The biocatalysis of tyrosinase was investigated in selected organic solvent media, using catechin as substrate. The results showed that the optimal enzymatic activity was obtained at pH 6.2, 6.6, 6.0 and 6.2 in heptane, toluene, dichloromethane and dichloroethane media, respectively. The kinetic studies indicated that the Km values were 5.38, 1.03, 2.52 and 4.03 mM, for the enzymatic reaction in heptane, toluene, dichloromethane and dichloroethane media, respectively, whereas the Vmax values were 12.2 x 10--4, 3.3 x 10--4, 14.7 x 10--4 and 12.0 x 10--4 deltaA mug protein--1 sec--1 , respectively. The results showed that the change in acetone concentration, used as co-solvent for the tyrosinase biocatalysis, from 5 to 30% (v/v) in the heptane medium resulted in a decrease of 4.3 to 96.7% in enzymatic activity. However, the presence of 12.5, 22.0 and 22.0% of acetone in the media of dichloromethane, dichloroethane and toluene resulted in a maximal increase in enzymatic activity of 42.6, 71.8 and 92.1%, respectively. Moreover, the biocatalysis of tyrosinase in dichloromethane and heptane reaction media, using model phenolic substrates was also investigated. The Km values for the tyrosinase biocatalysis in dichloromethane medium, using 4-methyl catechol, catechol and catechin as substrates, were 2.21, 2.36 and 2.52 mM, respectively, whereas the Vmax values were 5.1 x 10--4 , 6.0 x 10--4 and 14.7 x 10 --4 deltaA mug protein--1 sec --1, respectively. In addition, the Km values for tyrosinase biocatalysis in the heptane medium, using p-cresol, catechol and catechin as substrates, were 1.07, 4.32 and 5.38 mM, respectively, whereas the Vmax values were 0.8 x 10--4, 1.0 x 10 --4 and 12.2 x 10--3 deltaA mug protein--1 sec--1, respectively. The characterization of the end products resulting from the tyrosinase biocatalysis, using selected substrates, was carried out by spectrophotometeric scanning, differential scanning calorimetry and pyrolysis/gas chromatography coupled to

Polyphenol oxidases are important enzymes because of their role in food spoilage, oxidizing monophenols into o-diphenols and/or diphenols into the corresponding o-quinones. The resulting compounds are unstable and can rapidly form brown colored compounds, called melanins. Polyphenol oxidases, (PPOs) have been purified from several sources, particularly from fruits and vegetables. However, successful purification of PPO to homogeneity from plant sources has always been difficult. / The purification procedure of PPOs from cassava tuber consisted of (1) the preparation of cassava acetone powder; (2) the buffer extraction of the acetone powder to obtain a crude extract, followed by one of two possible purification procedures. The first consisted of ammonium sulfate fractionation, ion exchange chromatography on Mono-Q and gel filtration on Superdex G75 to yield two isoenzymes, PPO1 and PP02 having molecular weights of 71.8 +/- 6.0 and 69.6 +/- 1.5 kDa, respectively. The second purification procedure involved hydrophobic interaction chromatography (HIC) on phenyl-sepharose CL-4B followed by gel filtration on Superdex G75 to yield a single active PPO fraction of 68.3 +/- 2.8 kDa molecular weight. / The two isoenzymes obtained by ion exchange chromatography exhibited pH optima of 6.5 (PPO1) and 6.8 (PPO2) and were stable in the pH range of 7.5 to 10.0. These two isoenzymes had a temperature optimum of 30--40°C. PPO2 retained 65% of its original activity after heating at 50°C for 10 min whereas PPO1 was completely inactivated by the same treatment. The PPO fraction obtained by HIC purification exhibited a pH optimum of 7.5 with catechol and D,L-dopa as substrates and was stable in the pH range 4 to 8. Its temperature optima, were 20 and 30°C respectively with D,L-dopa and catechol as substrates and this PPO fraction was able to retain 80% of its original activity after heating at 50°C for 10 min. Unstable enzymes were obtained by the ion exchange chromatography purification procedure suggesting that artifacts were created. / Kinetic studies performed with the PPO fraction obtained by the HIC purification showed that catechol had the highest catalytic efficiency ratio. The Km values were 28.1, 5.27 and 3.72 mM for catechol, catechin and D,L-dopa, respectively. The PPO from the HIC purification procedure was inhibited by benzoic acid and p-coumaric acid and inactivated by diethyldithiocarbamate but not by EDTA. L-Cysteine, ascorbic acid and its derivatives (erythorbic acid and sodium erythorbate) were also inhibitors of the oxidation of catechol, catechin and D,L-dopa.

Thesis (M.App.Sc.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 2005. / Includes bibliographical references. Also available in a print form.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Vita. Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The potential of using mushroom polyphenol oxidase (EC 1.14.18.1) as a biocatalyst for the biotransformation of phenols to produce catechols in an aqueous medium was investigated. Polyphenol oxidase is characterised by two distinct reactions i.e., the ortho-hydroxylation of phenols to catechols (cresolase activity) and the subsequent oxidation of catechols to orthoquinones (catecholase activity). In order to facilitate the development of a process to produce catechols, the accumulation of catechol as a true intermediate product released in the reaction system needed to be investigated, as its release had been disputed due to the oxidation of catechols to o-quinones. Using LC-MS, catechol products were successfully identified as true intermediate products formed during biocatalytic reactions in water.

Polyphenolases (O-diphenol: O₂ oxidoreductase E.C. 1.10.3.1) have been isolated from a wide variety of plant and animal sources. This work deals with the isolation and characterization of polyphenolase from a previously unreported source, Pyrus communis, the common pear, horticultural variety D'Anjou. The chronometric method of assay was used, in which the enzymic oxidation of the substrate, usually catechol, is coupled to the oxidation of ascorbic acid and the time required to oxidize a specific amount of substrate is noted as the time required to colorize an external startch-iodide indicator. Various methods of isolation and purification were attempted. After a suitable isolation procedure was established, the enzyme was characterized by its substrate specificity, and its sensitivity to temperature, pH and inhibitors. Pear polyphenolase was characterized in particulate and soluble forms. The enzyme differs from other reported catechol oxidases in that it does not oxidize monophenols. A new spectrophotometric assay is described.

No description available.

No description available.

Master of Science / Department of Animal Sciences and Industry / Barry J. Bradford / Dairy cows undergo many homeorhetic adaptations during the transition to lactation. Although many of the physiological processes - including increased lipolysis and postpartum inflammation - are adaptive, exaggerated responses can contribute to metabolic disease and reduced milk production. L-carnitine has been shown to increase hepatic oxidation of fatty acids and reduce hepatic lipid accumulation in early lactation cows; however, L-carnitine is degraded in the rumen. An experiment using 4 ruminally-cannulated Holstein heifers in a split plot design demonstrated that the relative bioavailability of L-carnitine was greater when delivered abomasally than ruminally. There was a dose × route interaction and a route effect for increases in plasma carnitine above baseline, with increases above baseline being greater across all dose levels (1, 3, and 6 g L-carnitine/d) when infused abomasally compared to ruminally. A second experiment used 56 lactating Holstein cows in a randomized complete block design to evaluate 2 rumen-protected products (40COAT and 60COAT) compared to crystalline L-carnitine at doses targeting 3 and 6 g/d carnitine. Although crystalline and 40COAT were effective in linearly increasing carnitine concentrations, only subtle responses were seen for the 60COAT, which were less than that for crystalline carnitine in plasma, milk, and urine. Ineffectiveness of rumen-protected products to increase carnitine concentrations beyond crystalline may have been due to over-encapsulation that hindered liberation of the carnitine and its absorption in the small intestine. Although L-carnitine has the potential to reduce postpartum hepatic lipidosis, effective rumen protection of L-carnitine while maintaining intestinal availability needs further investigation. Plant polyphenols have anti-inflammatory properties and when administered during the transition period, have been shown to increase milk production. An experiment used 122 multiparous Holstein cows in a randomized block design to determine the effect of short term (5-d; SBE5) and long term (60-d; SBE60) administration of Scutellaria baicalensis extract (SBE)on whole-lactation milk yield, 120-d milk component yield, and early lactation milk markers of inflammation. Whole-lactation milk yield was increased for SBE60 compared to control, but was not different for SBE5 compared to control. Greater total pellet intake, milk lactose yield, and reduced SCC during wk 1-9 for SBE60 compared to control, all could have contributed to the observed sustained increase in milk yield. Milk production parameters were not different for SBE5 compared to control. No treatment effects were observed for BCS or milk markers of inflammation (haptoglobin) and metabolic function (β-hydroxybutyrate). Overall, long term administration of S. baicalensis effectively increased milk production, however the mechanism by which this was achieved is unknown. Although routes of administration to effectively achieve their physiological responses were different between L-carnitine (abomasal delivery) and SBE (feeding), both bioactive nutrients can improve the metabolic function of early lactation dairy cows.

by Suen Tsang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 96-103). / Abstract also in Chinese. / Chapter Chapter 1: --- Literature review --- p.1 / Chapter 1.1 --- "Straw mushroom, Volvariella volvacea" --- p.1 / Chapter 1.2 --- Problems which restrict the market of straw mushroom --- p.3 / Chapter 1.3 --- Non-enzymatic browning --- p.5 / Chapter 1.4 --- Enzymatic browning --- p.7 / Chapter 1.5 --- Impact of browning --- p.12 / Chapter 1.6 --- Mechanism of inhibition of PPO --- p.13 / Chapter 1.7 --- Sulfites --- p.13 / Chapter 1.8 --- Classification of PPO inhibitors based on chemical property --- p.14 / Chapter 1.9 --- Classification of PPO inhibitors based on inhibitory mechanism --- p.17 / Chapter 1.10 --- Physical methods for prolonging shelf-life --- p.18 / Chapter 1.11 --- Significance of this research --- p.20 / Chapter Chapter2: --- Characterization of PPO in straw mushroom --- p.21 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.22 / Chapter 2.2.1 --- PPO content in straw mushroom compared to other food sources: potato and pear --- p.22 / Chapter 2.2.2 --- "Optimal pH, enzyme kinetics and localization of PPO in straw mushroom" --- p.24 / Chapter 2.2.3 --- PPO isoenzymes in straw mushroom --- p.25 / Chapter 2.3 --- Results --- p.29 / Chapter 2.3.1 --- PPO content in straw mushroom compared to other food sources: potato and pear --- p.29 / Chapter 2.3.2 --- "Optimal pH, enzyme kinetics and localization of PPO in straw mushroom" --- p.29 / Chapter 2.3.3 --- PPO isoenzymes in straw mushroom --- p.32 / Chapter 2.4 --- Discussion --- p.43 / Chapter Chapter3: --- Several attempts to solve browning problem of straw mushroom --- p.55 / Chapter 3.1 --- Inhibitors of PPO in straw mushroom --- p.55 / Chapter 3.1.1 --- Investigation of inhibitors of PPO in straw mushroom --- p.55 / Chapter 3.1.1.1 --- Materials and methods --- p.55 / Chapter 3.1.1.2 --- Results --- p.56 / Chapter 3.1.2 --- The potential of using a combination of different PPO inhibitors --- p.58 / Chapter 3.1.2.1 --- Materials and methods --- p.58 / Chapter 3.1.2.2 --- Results --- p.59 / Chapter 3.1.3 --- Direct application of PPO inhibitors --- p.61 / Chapter 3.1.3.1 --- Materials and methods --- p.61 / Chapter 3.1.3.2 --- Results --- p.62 / Chapter 3.1.4 --- PPO and lipase content in straw mushroom under post harvest storage --- p.62 / Chapter 3.1.4.1 --- Materials and Methods --- p.74 / Chapter 3.1.4.2 --- Results --- p.75 / Chapter 3.2 --- Vacuum packaging --- p.75 / Chapter 3.2.1 --- Materials and methods --- p.75 / Chapter 3.2.2 --- Results --- p.78 / Chapter 3.4 --- Discussion --- p.78 / Chapter Chapter 4: --- Future work --- p.87 / Chapter 4.1 --- Suggested improvements of experiments --- p.87 / Chapter 4.2 --- Suggested experiment in future: application of calcium chloride --- p.88 / Chapter Chapter 5: --- Conclusion --- p.94 / References --- p.96