Tesis de Magíster en Alimentos, Mención Gestión, Calidad e Inocuidad de los Alimentos / La literatura describe métodos para el análisis posicional sn-2 en triacilgliceroles. El más usado se basa en la ruptura de los ácidos grasos en las posiciones sn-1 y sn-3 utilizando una lipasa pancreática, sin embargo, es un método laborioso y caro. De ahí, nace la necesidad de desarrollar un método más simple para el análisis de rutina. El objetivo de esta tesis es determinar indirectamente los ácidos grasos ubicados en la posición sn-2, con especial énfasis en los ácidos grasos poliinsaturados de cadena larga mediante un método enzimático alternativo utilizando la lipasa Rhizomucor miehei. En el desarrollo de la optimización del método se seleccionó el proceso más adecuado para purificar los triacilgliceroles utilizando una columna cromatográfica de vidrio, donde el óxido de aluminio actuó como compuesto adsorbente y el dietil éter como eluyente. Por otro lado, se optimizaron las condiciones de reacción enzimática con la lipasa del Rhizomucor miehei en manteca de cacao dada su composición de ácidos grasos (25,3; 36,8 y 33,4 g/100g para el ácido palmítico, esteárico y oleico, respectivamente) y su conocida distribución SOS, donde el ácido oleico insaturado ocupa preferentemente la posición sn-2. La optimización se realizó mediante un diseño estadístico donde las variables independientes fueron la temperatura, tiempo de reacción y cantidad de enzima, mientras que la dependiente fue la capacidad de desesterificación, expresada como porcentaje de corte de los ácidos grasos en posición sn-1 y sn-3. Los resultados indicaron que las condiciones óptimas para la actividad de la enzima fueron una temperatura de reacción de 61 °C, tiempo 10 min y la máxima cantidad de enzima que fue de 150 mg. Posteriormente, una muestra patrón de aceite de girasol enriquecida con trieicosapentanoina y tridocosahexanoina y un triacilglicerol estructurado con ácido caprílico y ácidos grasos poliinsaturados de origen marino fueron analizados en las condiciones de reacción óptimas previamente determinadas para la lipasa del Rhizomucor miehei. Los resultados obtenidos indicaron que la lipasa es capaz de cortar ácidos grasos en las posiciones sn-1 y sn-3, sin embargo, la velocidad de desesterificación está altamente influenciada por la composición en ácidos grasos de la matriz lipídica evaluada y por el posible efecto de equilibrio que provocan los productos que se generan en el transcurso de la reacción. En las matrices manteca de cacao y triacilglicerol estructurado fue posible determinar los ácidos grasos presentes en la posición sn-2 en forma indirecta, a partir de la determinación de los ácidos grasos presentes en las posiciones sn-1 y sn-3, mediante un método enzimático menos tóxico, simple y más rápido que el método oficial AOCS Ch 3-91 / The literature describes methods for positional analysis sn-2 triacylglycerols. The most used is based on the breakdown of fatty acids in the sn-1 and sn-3 positions using a pancreatic lipase, according to the official AOCS method Ch 3-91, however, is a laborious and expensive. Hence, it comes the need to develop a simpler method for routine analysis. The aim of this thesis is indirectly determining fatty acids located at the sn-2 position, with special emphasis on the long-chain polyunsaturated fatty acids by alternative enzymatic method using Rhizomucor miehei lipase. In the development of the optimization method the most suitable process to purify selected triacylglycerols using a glass chromatography column, where the aluminum oxide was the adsorbent and the eluent was diethyl ether. Furthermore, the enzyme reaction conditions were optimized with Rhizomucor miehei lipase cocoa butter given its fatty acid composition (25.3, 36.8 and 33.4 g / 100g for palmitic acid, stearic and oleic respectively) and known distribution SOS where unsaturated oleic acid preferably occupies the sn-2 position. The optimization was performed using a statistical design in which the independent variables were temperature, reaction time and amount of enzyme, while the dependent variable was the ability to des-esterification, expressed as a percentage cut of fatty acids in position sn-1 and sn -3. The results indicated that the optimum conditions for enzyme activity were a reaction temperature of 61 °C, time 10 min and the maximum amount of enzyme was 150 mg. Subsequently, a standard sample of sunflower oil and enriched with tri-eicosapentanoic and tri-docosahexanoic and a structured triacylglycerol with caprylic acid and polyunsaturated fatty acids of marine origin were analyzed in the optimal reaction conditions previously determined for the Rhizomucor miehei lipase. The results showed that the lipase is able to cut fatty acids in the sn-1 and sn-3 positions, however, the rate of des-esterification is highly influenced by the fatty acid composition of the lipid matrix and evaluated for the potential effect causing equilibrium products generated during the reaction. In cocoa butter and structured triacylglycerol it was possible to determine the fatty acids present in the sn-2 position indirectly from the determination of the fatty acids in the sn-1 and sn-3 positions, by an enzymatic method less toxic, simple and faster than the AOCS Official Method Ch 3-91 / Fondecyt

Apple pomace is a solid by-product acquired from pressing and crushing millions of tons of apple in juice-industries. It represents 25-30 % of the original fruit and consists of peels, seeds and pulp. This raw material has multiple applications due to its high carbohydrate and moisture content. This bachelor thesis evaluated the use apple pomace acquired from Herrljunga cider for the cultivation of a filamentous fungus to produce biomass and ethanol. Different pretreatment strategies were applied to the apple pomace to extract as much sugars as possible. Several batches were made by mixing pomace and distilled water at different ratios (g pomace per g water) and different water temperatures. Apple juice was produced by filtering soaked pomace using a fine fabric. Apple pomace suspensions were made by adding pomace and water without mixing it (non-homogenised) and homogenised suspensions by mixing with a kitchen blender. Some apple juice batches were pH adjusted to 5.5 to investigate the effect on the fungal growth. The batches were put in Erlenmeyer flasks, sterilised and inoculated with the fungal strain Rhizomucor that has been isolated from Indonesian leaves used for tempe preparation. The Erlenmeyer flasks were incubated in a water shake for 72 h. Samples were taken every 24 h to follow sugar and ethanol concentrations. The samples were analysed by HPLC (High-Performance Liquid Chromatography). The results showed that apple pomace suspension did not perform well compared to the apple juice since the suspension was too viscous and lacked oxygen for the fungus to grow properly in the solution. The apple juice did show a significant improvement compared to the suspension, however pH adjustment to 5.5 had a negative impact on the fungal growth. Cold pre-treatment with an apple pomace to water ratio of 1 g pomace /g water produced the most biomass, with a yield of 9.7 g biomass per kg dry apple pomace. For ethanol production, an apple pomace to water ratio of 1 g pomace /g water using hot water had the highest yield of 11.2 g ethanol per kg dry apple pomace. / Äppelpressmassa är en solid biprodukt, producerad genom att pressa och trycka milliontals ton äpple i bland annat juiceindustrier. Den kvarstående massan motsvarar 25-30 % av äpplet och består utav skal, frön och fruktkött. Denna råvara har många tillämpningar då den har ett högt kolhydrat- och vätskeinnehåll. Detta examensarbete utvärderade användningen av äppelpressmassa från Herrljunga cider för att odla en filamentös svamp i syfte att producera biomassa och etanol. Massan blev utsatt för olika förbehandlingar för att extrahera så mycket socker som möjligt. Olika satser gjordes genom att blanda äppelpressmassa med vatten i olika förhållanden (g äppelpressmassa per g vatten) och olika vattentemperaturer. Äppeljuice producerades genom att filtrera blöt massa med ett fint tyg. Suspensioner gjordes genom att tillsätta vatten till massan och inte blanda det (icke-homogent). En annan variant gjordes genom att blanda äppelpressmassa och vatten med en mixer (homogent). Några utav äppeljuicesatserna pH justerades till 5.5 för att se hur det påverkade svamptillväxten. Satserna flyttades till Erlenmeyer flaskor, steriliserades och ympades med den filamentösa svampen Rhizomucor. Erlenmeyer flaskorna flyttades till ett skakvattenbad för att jäsa i 72 timmar. Prover togs var 24:e timma för att se socker- och etanolkoncentrationerna ändras. Detta analyserades med hjälp utav en HPLC. Resultaten visade att suspensionssatserna inte presterade bra jämfört med äppeljuicesatserna. Detta misstänks bero på att innehållet var väldigt visköst och hade lågt syreinnehåll för svampen att kunna växa. Att justera pH till 5.5 för äppeljuicesatserna visade sig inte vara bra, de presterade sämre jämfört med justerade satser. Förbehandlingen med en äppelpressmassa till vatten förhållandet på 1 g äppelpressmassa/1 g kallt vatten producerade mest biomassa med ett utbyte av 9.7 g torr biomassa per kg torr äppelpressmassa. För etanolproduktion hade förbehandlingsmetoden med en äppelpressmassa till vatten förhållandet på 1 g äppelpressmassa/1 g varmt vatten högst utbyte med 11.2 g etanol per kg torr äppelpressmassa.

Syftet med detta examensarbete är att finna exoenzymer från Rhizopus sp, Mucor indicus och Rhizomucor pusillus som kan användas vid förbehandling av organiskt avfall. Syftet är även att finna kolkällor/energikällor som tidigare inte använts inom forskningen i ämnet resursåtervinning vid Högskolan i Borås.För att kunna undersöka vilka kolkällor mikroorganismerna bryter ner odlas dessa upp på agarplattor innehållande minimal-medium samt en specifik kolkälla. Efter fyra dagars inkubering i 30oC studerar man agarplattorna för att se om mikroorganismerna vuxit eller inte. Kan man urskilja tillväxt har de lyckats bryta ner kolkällan samt producera motsvarande exoenzym. Då vissa resultat är oklara odlas mikroorganismerna även i skakflaskor, detta för att se om det är själva agarn i agarplattorna som påverkar mikroorganismernas tillväxt.Resultatet visar att vissa mikroorganismer växer bättre än andra. Detta kan bero på kolkällornas struktur, det vill säga om de är komplicerade eller ej. Studerar man mikroorganismerna var för sig skiljer de sig lite åt. Rhizopus sp växer bäst på galaktan vilket indikerar att den lyckas producera exoenzymet galaktas. Mikroorganismen saknar produktion av exoenzym när den odlas på kolkällorna cellulosa och kitin.Studerar man mikroorganismen Mucor indicus har den bäst tillväxt på galaktan och potatismjöl, vilket indikerar att den producerar exoenzymerna galaktas samt α-amylas. Den kolkällan som ger sämst tillväxt är cellulosa.Rhizomucor pusillus har bäst tillväxt på galaktan samt triglycerider och producerar då exoenzymerna galaktas och lipas. Den lyckas inte bryta ner cellulosa eller kitin och saknar då produktion av exoenzymen cellulas samt kitinas.Både xylan och galaktan testas var för sig för att kunna dra slutsatser om någon produktion av exoenzymet hemicellulas finns. Detta görs eftersom det inte finns tillgång till något rent ämne med hemicellulosa. Xylan testas även endast för exoenzymet xylanas.En av de kolkällorna som gett minst tillväxt för alla de testade mikroorganismerna var cellulosa. För att styrka detta resultat odlas mikroorganismerna upp i skakflaskor, där ingen tillväxt skedde. Den lilla tillväxt som erhölls på agarplattorna tyder på att mikroorganismerna växer med den tillsatta agarn som kolkälla och inte utnyttjar själva kolkällan. Varför mikroorganismerna inte kan tillgodo se sig cellulosa kan bero på att cellulosa har en komplex struktur som gör den svår att bryta ner utan förbehandling.

The effects of temperature, reaction time, and substrate concentration on the incorporation of decanoic acid (DA) and alpha-linolenic acid (ALA), into cocoa butter, were compared, using an immobilized enzyme derived from Rhizomucor miehei. All variables had an effect on incorporation of DA and ALA into cocoa butter but effects were not equivalent for the two fatty acids. Thus, DA was not an adequate model fatty acid for the incorporation of ALA into cocoa butter. The highest ALA incorporation achieved was 77.3±1.3. Samples with ALA incorporated were prepared as “pure” and “blends”, and these exceeded the milk and dark chocolate Canadian Food and Drug Regulation guidelines for products making omega-3 fatty acid content claims. The highest %TAG content, 97.3±1.0%, was achieved for the 11.9wt% “blend” sample. Differential scanning calorimetry suggested that both “pure” and “blend” samples contained mainly form IV and V, with much smaller quantities of form II polymorphs.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Lipases, triacilglicerol Ãster hidrolases E.C. 3.1.1.3, sÃo enziÂmas que atuam nas ligaÃÃes Ãsteres de triacilglicerÃis, liberando Ãcidos orgÃnicos e glicerol. Podendo, em condiÃÃes microaquosas, catalisar a reaÃÃo reversa. Uma limitaÃÃo da utilizaÃÃo destas enzimas em processos industriais reside na falta de estabilidade operacional e na impossibilidade de sua reutilizaÃÃo na forma livre. O uso do sistema de organo-gÃis consiste em uma alternativa para a imobilizaÃÃo de enzimas, e para sua utilizaÃÃo na catÃlise enzimÃtica em meio orgÃnico. Neste sistema a enzima està localizada no centro micelar (centro aquoso) do organo-gel, eliminando o problemas como de estabilizar a enzima contra inativaÃÃo por um solvente nÃo-aquoso. O objetivo deste trabalho foi desenvolver derivados de lipases de Rhizomucor miehei imobilizadas em organo-gÃis à base de polÃmeros, visando à sÃntese de Ãsteres etÃlicos a partir de reaÃÃes de esterificaÃÃo de matÃrias-primas com elevado teor de Ãcidos graxos livres. Os suportes foram obtidos atravÃs de diferentes combinaÃÃes entre os componentes. Utilizaram-se polÃmeros gelatina (Gel), alginato (Alg) ou quitosana (Qui), fases orgÃnicas hexano (Hex) ou heptano (Hep) e os tensoativos dodecilsulfato de sÃdio (SDS) ou brometo de acetilmetilamÃnio (CTABr). Verificou-se a estabilidade tÃrmica da enzima na sua forma livre, determinando seu tempo de meia-vida. Na primeira etapa, foram produzidos derivados com e sem ativaÃÃo via glutaraldeÃdo 2% (v/v). A atividade enzimÃtica foi avaliada atravÃs hidrÃlise do p-nitrofenilbutirato (pNPB). Os derivados foram caracterizados quanto: fator de estabilidade a 60ÂC em relaÃÃo à enzima livre, eficiÃncia e rendimento de imobilizaÃÃo para assim determinar os melhores biocatalisadores. Dentre os catalisadores obtidos, os melhores apresentaram eficiÃncia de 4,1% e fator de estabilidade 30 vezes (Gel/SDS/Hex), eficiÃncia de 6,0% e fator de estabilidade 1,3 vezes (Alg/SDS/Hep) e eficiÃncia de 1,0% e fator de estabilidade de 2,3 vezes (Qui/SDS/Hep). Os suportes produzidos ativados com glutaraldeÃdo 2% (v/v) apresentaram baixas atividades e eficiÃncias, apesar de obterem valores bons de tempo de meia-vida e fator de estabilidade. Os derivados produzidos com o tensoativo CTABr apresentaram baixas atividades, eficiÃncias, tempo de meia-vida e fator de estabilidade. Na segunda fase, os derivados selecionados foram estudados quanto à carga mÃxima (50 U.g-1 a 500 U.g-1) de imobilizaÃÃo e eficiÃncia, nas temperaturas de 15ÂC e 25ÂC. Avaliou-se a aplicaÃÃo dos biocatalisadores na reaÃÃo de esterificaÃÃo do oleato de etila a partir de Ãcido oleico e etanol, variando a razÃo molar Ãcido/Ãlcool e utilizaÃÃo de agente dessecante (zeÃlitas). Verificou-se a estabilidade de estocagem sob 10ÂC por um perÃodo de 100 dias. Todos os derivados apresentaram melhores eficiÃncias utilizando carga de 50 U.g-1, apresentando valores de 4,2% e 4,8% (Gel/SDS/Hex), 2,0% e 2,3% (Alg/SDS/Hep) e 0,9% e 1,1% (Qui/SDS/Hep ) nas temperaturas de 15ÂC e 25ÂC, respectivamente. Nas reaÃÃes de esterificaÃÃo os derivados Gel/SDS/Hex e Alg/SDS/Hep obtiveram maiores conversÃes na razÃo molar Ãcido/Ãlcool 1:10, 72,9% e 16,9%, respectivamente. O derivado Qui/SDS/Hep obteve 80,0% de conversÃo na razÃo de 1:1. Com utilizaÃÃo de zeÃlitas o derivado Gel/SDS/Hex aumentou a conversÃo para 79,0% nas razÃes 1:1 e 1:5, os derivados Alg/SDS/Hep e Qui/SDS/Hep apresentaram decrÃscimo nas conversÃes. Durante os 100 dias de estocagem sob 10ÂC, os derivados Gel/SDS/Hex e Qui/SDS/Hep mantiveram atividade hidrolÃtica atà 40 dias, tendo um decrÃscimo ao longo do tempo. O derivado Alg/SDS/Hep obteve um tempo maior de 60 dias, apresentando tambÃm um decrÃscimo. / Lipases, triacylglycerol ester hydrolases EC 3.1.1.3, are enzymes that act on ester bonds of triacylglycerols, releasing organic acids and glycerol. May in microaquosas conditions, catalyze the reverse reaction. A limitation of using these enzymes in industrial processes is the lack of operational stability and the inability to re-use the free form. The use of organo-gels system is an alternative for the immobilization of enzymes and to their use in enzyme catalysis in organic media. In this system the enzyme is located in the micelle center (aqueous center) of the organo-gel, eliminating problems such as stabilizing the enzyme against inactivation by a non-aqueous solvent. The aim of this work was immobilize lipases from Rhizomucor miehei into organo - gels based on polymers for future application in ethyl esters synthesis through esterification of raw materials with high free fatty acids content. Supports were obtained using different combinations of components. It was used gelatin polymers (Gel), alginate (Alg) and / or chitosan (Chi), organic phases such as hexane (Hex) and heptane (Hep) and surfactants sodium dodecyl sulfate (SDS) or acetylmetylamonium bromide (CTABr). In the first step, derivatives were produced with and without glutaraldehyde 2% (v/v) activation. Enzymatic activity was measured by hydrolysis of p â nitrophenyl butyrate (PNPb). Biocatalysts were characterized as: stability at 60  C and compared to free enzyme, immobilization efficiency and yield factor, thus determining the best biocatalysts. Among the catalysts obtained, (Gel/SDS/Hex) showed the best efficiency of 4.1% , 30 âfold more stable; (Alg/SDS/Hep) with 6.0% efficiency , 1.3 âfold more stable and (Qui/SDS/Hep) with efficiency of 1.0 % , 1.3 âfold more stable than free lipase. Obtained supports activated with glutaraldehyde 2 % (v/v) showed lower activities and efficiencies, in despite of having good values for stability factor. Produced derivatives using surfactant CTABr presented low activity, efficiency and stability factor. In the second step, derivatives were analyzed as maximum load (50 U.g-1 a 500 U.g-1) enzyme immobilization and efficiency at 15  C and 25  C. It was evaluated biocatalysts application in ethyl oleate achievement in an esterification reaction, using oleic acid and ethanol, by varying molar ratio acid / alcohol with and without using of desiccant agent (zeolite) at 37  C and 24 h of reaction. Derivatives were submitted storage stability under 10  C studies, for a period of 100 days. All derivatives showed higher efficiencies using an initial enzyme loading of 50 U.g -1, with values of 4.2% and 4.8% (Gel/SDS/Hex), 2.0 % and 2.3 % (Alg/SDS/ Hep) and 0.9 % to 1.1% (Qui/SDS/Hep) at 15  C and 25  C, respectively. In esterification reactions, Gel/SDS/Hex and Alg/SDS/Hep derivatives showed higher conversions 72.9 % and 16.9 %, respectively, with molar acid / alcohol 1:10. The chemical derivative Qui/SDS/Hep presented 80.0 % conversion with molar acid / alcohol 1:1 ratio. Using zeolites, Gel/SDS/Hex conversion increased to 79.0 % using ratios of 1:1 and 1:5, the Alg/SDS/Hep and Qui/SDS/Hep presented a decreasing in conversions. During 100 days of storage at 10  C, Gel/SDS/Hex and Qui/SDS/Hep hydrolytic activity maintained up to 40 days and a decreasing during this period, however, Alg/SDS/ Hep achieved more than 60 days with activity.

<p> </p><p>A chemoselective catalyst is preferred in a chemical reaction where protecting groups otherwise are needed. The two lipases <em>Candida antarctica </em>lipase B and <em>Rhizomucor miehei</em> lipase showed large chemoselectivity ratios, defined as (<em>k_cat</em>/<em>K</em>_M)_OH/ (<em>k_cat</em>/<em>K</em>_M)_SH, in a transacylation reaction with ethyl octanoate as acyl donor and hexanol or hexanethiol as acyl acceptor (<strong>paper I</strong>). The chemoselectivity ratio of the uncatalyzed reaction was 120 in favour of the alcohol. Compared to the uncatalyzed reaction, the chemoselectivity was 730 times higher for <em>Candida antarctica </em>lipase B and ten times higher for <em>Rhizomucor miehei</em> lipase. The <em>K</em>_M towards the thiol was more than two orders of magnitude higher than the <em>K</em>_M towards the corresponding alcohol. This was the dominating contribution to the high chemoselectivity displayed by the two lipases. In a novel approach, <em>Candida antarctica </em>lipase B was used as catalyst for enzymatic synthesis of thiol-functionalized polyesters in a one-pot reaction without using protecting groups (<strong>paper II</strong>). Poly(e-caprolactone) with a free thiol at one of the ends was synthesized in an enzymatic ring-opening polymerization initiated with mercaptoethanol or terminated with either 3-mercaptopropionic acid or g-thiobutyrolactone.</p><p> </p>

Many industrially important fungi can grow in the form of small spherical pellets.Such pellets reduce the viscosity and enhances mass transfer rates in culture broths.The pelleted morphology also influences the fungus’s metabolism, directing it tospecific metabolites. The pellets are easily harvested from the broth and recycled.These properties makes pelleted morphology very attractive.The pelleted morphology of four Zygomycetes strains was studies. Several differentnutrient media used by other researchers for achieving pelleted growth was tested.The influence of eight factors on pelletization of Rhizopus sp. in a completely definedmedium was determined using a fractional central composite design and logistic regression.Pelleted growth of all four Zygomycetes was achieved, with very good results for twoRhizomucor sp. strains. A simple medium containing calcium carbonate was foundto induce pelletization with very high reproducibility.Immobilization of yeast cells was attempted in pellets of Rhizomucor. It was foundthat a flocculating yeast can be immobilized inside pellets of fungal mycelium, forming”biocapsules”. This is accomplished by first using a medium that induces pelletizationof the filamentous fungus and does not allow for growth of the yeast. Thepellets are then inoculated into a second medium that induces growth and flocculationof the yeast and inhibits further growth of the filamentous fungus.Non-flocculating yeasts could not be immobilized, suggesting that the flocculin proteinsin the cell wall of flocculating strains are important for proper immobilization.The flocculation and immobilization arises due to expression of several differentFLO-genes and the importance of these genes for successful immobilization isdiscussed.The results demonstrate that the morphology of Zygomycetes can be controlled andthat this may be useful in industrial fermentation. The new immobilization techniquereveals the great importance of flocculation and cell surface hydrophobicity. Usingyeast strains that express certain FLO-genes may be beneficial in fermentation oflignocellulosic hydrolysates.Microscopy techniques were developed that allows for high quality microphotographyof pellets and thin cross-sections of pellets and biocapsules. / Program: MSc in Resource Recovery - Industrial Biotechnology

Submitted by RONIVALDO RODRIGUES DA SILVA (rds.roni@yahoo.com.br) on 2016-03-01T13:46:53Z No. of bitstreams: 1 Tese Doutorado RONIVALDO R. SILVA.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-01T18:27:48Z (GMT) No. of bitstreams: 1 silva_rr_dr_sjrp.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) / Made available in DSpace on 2016-03-01T18:27:48Z (GMT). No. of bitstreams: 1 silva_rr_dr_sjrp.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) Previous issue date: 2016-02-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Aspártico (E.C. 3.4.23), cisteíno (E.C. 3.4.22) e serino peptidases (E.C. 3.4.21) são endopeptidases, cujos modos de ação são dependentes de resíduos de ácido aspártico, cisteína e serina presentes no sítio catalítico, respectivamente. Atualmente, vários estudos são realizados na busca por novas enzimas com relevantes propriedades bioquímicas para aplicação industrial. Neste contexto, nós propomos a produção de enzimas em bioprocesso submerso, purificação, estudo das propriedades bioquímicas e determinação da especificidade catalítica das peptidases secretadas pelos fungos filamentosos Rhizomucor miehei, Phanerochaete chrysosporium e Leptosphaeria sp. Inicialmente, após produção por bioprocesso submerso, estas enzimas foram purificadas utilizando cromatografias de exclusão molecular e troca iônica. Em ensaios de inibidores na atividade enzimática, notamos inibição das peptidases por pepstatina A (R. miehei), ácido iodoacético/N-Etilmaleimida (P. chrysosporium) e fluoreto de fenil metil sulfonila (Leptosphaeria sp), sendo então definidas como aspártico, cisteíno e serino peptidases, respectivamente. Por SDS-PAGE (12%), as massas moleculares foram estimadas em 37 kDa (aspártico), 23 kDa (cisteíno) e 35 kDa (serino). O máximo de atividade proteolítica foi alcançado em pH 5,5 e 55 ºC para peptidase aspártica secretada por R. miehei; pH 7 e faixa de temperatura 45-55 ºC para cisteíno peptidase secretada por P. chrysosporium, e pH 7 e 45 ºC para serino peptidase secretada por Leptosphaeria sp. Sob efeito de incubação a diferentes pH, a peptidase aspártica mostrou-se estável em condições ácidas (pH 3-5); cisteíno peptidase foi estável em ampla faixa de pH (pH 4-9), e serino peptidase mostrou-se mais estável em condições com tendências alcalinas e pH ligeiramente ácido (pH 5-9). Em todas estas faixas de pH citadas, as peptidases apresentaram atividade proteolítica acima de 80% por 1 hora de incubação. Quanto à estabilidade térmica, a cisteíno peptidase mostrou-se mais termoestável dentre as três enzimas e serino peptidase descreveu a menor tolerância à temperatura. Em incubação com agentes desnaturantes, observamos redução na atividade proteolítica sob efeito de surfactantes iônicos (0,02-1%): dodecil sulfato de sódio (SDS) e brometo de cetil-trimetil amônio (CTAB); íon cobre II (5 mM); Ditiotreitol (DTT) e guanidina (ambos na faixa de 10-200 mM) para todas as peptidases. Por último, em estudo de especificidade catalítica destas enzimas, observamos a preferência por aminoácidos aromáticos (F e W), básicos (K e R) e apolares (em particular, resíduo de metionina) para peptidase aspártica. Alta especificidade descrita por cisteíno peptidase, cuja preferência catalítica é notória por aminoácidos básicos (K, H e R), especialmente na posição P3 e lisina-dependência para catálise na posição P'3. Em serino peptidase, notamos maior aceitação por aminoácidos apolares (G, I, L, M e V), básicos (H e R) e polares neutros (N e Q) para as diferentes posições avaliadas no substrato. / Aspartic (EC 3.4.23), cysteine (EC 3.4.22) and serine peptidases (EC 3.4.21) are endopeptidases whose modes of action are dependent on aspartic acid, cysteine and serine residues present in the catalytic site, respectively. Currently, several studies are conducted in the search for new enzymes with relevant biochemical properties for industrial application. In this context, we propose the production of enzymes in submerged bioprocess, purification, the study of biochemical properties and determining the catalytic specificity peptidases secreted by the filamentous fungus Rhizomucor miehei, Phanerochaete chrysosporium and Leptosphaeria sp. Initially, after production submerged bioprocess, these enzymes have been purified using size-exclusion and ion exchange chromatographies. In the effect of inhibitors on enzyme activity, we note peptidase inhibition by pepstatin A (R. miehei), iodoacetic acid/ N-Ethylmaleimide (P. chrysosporium) and phenyl methyl sulfonyl fluoride (Leptosphaeria sp), suggesting that these enzymes are aspartic, cysteine and serine peptidases, respectively. For SDS-PAGE (12%), molecular weights were estimated at 37 kDa (aspartic), 23 kDa (cysteine) and 35 kDa (serine). Maximum proteolytic activity was achieved at pH 5.5 and 55 °C for aspartic peptidase secreted by R. miehei; pH 7 and temperature range 45-55 °C for cysteine peptidase secreted by P. chrysosporium and pH 7 and 45 °C for serine peptidase secreted by Leptosphaeria sp. Under incubation at different pH effect, aspartic peptidase was stable under acidic conditions (pH 3-5); cysteine peptidase was stable in wide pH range (pH 4-9), and serine peptidase was more stable under alkaline conditions and pH slightly acidic (pH 5-9). In all these pH ranges mentioned, peptidases showed proteolytic activity above 80% by 1 hour incubation. As regards the thermal stability, cysteine peptidase was more thermostable enzyme and serine peptidase described the lowest temperature tolerance. In incubation with denaturing agents, we observed a decrease in proteolytic activity under the effect of ionic surfactant (0.02-1%) sodium dodecyl sulfate (SDS) bromide and cetyl-trimethyl ammonium bromide (CTAB); copper (II) ion (5 mM); Dithiothreitol (DTT) and guanidine (both in the range of 10-200 mM) for all peptidases. Finally, the study of catalytic specificity of these enzymes, we found a preference for aromatic amino acids (F and W), basic (K and R) and nonpolar (in particular, methionine residue) to aspartic peptidase. High specificity described by cysteine peptidase, which a catalytic preference is notorious for basic amino acids (K, R and H), especially in position P3 and lysine-dependence for catalysis at position P'3. In serine peptidase, for different evaluated positions, we noticed greater acceptance by nonpolar amino acids (G, I, L, M and V), basic (M and R) and neutral polar (N and Q).

A chemoselective catalyst is preferred in a chemical reaction where protecting groups otherwise are needed. The two lipases Candida antarctica lipase B and Rhizomucor miehei lipase showed large chemoselectivity ratios, defined as (kcat/KM)OH / (kcat/KM)SH, in a transacylation reaction with ethyl octanoate as acyl donor and hexanol or hexanethiol as acyl acceptor (paper I). The chemoselectivity ratio of the uncatalyzed reaction was 120 in favour of the alcohol. Compared to the uncatalyzed reaction, the chemoselectivity was 730 times higher for Candida antarctica lipase B and ten times higher for Rhizomucor miehei lipase. The KM towards the thiol was more than two orders of magnitude higher than the KM towards the corresponding alcohol. This was the dominating contribution to the high chemoselectivity displayed by the two lipases. In a novel approach, Candida antarctica lipase B was used as catalyst for enzymatic synthesis of thiol-functionalized polyesters in a one-pot reaction without using protecting groups (paper II). Poly(e-caprolactone) with a free thiol at one of the ends was synthesized in an enzymatic ring-opening polymerization initiated with mercaptoethanol or terminated with either 3-mercaptopropionic acid or g-thiobutyrolactone.