Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 95-109).

Orientador: Elias Basile c / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-09T16:09:35Z (GMT). No. of bitstreams: 1 Godoi_PatriciaHelenade_M.pdf: 865544 bytes, checksum: 0a09a500bcd26f04fe7be64851f3522a (MD5) Previous issue date: 2007 / Resumo: Bromelina é uma enzima de origem vegetal obtida de diversas espécies da família Bromeliaceae, presente na casca, no talo e no fruto do abacaxi. O Brasil encontra-se entre um dos maiores produtores mundiais de abacaxi, ocupando o terceiro lugar no ranking mundial. O desenvolvimento de novas técnicas de extração e purificação da bromelina vem sendo bem explorado, entretanto, tornouse necessário um estudo de estabilidade desta protease, proporção pela qual a bromelina conserva sua conformação estrutural ou sua atividade quando sujeita à estocagem, isolamento e purificação ou várias outras manipulações físicas ou químicas, incluindo autodigestão, outras enzimas proteolíticas e aquecimento. Tornou-se de fundamental importância saber em que condições a bromelina se mantém estável, ou seja, ativa e por qual período de tempo, já que a meta mais significante da enzimologia aplicada é obter compostos úteis para biocatálise. Este trabalho apresenta um estudo das condições de pH e temperatura nas quais a Bromelina P.A em solução aquosa em concentração próxima ao do suco extraído da polpa da fruta, mantém-se ativa, ou seja, não desnaturada. A atividade enzimática da bromelina em solução foi medida através da hidrólise da caseína e a condição de pH e temperatura mais próxima da ideal foi determinada / Abstract: Bromelain is a vegetable enzyme found in many species of Bromeliaceae family, its present in pineapple skins, stem and fruit. Brazil is one of the world¿s largest producers of pineapples, its production being the third one in the world. The development of new extraction and purification processes of bromelain have been studied, however, its necessary a enzyme stabilization investigation, state that bromelain remains its structure or biological activity when stored, isolated, purified or any other manipulation, included autodigestion, proteolytic enzymes and heating. It became very important to know the conditions and the time which bromelain remain stabilized, active. In applied enzymology, the most significant goal is to achieve useful compounds by biocatalysis. This work presents a study about pH and temperature conditions which a bromelain aqueous solution, in the same concentration of a pineapple fruit extract, remains with biological activity. The bromelain aqueous solution enzyme activity was tested across the casein hydrolysis and the ideal pH and temperature was determinated. / Mestrado / Sistemas de Processos Quimicos e Informatica / Mestre em Engenharia Química

Orientadores: Elias Basic Tambourgi, Priscila Gava Mazzola / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-21T20:42:46Z (GMT). No. of bitstreams: 1 Bresolin_IaraRochaAntunesPereira_D.pdf: 1407939 bytes, checksum: 1259333d5753554819357dfada6831b4 (MD5) Previous issue date: 2013 / Resumo: A bromelina é uma enzima proteolítica encontrada em tecidos vegetais como casca, talo, fruta e folhas de espécies da família Bromeliaceae, incluindo o abacaxi (Ananas comosus). É uma enzima conhecida por seus efeitos terapêuticos, sendo usadas em tratamentos de vários problemas de saúde, como desordens digestivas, feridas, queimaduras e inflamações. Este trabalho teve como objetivo a purificação da enzima bromelina de casca de abacaxi e sua posterior aplicação em bases dermatológicas. Foi demonstrado que é possível purificar bromelina extraída de casca de abacaxi, um resíduo da indústria alimentícia, através de um processo de recuperação e purificação, incluindo precipitação por sulfato de amônio (40- 80%), seguido de dessalinização e liofilização, com 75% de recuperação da atividade. Cromatografia de troca iônica com dietilaminoetil-agarose (DEAE-Sepharose) separou polissacarídeos da enzima e esta foi recuperada na etapa de eluição, mantendo sua atividade enzimática. A enzima foi incorporada em creme e loção Lanette, gel de Carbopol e creme e loção da Chemyunion na concentração de 5 mg de enzima por mL de base dermatológica. Essas bases foram submetidas ao teste de centrifugação e teste de estabilidade acelerada durante 90 dias a 25ºC (com e sem a incidência de luz solar), 37ºC e 4°C, a fim de avaliar a estabilidade da bromelina nas bases dermatológicas. As formulações permaneceram estáveis quanto a suas características organolépticas (aspecto, cor, odor, sensibilidade ao tato) apenas quando mantidas a 4°C, com atividade remanescente de 84,9%, 73,8%, 95,5%, 77,7% e 72,3% após 90 dias de teste em creme e loção Lanette, gel de Carbopol e creme e loção da Chemyunion, respectivamente. Baseando-se nestes resultados, foi possível purificar bromelina de casca de abacaxi e incorporá-la em bases dermatológicas mantendo se sua atividade mais estável quando estas bases foram mantidas em geladeira a 4ºC / Abstract: Bromelain is a proteolytic enzyme found in vegetable tissues like peel, stem, fruit and leaves of the Bromeliaceae family including pineapple (Ananas comosus). Bromelain is known for its therapeutic effects, being useful in the treatment of several health problems such as digestive disorders, burns and inflammation. This work aimed the purification of the enzyme bromelain from pineapple peel for potential therapeutic application in dermatological bases. It was shown that it is possible to purify bromelain extracted from pineapple peel, a waste in food industry, in a downstream processing, including ammonium sulfate precipitation (40-80%), followed by desalting and freeze-drying with a 75% activity recovery. Ion exchange chromatography on diethylaminoethyl-agarose (DEAE-Sepharose) was able to separate polysaccharides from the enzyme, which was recovered in the elution step, maintaining its enzymatic activity. The enzyme was then incorporated in Lanette cream and lotion, Carbopol gel and Chemyunion cream and lotion at a concentration of 5 mg enzyme per mL of dermatological bases. These bases were subjected to centrifugation test and accelerated stability test during 90 days at 25°C (with and without sunlight), 37°C and 4°C, in order to evaluate bromelain stability in a dermatologic formulation. The formulations were stable as its organoleptic characteristics (appearance, color, smell and sensitivity to touch) only when kept at 4ºC with activity remaining 84.9%, 73.8%, 95.5%, 77.7 % and 72.3% after 90 days of testing in Lanette cream and lotion, Carbopol gel and Chemyunion cream and lotion, respectively. Based on the results, it was possible to purify bromelain from pineapple peel and to incorporate it in dermatological bases maintaining the activity stabler when these bases were kept in refrigerator at 4ºC / Doutorado / Sistemas de Processos Quimicos e Informatica / Doutora em Engenharia Quimica

Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.

Biofilms are characterized by surface attachment, structural heterogeneity; genetic diversity; complex community interactions and an extracellular matrix of polymeric substances (EPS). Biofilms deposit and adhere to all surfaces that are immersed in aqueous environments. EPS serves many functions including: facilitation of the initial attachment of bacterial cells to a surface; formation and maintenance of the micro colony; enables the bacteria to capture nutrients; causes biofouling; cell-cell communication and enhances bacterial resistance antimicrobial agents. EPS also function as a stabilizer of the biofilm structure and as a barrier against hostile environments. Extracelullar polymeric substances are composed of a wide variety of materials including polysaccharides, proteins, nucleic acid, uronic acid, DNA, lipid and even humid substances. EPS can be hydrophilic or hydrophobic depending on the structural components making up such EPS and the environmental conditions were the biofilms are developing. The exopolysachharides (EPS) synthesized by microbial cells vary greatly in their composition and in their chemical and physical properties within the bacterial strains. Due to variety in the structural components of the bacterial EPS, removal of biofilms by compounds that have no effects on the biofilm EPS would be difficult. Enzymes are proven to be effective in degrading biofilm EPS. The manner in which enzymes degrade the biofilm EPS is through binding and hydrolysis of the EPS components (proteins and carbohydrates) molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. The objectives of this study were to grow Pseudomonas fluorescens and mixed bacterial species biofilms in nutrient rich and nutrient limited medium conditions; to determine the EPS, protein and carbohydrate concentrations of the biofilm grown in rich and in limited nutrient conditions and to test the efficiency of protease and amylase enzymes for the degradation of the EPS and biofilm removal. In the results, there was a slight difference in the number of viable cells grown in biofilms that were fed than the cells of the unfed biofilms. As a result, the EPS, protein and carbohydrate concentrations were higher in the fed biofilms than the unfed biofilms. There are contradictory reports about the composition of EPS especially with the ratio of carbohydrate to protein. Some of these reports indicate that certain biofilms EPS have bigger proportion of proteins and some found polysaccharides to be the dominant composition of the EPS of the biofilms. Nonetheless, the quantity and the composition of the EPS produced by bacterial biofilms depend on a number of factors such as microbial species, growth phase and the type of limiting substrate. Enzymes were tested individually and in combination for the degradation of biofilm EPS. For efficient removal of biofilm, it is important that the structural components of the biofilm EPS should be known before application of the relevant enzymes. In this study, the test enzymes were effective for the degradation of the biofilm EPS except for the protease Polarzyme which had no activity. The reason for the inefficiency of Polarzyme may be due to its incompatibility with the specific protein structural components of the biofilm EPS tested in this study. The manner in which the enzymes degrade the biofilm EPS is through binding and hydrolysis of the protein and carbohydrate molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. In addition, the mode of enzymatic action will depend on the specific EPS components and this in turn will determine its efficacy. The protease enzymes tested individually and in combination were most effective for EPS degradation. The efficiency of the proteases may be due to their broad spectrum activity in degrading a variety of proteins acting partly as the multi structural components of Pseudomonas fluorescens and mixed bacterial species biofilm EPS. On the other hand, amylase enzymes tested individually and in combination was less effective for the EPS degradation. The structures of polysaccharides synthesized by microbial cells vary. Microbial exopolysaccharides are comprised of either homopolysachharides or heteoropolysaccharides. A number of lactic acid bacteria produce heteropolysaccharides and these molecules form from repeating units of monosaccharides including D- glucose, D- galactose, L- fructose, L- rhamnose, D- glucuronic acid, L- guluronic acid and D- mannuronic acid. The type of both linkages between monosaccharides units and the branching of the chain determines the physical properties of the microbial heteropolysaccharides. Due to a wide range of linkages and the complexity of polysaccharides structures, it would therefore be difficult for the amylases to break down the bond linkages and the monomers making up polysaccharides which determine the physical and chemical structure of the EPS. It was therefore not surprising that the amylase enzymes tested for the degradation of Pseudomonas fluorescens and mixed bacterial species biofilms, were less effective than the proteases. Hence, when the amylase enzymes were tested in combination with the protease enzymes, efficiency improved. It was therefore concluded that the protease enzymes were the primary remedial compounds and the amylase enzymes were the secondary remedial compounds. Conclusion If a compound or compounds capable of destroying all the structural components of different EPS that are produced by different biofilms growing under different conditions is found then the “city of microbes” (biofilms) would be destroyed permanently. If only an enzyme or enzymatic mixture capable of shutting down or deactivating the quorum sensing systems of different biofilm EPS could be found, then there would not be any formation of biofilms. In this study, protease enzymes tested individually and in combination were the most effective in the degradation of biofilm EPS than the amylase enzymes resulting in the reduction of large population of the biofilm cells attached on the substratum. Recommendation Amylase enzymes tested individually and in combination were less efficient for the degradation of the biofilm EPS and biofilm removal. This may be due to the complex structure of the exopolysaccharides synthesized by different biofilms. Also, the bond linkages between monosaccharides units and the branching of the chain complex the structures and as a result confer in the physical properties of the microbial biofilms. Hence, when the amylase enzymes were tested in combination with the protease enzymes, activity improved. For efficient degradation of biofilm EPS, it is therefore recommended that, protease and amylase enzymes should be tested in combination. In addition, the structure of the biofilm EPS should be investigated so that relevant enzymatic mixtures are tested for biofilm removal. / Thesis (PhD)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2020 / In nature, saccharification is done by a variety of microorganisms, secreting a variety of cellulase in addition to other proteins. Co-culturing enables the production of more efficient enzyme preparations that would mimic the natural decomposition of lignocelluloses. During the decay of banana (Musa paradisiaca) pseudostem, a potential feedstock for second-generation biofuels, there may be a number of microorganisms producing cellulolytic enzymes, and other factors, which in combination might decompose the lignocelluloses more efficiently. The aim of the study was to establish a microbial co-culture for the production of highly active cellulase preparations. Banana pseudostems (BPS) and microbial samples from decaying banana pseudostems were collected in the Mopani District Allesbeste Nursery, Limpopo Province, South Africa. Fungi and bacteria were isolated using CMC agar plates. The best cellulase producing fungi and bacteria were tested for cellulase activity in monocultures and in various combinations (fungi-fungi, fungi-bacteria, bacteria-bacteria, fungi-live bacterial cells and fungi-dead bacterial cells) in submerged fermentation, using Avicel™ as a carbon source. Solid-state fermentation was also performed using banana pseudostem as a carbon source. Zymography was done in studying the variety of cellulase in the secretions from co-cultures/ mixed cultures. Identification of the bacterial and fungal isolates from decomposing banana pseudostems was also done using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) or DNA sequencing. A mixed culture of fungi in combination with dead bacterial cells was the best combination to produce higher levels of endoglucosidase and β-glucosidase activities in both submerged fermentation and solid-state fermentation. During SmF, endoglucosidase was (0.229 after 144 h) and β-glucosidase (4.519 after 96 h) activities and SSF, endoglucosidase (12.793 after 48 h) and β-glucosidase (37.45 after 144 h). Endoglucosidase zymography showed that monocultures and co-cultures produced four active bands for endoglucanase, except for the monoculture Trichoderma longibrachiatum 1B that produced a faint or unclear band. The current study demonstrated that three fungal strains namely, T longibrachiatum 1B, Aspergillus fumigatus 5A, and Aspergillus flavus 2A and one bacterial strain Enterobacter asburiae 1 are capable of producing a variety of endoglucanases. It seems that a combination of fungi with dead cells could significantly improve endoglucosidase and v β-glucosidase activities. The use of A. fumigatus in mixed cultures is highly recommended in order to produce high levels of β-glucosidases, no matter the combination used. / Foodbev Seta

LD2668 .T4 1947 G3 / Master of Science

Call number: LD2668 .T4 1962 H53

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The production, purification and functional characterisation of feruloyl esterase from Aureobasidium pullulans were set as the primary objectives of this study. A further objective was to investigate a possible co-operative effect with other selected lignocellulolytic enzymes on substrates relevant to industry. In a comprehensive review, feruloyl esterases from various micro-organisms were compared both functionally and with regard to their primary structure, where applicable. Feruloyl esterases show intriguing differences in substrate specificity and sequence structure. Enzymes that are closely related regarding their amino acid sequence exhibit different substrate specificities. Sequence similarities can be found with a range of other enzyme families, including serine esterases, acetyl xylan esterases, lipases, tannases, glycosyl hydrolases and xylanases. More data on the three dimensional structure of feruloyl esterases as well as an examination of all available feruloyl esterases with the same substrates is necessary before structure-function relationships can be established and before the feruloyl esterases can be organized into discrete families based on ancestral origins. The highest production levels of feruloyl esterase by A. pullulans are achieved when grown on birchwood xylan. Expression was not repressed when glucose or xylose was present in the medium. However, free ferulic acid supplemented to the medium affected fungal growth and therefore did not increase feruloyl esterase activity. It is also suggested that the synthesis of feruloyl esterase is independently regulated from xylanase synthesis. Feruloyl esterase from A. pullulans acts on a- and l3-naphthyl acetate, as well as naphthol AS-D chloroacetate as substrates. Feruloyl esterase from A. pullulans was purified to homogeneity using ultrafiltration with high molecular weight cut-off, anion exchange, hydrophobic interaction and ultimately gel filtration chromatography. With a molecular weight of 210 kDa, the enzyme is the largest of the feruloyl esterases reported to date. Kinetic data was produced using both synthetic and natural substrates. A. pullulans feruloyl esterase shows properties similar to other fungal feruloyl esterases, especially from Aspergillus niger cinnamic acid esterase and Penicillium funiculosum feruloyl esterase B. The N-terminal sequence of A. pullulans feruloyl esterase was identified, but no similarities to known enzyme families were found. Peptide mass mapping did not reveal structural information. In an effort to evaluate the significance of feruloyl esterase from A. pullulans in the degradation of lignocellulose, dissolving pulp and sugar cane bagasse were selectively treated using feruloyl esterase and hemicellulolytic enzymes. The enzymatic degradation reaction was monitored using microdialysis sampling, anion exchange chromatography, online desalting and mass spectrometry. It has been shown, that feruloyl esterase activity together with xylanase activity releases monosaccharides from both substrates. Sugars of higher degree of polymerisation were not released, giving evidence for the recalcitrance of the material. The fibre architecture of the substrates was apparently not accessible to the enzymes and therefore complete hydrolysis was hindered. / AFRIKAANSE OPSOMMING: Die produksie, suiwering en funksionele karakterisering van feruloïel esterase afkomstig van Aureobasidium pullulans was die primêre doelwitte van hierdie studie. 'n Verdere doelwit was om vas te stelof daar 'n kooperatiewe effek met ander geselekteerde lignosellulitiese ensieme op substrate wat industrierelevant is, bestaan. Die feruloïel esterase van verskillende mikro-organismes is vanuit die oogpunt van funksie en primêre struktuur omvattend met mekaar vergelyk, waar toepaslik. Interessante verskille tussen die substraat spesifisiteit en volgordestruktuur van feruloïel esterase kan waargeneem word. Ensieme wat nou aanmekaar verwant is wat hul aminosuurvolgorde betref, het duidelik verskillende substraatspesifiteite. Volgordeverwantskap kan in 'n reeks van ander ensiemfamilies, insluitende serienesterase, asetielxilaanesterase, lipases, tannases, glikosielhidrolases en xilanases vasgestel word. Meer inligting oor die driedimensionele struktuur van feruloïel esterase asook 'n analise van al die beskikbare feruloïel esterase met dieselfde substrate is nodig voordat struktuur-funksie verwantskappe vasgestel kan word en voordat die feruloïel esterases in eie families op die grond van huloorsprong georganiseer kan word. Die hoogste produksie vlakke deur feruloïel esterase van A. pullulans word bekom deur dit op berkhoutxilaan te groei. Ekspressie was nie onderdruk wanneer glukose of xilose in die medium aanwesig was nie. Wanneer vrye feruliensuur by die medium bygevoeg is, is die fungale groei beïnloed en het die feruloïel esterase aktiwiteit nie vermeerder nie. Dit word ook voorgestel dat die sintese van feruloïel esterase onafhanklik deur xilanase sintese gereguleer word. Feruloïel esterase van A. pullulans reageer op a- en f3- naftolasetaat, asook naftol AS-D chloroasetaat as substrate. Feruloïel esterase van A. pullulans is tot homogeniteit deur ultrafiltrering met .n hoë molekulêre gewiggrens, anioonuitruiling, hidrofobiese interaksie en eindelik gelfiltrasie-chromatografie gesuiwer. Met 'n molekulêre gewig van 210 kDa, is die ensiem die grootste van die feruloïel esterases tot dusver beskryf. Kinetiese data is met behulp van sintetiese en natuurlike substrate geproduseer. A. pullulans feruloïel esterase het eienskappe wat vergelykbaar is aan die van ander fungal feruloïel esterases, veral die wat afkomstig is van Aspergillus niger sinnamiensuur esterase en Penicillium funiculosum feruloïel esterase B. Die N-terminale volgorde van A. pullulans feruloïel esterase is identifiseer maar geen ooreenkoms aan bekende ensiemfamilies kon vasgestel word nie. Peptiedmassakaartering kon ook geen strukturele inligting gee nie. Oplosbare pulp en suikerrietbagasse is geselekteerd met behulp van feruloïel esterase en lignosellulitiese ensieme behandel om die belang van feruloïel esterase van A. pullulans in die afbraak van lignosellulose vas te stel. Die hidroliese-reaksie is deur mikrodialise monsterneming, anioonuitruilingschromatografie, oplyn ontsouting en massaspektrometrie gemonitor. Wanneer die aktiwiteit van feruloïel esterase met die van xilanase gekombineer is, is monosakkariede deur albei substrate afgeskei. Suikers met 'n hoër graad van polimerisering is nie afgeskei nie, wat 'n bewys van die materiaal se weerstandbiedendheid is. Dit het geblyk asof die vesel-argitektuur van die verbruikte substraat nie toeganklik was vir ensieme nie en dus is algehele hidroliese verhinder.

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy