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The Pseudomonas aeruginosa algG gene product is a mannuronan C5-epimerase, which converts mannuronate to guluronate residues within the polysaccharide, alginate. The aim of this work was to purii' the epimerase and determine optimum reaction conditions for its enzymic activity. The epimerase was produced as an over-expressed fusion protein, GST/algG, which was purified by affinity chromatography. Subsequent cleavage of algO from GST produced a yield of 36 % purified epimerase. A poly mannuronate substrate for the epimerase was produced from a mutant strain of Ps. aeruginosa (FRD462). To enable the poly mannuronate to act as substrate for the epimerase it was deacetylated, deacetylation was confirmed by infrared spectroscopy. Activity of the purified epimerase was demonstrated by NMR spectroscopy, and by a coupled assay, which coupled the epimerase activity to the activity of a guluronate specific lyase. Lyase activity was shown to be dependent on environmental conditions and could therefore not be used in the coupled assay to determine optimal conditions for epimerase activity. An assay for epimerase activity was developed from the micro-phenol/sulphuric acid assay of Chang (Chang et aL, 1998). This assay showed activation of the epimerase at low levels of Ca2 (in the range 3 mM -18 mlvi) with higher concentrations showing inhibition. In contrast, higher concentrations of Mg 2 (of the order of 18 mlvi) were needed to induce epimerase activity but no inhibitory effects were observed. Activation of the enzyme was induced by K concentrations in the range 1.5 mM - 8 mlvi, whilst the presence of Ne appeared to have no effect on epimerase activity. These results also suggested that the mannuronan C5-epimerase brings about conversion of the mannuronate residues within the alginate chain by a preferred or processive attack on substrate. Substrate viscosity was found to have an effect on the rate of epimerisation; increased viscosity led to lower activity.

Milk coagulation consists of four overlapping phases: enzymic hydrolysis, micelle aggregation, gelation and syneresis. The objectives of this study were to determine the effects of added CaCl2 on milk coagulation and the relationship between enzymic hydrolysis and micelle aggregation with substrate at different concentrations. Addition of CaCl2 to milk is widely practiced in industry and in laboratories. This changes calcium concentration, pH and ionic strength. It is impossible to separate these three variables and investigate each one independently. Addition of low levels of CaCl2 shortens coagulation time and increases curd firming rate. Low levels of CaCl2 also accelerate the enzymic hydrolysis process. Calcium ions increase hydrolysis rate, but this effect is much smaller than that of lowered pH. Increase of ionic strength due to addition of CaCl2 has an adverse effect on enzymic hydrolysis. This dominates at high CaCl2 concentration, and the overall coagulation process slows down. Adding CaCl2 also promotes micelle aggregation. However, aggregation is retarded by high levels of added CaCl2. Results of this study show that about 90% of the κ-casein is hydrolyzed for diluted milk (1/3) to coagulate. Samples at normal concentration (12 g NDM/100 ml solution) require only 60% conversion of κ-casein to para-κ-casein. Addition of CaCl2 significantly decreases this percentage. This suggests a different aggregation and gelation process in samples containing added CaCl2 When pepstatin A is used to stop enzymic hydrolysis at different times, different degrees of κ-casein conversion are obtained. Micelles aggregate even at very low percentages of hydrolysis. Previous reports have stated that a micelle cannot participate in aggregation until almost all of its κ-caseins have been hydrolyzed.

The development of an automated instrument employing reflectance colorimetry was described. Several models were designed, assembled, and programmed to perform microbial and enzymic tests automatically. Samples were prepared manually or automatically by a Zymate™ II robot. These samples were incubated during the tests to maintain an optimum temperature for reactions and microbial growth. During incubation, color changes of appropriate indicator dyes in the sample/reagent mixtures were measured intermittently, recorded, and compared to previously defined end points. The computer-controlled instrument received data that related time of color changes with the initial numbers of microorganisms or the enzyme activity of the samples. Traditional pH and oxidation/reduction dyes were used. Suitable dyes and media were selected for fast estimation in the different assays studied . Applications of the instrument to evaluate raw milk for the total viable microbial count, abnormality, broad spectrum antibiotics, and coliforms were emphasized. The automated colorimeter system successfully quantitated total and coliform microflora in raw milk. Correlations between reflectance colorimetry and the spiral plate count method were .932 (using .12% TIC as indicator), .922 (using BCP as indicator), and .681 (using .04% TTC as indicator). A coefficient of correlation of .874 was obtained when reflectance colorimetry was compared with coliform numbers on violet red bile agar. The reflectance colorimetry system provided better precision than current reference methods. Preliminary incubation or larger sample volumes were required to estimate low numbers of microflora . Antibiotic residue detection was also evaluated using Lactococcus lactis ssp. cremoris UC 310+ with the automated colorimeter system. The following concentrations (ppb) could be detected: penicillin G:::;; 5, ampicillin ≤ 5, tetracycline ≤ 250 , sulfamethazine ≤ 30, streptomycin ≤ 1000, kanamycin ≤ 500, and chloramphenicol ≤ 500. Abnormal milk could be screened out by measuring the NAGase activity and chloride ion content in milk samples. Both methods had been integrated into the automated colorimeter system. The coefficient of correlation between somatic cell count and the NAGase activity as measured with the colorimeter was .802; a correlation of .792 could be obtained when chloride ion content was measured.

Выпускная квалификационная работа магистра состоит из 7 глав и посвящена бесферментному электрокаталитическому определению глюкозы в щелочной и нейтральной среде с использованием различных модификаторов на основе наноматериалов, а именно карбоксилированных многостенных нанотрубок, наночастиц серебра и наночастиц палладия. В работе приведены аналитические характеристики использованных модификаторов и обоснования выбора наилучшего модификатора. Для обеспечения селективности анализа были получены полимеры с молекулярными отпечатками глюкозы. / Master’s graduation work consists of 7 chapters and is devoted to the non-enzymatic electrocatalytic determination of glucose in an alkaline and neutral medium using various modifiers based on nanomaterials, namely, carboxylated multi-walled nanotubes, silver nanoparticles, and palladium nanoparticles. The paper presents the analytical characteristics of the used modifiers and the rationale for choosing the best modifier.

В настоящей работе для электрохимического определения глюкозы предложены электрохимические катализаторы на основе органических комплексов никеля (II) и рутения (III) в присутствии карбоксилизированных многостенных углеродных нанотрубок и полиэтилеимина. Исследована каталитическая активность комплексов рутения (III) и никеля (II) при их раздельном и совместном присутствии на рабочем электроде в электрохимическом окислении глюкозы. Описан алгоритм проведения процедуры электрокаталитического определения глюкозы с использованием модифицированных электродов. Рассчитаны аналитические характеристики модифицированных электродов для электрохимического определения глюкозы. Выбран модификатор с оптимальными характеристиками, с наивысшей чувствительностью. / In this work, for the electrochemical determination of glucose, we propose electrochemical catalysts based on organic complexes of nickel (II) and ruthenium (III) in the presence of carboxylated multi-walled carbon nanotubes and polyethyleneimine. The catalytic activity of the complexes of ruthenium (III) and nickel (II) was investigated in the case of their separate and joint presence on the working electrode in the electrochemical oxidation of glucose. An algorithm for carrying out the procedure for electrocatalytic determination of glucose using modified electrodes is described. The analytical characteristics of the modified electrodes for the electrochemical determination of glucose are calculated. The selected modifier with optimal characteristics, with the highest sensitivity.