Estera Szwajcer Dey

Flow injection analysis of mercury(II) based on enzyme inhibition and thermometric detection

An enzymatic procedure for the determination of mercury(II) is described, based on inhibition of invertase using glucose oxidase and catalase co-immobilised on controlled-pore glass (CPG) coupled to a thermometric continuous-flow sensor system to follow the invertase activity. A small amount of invertase (0.66 U ml−1) was incubated for a short time in sucrose solution and 20 μl of the mixture were injected into the enzyme thermistor system to give a temperature change corresponding to 100% enzyme activity. Addition of a mercury(II) sample to the mixture caused a decrease in the invertase activity, that allowed the determination of mercury(II) concentrations in the 5–80 ppb range with RSD ⩽ 0.74%. The analysis time was 2–6 min including incubation. The main advantages of this thermometric biosensor assay are as follows: simplicity, with no need for regeneration due to the use of a cheap, soluble sensing enzyme; robustness, with excellent reproducibility and repeatability; and long operational and storage stability of the enzymes involved in the detection system.

Poly(3-Hydroxybutyrate) Biosynthesis in the Biofilm of Alcaligenes eutrophus, Using Glucose Enzymatically Released from Pulp Fiber Sludge

ABSTRACT Glucose, enzymatically released from pulp fiber sludge, was combined with inorganic salts and used as a growth medium for Alcaligenes eutrophus, a gram-negative strain producing poly(3-hydroxybutyrate) (PHB). By controlling the concentrations of the inorganic salts in the growth medium, almost 78% of the cell mass was converted to pure PHB. Efforts were made to find conditions for bacterial growth in the form of a biofilm on a cheap and reusable carrier. A number of positively charged carriers were tested, and the anion exchanger DEAE-Sephadex A-25 was chosen as a microcarrier for packed-bed biofilm cultures of A. eutrophus. Conditions for attachment, growth, and detachment were established. Biofilm formation on the microcarrier is strongly dependent on the ionic strength of the attachment medium. In order to achieve formation of the biofilm and its recovery from the microcarrier, the ionic strengths of the attachment and the detachment media were varied. Low ionic strength was tested for attachment, and high ionic strength was tested for detachment. Although biofilm formation in the packed-bed reactor is limited, the volumetric yield of cells based on the void volume of the packed bed is comparable with the batch culture yield.

Protein Fraction of Barley Spent Grain as a New Simple Medium for Growth and Sporulation of Soil Actinobacteria.

A cheap value-added product, the protein fraction of barley spent grains is proposed as a source of a potential and economical cultivation medium. We showed that medium composed of protein fraction extract allows the isolation of actinobacteria, especially Streptomyces, from soil samples, and enhances the sporulation. It was used for the screening and production of the biologically active substances from actinobacteria.

Rye bran alkylresorcinols suppress adipocyte lipolysis and hormone-sensitive lipase activity

The effects of alkylresorcinols (ARs) isolated from rye bran on adipocyte lipolysis, hormone-sensitive lipase activity and phosphorylation and on phosphorylation of protein kinase A substrates were studied. Preincubation with ARs for 18 h suppressed catecholamine-stimulated lipolysis in 3T3-L1 adipocytes. Furthermore, phosphorylation of hormone-sensitive lipase (HSL), a key lipase responsible for stimulated lipolysis, and phosphorylation of protein kinase A substrates, were diminished after preincubation with ARs, whereas HSL protein expression was unaltered. ARs were also shown to inhibit HSL activity in an in vitro assay.

Fructose-selective calorimetric biosensor in flow injection analysis

A highly selective, interference free biosensor for the measurement of fructose in real syrup samples was developed. The assay is based on the phosphorylation of D(-)fructose to fructose-6-phosphate by hexokinase and subsequent conversion of fructose-6-phosphate to fructose-1,6-biphosphate by fructose-6-phosphate-kinase. The heat liberated in the second reaction is monitored using an enzyme thermistor. The major advantages of this biosensor are rapid and selective measurement of fructose without the need to eliminate glucose and inexpensive FIA-based, mediator-free calorimetric measurement suitable for regular fructose analysis. This method was optimised for parameters, such as pH, ionic strength, interference, operational stability and shelf life. Good and reproducible linearity (0.5-6.0 mM) with a detection limit of 0.12 mM was obtained. Fructose determination in commercial syrup samples and spiked samples confirmed the reliability of this set-up and technique. The biosensor gave reproducible results with good overall stability for continuous measurements over a period of three months besides a useful shelf life of six months. The method could be used for routine fructose monitoring in food samples.

Artificial carrier for oxygen supply in biological systems

Several poly (dimethylsiloxanes) (PDMS) copolymers of dimethylsiloxane (DMS) with ethylene or propylene oxide were tested as artificial carriers for the delivery of oxygen to biological systems. Copolymers with a DMS content of 33% or lower enhanced glucose oxidation by 200% in contrast to the 25% increase produced by the same concentration of perfluorodecalin. When 0.05% of the copolymer with 18% DMS was included in the growth media of Bacillus thuriginensis, the biomass (growth rate) increased 1.5-fold. With 0.1% of this copolymer, actinorhodin production by Streptomyces coelicolor A3 (2) occurred in half the normal time and with an increased yield. In conclusion, these PDMS copolymers are a good alternative to perfluorodecalin as oxygen carriers in biotechnological processes.

Differentiation of human serum samples by surface plasmon resonance monitoring of the integral glycoprotein interaction with a lectin panel

Bacterial infection and inflammation result in massive changes in serum glycoproteins. These changes were investigated by the interaction of the saccharide glycoprotein moiety with lectins. A panel of eight lectins (Canavalia ensiformis, Bandeiraea simplicifolia BS-I, Arachis hypogaea, Phytolacca americana, Phaseolus vulgaris, Artocarpus integrifolia, Triticum vulgaris and Pisum sativum) was used to differentiate human serum glycoproteins obtained from patients with various bacterial infections. Lectin functionalised sensing layers were created on gold-coated wafers and lectin-glycoprotein interactions were monitored by surface plasmon resonance. The interaction of the lectin panel with serum glycoproteins produces unique patterns. Principal component analysis (PCA) was used to analyse the patterns. The actual panel of eight lectins enabled discrimination between sera obtained from patients sick with bacterial infection and healthy patients. Extended lectin panels have the potential to distinguish between types of bacterial infection and identify specific disease state. (Less)

Isolation of anacardic acid from natural cashew nut shell liquid (CNSL) using supercritical carbon dioxide.

Solvent extracted cashew nut shell liquid (CNSL), conventionally known as natural CNSL, is a mixture of several alkenyl phenols. One of these alkenyl phenols is anacardic acid, which is present at the highest concentration. In view of anticipated industrial applications of anacardic acid, the objective of this work was to isolate anacardic acid from natural CNSL by supercritical carbon dioxide (scCO 2). In this study, the solubility data for natural CNSL in scCO 2 under a range of operating conditions of pressure (100, 200, and 300 bar), temperature (40 and 50 degrees C), and CO 2 flow rate (5, 10, and 15 g min (-1)) were established. The best scCO 2 working conditions were found to be 50 degrees C and 300 bar at a flow rate of 5 g min (-1) CO 2. Using 3 g of sample (CNSL/solid adsorbent = 1/2) under these scCO 2 conditions, it was possible to quantitatively isolate high purity anacardic acid from crude natural CNSL (82% of total anacardic acid) within 150 min. The anacardic acid isolated by scCO 2 was analyzed by different spectroscopic techniques (UV-vis, FT-IR, and (1)H NMR) and HPLC analysis, indicating that the anacardic acid isolated by scCO 2 has better quality than that obtained through a conventional method involving several chemical conversion steps.

Solubilisation of sludge by combined chemical and enzymatic treatment

In this study, the effects of cation-binding agents used alone and/or in combination with enzymes on solubilisation of municipal sludge and structure changes were investigated. Formic acid, citric acid, tartaric acid, EDTA, sodium tripolyphosphate (STPP), Zeolite A, sodium fluoride, sodium thiosulphate or sodium silicate were added to both biological and digested sludges. Citric acid (50 mmol/l) released the highest COD, amounting to 8 g/l from bio-sludge and 3 g/l from digested sludge. The highest specific dissolution rate was 0.5 g COD per mmol citric acid. COD released by STPP (50 mmol/l) was 3.3 g/l from bio-sludge and 2 g/l from digested sludge. STPP acted most efficiently to reduce suspended solids, 20% for digested and 40% for bio-sludge. The pre-treatment by the sequestering agents was followed by addition of three glycosidic enzymes. The used enzymes were more effective in hydrolysis of bio-sludge than in hydrolysis of the digested sludge. Additionally, after 4 h of incubation the remained enzymes activities in enzyme treated sludges were improved by up to 20%, indicating high stability of added enzymes.

Enhanced efficiency of industrial-scale anaerobic digestion by the addition of glycosidic enzymes

The results of the action of two glycosidic enzymes added to an anaerobic digester processing mixed wastewater sludge are reported in this paper. The experiment was performed at a continuously operating full-scale wastewater treatment plant during a 6-month period. The addition of the enzyme mixture resulted in improved gas production and dewatering properties. Dewatering experiments, both on enzyme-treated and reference sludge on the local centrifuges, indicated that the carbonaceous matter was decreased by 7% in enzyme-treated sludge, and the average dry solids increased from 27% (reference) to 31% in enzyme-treated sludge. The polymer dosage was decreased from 8 to 5 kg/t dry solids. Additionally, practical experiences were gathered concerning the transformation from static laboratory-scale batch experiments to a dynamic continuous industrial scale setup. Preliminary cost calculations showed beneficial aspects and economical feasibility of enzyme addition to an anaerobic sludge digestion process.