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.

Diss. (sammanfattning) Stockholm : Karol.inst., 2001. / Härtill 5 uppsatser.

Inaugural-Dissertation zur Erlangung der Doktorwürde der Philosophischen Fakultät der Ernst-Moritz-Arndt-Universität zu Greifswald, 1933.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xxiii, 184 p.; also includes graphics (some color). Includes bibliographical references (p. 166-184).

Thesis (Ph. D.)--Ohio State University, 1976. / Bibliography: leaves 170-175. Available online via OhioLINK's ETD Center

Made available in DSpace on 2014-06-11T19:31:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2001-02-21Bitstream added on 2014-06-13T20:22:36Z : No. of bitstreams: 1 lopes_am_dr_botfca.pdf: 1368701 bytes, checksum: 1173340690bed7ebab55414585d6b7b7 (MD5) / No presente estudo ratos machos, var. Wistar, com peso médio de 120g, divididos aleatoriamente em cinco grupos de cinco animais cada, receberam concentrações orais de linamarina extraída de mandioca com 87,3% de pureza equivalentes a 87,3; 174,6; 349,2 e 698,4 mg/kg peso vivo através de gavagem para determinação da DL50. Após a administração das soluções, os animais foram observados quanto a ocorrência de óbitos por 4 horas. Nesse período morreram todos os animais do grupo que recebeu 698,4 mg/kg peso vivo, 3 animais com dose de 349,2 mg/kg peso vivo e 2 animais com dose de 174,6 mg/kg peso vivo. Os sobreviventes foram observados por 14 dias e posteriormente sacrificados. Foram realizadas análises histopatológicas do cérebro, coração, pulmão, fígado, intestino delgado, músculo estriado, pâncreas, rim e baço. Estudou-se também a bioquímica sérica relativa aos níveis de proteína total, albumina, globulina, glicose e atividade das enzimas Alanina Transaminase (EC 2.6.1.2), Aspartatos Transaminase (EC 2.6.1.1) e Lactato Desidrogenase (EC 1.1.1.27), além do consumo de ração e peso corporal. Os animais mortos pelas maiores concentrações de glicosídeos apresentaram ao exame histopatológico, congestão generalizada no cérebro, fígado, rins e coração. Os sobreviventes, sacrificados aos 14 dias, não apresentaram sinais de anóxia em tecido nobres. Não foram observadas alterações significativas nos parâmetros bioquímicos estudados. O consumo de ração e peso corporal não foram afetados. Pelos resultados obtidos foi possível estabelecer que a DL50 oral de linamarina extraída foi 324,86l1,5 mg/kg/peso vivo, correspondendo a 35,35 mg de HCN/kg peso vivo. / In the present study the toxicity of linamarin, a cyonogenic glucoside extracted from cassava roots, was investigated in rats. Rondon bred male Wistar rats weighing 100-120g were given 100, 200, 400 800 mg linamarin/kg bw by stomach tube and observed for the occurrence of deaths. The purity of extracted linamarin was 87,3%. After 4 hours all the 5 animals that received 800 mg/kg b.w. have died; 3 out of 5 rats on 400 mg/kg b.w. and 2 out 5 animals that ingested 200 mg/kg b.w. have died. The survivors were observed for 14 days and sacrificed for evaluation of srum biochemistry (total protein albumin, globulin, glucose, alanine aminotransferase, aspartate aminotransferase and lactato dehydrogenase) and post-mortem examination. Food intake was registered daily and body weight registred twice a week. The oral LD50 determined was 324.86l1.5 mg/kg b.w. There was generalised congestion in the brain, heart, liver and kidney in the animals that died from the toxic levels of linamarin. No significant changes in serum biochemistry levels were observed in these animals. The rats that survived until 14 days did not present significant changes in serum biochemistry nor histopathological lesions. The results of the present study indicate that LD50 of linamarin is higher than the safe levels set by the Codex Alimentarius Comission of FAO/WHO.

Resumo: No presente estudo ratos machos, var. Wistar, com peso médio de 120g, divididos aleatoriamente em cinco grupos de cinco animais cada, receberam concentrações orais de linamarina extraída de mandioca com 87,3% de pureza equivalentes a 87,3; 174,6; 349,2 e 698,4 mg/kg peso vivo através de gavagem para determinação da DL50. Após a administração das soluções, os animais foram observados quanto a ocorrência de óbitos por 4 horas. Nesse período morreram todos os animais do grupo que recebeu 698,4 mg/kg peso vivo, 3 animais com dose de 349,2 mg/kg peso vivo e 2 animais com dose de 174,6 mg/kg peso vivo. Os sobreviventes foram observados por 14 dias e posteriormente sacrificados. Foram realizadas análises histopatológicas do cérebro, coração, pulmão, fígado, intestino delgado, músculo estriado, pâncreas, rim e baço. Estudou-se também a bioquímica sérica relativa aos níveis de proteína total, albumina, globulina, glicose e atividade das enzimas Alanina Transaminase (EC 2.6.1.2), Aspartatos Transaminase (EC 2.6.1.1) e Lactato Desidrogenase (EC 1.1.1.27), além do consumo de ração e peso corporal. Os animais mortos pelas maiores concentrações de glicosídeos apresentaram ao exame histopatológico, congestão generalizada no cérebro, fígado, rins e coração. Os sobreviventes, sacrificados aos 14 dias, não apresentaram sinais de anóxia em tecido nobres. Não foram observadas alterações significativas nos parâmetros bioquímicos estudados. O consumo de ração e peso corporal não foram afetados. Pelos resultados obtidos foi possível estabelecer que a DL50 oral de linamarina extraída foi 324,86l1,5 mg/kg/peso vivo, correspondendo a 35,35 mg de HCN/kg peso vivo. / Abstract: In the present study the toxicity of linamarin, a cyonogenic glucoside extracted from cassava roots, was investigated in rats. Rondon bred male Wistar rats weighing 100-120g were given 100, 200, 400 800 mg linamarin/kg bw by stomach tube and observed for the occurrence of deaths. The purity of extracted linamarin was 87,3%. After 4 hours all the 5 animals that received 800 mg/kg b.w. have died; 3 out of 5 rats on 400 mg/kg b.w. and 2 out 5 animals that ingested 200 mg/kg b.w. have died. The survivors were observed for 14 days and sacrificed for evaluation of srum biochemistry (total protein albumin, globulin, glucose, alanine aminotransferase, aspartate aminotransferase and lactato dehydrogenase) and post-mortem examination. Food intake was registered daily and body weight registred twice a week. The oral LD50 determined was 324.86l1.5 mg/kg b.w. There was generalised congestion in the brain, heart, liver and kidney in the animals that died from the toxic levels of linamarin. No significant changes in serum biochemistry levels were observed in these animals. The rats that survived until 14 days did not present significant changes in serum biochemistry nor histopathological lesions. The results of the present study indicate that LD50 of linamarin is higher than the safe levels set by the Codex Alimentarius Comission of FAO/WHO. / Orientador: Marney Pascoli Cereda / Coorientador: Maria Aparecida Marchesan Rodrigues / Banca: Claudio Cabello / Banca: Ely Carvalho V. de Moura / Banca: Ana Catarina Cataneo / Banca: Magali Leonel / Doutor

Please read the abstract in the section 00front of this document / Dissertation (M Inst Agrar (Agricultural Economics))--University of Pretoria, 2006. / Agricultural Economics, Extension and Rural Development / unrestricted

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