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    Carbohydrate effects on the inducement of the arginine deiminase pathway enzymes in wine lactic acid bacteria : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1998) Church, Peter R F
    Characterised by a fermentative sugar metabolism resulting in lactic acid as a major end product, the lactic acid bacteria (LAB) may be isolated from a broad range of sources. Dairy products, fermented vegetables, meats and baking products such as sourdough bread involve these organisms in a consistent and intentional manner in present times, no matter how accidental or fortuitous their initial involvement may have been. Alcoholic beverages such as beer, cider and, most pertinently here, wine are also affected by the presence of particular LAB. As conditions differ between nutrient environments so do the LAB found in wine differ to those isolated elsewhere - being both ethanol tolerant to the degree where growth is capable in 10% v/v ethanol and aciduric, able to maintain an active presence at acidic levels as great as pH 4 or less. This ability to remain viable during the primary yeast fermentation of juice into wine leads to these LAB being of no small practical interest in the wine industry. The process of malolactic fermentation (MLF) involves the wine LAB altering the law materials present in the juice and wine further, increasing the intricacies of the winemaking and final product. Primarily encouraged due to its effect of reducing wine acidity, MLF also alters flavour and aroma in what is generally thought to be an advantageous manner when applied correctly. Another factor thought to be of significance is an increase in biological stability. Found, for example, among the lactobacilli, pediococci and leuconostocs, the wine LAB are classed as either homofermentative or heterofermentative. Homofermenters commonly produce two moles of lactic acid per mole of glucose fermented, while heterofermenters form one mole each of lactic acid and carbon dioxide and varied quantities of ethanol and acetic acid from one mole of glucose. Natural or chance occurrences of wine LAB, whether as part of the microbiological community on the raw materials or from other sources - such as inoculation from contaminated equipment - were the original manner in which these organisms were introduced into the vinification equation. With the predilection towards quality control, standardisation and safety in the present day, the use of pure microbial starter cultures to initiate MLF has become increasingly widespread. In order to optimise the manipulation of wine LAB in both the laboratory and industry a thorough insight into their physiology and metabolism is an obvious necessity. Certain areas of interest have undergone more intensive study than others, with, for example, the catabolism of carbohydrates in both wine (Davis et al., 1986) and model wine systems (Liu et al., 1995a) having had a considerable amount of research compared to less primary sources of energy such as nitrogen metabolism.
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    Arginine metabolism in malolactic wine lactic acid bacteria and its oenological implications : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 1993) Liu, Shao-Quan
    L-Arginine is a major amino acid found in grapes and wine which is degraded by some wine lactic acid bacteria (LAB). The mechanism of this degradation and its oenological implications were examined in this research. It was found that wine LAB able to degrade arginine do so by means of the arginine deiminase pathway, demonstrated by measuring the enzyme activitie since l l-free extracts: arginine deiminase, ornithinetrans carbamylase and carbamate kinase. These enzymes we represent in most heterofermentative lactobacilli and leuconostocs, but were absent in homofermentative lactobacilli and pediococci. The presence of arginine increased the activities of arginine deiminase pathway enzymes in heterofermenters, but failed to induce these enzymes in homofermenters even under conditions of low glucose concentration (1g/L). Glucose did not repress arginine utilisatio n but fructose appeared to do so, as fructose and arginine were metabolised sequentially, with arginine being metabolised mainly after utilisation of the fructose. D etailed studies o n Leuconostoc oenos OENO, Lactobacillus b uchneri CUC-3 and Lactobacillus brevis 250 showed that arginine was converted stoichiometrically to ammonia and ornithine as the major end-products and that arginine catabolism could supply energy (ATP) to support growth. lt was also demonstrated that citrulline was excreted during arginine catabolism by both the lactobacilli and the leuconostoc. Some of the excreted citrulline was reassimilated and catabolised after arginine depletion by the lactobacilli, but not by the leuconostoc. The implication of citrulline excretion for the wine industry was explored by studying the formation of the carcinogen ethyl carbamate (urethane) in a synthetic wine and a white wine, since citrulline is a known precursor of ethyl carbamate. During growth of Le. oenos OENO and Lb. buchneri CUC-3 in the synthetic wine and wine, significant amounts of ethyl carbamate were found i n the two wine types upon heat treatment of samples. The formatio n of ethyl carbamate correlated well with arginine degradation and citrulline excretion. Citrulline excretion during arginine degradation is of concern to the winemaker, since the reaction of citrulline and ethanol to form ethyl carbamate has been shown by other workers to occur even at normal wine storage temperatures. Winemakers, therefore , should avoid using argininedegrading LAB starter cultures for inducing malolactic fermentation (MLF). In addition, spontaneous MLF in wine by undefined LAB strains should be discouraged, as this may lead to formation of ethyl carbamate precursors. Ammonia detection with Nessler's reagent provides a simple, rapid test to assess arginine degradation by wine LAB in a complex medium, but is useful only for strains showing strong ammonia formation . The more sensitive enzymatic determination of ammonia is required for strains showing weak ammonia formation.
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    Nitrogen metabolism in Haemonchus contortus and Teladorsagia circumcincta : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
    (Massey University, 2012) Umair, Sallah
    This is the first study to characterise proline, arginine and lysine metabolism in homogenates of L3 and adult Haemonchus contortus and Teladorsagia circumcincta. The properties of glutamate dehydrogenase (GDH), glutamate synthase and the GABA shunt were also compared in the two species. The kinetic properties of 26 enzymes were determined. The gene encoding T. circumcincta GDH was sequenced and recombinant TcGDH expressed and biochemically characterised. The ornithine-glutamate-proline pathway was fully functional. The mammalian α-AAA (saccharopine) and pipecolate pathways of lysine catabolism, but not the bacterial enzymes lysine dehydrogenase and decarboxylase, were present in adult worms. The pipecolate pathway was incomplete in L3 of both species, as Pip2CR activity was undetectable. Unusually, lysine ketoglutarate reductase and saccharopine dehydrogenase, Δ1-pyrroline-5-carboxylate synthase and reductase were able to use both cofactors. The glutamine synthetase-glutamate synthase pathway of ammonia incorporation into glutamate was present, except in L3 H. contortus. T. circumcincta GDH was cloned, purified and characterised and the predicted protein sequence was very similar to H. contortus GDH. T. circumcincta recombinant and H. contortus homogenate GDH were both dual co-factor specific, although the latter had 50% greater activity with NAD+/H as cofactor. GDH activity was inhibited by GTP and stimulated by ADP whereas ATP either inhibited or stimulated depending on the concentration and direction of the reaction. The GABA shunt enzymes glutamate decarboxylase and succinic semialdehyde dehydrogenase was not detected in homogenates of whole L3 or adult H. contortus or T. circumcincta. Neither parasite had a full functional ornithine urea cycle, nor appeared to use bacterial pathways to covert arginine to ornithine. NOS were demonstrated histochemically in nerves of adult H. contortus, but was undetectable in homogenates of both species. There was species variation in polyamine metabolism: T. circumcincta used arginase to form ornithine, followed by decarboxylation by ODC, while in H. contortus there was the additional pathway of first decarboxylation by ADC to form agmatine, then hydrolysis by agmatinase to putrescine. The present study helped in the better understanding of nitrogen metabolism and these enzymes can be useful targets if they differ antigenically from the host, provided the enzyme is accessible to blockage by immune effectors.
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    Intra-molecular lysine-arginine derived advanced glycation end-product cross-linking in Type I collagen: A molecular dynamics simulation study.
    (2016-11) Collier TA; Nash A; Birch HL; de Leeuw NH
    Covalently cross-linked advanced glycation end products (AGE) are among the major post-translational modifications to proteins as a result of non-enzymatic glycation. The formation of AGEs has been shown to have adverse effects on the properties of the collagenous tissue; they are even linked to a number of age related disorders. Little is known about the sites at which these AGEs form or why certain sites within the collagen are energetically more favourable than others. In this study we have used a proven fully atomistic molecular dynamics approach to identify six sites where the formation of the intra-molecular 3-deoxyglucosone-derived imidazolium cross-link (DOGDIC) is energetically favourable. We have also conducted a comparison of these positions with those of the more abundant glucosepane cross-link, to determine any site preference. We show that when we consider both lysine and arginine AGEs, they exhibit a prevalence to form within the gap region of the collagen fibril.