of the endoxylanase XYN3A4 from the anaerobic fungi Neocallimastix frontalis. The effects of gene fusion and immobilization on metal-chelate matrix are also compared for the reference enzymes XYN3, XYN3A, XYN4 used for the construction of the fusion protein XYN3A4. The influence of the metal ion in the immobilization process was first investigated and best immobilization yields were obtained with the Cu(II) ion whereas best coupling efficiencies were reached with the Ni(II) ion. It was also observed that XYN3, XYN3A and XYN34 had a lower rate of hydrolysis when immobilized on Ni(II)-IDA and more difficulties to accomodate small substrates than the soluble enzymes. Nevertheless, a major difference was noted during the hydrolysis of birchwood xylan and it appears that the reaction using the immobilized XYN3A4 chimeric enzyme leads to the accumulation of a specific product.     

The early ontogeny of digestive and metabolic enzyme activities in two commercial strains of arctic charr (Salvelinus alpinus L.)

The extent to which growth performance is linked to digestive or energetic capacities in the early life stages of a salmonid species was investigated. We compared two strains of Arctic charr known to have different growth potentials during their early development (Fraser and Yukon gold). Trypsin, lipase, and amylase activities of whole alevins were measured at regular intervals from hatching through 65 days of development. To assess catabolic ability, we also measured five enzymes representing the following metabolic pathways: amino acid oxidation (amino aspartate transferase), fatty acid oxidation (beta-hydroxy acyl CoA-dehydrogenase), tricarboxylic acid cycle (citrate synthase), glycolysis (pyruvate kinase), and anaerobic glycolysis (lactate dehydrogenase). The measurement of these enzyme activities in individual fish allowed a clear evaluation of digestive capacity in relation to energetic demand. We also compared triploid and diploid individuals within the Yukon gold strain. For the whole experimental period, diploid Yukon gold fish exhibited the highest growth rate (1.08+/-0.18% length/day) followed by triploid Yukon gold fish (1.00+/-0.28% length/day) and finally Fraser strain fish (0.84+/-0.28% length/day). When differences in enzyme activities were observed, the Fraser strain showed higher enzyme activities at a given length than the Yukon gold strain (diploid and triploid). Higher growth performance appears to be linked to lower metabolic capacity. Our results suggest that fish may have to reach an important increase in the ratio of digestive to catabolic enzyme activities or a leveling off of metabolic enzyme activities before the onset of large increases in mass.     

The Saccharomyces cerevisiae alcohol acetyl transferase gene ATF1 is a target of the cAMP/PKA and FGM nutrient-signalling pathways

The ATF1-encoded Saccharomyces cerevisiae yeast alcohol acetyl transferase I is responsible for the formation of several different volatile acetate esters during fermentations. A number of these volatile esters, e.g. ethyl acetate and isoamyl acetate, are amongst the most important aroma compounds in fermented beverages such as beer and wine. Manipulation of the expression levels of ATF1 in brewing yeast strains has a significant effect on the ester profile of beer. Northern blot analysis of ATF1 and its closely related homologue, Lg-ATF1, showed that these genes were rapidly induced by the addition of glucose to anaerobically grown carbon-starved cells. This induction was abolished in a protein kinase A (PKA)-attenuated strain, while a PKA-overactive strain showed stronger ATF1 expression, indicating that the Ras/cAMP/PKA signalling pathway is involved in this glucose induction. Furthermore, nitrogen was needed in the growth medium in order to maintain ATF1 expression. Long-term activation of ATF1 could also be obtained by the addition of the non-metabolisable amino acid homologue beta-L-alanine, showing that the effect of the nitrogen source did not depend on its metabolism. In addition to nutrient regulation, ATF1 and Lg-ATF1 expression levels were also affected by heat and ethanol stress. These findings help in the understanding of the effect of medium composition on volatile ester synthesis in industrial fermentations. In addition, the complex regulation provides new insights into the physiological role of Atf1p in yeast.     

Method for monitoring alginate released in biological fluids by high-performance anion-exchange chromatography with pulsed amperometric detection

The use of alginate-entrapped cells in cell therapy requires a method for monitoring possible released compound within biological fluids following either their implantation or inoculation in artificial organs. Oligomannuronic and oligoguluronic acids were prepared by enzymatic depolymerization with alginate lyase from Pseudomonas alginovora, characterized by high-performance anion-exchange chromatography with pulsed amperometric detection and quantitated in human, pig and rabbit blood, urine and tissue samples. The method was tested for linearity and detection limit, accuracy, intra- and inter-day precision. The limit of detection was 3 microgram/ml in both urine and plasma and 5 mg/g of tissues. The relative standard deviations (RSDs) of intra-day precision were 6.0-16.6% and 4.8-8.7% in plasma and urine, respectively; the RSDs of inter-day precision were 5.1-14.4% and 5.0-11.6% in plasma and urine, respectively. Thus, this method appears suitable for the measurement of released alginate from entrapped cells used in cell therapy.     

Automated large-volume sample stacking procedure to detect labeled peptides at picomolar concentration using capillary electrophoresis and laser-induced fluorescence detection

We have developed an automated large-volume sample stacking (LVSS) procedure to detect fluorescein isothiocyanate-labeled peptides in the picomolar range. The injection duration is 10 min at 50 mbar to fill 62% of the capillary volume to the detection cell. The calculated limit of detection (S/N=3), filling 1% of the capillary volume, is 74 pM for bradykinin and 45 pM for L-enkephalin with samples diluted in water and analyzed in a 50 mM borate buffer, pH 9.2. With the automated LVSS system, the limits of detection are 7 pM for bradykinin, 3 pM for L-enkephalin and 2 pM for substance P. LVSS is shown to be quantitative from 500 to 10 pM.     

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.     

Microphytobenthos assemblage mapping by spatial visible-infrared remote sensing in a shellfish ecosystem

The aim of this work is to assess the use of (SPOT) multispectral visible infrared remote sensing to study microphytobentos assemblages in a shellfish ecosystem (Bay of Bourgneuf, France). SPOT satellite images (acquired at low tide in spring or autumn between 1986 and 1998) were calibrated using in situ radiometric data, and the normalised vegetation index (NDVI) obtained from these images showed microphytobenthos on bay mudflats. Proliferation was mainly along a north-south strip, essentially localised around the +2 m isobath and covering a surface area of 19 to 25% of the total mudflat area studied (420 to 550 ha). Three factors seem to be responsible for the spatial structure of the assemblages: bathymetry, nutrient input from the Falleron River and its channel, and the location of oyster-farming areas. Although spatial and spectral resolutions of multispectral remote sensing data have certain limitations, this approach opens up a new field of application for hyperspectral remote sensing, particularly for synoptic mapping of biomass distribution.     

Heterologous protein production and delivery systems for Lactococcus lactis

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed.