Thesis (M.S.)--University of Michigan, 1970.

Thesis (M.S.)--University of Michigan, 1970.

Thesis (Ph. D.)--Illinois State University, 1974. / Title from title page screen, viewed Oct. 26, 2004. Dissertation Committee: Anthony E. Liberta (chair), T.I. Chuang, H. Huizinga, A. Richardson, E. Willis. Includes bibliographical references (leaves 87-90) and abstract. Also available in print.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Vita. Includes: Comparison of the [alpha]-glucosides of Saccharomyces produced in response to five non-allelic maltose genes / Harlyn O. Halvorson, Sara Winderman and John Gorman. Reprinted from Biochimica et biophysica acta, vol. 67 (1963), p. 42-53 -- Relationship between protein and nucleic acid synthesis in Pseudomonas azotogensis grown in hexetidine / John Gorman and Harlyn Halvorson. Reprinted from Archives of biochemistry and biophysics, vol. 84, no. 2 (Oct. 1959), p. 462-470 -- The abnormal pattern of protein synthesis in Pseudomonas azotogensis in the presence of hexetidine / H.O. Halvorson and John Gorman. Reprinted from Experimental cell research, vol. 17, p. 522-524. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (MSc (Genetics. Institute for Plant Biotechnology (IPB)))--Stellenbosch University, 2008. / Dextran is a high value glucose polymer used in medicine and an array of laboratory techniques. It is synthesised by lactic-acid bacteria from sucrose but has also reportedly been produced by Gluconobacter oxydans (G. oxydans) from a range of maltooligosaccharides (MOS) via the action of dextrin dextranase (DDase). In this study the presence of DDase is investigated in two G. oxydans strains (ATCC 621H and ATCC 19357) and shown to be present in the ATCC 19357 strain, but not in the ATCC 621H strain. The enzyme was partially purified from the ATCC 19357 strain, and its kinetic properties investigated. The partially purified protein was also digested with trypsin, and de novo peptide sequences obtained from it. Several attempts were made to obtain the gene coding for the DDase. These include amplifying an open reading frame from the G. oxydans genome coding for a glycosyltransferase with the approximate molecular weight of the DDase, using the peptide sequences obtained from the partially purified protein to design degenerate PCR primers and the production of a genomic DNA library for functional screening in E. coli. None of these approaches led to the successful isolation of the extracellular DDase sequence.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2018-04-19T12:33:37Z No. of bitstreams: 1 texto completo.pdf: 2096061 bytes, checksum: bc307da4c22fbfa9e0230bdede6f8c29 (MD5) / Made available in DSpace on 2018-04-19T12:33:37Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2096061 bytes, checksum: bc307da4c22fbfa9e0230bdede6f8c29 (MD5) Previous issue date: 2018-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fungos e plantas produzem enzimas que possuem as mais diversas aplicações biotecnológicas. Uma possível e promissora aplicação dessas enzimas é o controle de nematoides, em substituição aos nematicidas químicos prejudiciais à saúde humana e animal, ao meio ambiente e à microbiota do solo. Outra possível aplicação das enzimas de fungos é o uso da dextranase na redução de dextrana na indústria sucroalcooleira. Dessa forma, o presente trabalho teve como objetivo investigar a produção de enzimas pelo fungo Pleurotus eryngii e pelas plantas Euphorbia milii e E. trigona, purificar e caracterizar as enzimas de E. milii, E. trigona e Pochonia chlamydosporia, e testar a atividade nematicida das enzimas das plantas E. milii, E. trigona e do fungo P. eryngii. O fungo P. eryngii e seu extrato reduziram significativamente (p<0,01) o número de larvas intactas de Panagrellus sp. após 24 horas de tratamento em 60 e 90%, respectivamente. Este efeito não está relacionado à atividade enzimática, mas sim à presença de outros metabólitos. Os ovos de Meloidogyne javanica, quando tratados com o extrato de P. eryngii, mostraram redução de 53% (p<0,01) no número de ovos intactos. A redução de ovos intactos de M. javanica é atribuída à atividade enzimática, uma vez que o extrato apresentou atividade quitinolítica e proteolítica de, respectivamente, 32,74 e 3,57 U/mL. A purificação do látex de E. trigona por cromatografia de exclusão molecular permitiu a purificação de três proteases distintas. O peso molecular das proteases foi estimado em aproximadamente: 36, 31 e 29 kDa, para a trigonina 1, 2 e 3, respectivamente. O pH e a temperatura que proporcionaram maior atividade de protease foram 4,0, 6,0 e 9,0, e temperatura de 70 °C. As proteases foram inibidas por fluoreto de fenilmetilsulfonila e iodoacetamida. O pool das três proteases presentes no látex de E. trigona reduziram significativamente (p<0,01) o número de J 2 vivas de M. incognita em 96% após 24 horas de tratamento. A purificação parcial das proteases presentes no látex de E. milii indicou que estas proteases correspondem à milina e miliina. Estas enzimas reduziram em 65,59% e 96,46% o número de larvas de Panagrellus redivivus após 24 e 48 h, respectivamente (p<0,01). A dextranase produzida pelo fungo P. chlamydosporia foi purificada à homogeneidade em duas etapas, com rendimento de 152%, fator de purificação de 6,84 e atividade específica de 358,63 U/mg. Seu peso molecular foi estimado por SDS-PAGE em 64 kDa. A enzima apresentou maior atividade a 50 °C e pH 5,0, usando tampão citrato-fosfato 100 mM, foi inibida por Ag 1+ , Hg 2+ , Cu 2+ , Mg 2+ , e apresentou K M de 1,77 g/L. A dextranase madura é composta por 585 resíduos de aminoácidos, com peso molecular predito de 64,38 kDa e pI 5,96. Esta dextranase demonstrou forte semelhança filogenética em comparação à dextranase de Trichoderma harzianum. Sua estrutura consiste de dois domínios: o primeiro composto por 15 cadeias beta, e o segundo composto por uma β-hélice paralela. Esses resultados evidenciam o potencial biotecnológico desses fungos, plantas e suas enzimas extracelulares informações acerca no da controle dextranase de nematoides, produzida pelo e fornecem fungo P. chlamydosporia, que podem ser utilizadas para futuras aplicações industriais desta enzima. / Fungi and plants produce enzymes that have several biotechnological applications. A possible and promising application of these enzymes is the control of nematodes, replacing chemical nematicides harmful to human and animal health, to the environment and to the soil microbiota. Another possible application of fungi enzymes is the use of dextranase to reduce dextran content in sugar industry. The objective of this work was to investigate the production of enzymes by Pleurotus eryngii, Euphorbia milii and E. trigona, to purify and characterize the enzymes from E. milii, E. trigona and Pochonia chlamydosporia, and to test the nematicidal activity of E. milii, E. trigona and P. eryngii. P. eryngii fungus and its extract significantly reduced (p<0.01) the number of intact Panagrellus sp. larvae after 24 hours treatment in 60 and 90%, respectively. This effect is not related to enzymatic activity, but to the presence of other metabolites. M. javanica eggs, when treated with P. eryngii extract, showed a 53% reduction (p<0.01) in the number of intact eggs. The M. javanica intact eggs reduction is attributed to enzymatic activity, once the extract showed proteolytic and chitinolytic activities of, respectively, 32.74 and 3.57 U/mL. The purification of E. trigona latex with size-exclusion chromatography allowed partially purification of three distinct proteases. The molecular weight of proteases was estimated at approximately: 36, 31 and 29 kDa, for trigonin 1, 2 and 3, respectively. The pH and temperature that provided highest protease activity were 4.0, 6.0 and 9.0, and temperature of 70 °C. The proteases were inhibited by phenylmethylsulfonyl fluoride and iodoacetamide. The pool of three proteases present in E. trigona latex reduced significantly (p<0.01) the number of live M. incognita J 2 in 96% after 24 hours treatment. The partial purification of the proteases present in E. milii latex indicated that these proteases are milin and miliin. These enzymes reduced the Panagrallus redivivus larvae number by 65.59 % and 96.46 % after 24 and 48 hours, respectively (p<0.01). Dextranase produced by the fungus P. chlamydosporia was purified to homogeneity in two steps, with a yield of 152 %, purification factor of 6.84 and specific activity of 358.63 U/mg. Its molecular weight was estimated by SDS-PAGE at 64 kDa. The enzyme presented higher activity at 50 °C and pH 5.0, using 100 mM citrate-phosphate buffer, was inhibited by Ag 1+ , Hg 2+ , Cu 2+ , Mg 2+ , and presented K M of 1.77 g/L. Mature dextranase is composed of 585 amino acids residues, with a predicted molecular weight of 64.38 kDa and pI 5.96. This dextranase showed a strong phylogenetic similarity when compared to Trichoderma harzianum dextranase. Its structure consists of two domains: the first composed by 15 β strands, and the second composed by a right-handed parallel β-helix. These results evidence the biotechnological potential of these fungi, plants and their extracellular enzymes to nematodes control, and provide information about the dextranase produced by P. chlamydosporia fungus, which can be used for future industrial applications of this enzyme.

<p>Glycosidases hydrolyse the glycosidic bond in carbohydrates. Structural studies of three glycosidases with different substrate specificities are presented in this work.</p><p>Dextranase catalyzes the hydrolysis of <i>α</i>-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from <i>Penicillium minioluteum</i> was solved in the apo-enzyme (1.8 Å resolution) and product-bound (1.65 Å resolution) forms. The main domain of the enzyme is a right-handed β-helix, which is connected to a β-sandwich domain at the N-terminus. Using NMR spectroscopy the reaction course was shown to occur with net inversion at the anomeric carbon. A new clan is suggested that links glycoside hydrolase (GH) families 28 and 49.</p><p>Endo-<i>β</i>-1,4-D-mannanase catalyzes the depolymerization of <i>β</i>-1,4-mannan polymers. The structure of endo-1,4-<i>β</i>-mannanase Man5A from blue mussel <i>Mytilus edulis</i> has been determined at 1.6 Å resolution. Kinetic analysis of Man5A revealed that the enzyme requires at least 6 subsites for efficient hydrolysis. The architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that Man5A represents a new subfamily in GH 5. </p><p>Both the Dex49A and the Man5A structures were determined by multiple-wavelength anomalous diffraction using the selenium <i>K</i>-edge with selenomethionyl enzymes expressed in the yeast <i>Pichia pastoris</i>.</p><p>Endoglucanase Cel6A from <i>Thermobifida fusca</i> hydrolyzes the <i>β</i>-1,4 linkages in cellulose. The structure of the catalytic domain of Cel6A from <i>T. fusca</i> in complex with a non-hydrolysable substrate analogue has been determined to 1.5 Å resolution. The glycosyl unit in subsite –1 was sterically hindered by Tyr73 and forced into a distorted ²S_O conformation. In the enzyme where Tyr73 was mutated to a serine residue the hindrance was removed and the glycosyl unit in subsite –1 had a relaxed ⁴C₁ chair conformation.</p>

Glycosidases hydrolyse the glycosidic bond in carbohydrates. Structural studies of three glycosidases with different substrate specificities are presented in this work. Dextranase catalyzes the hydrolysis of α-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from Penicillium minioluteum was solved in the apo-enzyme (1.8 Å resolution) and product-bound (1.65 Å resolution) forms. The main domain of the enzyme is a right-handed β-helix, which is connected to a β-sandwich domain at the N-terminus. Using NMR spectroscopy the reaction course was shown to occur with net inversion at the anomeric carbon. A new clan is suggested that links glycoside hydrolase (GH) families 28 and 49. Endo-β-1,4-D-mannanase catalyzes the depolymerization of β-1,4-mannan polymers. The structure of endo-1,4-β-mannanase Man5A from blue mussel Mytilus edulis has been determined at 1.6 Å resolution. Kinetic analysis of Man5A revealed that the enzyme requires at least 6 subsites for efficient hydrolysis. The architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that Man5A represents a new subfamily in GH 5. Both the Dex49A and the Man5A structures were determined by multiple-wavelength anomalous diffraction using the selenium K-edge with selenomethionyl enzymes expressed in the yeast Pichia pastoris. Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the β-1,4 linkages in cellulose. The structure of the catalytic domain of Cel6A from T. fusca in complex with a non-hydrolysable substrate analogue has been determined to 1.5 Å resolution. The glycosyl unit in subsite –1 was sterically hindered by Tyr73 and forced into a distorted 2SO conformation. In the enzyme where Tyr73 was mutated to a serine residue the hindrance was removed and the glycosyl unit in subsite –1 had a relaxed 4C1 chair conformation.