Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Water-soluble deep-cavity cavitands provide a rare opportunity to study self-assembly driven by the hydrophobic effect. These molecular hosts dimerize in the presence of certain guest molecules to form water-soluble molecular capsules. These systems have given rise to numerous novel chemical phenomena and have potential use in drug delivery. The host octaacid (OA) has been particularly well-characterized, but studies are limited to basic pH because of limited host solubility. Herein we report an improved synthesis of OA and the syntheses of three new water-soluble deep-cavity cavitands. The new hosts are soluble at neutral pH, increasing relevance for biological studies. The new syntheses are versatile enough to apply to the synthesis of additional water- soluble cavitands in the future. We also describe preliminary characterization of the molecular recognition properties of the new hosts. Binding of organic guest molecules to form 1:1 host:guest complexes and 2:1 host:guest capsules was qualitatively similar to that of OA. However, binding of anions spanning the Hofmeister series revealed interesting new behavior. The new hosts bound a wider range of anions inside the hydrophobic pocket with much higher association constants. Moreover, external binding of several anions to the cavitand pendant feet was observed. Looking towards biological applications, we desired to learn how these molecules interact with phospholipid membranes. Six water-soluble cavitands were tested for their ability to permeabilize liposomal POPC membranes. One host showed very high potency in permeabilizing membranes, while three other hosts showed moderate activity. Host binding of POPC was found to be at least one factor in host-induced permeabilization. A requenching assay to determine leakage mechanism strongly supported all-or-none leakage, whereby some vesicles lose all contents while others lose none. These results suggest that these cavitands induce partial transient leakage of vesicles by the formation of transient membrane pores. These findings show potential for the use of these hosts as drug delivery carriers, antimicrobial compounds, and tools in membrane alteration studies.

Este trabalho teve como objetivo estudar a permeabilização celular de Candida guilliermondii FTI 20037 empregando processos químicos (agentes tensoativos) e físicos (congelamento-descongelamento) e verificar o potencial uso das células permeabilizadas na redução de xilose em xilitol. Os ensaios de permeabilização empregaram suspensão celular de 2 g/L, temperatura de 30ºC e pH 7. Para os processos químicos foram avaliados CTAB (Brometo de cetiltrimetilamônio) e Triton X-100 e os ensaios foram realizados empregando metodologia do planejamento experimental. O monitoramento da permeabilidade celular foi realizado através da dosagem in situ e no sobrenadante da enzima glicose-6-fosfato desidrogenase (G6PD), selecionada como marcador do tratamento. As enzimas xilose redutase (XR) e xilitol desidrogenase (XD) também foram dosadas. A permeabilização de C. guilliermondii com CTAB permitiu a dosagem in situ de G6PD e XD, mas não de XR. As três enzimas avaliadas não foram detectadas no sobrenadante. As condições que promoveram máxima permeabilidade celular (0,41 mM de CTAB, 200 rpm de agitação e 50 min de tempo de contato) resultaram em níveis in situ de G6PD de 283,4 ± 60,7 U/L e 122,4 ± 15,7 U/gcélulas. Nestas condições de tratamento, o CTAB influenciou negativamente a atividade catalítica de G6PD, XR e XD presentes no homogenato das células rompidas (não tratadas). O estudo de permeabilização celular com Triton X-100 mostrou que o tensoativo foi pouco efetivo, permitindo a dosagem in situ apenas da G6PD. As condições que promoveram máxima permeabilidade celular, ou seja, 2,78 mM de Triton X-100, 200 rpm de agitação e 50 min de tempo de contato, resultaram em níveis in situ de G6PD de 44,7 ± 0,0 U/L e 16,9 ± 0,0 U/gcélulas. Nestas condições, Triton X-100 não afetou a atividade catalítica de G6PD, XR e XD presentes no homogenato das células rompidas (não tratadas). O processo físico de permeabilização consistiu no congelamento da suspensão celular (-18ºC) por período de 48h, seguido do descongelamento em banho-maria (30ºC). Este tratamento permitiu a dosagem in situ das enzimas G6PD (108,7 ± 3,8 U/L e 54,3 ± 1,9 U/ gcélulas) e XR (26,4 ± 0,1U/L e 13,2 ± 0,1 U/gcélulas), mas não da XD. O tratamento não foi suficiente para liberar G6PD, no entanto, cerca de 60% da atividade total de XR foi detectada no sobrenadante (47,1 ± 0,4 U/L e 23,6 ± 0,2 U/gcélulas). Os ensaios de biotransformação mostraram que, nas condições avaliadas, a conversão de xilose em xilitol foi dependente do tipo de tratamento de permeabilização do biocatalisador. Os ensaios de cultivo mostraram que o tratamento das células com Triton X-100 não foi suficiente para causar perda de viabilidade e atividade metabólica de C. guilliermondii, enquanto o congelamento-descongelamento promoveu perda parcial da viabilidade celular. O tratamento das células com CTAB foi mais agressivo, causando a perda total de viabilidade celular. Foi também verificado que resting cells (células em estado de repouso) de C. guilliermondii sem tratamento e permeabilizadas com Triton X-100 foram capazes de converter xilose em xilitol com rendimento de ~60%, após 10 h de reação. Com o presente trabalho pode se concluir que os métodos estudados podem ser especialmente úteis para a determinação in situ de G6PD. Além disto, a utilização de células permeabilizadas pode ajudar a superar os problemas/custos associados com a extração e purificação das enzimas e conseqüentemente contribuírem para o desenvolvimento de uma tecnologia de baixo custo para a produção de xilitol. / This work describes the effect of the surfactants (CTAB and Triton X-100) and freezing-thawing treatment on the permeabilization of C. guilliermondii cells. The potential use of these cells (unpermeabilized and permeabilized by CTAB, Triton X-100 and freezing-thawing treatment) was also evaluated. Response surface methodology was used to investigate the effect of different parameters (detergent concentration, agitation and treatment time) on the permeabilization of C. guilliermondii cells. The experimentation was aimed to find the values of process variables to achieve maximal glucose-6-phosphate dehydrogenase (G6PD) activity in situ. The intracellular G6PD of the C. guilliermondii could not be detected in intact (unpermeabilized) whole cells. However, on treatment of C.guilliermondii with detergents (CTAB and Triton X-100) and freeze-thawing, the G6PD activity could be measured.The effectiveness of detergent permeabilization of C.guilliermondii cells was dependent on its concentration and exposure time. Maximum permeabilization, measured in terms of assayable G6PD activity in situ, was obtained when the cells were treated with CTAB. Triton X-100 and freeze-thawing were also found to permeabilize the cells, but to a lesser degree than CTAB. The optimum operating conditions for permeabilization process were 0.41 mM (CTAB) or 2.78 mM (Triton X-100) under agitation of 200 rpm at 30ºC temperature and process duration of 50 min and pH 7. At these conditions of process variables, the maximum value of enzyme activity was found to be 283.4 ± 60.7 U/L (122.4 ± 15.7 U/gcells) and 44.7 ± 0.0 U/L (16.9 ± 0.0 U/gcells) for permeabilized cells with CTAB and Triton X-100, respectively. The Triton X-100 was not enough to cause loss of viability and metabolic activity of C. guilliermondii. Freezing-thawing treatment promoted partial loss of cell viability. On the other hand the cells treated with CTAB were totally affected. The biotransformation of xylose to xylitol was studied by employing C. gulliermondii FTI 20037 in two different forms namely unpermeabilized cells and permeabilized cells. The maximum xylitol yield of about 60% was observed with unpermeabilized yeast cells and Triton X-100 permeabilized cells after 10 h of reaction time. In conclusion, surfactants and freezing-thawing treatment provides a simple and mild procedure for C.guilliermondii permeabilization. The method may be especially useful for the in situ determination of G6PD. Response surface methodology was found effective in optimizing and determining the interactions among process variables for the permeabilization process. The use of permeabilized cells can help to overcome the problems/costs associated with enzyme extraction and purification from yeast cells and in the development of a low-cost technology for xylitol production.

Supercritical fluids have unique properties which may make them ideal as reaction media for biotransformation or extractive solvents. Supercritical fluids are ideal for reducing diffusivity limitations over conventional fluids. Depending on the polarity of the fluid, a supercritical fluid can be similar to conventional organic solvents, but with few of the environmental drawbacks. The use of supercritical fluids in enzymatic research has the advantage of removing mass transport limitations so that they can act as a suitable solvent. In this study, four permeabilization techniques were compared: control, toluene, supercritical carbon dioxide, and freeze/thaw cycles. The model cell systems studied were Z. mobilis and E. coli. The cells were analyzed for lipid profiles, recovery of proteins and enzymatic activity. The use of supercritical carbon dioxide may not be the most effective of the treatments based on total protein or enzyme recovery since the greatest protein and enzyme recovery was with the freeze/thaw treatment. However, it can be selective in removing cofactors from Z. mobilis enabling sorbitol production and minimizing side reactions. In this application, supercritical carbon dioxide does show an advantage over the freeze/thaw treatment. Aspects of the mechanism of permeabilization were investigated based on the lipid profiles of the cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The SEM and AFM show changes of the cell surface morphology which indicate that the treatments affect the cellular surface. The use of supercritical carbon dioxide as a reaction medium was investigated. Minute quantities of sorbitol were produced when Z. mobilis and sugars were placed in a supercritical carbon dioxide environment over a period of 24 hours.

Supercritical fluids have unique properties which may make them ideal as reaction media for biotransformation or extractive solvents. Supercritical fluids are ideal for reducing diffusivity limitations over conventional fluids. Depending on the polarity of the fluid, a supercritical fluid can be similar to conventional organic solvents, but with few of the environmental drawbacks. The use of supercritical fluids in enzymatic research has the advantage of removing mass transport limitations so that they can act as a suitable solvent. In this study, four permeabilization techniques were compared: control, toluene, supercritical carbon dioxide, and freeze/thaw cycles. The model cell systems studied were Z. mobilis and E. coli. The cells were analyzed for lipid profiles, recovery of proteins and enzymatic activity. The use of supercritical carbon dioxide may not be the most effective of the treatments based on total protein or enzyme recovery since the greatest protein and enzyme recovery was with the freeze/thaw treatment. However, it can be selective in removing cofactors from Z. mobilis enabling sorbitol production and minimizing side reactions. In this application, supercritical carbon dioxide does show an advantage over the freeze/thaw treatment. Aspects of the mechanism of permeabilization were investigated based on the lipid profiles of the cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The SEM and AFM show changes of the cell surface morphology which indicate that the treatments affect the cellular surface. The use of supercritical carbon dioxide as a reaction medium was investigated. Minute quantities of sorbitol were produced when Z. mobilis and sugars were placed in a supercritical carbon dioxide environment over a period of 24 hours.

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Orientadores: Francisco Maugeri Filho, Susana Juliano Kalil / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-17T02:58:06Z (GMT). No. of bitstreams: 1 Manera_AnaPaula_D.pdf: 2066521 bytes, checksum: 108d932fdf16225bc7cd32688323bd02 (MD5) Previous issue date: 2010 / Resumo: Galacto-oligossacarídeos (GOS) são carboidratos não digeríveis por humanos, produzidos a partir da lactose por ação da enzima ?-galactosidase. São considerados ingredientes prebióticos e possuem propriedades favoráveis à saúde dos consumidores. Este trabalho teve como objetivo o estudo da produção de GOS a partir de células permeabilizadas de Kluyveromyces marxianus CCT 7082. A primeira etapa foi a otimização das condições de cultivo da levedura para a produção da enzima ?-galactosidase empregando como substratos os subprodutos agroindustriais, soro de queijo e água de maceração de milho, obtendo 1400 U/gcel em 24 h de fermentação. Em seguida estudou-se a permeabilização das células da levedura. Foram testados sete agentes permeabilizantes: etanol, isopropanol, butanol, acetona, brometo de cetiltrimetilamônio, Tween 80 e Triton X-100, tendo sido selecionado o isopropanol, para a etapa de otimização do processo de permeabilização, onde se avaliou o efeito da relação biomassa/isopropanol e da temperatura. Na caracterização da enzima, a ß-galactosidase das células permeabilizadas apresentou pH ótimo de 6,6 e temperatura ótima de 50°C, sendo esta mais estável no pH 7,0 e na temperatura de 30ºC. A energia de desnaturação foi 81,6 Kcal/mol. A cinética enzimática da enzima seguiu o modelo de Michaelis-Menten. O estudo da síntese de GOS, através de delineamentos experimentais, empregando as células permeabilizadas, resultou em 83 g/L de GOS. O emprego de fluidos pressurizados como meio reacional de reações enzimáticas podem favorecer a solubilidade dos compostos, as transferências de massa das reações, e aumentar a atividade e estabilidade de enzimas, assim sendo, estudou-se o comportamento da atividade enzimática da enzima das células permeabilizadas tratadas nessas condições. Foi realizado um delineamento composto central para cada fluido (n-butano, propano, CO2), sendo observado um aumento na atividade residual, em todos os ensaios dos delineamentos, de 110 a 211% dependendo do fluido. O tratamento com n-butano resultou na maior estabilidade da enzima: após 3 semanas de armazenamento a 10°C a enzima tratada manteve 96% de sua atividade. Estas células tratadas a alta pressão foram aplicadas na síntese de GOS em reator batelada a pressão atmosférica. Obteve-se aproximadamente 75 g/L de GOS para os três fluidos pressurizados e para as células permeabilizadas sem tratamento. Porém, a quantidade necessária de enzima (em gramas de células) para se obter a mesma atividade enzimática, foi bem menor para as enzimas tratadas a alta pressão, tendo em vista o aumento da atividade enzimática após o tratamento. Na etapa seguinte, estudou-se a síntese de GOS em reator batelada empregando como meio reacional fluidos pressurizados (n-butano, propano, CO2). Realizou-se um delineamento composto central para cada fluido, obtendo-se entre 65 e 83,4 g/L de GOS, dependendo do meio reacional. De acordo com os resultados deste trabalho, usando células de K. marxianus CCT 7082, pôde-se definir a metodologia empregando as células permeabilizadas, tratadas a alta pressão com n-butano e síntese em reator batelada a pressão atmosférica como a mais promissora para a produção de GOS / Abstract: Galactooligosaccharides (GOS) are humans non-digestible carbohydrates, produced from lactose by the action of the enzyme ?-galactosidase. They are considered prebiotic ingredients and have beneficial properties to the health of consumers. This work aimed the study of galacto-oligosaccharide production from permeabilized cells of Kluyveromyces marxianus CCT 7082. The first step was to optimize the yeast culture conditions in order to produce the ?-galactosidase enzyme, employing as substrates by-products from agriculture industries, such as cheese whey and corn steep liquor, it were obtained 1400 U/g in 24 h of fermentation. The next step was to study the yeast cell permeabilization. Seven permeabilizant agents were tested: ethanol, isopropanol, butanol, acetone, cetyl-trimethylammonium bromide, Tween 80 and Triton X-100. Isopropanol was selected for the optimization step of the permeabilization process, where the effect of the biomass/isopropanol ratio and the temperature on cell permeabilization were evaluated. Afterward, the ?-galactosidase from permeabilized cells was characterized presenting the optimum pH of 6.6 and the optimum temperature 50°C. The enzyme was more stable at pH 7.0 and 30°C temperature. The denaturation energy was 81.6 Kcal/mol. The enzyme kinetics followed Michaelis-Menten model. The GOS synthesis, studied through experimental designs, employing the permeabilized cells, resulted in 83 g/L of GOS. The use of pressurized fluids as a reactional medium for enzymatic reactions can help the components solubility and the mass transferences of the reactions and to increase the enzymes activity and stability. Therefore, the behavior of the enzymatic activity of the permebialized cell enzymes, treated with pressurized fluids, was studied. A central composite design was performed for each pressurized fluid, and it was observed an increase on residual activity for all pressurized fluids, in all designs essays, from 110 a 211%, depending on the fluid. The enzyme treated with n-butane resulted in the highest enzyme stability. After 3 weeks of storage at 10°C the enzyme kept 96% of activity. These cells treated at pressure were employed at GOS synthesis in batch reactor at atmospheric pressure. Around 75 g/L of GOS were obtained for all three pressurized fluids, as well as for the enzyme without treatment. However, the amount of the enzyme needed (in g of cells) to obtain the same enzymatic activity was lower in the case of the enzymes treated at high pressure, due to the increase of enzymatic activity after the treatment. In the next step of the work, the GOS synthesis was studied in batch mode, using as a reactional medium pressurized fluids (n-butane, propane, CO2). A central composite design for each pressurized fluid was carried out, obtaining between 65 g/L and 83 g/L of GOS, depending on reactional medium. According to the results of this work, using cells of K. marxianus CCT 7082, it can be defined that the methodology of permeabilized cells, treated at high pressure with n-butane and synthesis in atmospheric pressure reactor, is the most promising one for GOS production / Doutorado / Doutor em Engenharia de Alimentos

Este trabalho teve como objetivo estudar a permeabilização celular de Candida guilliermondii FTI 20037 empregando processos químicos (agentes tensoativos) e físicos (congelamento-descongelamento) e verificar o potencial uso das células permeabilizadas na redução de xilose em xilitol. Os ensaios de permeabilização empregaram suspensão celular de 2 g/L, temperatura de 30ºC e pH 7. Para os processos químicos foram avaliados CTAB (Brometo de cetiltrimetilamônio) e Triton X-100 e os ensaios foram realizados empregando metodologia do planejamento experimental. O monitoramento da permeabilidade celular foi realizado através da dosagem in situ e no sobrenadante da enzima glicose-6-fosfato desidrogenase (G6PD), selecionada como marcador do tratamento. As enzimas xilose redutase (XR) e xilitol desidrogenase (XD) também foram dosadas. A permeabilização de C. guilliermondii com CTAB permitiu a dosagem in situ de G6PD e XD, mas não de XR. As três enzimas avaliadas não foram detectadas no sobrenadante. As condições que promoveram máxima permeabilidade celular (0,41 mM de CTAB, 200 rpm de agitação e 50 min de tempo de contato) resultaram em níveis in situ de G6PD de 283,4 ± 60,7 U/L e 122,4 ± 15,7 U/gcélulas. Nestas condições de tratamento, o CTAB influenciou negativamente a atividade catalítica de G6PD, XR e XD presentes no homogenato das células rompidas (não tratadas). O estudo de permeabilização celular com Triton X-100 mostrou que o tensoativo foi pouco efetivo, permitindo a dosagem in situ apenas da G6PD. As condições que promoveram máxima permeabilidade celular, ou seja, 2,78 mM de Triton X-100, 200 rpm de agitação e 50 min de tempo de contato, resultaram em níveis in situ de G6PD de 44,7 ± 0,0 U/L e 16,9 ± 0,0 U/gcélulas. Nestas condições, Triton X-100 não afetou a atividade catalítica de G6PD, XR e XD presentes no homogenato das células rompidas (não tratadas). O processo físico de permeabilização consistiu no congelamento da suspensão celular (-18ºC) por período de 48h, seguido do descongelamento em banho-maria (30ºC). Este tratamento permitiu a dosagem in situ das enzimas G6PD (108,7 ± 3,8 U/L e 54,3 ± 1,9 U/ gcélulas) e XR (26,4 ± 0,1U/L e 13,2 ± 0,1 U/gcélulas), mas não da XD. O tratamento não foi suficiente para liberar G6PD, no entanto, cerca de 60% da atividade total de XR foi detectada no sobrenadante (47,1 ± 0,4 U/L e 23,6 ± 0,2 U/gcélulas). Os ensaios de biotransformação mostraram que, nas condições avaliadas, a conversão de xilose em xilitol foi dependente do tipo de tratamento de permeabilização do biocatalisador. Os ensaios de cultivo mostraram que o tratamento das células com Triton X-100 não foi suficiente para causar perda de viabilidade e atividade metabólica de C. guilliermondii, enquanto o congelamento-descongelamento promoveu perda parcial da viabilidade celular. O tratamento das células com CTAB foi mais agressivo, causando a perda total de viabilidade celular. Foi também verificado que resting cells (células em estado de repouso) de C. guilliermondii sem tratamento e permeabilizadas com Triton X-100 foram capazes de converter xilose em xilitol com rendimento de ~60%, após 10 h de reação. Com o presente trabalho pode se concluir que os métodos estudados podem ser especialmente úteis para a determinação in situ de G6PD. Além disto, a utilização de células permeabilizadas pode ajudar a superar os problemas/custos associados com a extração e purificação das enzimas e conseqüentemente contribuírem para o desenvolvimento de uma tecnologia de baixo custo para a produção de xilitol. / This work describes the effect of the surfactants (CTAB and Triton X-100) and freezing-thawing treatment on the permeabilization of C. guilliermondii cells. The potential use of these cells (unpermeabilized and permeabilized by CTAB, Triton X-100 and freezing-thawing treatment) was also evaluated. Response surface methodology was used to investigate the effect of different parameters (detergent concentration, agitation and treatment time) on the permeabilization of C. guilliermondii cells. The experimentation was aimed to find the values of process variables to achieve maximal glucose-6-phosphate dehydrogenase (G6PD) activity in situ. The intracellular G6PD of the C. guilliermondii could not be detected in intact (unpermeabilized) whole cells. However, on treatment of C.guilliermondii with detergents (CTAB and Triton X-100) and freeze-thawing, the G6PD activity could be measured.The effectiveness of detergent permeabilization of C.guilliermondii cells was dependent on its concentration and exposure time. Maximum permeabilization, measured in terms of assayable G6PD activity in situ, was obtained when the cells were treated with CTAB. Triton X-100 and freeze-thawing were also found to permeabilize the cells, but to a lesser degree than CTAB. The optimum operating conditions for permeabilization process were 0.41 mM (CTAB) or 2.78 mM (Triton X-100) under agitation of 200 rpm at 30ºC temperature and process duration of 50 min and pH 7. At these conditions of process variables, the maximum value of enzyme activity was found to be 283.4 ± 60.7 U/L (122.4 ± 15.7 U/gcells) and 44.7 ± 0.0 U/L (16.9 ± 0.0 U/gcells) for permeabilized cells with CTAB and Triton X-100, respectively. The Triton X-100 was not enough to cause loss of viability and metabolic activity of C. guilliermondii. Freezing-thawing treatment promoted partial loss of cell viability. On the other hand the cells treated with CTAB were totally affected. The biotransformation of xylose to xylitol was studied by employing C. gulliermondii FTI 20037 in two different forms namely unpermeabilized cells and permeabilized cells. The maximum xylitol yield of about 60% was observed with unpermeabilized yeast cells and Triton X-100 permeabilized cells after 10 h of reaction time. In conclusion, surfactants and freezing-thawing treatment provides a simple and mild procedure for C.guilliermondii permeabilization. The method may be especially useful for the in situ determination of G6PD. Response surface methodology was found effective in optimizing and determining the interactions among process variables for the permeabilization process. The use of permeabilized cells can help to overcome the problems/costs associated with enzyme extraction and purification from yeast cells and in the development of a low-cost technology for xylitol production.