A recently developed and validated method for simultaneous determination of 17 inorganic and organic arsenic compounds in marine biota has been successfully applied to routine analysis of different ...food products, including fish, shellfish, edible algae, rice, and other types of grain. During one year, approximately 250 food samples were analyzed, mostly fish and rice. Long-term stability and robustness of the system was observed and reproducible results for certified reference materials were ensured by means of control charts. The separation was performed by ion-pair chromatography on an anion-exchange column to separate anionic, neutral, and cationic arsenic species in one chromatographic run. Hyphenation to ICP-MS allowed element-specific and sensitive detection of the different arsenic species with a detection limit as low as 8 ng As L(-1 )in the sample extract, which is equivalent to 2 ng As g(-1) in the original sample. Special emphasis was laid on the analysis of marine algae and rice samples. These food types can contain elevated levels of the very toxic inorganic arsenic species (up to 90% in rice) and therefore are the focus of interest in the food industry. In marine algae, inorganic arsenic was mainly present as arsenate whereas in rice arsenite predominated.
Because of their ubiquity in the aquatic environment, the antifouling agent tributyltin (TBT) and other organotins (OTs) accumulate through the food chain, resulting in the occurrence of OTs in ...seafood products. Despite a high number of studies on the negative impact of TBT in female prosobranch gastropods, few works exist in Europe reporting the levels of these compounds in edible parts of marine organisms used in Human diet. Therefore, within the scope of an EU project OT-SAFE the levels of several OTs were evaluated in the most relevant seafood products for Portuguese consumers. Butyltins (BTs) have been detected in all analysed groups (fish, crustaceans, bivalves, cephalopods), whereas triphenyltin, tricyclohexyltin, monooctyltin and dioctyltin could not be detected and tetrabutyltin was present above detection limits in a single sample. In general, levels of BTs in edible parts of fish, crustaceans and cephalopods collected in Portuguese markets during this study are in the lower range of that reported for these animal groups from other locations (i.e. below 30ngg−1 wet weight). In contrast, moderate to high concentrations have been observed in bivalves (up to 275ng TBTg−1 wet weight). While most samples showed TBT plus DBT levels below the tolerable average residue levels (TARL), which may indicate low risk for consumer, four bivalve samples displayed BT levels above TARL, thus indicating that higher bivalve consumer groups may be at risk. The results found are discussed in relation to the potential risk for consumers and integrated with recent finds on the molecular targets of OTs in mammals.
Hyaluronic acid (HA), with diverse cosmetic and medical applications, is the natural glycosaminoglycan product of HA synthases. Although process and/or metabolic engineering are used for industrial ...HA production, the potential of protein engineering has barely been realised. Herein, knowledge‐gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain‐length specificity and a twofold increase in mass‐based turnover number. Seven improved pmHAS variants out of 1392 generated by error‐prone PCR were identified; eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (<4.7 MDa), through an engineered pmHAS variant in a cell‐free system, was synthesised. Computational studies showed that substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increase the flexibility of the N‐terminal region of pmHAS. Taken together, these findings suggest that the N terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.
The more the merrier: Engineered hyaluronan synthase from P. multocida (pmHAS) exhibits improved N‐terminal region flexibility and enhanced hyaluronic acid (HA) polymerising activity. The production of high‐molecular‐weight HA at twice the normal output can now be achieved; this demonstrates the employability of protein engineering in HA production.
Hydrophilic poly(ethylene glycol)-based monoliths were synthesized in the spin-tip format for high-throughput applications via pulsed electron beam irradiation. Monoliths with a homogeneous porous ...structure and a total porosity of 69% were obtained. The cross-linked polymeric structure was further mechanically stabilized via the incorporation of silica nanoparticles. Amino-functionalization of the monoliths was accomplished by a straightforward, water-based, one-step approach that entailed the electron-beam irradiation-induced grafting of poly(allylamine). The amine functionalized spin columns showed very low unspecific protein adsorption and were successfully applied as adsorbents in lectin affinity chromatography for the purification of ovalbumin. The novel columns also outperformed a commercially available system.
Arsenic speciation is of increasing interest to the food industry, as concerns about high total arsenic concentrations in food can often be alleviated to a great extent if the ratio of toxic, less ...toxic and non-toxic arsenic compounds in the sample is known. The lipid matrix of fish oil is a challenge in the determination of arsenic species, as current methods for this type of analysis require the analyte to be water-soluble. In this study, two sample preparation techniques were applied. One the one hand water-soluble species were extracted with methanol/water, on the other, acid digestion was applied to release lipid-soluble arsenic compounds into the aqueous phase. Ion chromatography - inductively coupled plasma mass spectrometry (IC-ICP-MS) was used for separation and sensitive element-specific detection of arsenic compounds. Additional experiments, including alkaline hydrolysis, were carried out to find out more about the type of lipids arsenic is bound to in fish oil. Up to eight different arsenic species were detected and quantified in fish oil with dimethylarsinate being the major compound both in the aqueous extract and in the acid digest. No inorganic arsenic was detected in the aqueous extract, and the maximum concentration of arsenate determined in the acid digest was 0.05 mu g g super(-1). The total arsenic concentration determined by ICP-MS ranged from <0.1 to 5 mu g g super(-1). With regard to the mass balance, approximately 1% of the total arsenic content was extractable with methanol/water, whereas the sum of arsenic species quantified after acid digestion yielded 85-100% of the total arsenic content. It was confirmed that the large fraction of arsenic in fish oil not extractable on an aqueous basis consists of organoarsenic compounds. This new approach in sample preparation makes the complete characterization of the arsenic content in the sample possible with regard to the respective species, providing necessary information required for risk assessment.
