No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Cutinases (EC 3.1.1.74) são enzimas que catalisam a hidrólise da cutina, um biopoliéster insolúvel que compõe a estrutura da cutícula das plantas. Essas enzimas possuem potencial na síntese de triglicerídeos, polímeros, surfactantes, em indústrias química, farmacêutica e agroquímica. As cutinases têm sido aplicadas na modificação de superfícies de polímeros, facilitando a degradação desses compostos. O objetivo deste trabalho foi produzire caracterizar cutinases por Escherichia colimodificadas geneticamente para aplicação no tratamento de politereftalato de etileno (PET). E. coli CUT e E. coli CUT-N1foram cultivadas em Lysogenybroth (LB) na presença de 100 μg/mL deampicilina e isopropil β-D-1- tiogalactopiranosídeo (IPTG) como indutor. As atividades de cutinases foram determinadas na presença do paranitrofenilbutirato (pNPB). A máxima atividade de cutinases foi 1,4 UI/mL, determinada no líquido metabólicolivre de células, produzido por E. coli CUT-N1.Os líquidos metabólicoscom atividade cutinaseproduzidos por E. coli CUT e E. coli CUT-N1foram concentrados duas vezes por ultrafiltração e formulados com conservantes microbiológicos e substâncias estabilizadoras de estruturas protéicas. As cutinases apresentaram pH ótimo igual a 7,0 e estabilidade térmicaa30 - 50 C.A adição de (NH4)2SO4, em concentrações inferiores a 10 % estabilizou a atividade de cutinasesdurante 60 dias, a 28 C. As cutinases produzidas pelas culturas de E. coli degradaramo plástico politereftalato de etileno (PET), com perda de massa de 0,90 %. Cutinases microbianas recombinantes são uma alternativa para aplicação em tratamento biológico de plásticos. / Cutinase (EC 3.1.1.74) are enzymes that catalyze the hydrolysis of cutin, an insoluble biopolyester that compound the cuticle of plants. These enzymes have potential in the synthesis of triglycerides, polymers, surfactants in chemical, pharmaceutical and agrochemical industries. Cutinases have been applied to the surface modification of polymers, facilitating the degradation of these compounds. The aim of this work was to produce and characterize cutinases by Escherichia coli genetically engineered for use in the treatment of polyethylene terephthalate (PET). E. coli CUT and E. coli CUT-N1 were grown in Lysogeny broth (LB) in the presence of 100 μg/ml ampicillin and isopropyl β-D-1-tiogalactopiranosídeo (IPTG) as inducer. Cutinases activities were determined in the presence of p-nitrophenyl butyrate (p- NPB). The maximum cutinase activity was 1.4 U/mL, determined in the cell-free metabolicliquid, produced by E. coli CUT-N1. The metabolic liquid with activity of cutinase produced by E. coli CUT and E. coli CUT-N1 were concentrated two times by ultrafiltration and formulated with microbial preservatives and stabilizing substances of protein structures. Cutinases had optimum pH equal to 7.0 and thermal stability at 30 - 50 C. The addition of (NH4)2SO4 at concentrations less than 10% stabilized cutinase activity for 60 days at 28 C. Cutinases produced by cultures of E. coli degraded the plastic polyethylene terephthalate (PET) with a weight loss of 0.90%. Recombinant microbial cutinases are an alternative for application in biological treatment of plastics.

Orientador: Gabriela Alves Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T02:54:59Z (GMT). No. of bitstreams: 1 Fraga_LairaPriscila_M.pdf: 554432 bytes, checksum: 56119a190bf37124ca4c590d3059597c (MD5) Previous issue date: 2008 / Resumo: Cutinases são enzimas amplamente encontradas na natureza, podendo ser obtidas de fontes vegetais ou microorganismos. Trata-se de uma esterase que atua tanto como esterase quanto como lipase, capaz de hidrolisar uma ampla variedade de ésteres sintéticos e triglicerídeos. Este perfil de atividade oferece-lhe um número de aplicações importantes nas indústrias como detergentes de roupas e louças, alimentos, fármacos, agroquímicos e cosméticos. Utilizando a fermentação em estado sólido (FES) é possível a produção de uma enzima com características interessantes de catálise, com baixo custo de produção e bom rendimento, e que possa ser empregada na indústria de modo geral. Este trabalho utilizou FES para produção de cutinase por Fusarium oxysporum utilizando sub-produtos agrícolas visando à seleção dos melhores meios e indutores para produção da enzima (utilizando fontes de carbono e minerais), otimização das condições de cultivo (avaliando os parâmetros temperatura e quantidade de água do meio além do estudo do inóculo), produção de cutinases utilizando diferentes substratos (farelo de trigo, casca de soja, farelo de arroz e produzida por meio líquido) e comparação entre eles, além da aplicação das enzimas em reações de esterificação para separação enantiomérica de substâncias racêmicas como o 2-octanol e o Ibuprofeno. De um modo geral o trabalho mostrou a produção de enzima de grande interesse comercial usando substratos diferentes e seu comportamento frente a reações de esterificação. Três meios sólidos foram selecionados (farelo de trigo, casca de soja e farelo de arroz) e suas condições de cultivo otimizadas (temperatura 28ºC e quantidade de água do meio 100%). Nos testes de aplicação das cutinases, para resolução enantiomérica do 2-octanol todas as cutinases tiveram bons valores de esterificação e enantioseletividade com destaque para farelo de trigo (76,4% de esterificação após 200 horas) e casca de soja (71,9% de esterificação após 200 horas). Para resolução do fármaco racêmico Ibuprofeno as cutinases mais efetivas foram as produzidas por farelo de arroz (5,1% de conversão após 200 horas) e meio líquido (2,9% de conversão após 200 horas). Esses resultados mostraram a diferença entre o comportamento de cutinases, do mesmo microorganismo, produzidas em diferentes meios, e a grande possibilidade de utilização de cutinases para processos de interesse industrial / Abstract: Cutinases enzymes are often found in nature and may be obtained from plant sources or microorganisms. This is an enzyme that acts both as esterases and as lipases, capable of hydrolyzed a several variety of synthetic esters and triglycerides. This activity profile gives a number of important applications in industries such detergents, clothes and dishes, foods, drugs, chemicals and cosmetics. Using solid state fermentation SSF it is possible production of an enzyme interesting of catalysis with low production cost and good yields, and it can be employed in the industries. This work used SSF for the cutinase production by Fusarium oxysporum using agro-industrial sub-products to find selection of the best media and inducer to enzyme production (using sources of carbon and minerals), optimization of fermentation conditions (modifying the parameters temperature and water amount and after the inoculum study), cutinases production using different substrates (wheat bran, soy rind, rice bran and produced by liquid medium), comparison among them and enzymes application in esterification reactions to enantiomeric separation of racemic substances such 2-octanol and drug Ibuprofen. In general the work showed enzyme production of great commercial interest using various substrates and their behavior in esterification reactions. Three solid media were selected (wheat bran, soy rind, and rice bran) and their fermentation conditions optimized (temperature 28ºC and water amount 100%). In tests of cutinases application for enantiomeric resolution of 2-octanol all cutinases had good values to esterification and enantioselectivity and the best results was using wheat bran (76.4% of esterification after 200 hours) and soy rind (71.9% of esterification after 200 hours). The test using racemic drug Ibuprofen the more effective resolution was found with cutinase produced by rice bran (5.1% of esterification after 200 hours) and liquid medium (2.9% of esterification after 200 hours). These results showed the difference among behavior of cutinases using same strain, produced by different media, and the great possibility of cutinases with interesting catalysis characteristics and it can be employed in the industry / Mestrado / Mestre em Ciência de Alimentos

Orientadores: Gabriela Alves Macedo, Patricia de Oliveira Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-16T15:43:03Z (GMT). No. of bitstreams: 1 Speranza_Paula_M.pdf: 2790177 bytes, checksum: c48b32a4db9fbe192ae18d8080a3c523 (MD5) Previous issue date: 2010 / Resumo: A cutinase (E.C. 3.1.1.74) é uma enzima que catalisa a hidrólise de ligações ésteres da cutina. A cutina é um biopolímero insolúvel em água, composto de ácidos graxos com C16 e C18, e que está presente na superfície externa das partes aéreas das plantas. Estudos têm sido realizados com o objetivo de caracterizar bioquimicamente cutinases produzidas por diferentes processos fermentativos, micro-organismos e meios de cultivo. As cutinases têm-se mostrado bastante estáveis em diferentes condições de análise. O conhecimento das condições ótimas de ação desta classe de enzimas, assim como dos fatores que afetam a sua atividade são de extrema importância para se compreender melhor a atuação e as alterações sofridas por estas enzimas em diferentes condições de estocagem e trabalho. Tendo em vista estas observações, o objetivo deste estudo foi caracterizar bioquimicamente e comparar as cutinases produzidas por Fusarium oxysporum em quatro meios de cultivo sólidos distintos, compostos por farelo de trigo, casca de soja, farelo de arroz e torta de pinhão manso. A caracterização de cada uma dessas enzimas mostrou que estas apresentam temperatura ótima entre 30 e 37°C, temperatura de estabilidade de 30°C para as enzimas produzidas em meios com de farelo de trigo e farelo de arroz e 37°C para as enzimas produzidas em meios com casca de soja e torta de pinhão manso. O pH ótimo de ação das quatro enzimas mostrou-se levemente alcalino, sendo igual a 8,0 nas enzimas produzidas em meios com farelo de trigo, casca de soja e farelo de arroz e 9,0 para a enzima produzida em meio com torta de pinhão manso. O pH em que as quatro enzimas foram mais estáveis foi igual a 6,0. As quatro enzimas apresentaram boa afinidade pelo substrato, com valores de constante cinética (km) que variaram de 0,25 a 0,65 mM. A enzima produzida em meio com casca de soja apresentou a maior velocidade máxima (vmax), 7,86 U/mL. A maioria dos sais minerais testados inativaram ou ativaram pouco as enzimas produzidas em meios com farelo de trigo, casca de soja e farelo de arroz, enquanto que a enzima produzida em meio com torta de pinhão manso foi bastante ativada na presença da maioria dos sais minerais. As enzimas de farelo de trigo, casca de soja e torta de pinhão manso apresentaram maior especificidade por ésteres de cadeia carbônica média (p-nitrofenil caprilato), enquanto que a enzima produzida em meio com farelo de arroz apresentou maior especificidade por ésteres de cadeia curta (p-nitrofenil butirato). A enzima produzida em meio com torta de pinhão manso foi bastante ativa na presença de solventes orgânicos, especialmente o hexano. A eletroforese por SDS-PAGE mostrou uma banda de 27-30 kDa nas enzimas produzidas em meios com farelo de trigo, casca de soja e torta de pinhão manso. Em relação as suas propriedades enantiosseletivas, a enzima produzida em meio com casca de soja foi a que apresentou melhor resultado (E=5,9) / Abstract: Cutinase (E.C. 3.1.1.74) is an enzyme that catalyzes the hydrolysis of esters bonds in cutin. Cutin is a biopolymer insoluble in water composed of fatty acids C16 and C18 and found in the outside surface of the plants¿ aerial parts. Studies have been conducted with the aim of biochemically characterizing the cutinases produced by different fermentation conditions, microorganisms and growth medium. Cutinases have proved to be very stable in different conditions of analysis. The knowledge of optimum conditions of this class of enzymes, as well as of the factors that affect their activity are very important in order to understand the action of such enzymes and the alterations they go through in different conditions of storage and use. Considering that, the objective of this study is to biochemically characterize and compare cutinases produced by Fusarium oxysporum in four different solid growth medium composed of wheat bran, soy rind, rice bran and Jatropha curcas seed cake. The characterization of each one of these enzymes showed that they present the optimum temperature between 30 and 37°C, stability temperature at 30°C for wheat bran and rice bran and 37°C for soy rind and Jatropha curcas seed cake. The optimum pH of action of the four enzymes was little alkaline: 8.0 for the enzymes produced by wheat bran, soy rind and rice bran and 9.0 for the enzyme produced by Jatropha curcas seed cake. The most stable pH for the four enzymes was 6.0. The four enzymes showed good affinity by the substrate, with values of km ranging from 0.25 to 0.65 mM. The enzyme produced in soy rind showed higher value of vmax 7.86 U/mL. Most of the tested mineral salts inactivated or activated just a little the enzymes produced by wheat bran, soy rind and rice bran, whereas the enzyme produced by Jatropha curcas seed cake was very activated in the presence of most of the mineral salts. The enzymes of wheat bran, soy rind and Jatropha curcas seed cake showed more specificity for mediumlength carbonic chain esters (p-NPC), whereas the enzyme of rice rind showed more specificity for short-length chain esters (p-NPB). The enzyme of Jatropha curcas seed cake was very activated by the presence of organic solvents, especially hexane. The electrophoresis by SDS-PAGE showed a band of 27-30 kDa in the enzymes produced by wheat bran, soy rind and Jatropha curcas seed cake. Regarding enantioselectivity proprieties the enzyme produced in soy rind showed the best result (E= 5.9 ) / Mestrado / Mestre em Ciência de Alimentos

Made available in DSpace on 2017-06-01T18:20:39Z (GMT). No. of bitstreams: 1 daniela_silva_gomes.pdf: 1405073 bytes, checksum: 6c85347ca66829fdd4d81eaf6bfc02f3 (MD5) Previous issue date: 2013-02-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Cutinase (EC 3.1.1.74) are enzymes that catalyze the hydrolysis of cutin, an insoluble biopolyester that compound the cuticle of plants. These enzymes have potential in the synthesis of triglycerides, polymers, surfactants in chemical, pharmaceutical and agrochemical industries. Cutinases have been applied to the surface modification of polymers, facilitating the degradation of these compounds. The aim of this work was to produce and characterize cutinases by Escherichia coli genetically engineered for use in the treatment of polyethylene terephthalate (PET). E. coli CUT and E. coli CUT-N1 were grown in Lysogeny broth (LB) in the presence of 100 μg/ml ampicillin and isopropyl β-D-1-tiogalactopiranosídeo (IPTG) as inducer. Cutinases activities were determined in the presence of p-nitrophenyl butyrate (p- NPB). The maximum cutinase activity was 1.4 U/mL, determined in the cell-free metabolicliquid, produced by E. coli CUT-N1. The metabolic liquid with activity of cutinase produced by E. coli CUT and E. coli CUT-N1 were concentrated two times by ultrafiltration and formulated with microbial preservatives and stabilizing substances of protein structures. Cutinases had optimum pH equal to 7.0 and thermal stability at 30 - 50 °C. The addition of (NH4)2SO4 at concentrations less than 10% stabilized cutinase activity for 60 days at 28 °C. Cutinases produced by cultures of E. coli degraded the plastic polyethylene terephthalate (PET) with a weight loss of 0.90%. Recombinant microbial cutinases are an alternative for application in biological treatment of plastics. / Cutinases (EC 3.1.1.74) são enzimas que catalisam a hidrólise da cutina, um biopoliéster insolúvel que compõe a estrutura da cutícula das plantas. Essas enzimas possuem potencial na síntese de triglicerídeos, polímeros, surfactantes, em indústrias química, farmacêutica e agroquímica. As cutinases têm sido aplicadas na modificação de superfícies de polímeros, facilitando a degradação desses compostos. O objetivo deste trabalho foi produzire caracterizar cutinases por Escherichia colimodificadas geneticamente para aplicação no tratamento de politereftalato de etileno (PET). E. coli CUT e E. coli CUT-N1foram cultivadas em Lysogenybroth (LB) na presença de 100 μg/mL deampicilina e isopropil β-D-1- tiogalactopiranosídeo (IPTG) como indutor. As atividades de cutinases foram determinadas na presença do paranitrofenilbutirato (pNPB). A máxima atividade de cutinases foi 1,4 UI/mL, determinada no líquido metabólicolivre de células, produzido por E. coli CUT-N1.Os líquidos metabólicoscom atividade cutinaseproduzidos por E. coli CUT e E. coli CUT-N1foram concentrados duas vezes por ultrafiltração e formulados com conservantes microbiológicos e substâncias estabilizadoras de estruturas protéicas. As cutinases apresentaram pH ótimo igual a 7,0 e estabilidade térmicaa30 - 50 °C.A adição de (NH4)2SO4, em concentrações inferiores a 10 % estabilizou a atividade de cutinasesdurante 60 dias, a 28 °C. As cutinases produzidas pelas culturas de E. coli degradaramo plástico politereftalato de etileno (PET), com perda de massa de 0,90 %. Cutinases microbianas recombinantes são uma alternativa para aplicação em tratamento biológico de plásticos.

In Tumoren an Arabidopsis thaliana, induziert über Agrobacterium tumefaciens (Stamm C58), ist von den 49 bekannten Lipidtransferproteinen (LTPs) nur die Expression von LTP2 stark erhöht (Deeken et al., 2006). Mutanten ohne LTP2-Transkripte (ltp2KO) entwickeln deutlich kleinere Tumore als der Wildtyp. Durch die permanenten Zellstreckungs- und Dehnungsprozesse besitzen Tumore keine intakte Epidermis (Efetova et al., 2007). Dies wiederum führt zum Verlust einer vollständigen Cuticula-Schicht, welche von der Epidermis produziert wird und dieser als Barriere zur Umwelt aufgelagert ist. Um den transpirationsbedingten Wasserverlust zu minimieren, werden in Tumoren langkettige Aliphaten in die äußeren Zellschichten eingelagert (Efetova et al., 2006). Ein ähnliches Szenario findet um Verwundungsareale statt (Kolattukudy et al., 2001). Die Gen-Expression von LTP2 wird nicht durch tumorinduzierende Agrobakterien ausgelöst. Faktoren wie Verwundung, sowie die Applikation des Trockenstress-Phytohormons Abscisinsäure (ABA) begünstigen die LTP2-Gen-Expression positiv. Außerdem ist der LTP2-Promotor in Gewebe aktiv, in welchem sekundäre Zellwandmodifikationen auftreten, sowie insbesondere in Abscissionsschichten von welkenden Organen. Ungerichtete Lipidanalysen der ltp2KO-Mutante im Vergleich zum Wildtyp zeigten nur signifikante Veränderungen in der Menge definierter Sphingolipide – obwohl bislang eine Beteiligung von LTP2 am Transfer von Phospholipiden postuliert wurde. Allerdings kann das LTP2-Protein, wie Protein-Lipid-Overlay-Analysen demonstrierten, weder komplexen Sphingolipide noch Sphingobasen binden. Neben Sphingobasen sind auch langkettige Fettsäuren Bestandteile von Sphingolipiden und diese sind wiederum Bindepartner von LTP2. Um eine eventuelle Beteiligung von LTP2 an der Bildung von Suberin von Tumoren zu zeigen, wurde dieses analysiert. Die GC-MS-Analysen des Tumor-Suberins haben jedoch veranschaulicht, dass durch das Fehlen von LTP2-Transkripten das Lipidmuster nicht beeinträchtigt wird. Eine Überexpression von LTP2 im gesamten Kormophyten war trotz drei unabhängiger experimenteller Ansätze nicht möglich. Daher wurde das Protein ektopisch in epidermalen Zellen exprimiert (CER5Prom::LTP2). Die Transgenen CER5Prom::LTP2 wiesen einige morphologische Besonderheiten auf, wie verminderte Oberflächenhydrophobizität, aberrante Blüten- und Blattmorphologien etc., die typisch für Wachsmutanten sind. GC-MS-Analysen der cuticulären Wachse dieser transgenen Pflanzen zeigten, einen erhöhten Gehalt an C24- und C26-Fettsäuren, wohingegen die korrespondierenden Aliphaten wie Aldehyde und Alkane dezimiert waren. Unterstützend zeigten Lokalisationsanalysen, dass das LTP2-Protein an/in der Plasmamembran assoziiert ist. Somit kann die These aufgestellt werden, dass LTP2 langkettigen, unverzweigten Aliphaten (Fettsäuren) an der Grenzfläche Plasmamembran/Zellwand transferiert, die zur Versieglung und Festigung von Zellwänden benötigt werden. / Out of 49 known lipid transfer protein (LTP) only the expression of LTP2 is highly increased in tumors induced on Arabidopsis thaliana via Agrobacterium tumefaciens (strain C58; Deeken et al., 2006). Mutants with no LTP2 transcripts (ltp2KO) develop significantly smaller tumors than the wild-type. Due to the permanent cell stretch and elongation processes tumors do not possess an intact epidermal layer (Efetova et al., 2007). This leads to the loss of a complete cuticle layer, which is produced by the epidermis and builds up a barrier to the environment. To minimize the transpirational water loss, long-chain aliphatic compounds are incorperated into the outer cell layers of tumors (Deeken et al., 2006). The gene expression of LTP2 is not triggered by tumor-inducing agrobacteria. Instead, factors such as wounding and the application of the phytohormone abscisic acid (ABA) induce the LTP2 gene expression. In addition, the LTP2 promoter is highly active in tissue, in which secondary cell wall modifications occur, and in the abscission zone of wilting organs. Untargeted lipid analyzes of ltp2KO mutant in comparison to the wild type showed significant changes in the amount of defined sphingolipids only - although the involvement of LTP2 has been postulated for the transfer of phospholipids. However, the LTP2 protein, as protein-lipid overlay analysis demonstrated, binds neither complex sphingolipids nor sphingobases. Instead LCFAs, which are part of sphingolipids are binding partners of LTP2. In order to show a possible involvement of LTP2 in the formation of tumor-suberin GC-MS analyzes were performed. These demonstrated that the composition of the lipid-pool is not altered in ltp2KO plants. Overexpression of LTP2 was not possible in spite of three independent experimental approaches. The protein was instead expressed ectopically in epidermal cells (CER5Prom::LTP2). The transgenes CER5Prom::LTP2 showed some morphological abnormities, such as reduced surface hydrophobicity, aberrant flowers and leaf morphologies, which are typical for wax mutants. GC-MS analyzes of the cuticular wax of those transgenic lines revealed an increased amount of C24- and C26- fatty acids. Furthermore LTP2 was localized at the plasma membrane. Thus, this thesis proposes a role of LTP2 in the transfer of long chain, unbranched aliphatics (fatty acids), which are needed to seal up and strengthen cell walls at the interface plasma membrane and cell wall.

Färgning av polyester (PET) sker i temperaturer omkring 130℃ och som en följd av den höga värmen migrerar oligomerer ut ur PET-fibern. Dessa oligomerer skapar därefter problem i form av vita fällningar och avlagringar, som leder till en reducering av maskinernas effektivitet, samt försämring av materialets utseende. En vanligt förekommande metod för att begränsa problemet är att tillsätta hjälpkemikalier i färgbadet. Novozymes A/S hävdar att genomförda studier med PET-garn och enzymet cutinase NS59038 har visat en minskning av det vita damm som kan ses på garnet i samband med migration av oligomerer. Eftersom enorma mängder PET produceras globalt varje år skulle endast en liten minskning av mängden kemikalier innebära en stor skillnad. Kemikalier som ersätts med andra kemikalier kan ifrågasättas om det är en bra metod eller om det bestrider syftet. Ur ett hållbarhetsperspektiv kan enzymer ses som ett bättre alternativ eftersom de enligt Jajpura (2017) är biologiskt hållbara, formar sällan någon biprodukt och dess reaktion kräver oftast mildare förhållanden. Hjälpkemikalien Sera Con P-NSI används av Ludvig Svensson AB som färgar garn av den flamskyddade polyesterfibern Trevira CS. Syftet med projektet har därmed varit att undersöka om enzymet cutinase NS59038 är ett alternativ till hjälpkemikalien Sera Con P-NSI, för att reducera de problem som uppstår med oligomerer i färgningsprocessen med Trevira CS. Garnets egenskaper har jämförts genom visuell analys med mikroskop, viktförändring, reflektionsspektrofotometer och dragprovning. Metoder som använts i syfte att detektera oligomerer har varit FTIR, UV-vis spektrofotometer och svart svavelfilterpapper. Utifrån genomförda analyser av garnets vikt, styrka, färgupptagning och visuell bedömning har det inte kunnat konstateras att cutinase NS59038 skulle vara ett alternativ till Sera Con P-NSI. Resultat från analysmetoderna visade skillnader i medelvärde, men inget samband mellan val av färgrecept och garnets egenskap har detekterats. De olika färgningsförsöken visade inte någon förekomst av fällningar/avlagringar som med använda testmetoder har kunnat bekräftas som oligomerer. Resultaten bedömdes därmed inte som tillräckliga för att konstatera om cutinase NS59038 påverkar garnets kvalitet i jämförelse med hjälpkemikalien Sera Con P-NSI. / In the dyeing process of polyester (PET) the temperature goes up to around 130℃ and as a result oligomers migrate out of the PET fiber. These oligomers create problems as white precipitates that deposits on the material and the inside of machines. This leads to a reduction in machine efficiency, as well as a change of material appearance. One common method for limiting the problem is to add help chemicals in conjunction with the dyeing process. According to results from previous studies, Novozymes A/S claims that the enzyme cutinase NS59038 has reduced the white dust on the yarn of polyester. Based on the enormous quantity of PET that are produced every year, even a small reduction of the chemicals that are used would mean a huge difference. Chemicals that replace other chemicals can be questioned whether or not it can be seen as an alternative or if it disputes the purpose. From a sustainable point of view enzymes could according to Jajpura (2017) be seen as a better alternative because they are biodegradable, their reaction seldom form any byproduct and often requires less energy. The help chemical Sera Con P-NSI is used by Ludvig Svensson AB and is added to the dye bath in the dyeing process of the specific polyester Trevira CS (a flame retardant polyester fiber). The purpose of the project has thus been to investigate whether the enzyme cutinase NS59038 is an alternative to the help chemical Sera Con P-NSI, to reduce the problems with oligomers in the dyeing process of Trevira CS. The properties of the yarn have been compared by visual analysis with microscope, weight change, measurement of the color change with spectrophotometer and tensile strength. Methods used to detect oligomers have been FTIR, UV-vis spectrophotometer and black sulfur filter paper. Based on the analysis of the weight of the yarn, strength, color uptake and visual assessment, it can not be established that cutinase NS59038 would be an alternative to Sera Con P-NSI. Results from the analysis methods show differences in the mean, but no connection between the choice of dye recipe and the properties of the yarn can be detected. None of the precipitates that was predicted to arise on the surface of the yarn was ever detected. The results are therefore not considered sufficient to determine whether cutinase NS59038 affects the quality of the yarn compared to the help chemical Sera Con P-NSI.

<p><i>S. sclerotiorum</i> has been studied for over 100 years. Despite this, a definite resistance mechanism to this plant pathogen remains to be identified. Researchers continue to examine the <i>S. sclerotiorum</i> life cycle to identify stages where effective disease management strategies can be applied. The development of molecular tools has allowed for a better understanding of the pathogen and created new opportunities for research on plant-pathogen interactions.</p> <p>Most of the past research on pathogenicity factors produced by this pathogen, such as hydrolytic enzymes, studied them in isolation. This thesis examines how <i>S. sclerotiorum</i> pathogenicity factors, including cutinases, polygalacturonases and necrosis-inducing peptides, work in concert during the infection. The first study explored processes for cuticle penetration leading to the identification of the gene encoding S. sclerotiorum cutinase A and the characterization of the factors that govern its expression during the infection. The second study investigated how the pathogen penetrates the cell wall and proliferates within the host. In this regard, the mechanism with which expression of <i>S. sclerotiorum</i> polygalacturonase genes is regulated was elucidated. The interplay with host polygalacturonase inhibitor proteins was also demonstrated and related to the mechanisms of host resistance. The third study examined factors involved in tissue necrosis and two necrosis-inducing proteins were characterized. This study also unraveled part of the signaling mechanisms that allow for the pathogen to regulate pathogenicity gene expression during the infection. The signaling mechanisms were found to involve calcium, cAMP and at least one <i>S. sclerotiorum</i> mitogen activated protein kinase (SMK3) working in concert to coordinate the infection process. SMK3 was found to play a major role in a variety of vital functions, such as mycelial branching, infection cushion formation and sclerotia production. Genetic transformation of <i>S. sclerotiorum</i> was required to enable certain aspects of this study. My approach to this led to the development of a highly efficient method to isolate homokaryotic lines of filamentous fungi. In conclusion, this thesis has advanced the understanding of <i>S. sclerotiorum</i>-host interactions and identified a number of factors involved in pathogenesis.</p>

<p><i>S. sclerotiorum</i> has been studied for over 100 years. Despite this, a definite resistance mechanism to this plant pathogen remains to be identified. Researchers continue to examine the <i>S. sclerotiorum</i> life cycle to identify stages where effective disease management strategies can be applied. The development of molecular tools has allowed for a better understanding of the pathogen and created new opportunities for research on plant-pathogen interactions.</p> <p>Most of the past research on pathogenicity factors produced by this pathogen, such as hydrolytic enzymes, studied them in isolation. This thesis examines how <i>S. sclerotiorum</i> pathogenicity factors, including cutinases, polygalacturonases and necrosis-inducing peptides, work in concert during the infection. The first study explored processes for cuticle penetration leading to the identification of the gene encoding S. sclerotiorum cutinase A and the characterization of the factors that govern its expression during the infection. The second study investigated how the pathogen penetrates the cell wall and proliferates within the host. In this regard, the mechanism with which expression of <i>S. sclerotiorum</i> polygalacturonase genes is regulated was elucidated. The interplay with host polygalacturonase inhibitor proteins was also demonstrated and related to the mechanisms of host resistance. The third study examined factors involved in tissue necrosis and two necrosis-inducing proteins were characterized. This study also unraveled part of the signaling mechanisms that allow for the pathogen to regulate pathogenicity gene expression during the infection. The signaling mechanisms were found to involve calcium, cAMP and at least one <i>S. sclerotiorum</i> mitogen activated protein kinase (SMK3) working in concert to coordinate the infection process. SMK3 was found to play a major role in a variety of vital functions, such as mycelial branching, infection cushion formation and sclerotia production. Genetic transformation of <i>S. sclerotiorum</i> was required to enable certain aspects of this study. My approach to this led to the development of a highly efficient method to isolate homokaryotic lines of filamentous fungi. In conclusion, this thesis has advanced the understanding of <i>S. sclerotiorum</i>-host interactions and identified a number of factors involved in pathogenesis.</p>

In this thesis we have employed two computational approaches, temperature-dependent molecular dynamics (MD) and normal mode analysis (NMA), to gain insights into the structureunction relationships between three structurally-related proteins, each possessing a central alpha/beta core. The three proteins studied here are: pnbCE from Bacillus subtilis, cutinase from Fusarium solani - both belong to the serine hydrolase family - and TTHA1554, from the thermophile Thermus thermophilus. Mutations at the gate residue 362, located at the side-door of the pnbCE enzyme, are known to alter the catalytic activity of this enzyme. In this work the modifications induced by mutating LEU362 on the structural and dynamical properties of pnbCE are also explored. From MD simulations at several temperatures, we propose a mechanism by which mutations at position 362 of pnbCE affect the stability and functionality of this enzyme. We have identified two coil residues, SER218 and GLN276, whose interactions with residue 362 in wild-type and mutant pnbCE enzymes control the dynamics of the side-door domain of pnbCE. A hydrogen bond between the GLN276 and ARG362 residues in the arginine substituted (L362R) pnbCE mutant enzyme appears to be responsible for locking the sidedoor domain region of the L362R enzyme, thus lowering the catalytic rates of the L362R mutant pnbCE enzyme compared to the wild-type. Similarly, a hydrogen bond formed between SER218 and ARG362 in L362R provides thermal stability to the arginine substituted mutant enzyme. This hydrogen bond is not as prevalent in the wild-type or other mutated pnbCEs, making them prone to structural fluctuations upon increasing temperature. The predominant lowrequency mode, obtained from normal mode analysis, reveals a collective scissor-like motion of residues surrounding the openings to the active site that validates the results of MD simulations on pnbCE systems. The collective motion of large loops also appear in the lowrequency modes of cutinase and TTHA1554, which correspond to particularly mobile regions in these proteins. An attempt to locate a putative active site of the thermophilic protein TTHA1554 was inconclusive. In general, useful comparisons of the flexibility, stability, and dynamic changes were calculated for the three selected proteins.