Characterization, Immobilization, and Polymer Related Applications of Watermelon Seed Powder, a Practical Source of Urease Enzyme

Urease enzyme was crystallized almost a century ago, and to this day its intrinsic stability is not ideal for everyday applications. This work introduces a new process by which a naturally encapsulated material, watermelon seed powder (WMSP), is characterized for its stability and activity. WMSP enzymatic activity has been measured for over a year at various storage conditions—exposed to ambient atmosphere for a year, WMSP retained above 90% activity. In aqueous conditions, the enzyme maintained above 60% activity after two months; with the addition of a preservative that number stays at about 90%. There is a pH shift of the maximum activity at 8.1 pH in addition to a broadening of the activity curve allowing for a wider range of reactions. In Chapter 3, immobilization techniques were employed on these powders, enhancing their usability and in turn presented some interesting dynamics. One of the techniques physically encapsulated the WMSP inside a porous resin, essentially creating a urea-ammonia exchange resin. These particles were subject to 10 cycles of urea hydrolysis, without any change in the urea-urease clock reaction. The agar immobilized WMSPs exhibited a behavior called quorum sensing. That is a biological communication process by which bacteria express certain genes when a “quorum” or sufficient number are in proximity of each other. With certain parameters, the particles will not clock individually, but when in a cluster of particles, the autocatalysis occurs. This property was then coupled with a hydrogel polymerization that gels in basic conditions. The WMSP particles in principle are biofilm generating analogues. Lastly, Chapter 4 presents an application of WMSP as a base generator for several adhesive formulations and outlines some potential future uses of WMSP

Research on applied bioelectrochemistry First quarterly progress report, 14 Mar. - 30 Jun. 1963

Optimum use of human waste as electrochemical fuels by urea bacterial organism conversion

Studies on the structure and function of plant protein inhibitors of trypsin and α-amylase

Studies of the primary structure of protein inhibitors of proteolytic enzymes and α-amylase were undertaken. The complete amino acid sequence of an α-amylase inhibitor and a bifunctional trypsin/α - amylase inhibitor from seeds of ragi (Eleusine coracana Gaertn.) and of the proteinase inhibitor PI-I from the Tracy cultivar of soybean (Glycine max L. Merr) were determined. The bifunctional trypsin/ α-amylase inhibitor from ragi seeds was shown to be a single polypeptide of 122 amino acids with a molecular weight of 13400. The two reactive (trypsin inhibitory) sites were also determined. Sequence comparisons revealed that this inhibitor seems to be divergently related to other trypsin and α-amylase inhibitors and also to the reserve protein from castor bean. In addition it is proposed that a new inhibitor family should be added to the existing ones (Laskowski and Kato, 1980) to accomodate this bifunctional inhibitor and its related proteins. The secondary structure of this inhibitor was also predicted. The α-amylase inhibitor from ragi seeds was shown to be a single polypeptide of 95 amino acids with a molecular weight of 9300. The existence of two homologous regions in the amino acid sequence of this inhibitor seemed, to indicate that the inhibitor molecule has arisen by a process of gene duplication. Sequence comparisons revealed that this inhibitor has no homology to any other α-amylase inhibitors, proteolytic enzyme inhibitors or any other plant protein of known primary structure. In addition the secondary structure of the α-amylase inhibitor from ragi seeds was also predicted. The amino acid sequence of the proteinase inhibitor PI-I from the Tracy cultivar of soybean (Glycine max L. Merr) was shown to be identical to another proteinase inhibitor PI-II) from the same cultivar, which have been previously sequenced by Kashlan(1980)

The role of allantoinase in soybean (Glycine max L.) plants

Soybean and related legumes export symbiotically-fixed nitrogen from the nodules to the leaves as ureides. The ureide allantoin is hydrolyzed by allantoinase to allantoate then further degraded by other enzymes, releasing ammonia and carbon dioxide. This study aimed to identify allantoinase genes in soybean and their gene expression as well as enzyme activity patterns. The effects of water limitation and allantoin treatment on the expression and activity of allantoinase in N2-fixing plants were also evaluated. Enzyme activity and ureide content were evaluated using a spectrophotometric assay. Real time RT-PCR was used to quantify the amount of gene products. Four allantoinase genes were identified and were expressed, with GmALN1 and 2 constantly expressed at higher levels. In seedlings, allantoinase was found to be actively synthesized more in cotyledons than in the embryonic axes, as seen by early enzyme activity and higher GmALN 1 and 2 transcript levels. Allantoate produced in these tissues appeared to be mobilized to the developing axes. GmALN1 and 2 were implicated in post-germination nitrogen assimilation during early seedling growth, while GmALN3 and 4 were consistently expressed at very low levels, with an exception in nodules. Transcript abundance in the nodules of N2-fixing plants, supported by the high enzyme activity and ureide content observed, suggested an important role in the synthesis and transport of allantoate in these tissues. Allantoinase was also detected in non-fixing tissues but may play a different role in these tissues, most probably functioning in the turnover and salvage of purine nucleotides. The effect of exogenous allantoin during water limitation was investigated. The addition of allantoin prior to water limitation seemed to change the sensitivity of soybean to such stress, prolonging its ureide catabolic activity at least up to 5 days without water. Results of this study will aid in our understanding of how ureide catabolism is regulated during soybean development. This information may help address problems in legume crop improvement specifically in enhancing N2-fixation and yield capacity and in coping with water limitation stress

The fat and lipase content of the liver

TypescriptContains several folded charts.The object of this thesis is to ascertain if any broad correlation exists between the lipase content of an organ and its state of fat metabolism. The author has chosen the liver as an organ, with its lipase content and fat metabolism as the two factors under comparison.Includes bibliographical reference

Non-ionic nitrogen nutrition of plants : nutrient uptake and assimilation and proton extrusion during utilization of urea or symbiotically fixed nitrogen

This thesis encompasses six papers, dealing with mainly ionic balance aspects of non-ionic nitrogen nutrition of plants. In most cases urea nutrition or symbiotic N 2 -fixation were compared with NH 4+ - or NO 3- -supply with respect to nutrient uptake and assimilation.From ionic balance and proton release data it was established that maize and sugar-beet plants are able to absorb urea as an undestructed molecule. Results of xylem sap analyses learned that urea, like NH 4+ , is almost quantitatively metabolized in the roots.Complete ionic uptake balances, including direct measurements of respective H + - and OH - /HCO 3- -release from the roots of N 2 -fixing and NO 3- -supplied pea plants are presented. Excess nutrient cation over anion uptake and hence H + -release by N 2 -fixing plants increased at higher pH of the nutrient solution. When such plants were grown in soil, cation uptake also exceeded anion uptake, but root growth was severely reduced at low soil pH. This effect could be eliminated completely by liming. Root growth was not inhibited when NO 3- was the form of N-nutrition.In soils, mineralized N may confuse the comparison between NO 3- -nutrition and N 2 -fixation. It is suggested that the relative contribution of N 2 -fixation to the total N- accumulation in plants reflects the point of time at which ( 15 N-)NO 3- in the soil was depleted and the N 2 -fixing process started.Different ionic uptake patterns of plants in relation to the form of nitrogen nutrition necessarily invoke essential differences in both inorganic and organic chemical composition of the xylem sap of these plants. Complete xylary ionic balances and data about partitioning of the nitrogenous compounds In xylem saps allowed the conclusion that N 2 -fixing pea plants belong to the group of amidetransporting legumes and that in NO 3- -supplied pea plants no phloem transport of cation-organate is necessary for. the regulation of Intracellular pH and electroneutrality

Carbonyl Compounds in Manuka Honey:: Antibacterial Activity, Reactions and Metabolic Transit

New Zealand is the world’s third-largest honey exporter by value behind China and Argentina and honey accounts for up to 80 % of New Zealand’s exports. However, it is only the 16th biggest global supplier by volume. Manuka honey from New Zealand is sold for premium prices and merchandised for its health benefits. Because of its exceptional antibacterial effect, there is a strong market demand and the price for a kilogram of manuka honey has tripled in recent years (Ministry for Primary Industries 2015). When consumers are willing to pay prices up to 200 €/kg manuka honey, the risk of misleading advertisement and intended fraud increases. This thesis aims to further characterize manuka honey and contribute to the development of a manuka honey definition. The first part deals with the antibacterial activity of manuka honey. The effect of manuka honey is mainly due to methylglyoxal, whereas the effect of non-manuka honeys is primarily caused by hydrogen peroxide. The objective is to develop a method to quantify the effect solely due to one of the respective chemical compounds and compare their effectiveness. Finally, an evaluation of the contribution of methylglyoxal and hydrogen peroxide to the inhibitory effect of honey should be given. The second part deals with chemical reactions of carbonyl compounds in honey. Because of the reactive nature of carbonyl compounds, the formation of specific glycation compounds in honey is assumed. Since the carbonyl profile of manuka honey differs remarkably from non-manuka honeys, the reaction products are expected to vary widely. Specific compounds, solely present in manuka honey, could serve as quality control parameters to ensure manuka honey authenticity. The final part deals with the metabolism of food-derived carbonyl compounds. Carbonyl compounds, like methylglyoxal or 3-deoxyglucosone are discussed to be potentially toxic to human tissues. Until now, only little is known about the impact of the diet on the physiological carbonyl-load and the metabolism of carbonyl compounds. With the help of nutrition studies and the analysis of body fluids, the question of metabolic transit of carbonyl compounds shall be addressed. The antibacterial studies showed that bacterial species are affected differently by bioactive compounds present in honey. Methylglyoxal (MGO), which is solely present in manuka honeys and hydrogen peroxide, which is formed in most conventional honeys by glucose oxidase, are strong inhibitors of the growth of S. aureus and E. coli. The strain of P. aeruginosa used for this work was not inhibited by MGO, whereas B. subtilis was not inhibited by hydrogen peroxide. To compare and quantify the effect of MGO and hydrogen peroxide, a mathematic model was created. By comparing the slopes of the linearized dose-response curves, it was found that S. aureus, E. coli and P. aeruginosa were more sensitive to hydrogen peroxide than to MGO. However, the natural amounts of MGO in honey are higher than the formation of hydrogen peroxide. Although most bacteria are more sensitive to hydrogen peroxide, MGO is the predominantly antibacterial compound in honey, because of its higher concentrations compared to hydrogen peroxide formation. The inclusion of manuka honey in α-cyclodextrin had only minor consequences on bioavailability and antibacterial activity. The commercial product “Cyclopower” (α-cyclodextrin with manuka honey) does not enhance the antibacterial activity of manuka honey on S. aureus, E. coli and P. aeruginosa. With the help of the newly developed quantitative model, it was shown that the growth of B. subtilis is synergistically inhibited with cyclopower compared to manuka honey and α-cyclodextrin alone. The study of bacterial enzymes as possible targets for bacterial inhibition with manuka honey revealed that MGO and DHA inhibited jack bean urease, which was used as a model for Helicobacter pylori urease. The concentration of MGO and DHA in manuka honey positively correlated with its urease inhibition. Conventional honeys, which lack MGO and DHA, showed significantly less urease inhibition. Based on the unique presence of MGO, manuka honey has extraordinary effects on bacteria, which might lead to further application to fight the emerging crisis of antibacterial resistance to antibiotics. Until now, there is no consistent definition for the term “genuine manuka honey”. In the present work, an approach based on unique chemical reactions in manuka honey was followed. It was shown that the exceptional high amounts of MGO induced the formation of 2-acetyl-1-pyrroline (2-AP). In manuka honey containing ≥ 250 mg/kg MGO, the 2-AP concentration was significantly increased compared to conventional honey. Moreover, honey proteins form MGO-derived reactions products, which were studied by measuring the molecular size of honey proteins. Manuka honey proteins significantly shifted to high molecular weights (HMW) with a size above 510 kDa. The amount of HMW protein in non-manuka honey was significantly lower. The cleavage of disulphide bonds led to a decrease of HMW fraction of conventional honeys but not of manuka honeys. It is hypothesized that MGO cross-linking of proteins is mainly responsible for the formation of HMW adducts in manuka honey. The formation of HMW adducts was also shown with fluorescence analysis, whereby manuka honey proteins had higher fluorescence intensities at λex=350 nm and λem=450 nm compared to non-manuka honeys. The artificial addition of MGO and its precursor dihydroxyacetone (DHA) to a non-manuka honey did not lead to an increased fluorescence up to the level of commercial manuka honeys. The MGO-derived modifications of proteins were further studied by quantifying the protein-bound Maillard reaction products N-ε-carboxyethyllysine (CEL) and methylglyoxal-derived hydroimidazolone 1 (MG-H1) after enzymatic hydrolysis of honey proteins and LC-MS/MS analysis. Their amount was significantly higher in manuka compared to conventional honeys and correlated with the MGO content of the honey. Most of the MGO-derived reactions could be simulated by spiking a conventional honey or a low MGO manuka honey with artificial MGO and subsequent storage at elevated temperatures. Higher storage temperatures were associated with a quick increase of 5-hydroxymethylfurfuraldehyd (HMF). The HMF level in honey is used as a quality parameter and should not exceed 40 mg/kg (Codex Alimentarius Commission, 2001). High concentrations of HMF may point to a fraudulent addition of MGO and the production of artificial high-price manuka honey products. Taken together, the Maillard reaction in honey could be used to control the natural origin of MGO and DHA. The consumption of honey and especially manuka honey exposes humans to high levels of dietary dicarbonyl compounds like MGO and 3-deoxyglucosone (3-DG). Both compounds were discussed as potential risk factors for the development of age-related diseases. The simulated digestion of manuka honey in the presence of gastric and ileal fluids showed that only 9 % of the initial concentration can be recovered after 8 h. The honey matrix had no stabilising effect on MGO compared to a synthetic MGO solution. In contrast to MGO, the manuka honey compound DHA was stable during all simulated digestion steps. The complexation of MGO with α-cyclodextrin did not enhance the stability of MGO. The metabolic transit of dietary MGO and 3-DG was further studied with an intervention study with healthy volunteers, who collected their daily urine. It was shown that urinary concentrations of 3-DG and its less reactive metabolites 3-deoxyfructose (3-DF) and 2-keto-3-deoxygluconic acid (3-DGA), but not MGO, were influenced by the diet. During the intervention studies, up to 40 % of dietary 3-DG was recovered as the sum of 3-DG, 3-DF and 3-DGA. The metabolite 3-DGA only played a minor role in the metabolism of dietary 3-DG in comparison to 3-DF. The concentrations 3-DF and 3-DGA in plasma only increased after the consumption of dietary 3-DG and not after the uptake of carbohydrate rich meals in general. This led to the conclusion that dietary 3-DG is effectively metabolized to 3-DF extracellularly on the apical site of the intestinal epithelium and is resorbed slowly into the circulation. In contrast, 3-DG, which is formed (intracellularly) postprandial from glucose, bypasses this metabolic system and cannot be metabolized as rapidly to 3-DF. Preliminary results obtained with saliva instead of urine as a bio fluid to study the dietary influence of dicarbonyl compounds, confirmed the hypothesis. Based on the present results, dietary dicarbonyl compounds are effectively metabolized during digestion