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CHAPTER ONE reviews the literature, discussing aspects of transition metal mediated asymmetric catalysis in the presence of enantiomerically pure ligands. CHAPTER TWO discusses the asymmetric addition of dialkyl-zinc reagents to aromatic aldehydes. The work presented is particularly concerned with the design and construction of enantiomerically pure oxazoline ligands tethered to alcohols These ligands have proved effective in the acceleration of the alkylation reaction and are able to influence good levels of asymmetric induction in the resultant secondary alcohol products CHAPTER THREE examines the electronic (and steric) effects of enantiomerically pure oxazoline ligands for the palladium catalysed allylic substitution reaction. Using ligands possessing two electronically different donor atoms, it is possible to create electronic distortion upon the intermediate allyl complex. In doing so it is possible to direct nucleophilic addition to one carbon centre preferentially to the other, resulting in asymmetric induction. Manipulation of these ligands enables control in the extent of electron distortion inflicted upon the allyl complex and consequently influences the levels of enantioselectivity observed. CHAPTER FOUR investigates the ability of hydrolytic enzymes to kinetically resolve a series of allylic acetates, under varying conditions. Lipases appeared superior to esterases for the substrates employed. In particular cis-3-acetoxy-5-carbomethoxycyclohexene was smoothly resolved m high yield and enantioselectivity. CHAPTER FIVE reports on the potentiality of a dynamic resolution of allylic acetates, using hydrolytic enzymes in the presence of a palladium catalyst. A proposed mechanism is discussed. Initial results are promising, however, the sensitivity of the reaction is realised and optimisation of conditions still needs to be addressed.

O processamento de celulose em moinho de bolas de porcelana produz um pó no qual se detecta a presença de radicais livres, por Ressonância Paramagnética Eletrônica. O espectro obtido e complexo e pode ser decomposto em pelo menos dois sinais: uma linha instável, eliminada apôs alguns dias de exposição ao ar, e outra estável, que decai lentamente por aquecimento a 400-500ºC, e desaparece a 900ºC. A moagem simultânea de celulose com amido, acrilamida, azul de dextrana e sacarose produz celulose quimicamente modificada. Por exemplo, no caso da acrilamida, dosagem pelo método kjeldahl revela a presença de ate 0,65% de nitrogênio no material obtido apôs lavagens exaustivas. A celulose moída na presença de vários reagentes foi submetida a ensaios de hidrólise e em alguns casos, mostrou-se significativamente mais suscetível ao ataque enzimático do que a celulose simplesmente moída. Os resultados deste trabalho mostram que a conversão enzimática de celulose em açucares redutores e sensível a uma pequena modificação no pré-tratamento mecanoquímico. / Cellulose processing on porcelain ball mill produce a powder in which one can detect free radicals by means of Electronic Paramagnetic Ressonance. The spectrum attained is complex and it may be decomposed in at least two signals: an unstable line that is eliminated after some days of exposition tc air and another stable line, which slowly decays due to heating at 400-500ºC and disapears at 900ºC. Cellulose simultaneously milled with starch, acrylamide, dextran blue and saccharose produce chemically modified cellulose. For instance, when using acrylamide one can detect, by means of the Kjeldhal method, the existence of nitrogen up to 0.65% in the material obtained after exhaustive washings. In some cases cellulose milled with several reactants seemed to be significantly more susceptible to the enzymatic attack during hydrolysis than cellulose milled without any reactant. The results of this work show that the enzymatic conversion of cellulose into reducible sugars is sensible to a little change in the mechano-chemical pre-treatment.

O processamento de celulose em moinho de bolas de porcelana produz um pó no qual se detecta a presença de radicais livres, por Ressonância Paramagnética Eletrônica. O espectro obtido e complexo e pode ser decomposto em pelo menos dois sinais: uma linha instável, eliminada apôs alguns dias de exposição ao ar, e outra estável, que decai lentamente por aquecimento a 400-500ºC, e desaparece a 900ºC. A moagem simultânea de celulose com amido, acrilamida, azul de dextrana e sacarose produz celulose quimicamente modificada. Por exemplo, no caso da acrilamida, dosagem pelo método kjeldahl revela a presença de ate 0,65% de nitrogênio no material obtido apôs lavagens exaustivas. A celulose moída na presença de vários reagentes foi submetida a ensaios de hidrólise e em alguns casos, mostrou-se significativamente mais suscetível ao ataque enzimático do que a celulose simplesmente moída. Os resultados deste trabalho mostram que a conversão enzimática de celulose em açucares redutores e sensível a uma pequena modificação no pré-tratamento mecanoquímico. / Cellulose processing on porcelain ball mill produce a powder in which one can detect free radicals by means of Electronic Paramagnetic Ressonance. The spectrum attained is complex and it may be decomposed in at least two signals: an unstable line that is eliminated after some days of exposition tc air and another stable line, which slowly decays due to heating at 400-500ºC and disapears at 900ºC. Cellulose simultaneously milled with starch, acrylamide, dextran blue and saccharose produce chemically modified cellulose. For instance, when using acrylamide one can detect, by means of the Kjeldhal method, the existence of nitrogen up to 0.65% in the material obtained after exhaustive washings. In some cases cellulose milled with several reactants seemed to be significantly more susceptible to the enzymatic attack during hydrolysis than cellulose milled without any reactant. The results of this work show that the enzymatic conversion of cellulose into reducible sugars is sensible to a little change in the mechano-chemical pre-treatment.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: β-fructofuranosidases are hydrolytic enzymes that act on sucrose to yield the products glucose and fructose. Under high substrate conditions these enzymes display fructosyltransferase activity which results in the synthesis of fructooligosaccharides (FOS). Some enzymes display higher propensities for FOS synthesis than others, with the determinants of this activity remaining unclear. The consumption of FOS produces a prebiotic effect that positively alters the composition of the colonic microflora, and as a result is linked to improved human and animal health. The increased demand for FOS has necessitated the industrial production of these nutraceuticals. In enzymatic sucrose biotransformation processes operating at high substrate loading and temperatures between 50 and 60°C, β-fructofuranosidase activity is negatively influenced by glucose product inhibition and thermal instability. The aim of this study was therefore to engineer the Aspergillus japonicus β-fructofuranosidase, FopA, to improve a FOS synthesis bioprocess. A dual approach was employed to engineer FopA so as to increase the probability of obtaining an improved enzyme variant(s). A random mutagenesis approach was applied to harness the potential of the randomness of introduced mutations as precise structural knowledge of the enzyme regions involved in the phenotypic presentation of product inhibition, specific activity and thermal stability was unavailable. A semi-rational approach afforded the additional opportunity to reduce the number of variants to be screened, yet theoretically increased the functional content of the library. This study details the development of a method to rapidly quantify FOS using Fourier transform mid infrared attenuated total reflectance spectroscopy and multivariate data analysis. The method offers improvements over conventionally used high performance liquid chromatography in terms of reduced sample analysis times and the absence of toxic waste products. This is the first report on the direct screening of an enzyme variant library for FOS synthesis to identify improved variants and will significantly support future engineering of β-fructofuranosidases using random mutagenesis approaches. The random mutagenesis approach yielded a variant displaying limited relief from glucose inhibition. At the peak difference in performance, the variant produced 28% more FOS from the same amount of sucrose, when compared to the parent. The semi-rational approach, using a combined crystal structure and evolutionary-guided approach, yielded a four amino acid combination variant displaying improved specific activity and thermostability that was able to reduce the time to completion of an industrial-like FOS synthesis reaction by 26%. The positive outcome of the semi-rational approach showed that engineering loops regions in an enzyme is a feasible strategy to improve both specific activity and thermostability, most probably due to the modification of enzyme structural flexibility. A bioinformatic tool that enables the identification of β-fructofuranosidases displaying high-level FOS synthesis from protein sequence alone was also developed during the study. These investigations revealed conserved sequence motifs characteristic of enzymes displaying low- and high-level FOS synthesis and a structural loop, unique to the latter group, that were readily applicable identifiers of FOS synthesis capacity. The tool presented may also be useful to improve the understanding of the structure-function relationships of β-fructofuranosidases by facilitating the identification of variations in groups of enzymes that have been functionally sub-classified. / AFRIKAANSE OPSOMMING: β-fruktofuranosidases is hidrolitiese ensieme wat op sukrose inwerk en glukose en fruktose as produkte vorm. Onder toestande met hoë substraatkondisies vertoon hierdie ensieme fruktosieltransferase-aktiwiteit wat tot die sintese van frukto-oligosakkariede (FOS) lei. Sommige ensieme neig na ʼn hoër FOS-sintese as ander, maar die bepalende faktore vir hierdie aktiwiteit is nog onbekend. Die verbruik van FOS veroorsaak ʼn prebiotiese effek wat die samestelling van kolon mikroflora positief beïnvloed en met verhoogde mens- en dieregesondheid verbind word. Die verhoogde aanvraag vir FOS het die industriële produksie van hierdie nutraseutiese middel genoodsaak. Tydens ensiemgedrewe sukrose-biotransformasieprosesse by hoë substraatladings en temperature tussen 50 en 60 °C, word β-fruktofuranosidase-aktiwiteit negatief deur glukose produkonderdrukking en termiese onstabiliteit beïnvloed. Die doel van hierdie studie was dus om die Aspergillus japonicus β-fruktofuranosidase, FopA, vir ʼn verbeterde FOS-sintese bioproses te manipuleer. ʼn Tweeledige benadering is vir FopA manipulasie gevolg om die waarskynlikheid van verbeterde variant(e) te verhoog. ʼn Lukrake mutagenese benadering, wat die potensiaal van ingevoegde mutasie ewekansigheid inspan, is in die lig van onvoldoende akkurate kennis van die strukturele gedeeltes betrokke by produkinhibisie-, spesifieke aktiwiteit- en termiese stabiliteit fenotipes gevolg. Die toepassing van ʼn semi-rasionele benadering het ook geleentheid vir die sifting van ʼn kleiner variantbibioloteek geskep, terwyl die funksionele inhoud teoreties verhoog word. Die studie beskryf die ontwikkeling van ʼn metode vir die vinnige kwantifisering van FOS, gebaseer op Fourier transform middel infrarooi geattenueerde totale refleksie spektroskopie en meerveranderlike data-analise. Dit is die eerste melding van ʼn direkte sifting van ʼn ensiemvariantversameling vir FOS-sintese om verbeterde variante te identifiseer, en kan die toekomstige manipulasie van β-fruktofuranosidases deur middel van lukrake mutagenese-benaderings beduidend ondersteun. Die lukrake mutagenese-benadering het ʼn variant met beperkte opheffing van glukose-onderdrukking gelewer. By die punt waar die prestasie die meeste verskil, het die variant 28% meer FOS vanaf dieselfde hoeveelheid sukrose geproduseer in vergelyking met die ouer-ensiem. Die semi-rasionele benadering, gegrond op ʼn kombinasie van kristalstruktuur en evolusionêre-geleide benaderings, het ʼn vier-aminosuurkombinasie variant met hoër spesifieke aktiwiteit en termostabiliteit gelewer wat die voltooiingstyd van ʼn tipiese industriële FOS sintesereaksie met 26% kon verkort. Die positiewe uitkoms van die semi-rasionele benadering het aangedui dat manipulasie van die lusgedeeltes in ʼn ensiem ʼn lewensvatbare strategie is om beide spesifieke aktiwiteit en termostabiliteit te verbeter, moontlik as gevolg van wysigings in die buigsaamheid van die ensiemstruktuur. ʼn Bioïnformatika-hulpmiddel vir die identifikasie van β-fruktofuranosidases met hoë vlakke van FOS-sintese op grond van proteïenvolgordes is ook tydens die studie ontwikkel. Motiewe met gekonserveerde volgordes kenmerkend van lae- en hoë-vlak FOS-produserende ensieme en ʼn strukturele lus, uniek tot die laasgenoemde groep, is tydens die ondersoek onthul wat as maklike identifiseerders van FOS-sintesekapasiteit kan dien. Die voorgestelde hulpmiddel kan ook nuttig wees om die struktuur-funksie-verwantskap van β-fruktofuranosidases beter te verstaan deur die identifikasie van variasie in ensiemgroepe wat funksioneel gesubklassifiseer is.

Stockholm Water is a water and sewage company with Henriksdal as one of two wastewater treatment plants (WWTPs). At Henriksdal wastewater sludge generated in the wastewater treatment process is digested which generate biogas; a mixture of mainly methane and carbon dioxide. If purified to methane content of 96 - 98 % this gas is called biomethane. Biogasmax is a project aiming to reduce the use of fossile fuels in Europe by providing that biogas is a good technical, economical and environmental alternative as vehicle fuel. The specific aim for Stockholm Water is to increase the biogas production at the existing plant in Henriksdal. Enzymatic treatment of wastewater sludge is an innovative technique earlier proofed to increase the biogas production from wastewater sludge with up to 60 %. The enzyme activity is in turn proven to significantly increase in the presence of a cation binding agent. One aim with this thesis was to investigate if the sludge from Henriksdal wastewater treatment process at all is affected of enzymatic treatment in presence of a cation binding agent since this has shown to have some significance. The chemical oxygen demand (COD) was measured in the liquid phase of sludge after treatment and used as a measurement of treatment effect. Another aim of this thesis was to look into the possibility to increase the methane production from sludge at Henriksdal WWTP. This was investigated through batch laboratory digestion tests. The sludge from Henriksdal WWTP was shown to be a good substrate for the enzymes added. COD in the liquid phase was increased with 17 – 32 % depending on the dose of enzymes and sodium citrate added. Digestion of sludge with a total addition of 18.6 mg enzymes per 1 g total solids (TS) and a concentration of 5 mM sodium citrate increased the methane production with almost 18 % compared to untreated sludge. This equals an increase of 18.3 % when converted to represent a totally blended and continuous digestion chamber at Henriksdal WWTP. The increased methane production also results in a sludge reduction out from the digestion chambers. The increased methane production and sludge reduction though does not fulfil the increased costs for the enzymes and sodium citrate applied. These doses must be decreased and the costs for both enzymes and sodium citrate must be reduced for this technique to be economically feasible in a full scale operation.

<p>Stockholm Water is a water and sewage company with Henriksdal as one of two wastewater treatment plants (WWTPs). At Henriksdal wastewater sludge generated in the wastewater treatment process is digested which generate biogas; a mixture of mainly methane and carbon dioxide. If purified to methane content of 96 - 98 % this gas is called biomethane.</p><p>Biogasmax is a project aiming to reduce the use of fossile fuels in Europe by providing that biogas is a good technical, economical and environmental alternative as vehicle fuel. The specific aim for Stockholm Water is to increase the biogas production at the existing plant in Henriksdal. Enzymatic treatment of wastewater sludge is an innovative technique earlier proofed to increase the biogas production from wastewater sludge with up to 60 %. The enzyme activity is in turn proven to significantly increase in the presence of a cation binding agent.</p><p>One aim with this thesis was to investigate if the sludge from Henriksdal wastewater treatment process at all is affected of enzymatic treatment in presence of a cation binding agent since this has shown to have some significance. The chemical oxygen demand (COD) was measured in the liquid phase of sludge after treatment and used as a measurement of treatment effect. Another aim of this thesis was to look into the possibility to increase the methane production from sludge at Henriksdal WWTP. This was investigated through batch laboratory digestion tests.</p><p>The sludge from Henriksdal WWTP was shown to be a good substrate for the enzymes added. COD in the liquid phase was increased with 17 – 32 % depending on the dose of enzymes and sodium citrate added. Digestion of sludge with a total addition of 18.6 mg enzymes per 1 g total solids (TS) and a concentration of 5 mM sodium citrate increased the methane production with almost 18 % compared to untreated sludge. This equals an increase of 18.3 % when converted to represent a totally blended and continuous digestion chamber at Henriksdal WWTP. The increased methane production also results in a sludge reduction out from the digestion chambers. The increased methane production and sludge reduction though does not fulfil the increased costs for the enzymes and sodium citrate applied. These doses must be decreased and the costs for both enzymes and sodium citrate must be reduced for this technique to be economically feasible in a full scale operation.</p>

Enzimas lipolíticas possuem enorme potencial biotecnológico. O objetivo foi prospectar genes para a codificação de enzimas lipolíticas em biblioteca metagenômica com 4224 clones. A atividade lipolítica foi avaliada pela formação de halo ao redor das colônias através do cultivo dos clones em meio de cultura suplementado com tributirina, sendo positiva para 30 clones, e dois foram selecionados e tiveram o DNA sub-clonado. Os DNAs das sub-bibliotecas foram sequenciados, gerando um contig completo para o clone PL28.F10, que foi comparado com as sequências do banco NCBI. Uma ORF codificadora de esterase/lipase de 303 aminoácidos e 61% de identidade com micro-organismo não cultivável foi encontrada. Árvores filogenéticas indicam que o clone possui a ORF15 mais próxima da família IV das esterases/lipases. Foi possível identificar os sítios ativos representativos da família, confirmando o resultado das árvores filogenéticas. Com sequências já patenteadas, a ORF15 é um grupo irmão das sequências de esterases/lipases da BASF e de uma proteína não identificada da CAMBIA. / Lipolytic enzymes have show enormous biotechnological potential. The work was done to find genes which codify lipolytic enzymes in a metagenomic library with 4224 clones. Clones were selected according to lipolytic activity and were assessed by cultivation in medium supplemented with tributyrin. Assessment was done by observation of halos formed around the colonies, with 30 clones producing halos. Of these, two were selected. DNA from the sub libraries was sequenced, generating a complete contig for clone PL28.F10 that was compared to sequences from the NCBI. An ORF of 303 amino acids with 61% of identity with uncunturable microorganism were found. The clone presented the ORF15 similar to that of lipolytic enzyme family IV. The alignments made possible the identification of active sites which represent the family, confirming the results obtained with the construction of the cladograms. The ORF15 showed similarities to patented BASF esterase/lipase and an unnamed protein of CAMBIA.

A banana é considerada um bom modelo de estudo para a transformação amido-sacarose, já que acumula um teor alto de amido durante o desenvolvimento, que é degradado durante o amadurecimento. Já foram detectadas em polpa de banana atividade e proteína relativa a várias enzimas supostamente envolvidas no processo de degradação do amido. Entre elas, a &#945;-amilase, a &#946;-amilase, a amido fosforilase e as glucano-água-diquinases (GWD). Estas enzimas estão envolvidas em dois processos distintos de degradação de amido em plantas: o dependente da ação inicial da &#945;-amilase e o dependente da fosforilação do grânulo pela GWD e PWD e posterior ação da &#946;-amilase. A dificuldade do estabelecimento da participação efetiva de cada enzima no processo de degradação do amido está associada a muitos fatores, entre eles a não-correlação entre atividade e real envolvimento em um processo, e a acessibilidade da enzima ao seu substrato. Aliado ao estudo da morfologia do grânulo de amido e suas modificações sofridas durante o processo de degradação que ocorre durante o amadurecimento do fruto, estudos in vitro que simulem a ação da enzima sobre o seu substrato poderiam ser mais efetivos no estabelecimento da real ação de dada enzima sobre o suposto substrato. Tentativas no sentido de obter as proteínas relativa à degradação não foram bem sucedidas. Assim, os ensaios de grânulos de amido isolados versus enzimas foram feitos com &#945;-amilase e &#946;-amilase comerciais. O grau de fosforilação da amilopectina nas posições Glic-6 e Glic-3 foi determinado, condição necessária para o início da degradação do grânulo pela &#946;-amilase. Os resultados mostraram que os grânulos de amido isolados de bananas recém colhidas, ou verdes, já estão fosforilados e as enzimas responsáveis por esta fosforilação estão associadas aos grânulos. Após 72 h de incubação dos grânulos de amido com as enzimas hidrolítica, os grânulos foram separados do tampão contendo as enzimas e os produtos de hidrólise. Os sobrenadantes foram analisados por cromatografia líquida acoplada a detector amperométrico e os grânulos por Microscopia Eletrônica de Varredura (MEV) e microscopia de força atômica (MFA). Os resultados mostraram que a &#945;-amilase hidrolisa preferencialmente regiões amorfas dos grânulos, com predominância de amilose, expondo as regiões mais cristalinas dos anéis de crescimento, enquanto que a &#946;-amilase parece atuar preferencialmente nas regiões cristalinas dos grânulos, degradando os bloquetes, que são formados por amilopectina. Pode-se concluir que ambas as enzimas parecem ser importantes no processo de degradação do amido da banana, com diferentes especificidades. / Banana is considered a good model to study the starch-sucrose metabolism, since it accumulates a high starch content during development, which is degraded during fruit ripening. It have been detected in banana pulp some proteins and activities of several enzymes supposedly involved in starch degradation process. Among them, &#945;-amylase, &#946;-amylase, starch phosphorylase and glucan-water-diquinases (GWD). These enzymes are involved in two separate processes of starch degradation in plants: the initial action of &#945;-amylase dependent, and the starch granule phosphorylation by GWD and PWD enzymes and subsequent action of &#946;-amylase. The difficulty of establishing the effective participation of each enzyme in the starch degradation process is associated with many factors, including the lack of correlation between real activity and involvement in the process, and accessibility of the enzyme to its substrate. Allied to study the morphology of the starch granule and its modifications suffered during the process of degradation, which occurs during the fruit ripening, in vitro studies that simulate the action of the enzyme on its substrate could be more effective in establishing the real action of a given enzyme on the argued substrate. However, attempts to obtain the proteins related to the degradation process were unsuccessful. Thus, assays of isolated starch granules versus enzymes were made with commercial &#945;-amylase and &#946;-amylase enzymes. The degree of phosphorylation of amylopectin in the Gluc-6 and Gluc-3 positions was determined, a necessary condition for the start of degradation by &#946;-amylase enzyme. The results showed that the starch granules isolated from freshly harvested bananas, or green, are already phosphorylated and the enzymes responsible for this phosphorylation is associated with the starch granules surface. After 72 h incubation of the starch granules with the hydrolytic enzymes, the granules were separated from the buffer containing the enzymes and the hydrolysis products. The supernatants were analyzed by liquid chromatography coupled with amperometric detector and the granules were visualized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The results showed that the &#945;-amylase preferentially hydrolyzes amorphous regions of the granule, especially amylose, exposing more crystalline regions of the growth rings, whereas &#946;-amylase appears to act preferentially on crystalline regions of the granule, degrading blocklets that consist of amylopectin. It can be concluded that both enzymes appear to be important in the banana starch degradation process, with different specificities.