Screening of Supports for the Immobilization of β-Glucosidase

A set of supports were screened for the immobilization of a partially purified extract of β-glucosidase from Aspergillus sp. These supports, namely, Eupergit, Amberlite, alginate, gelatin, polyvinyl alcohol- (PVA-) based matrices (Lentikats), and sol-gel, have proved effective for the implementation of some other enzyme-based processes. The initial criterion for selection of promising supports prior to further characterization relied on the retention of the catalytic activity following immobilization. Based on such criterion, where immobilization in sol-gel and in Lentikats outmatched the remaining approaches, those two systems were further characterized. Immobilization did not alter the pH/activity profile, whereas the temperature/activity profile was improved when sol-gel support was assayed. Both thermal and pH stability were improved as a result of immobilization. An increase in the apparent KM (Michaelis constant) was observed following immobilization, suggesting diffusion limitations

Avaliação de agentes precipitantes para produção de Agregados de ligação cruzada (Cleas) da enzima ?-glicosidase produzida por Aspergillus niger / Evaluation of precipitating agents for the production of cross-linked aggregates (Cleas) of the enzyme ?-glycosidase produced by Aspergillus niger

As enzimas já se apresentam como uma realidade em muitos processos industriais, tornando-os menos nocivos ao meio-ambiente através de sua especificidade, reduzindo produção de sub-produtos e etapas de purificação. Entretanto, para muitas aplicações a perda da estabilidade, assim como o custo se apresentam como entraves para novas aplicações. A técnica de imobilização de enzimas é uma das tecnologias que podem minimizar estes entraves, permitindo a reutilização das mesmas e aumentando sua rigidez e estabilidade operacional. Dentre as técnicas de imobilização, a técnica de Cleas que consiste na reticulação da enzima sem a necessidade de um suporte sólido, através de ligações cruzadas utilizando um agente bifuncional vem chamando atenção devido a combinação de purificação e imobilização em um único estágio. O objetivo desse trabalho foi avaliar diferentes solventes na precipitação da enzima ?-glicosidase para posterior produção de Cleas, os solventes avaliados foram etanol, propanona, metanol, 2-propanol, n-butanol e éter-etílico. Após a precipitação foi realizada a determinação da atividade enzimática e da concentração de proteínas nos sobrenadantes e precipitados. Os autores concluíram que o solvente que apresentou resultados mais promissores para a produção de Cleas de ?-glicosidase, foi o solvente 2-propanol que propiciou um maior teor de proteínas em conjunto com maior atividade catalítica no  precipitado, permanecendo com 94,4% da atividade residual

Determination and characterization on sugarcane starch and adequacy of the methodology for the residual a-amylase determination in raw sugar

Orientador: Helia Harumi SatoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: O presente trabalho visou à determinação da quantidade de amido em caldo de algumas variedades de cana-de-açúcar (RB86-7515, SO83-2847, RB72-454, SP80-3280 e RB85-5536) durante uma safra (período de Abril a Novembro de 2007), estudo de algumas características do amido de cana-de-açúcar e a adequação de metodologia para a determinação de atividade de a-amilase residual em açúcar bruto. A variedade de cana-de-açúcar RB86-7515 apresentou maior teor de amido durante toda safra entre as variedades analisadas. Não foi observado correlação entre o índice pluviométrico e as temperaturas da região e o teor de amido encontrado nas variedades de cana-de-açúcar. Na análise dos grânulos de amido por microscopia eletrônica de varredura (MEV) o amido de cana-de-açúcar apresentou forma esférica e diâmetro na faixa de 1-3 mm, tamanho muito inferior quando comparado ao amido de batata, mandioca e milho. Os amidos de cana-de-açúcar, milho, batata e amido solúvel complexados com iodo apresentaram maior absorção na faixa de 540 a 620 nm. O amido de cana-de-açúcar in natura mostrou maior susceptibilidade à enzima glicoamilase em relação aos amidos de milho ceroso, mandioca e batata. O amido de cana-de-açúcar mostrou susceptibilidade à enzima amilolítica desramificante pululanase que hidrolisa as ligações a-1,6 glicosídicas de modo similar ao amido de arroz ceroso, que apresenta alto teor de amilopectina. O amido de cana-de-açúcar mostrou susceptibilidade às a-amilases de Bacillus subtilis, Bacillus licheniformis e Aspergillus oryzae de modo similar aos outros amidos testados produzindo glicose; maltose; maltotriose; maltotetraose, maltopentaose e a-dextrinas limite. Para a adequação da metodologia para a determinação de a-amilase em açúcar bruto foram testados os métodos de Bernfeld baseado na determinação de açúcares redutores, método Iodométrico baseado na descoloração do complexo amido-iodo, método de Phadebas baseado na hidrólise do amido complexado com corante Cibacron Blue F3GA e o método usando-se o substrato BPNPG7 (benzylidene blocked r-nitrophenyl maltoheptaoside, Megazyme, Ireland) baseado na hidrólise do substrato e liberação do r-nitrofenol. O método de Bernfeld se mostrou mais adequado para a quantificação de a-amilase residual em açúcar brutoAbstract: The aim of this work was to quantitatively determine sugarcane starch in five varieties of sugarcane (RB86-7515, SP83-2847, RB72-454, SP80-3280 and RB85-5536) during one harvest (May to November 2007), and study some of the characteristics of sugarcane starch. The variety RB86-7515 showed a higher starch content than the other varieties analyzed throughout the entire harvest. No correlation between the temperature, pluviometric index and starch level was found in any of the samples of sugarcane juice. The sugarcane starch granules examined under the scanning electron miscroscope showed a spherical shape with a diameter between 1-3 mm, smaller than the starch granules of maize and cassava. The sugarcane, corn, potato and soluble starches formed complexes with iodine, which showed greater absorption in the range from 540 to 620 nm. The crude sugarcane starch showed greater susceptibility to the enzyme glucoamylase than the waxy corn, cassava and potato starches. The sugarcane starch showed a susceptibility to debranching by the amylolytic enzyme pululanase, which hydrolyzed the a-1,6 glucosidic linkages in a way similar to waxy corn starch, which contains a high amount of amylopectin. The sugarcane starch showed susceptibility to the a-amylases from Bacillus subtilis, Bacillus licheniformis and Aspergillus oryzae, similar to the other starches tested, producing glucose, maltose, maltotriose, maltotetraose, maltopentaose and a-limit dextrins. This work aimed to adequate the methodologies used to determine residual a-amylase in raw sugar. The Bernfeld method is based on the determination of the reducing sugars by an iodometric method based on discoloration of the starch-iodine complex. The Phadebas method is based on the hydrolysis of the starch polymer dyed with Cibachron Blue F3GA, and the method using BPNPG7 r ¿ nitrophenyl maltoheptaoside (benzylidene blocked maltoheptaoside, Megazyme, Ireland) is based on the hydrolysis of the substrate, releasing p-nitrophenol. These three methods were tested for the determination of residual a-amylase in raw sugar, and the Bernfeld method was shown to be more adequateMestradoMestre em Ciência de Alimento

Study of production, immobilization and application of ß-glucosidase from Aspergillus sp

Orientador: Hélia Harumi SatoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: O presente trabalho visou o estudo da produção da ?-glicosidase pela linhagem de Aspergillus sp. utilizando-se resíduos agrícolas como o farelo de trigo, casca de maracujá e bagaço de cana-de-açúcar, a imobilização da enzima em diferentes suportes como lentes PVA - Lentikats®, sol-gel, Eupergit, Amberlite, gelatina e alginato de cálcio, e a aplicação da enzima livre e imobilizada na conversão de isoflavonas glicosiladas de soja em isoflavonas agliconas. O fungo foi identificado como Aspergillus niger LBA 02. O extrato enzimático bruto obtido de A. niger LBA 02 apresentou atividade de ?-glicosidase, CM -celulase, amilase, poligalacturonase e pectinase. Foi obtida maior atividade de ?-glicosidase (44,81 U/g) na fermentação da linhagem A. niger LBA 02 em meio semissólido composto por 25 g de farelo de trigo e 5,7 mL de água destilada, após 240 h de fermentação a 30°C. Os efeitos da adição do extrato de levedura e dos sais KH2PO4, NH4NO3, MgSO4.7H2O, no meio de farelo de trigo, para a produção de ?-glicosidase por A. niger LBA 02 não foram significativos nos níveis estudados. Os resíduos casca de maracujá e bagaço de cana-de-açúcar adicionados no meio de farelo de trigo não atuaram como indutores da produção de ?-glicosidase, nos níveis estudados. Dentre os 7 métodos de imobilização testados, as técnicas de sol-gel e lentes PVA - Lentikats® apresentaram melhores resultados para a imobilização da enzima ?-glicosidase. A enzima livre apresentou atividade ótima a 65°C e pH 4,5, enquanto a enzima imobilizada em sol-gel mostrou atividade ótima na faixa de 60 - 65°C. A temperatura de 50°C foi fixada como temperatura ótima de trabalho para a enzima imobilizada em lentes PVA - Lentikats®, pois acima da temperatura de 60°C ocorreu a fusão das lentes. A imobilização da ?-glicosidase não alterou o pH ótimo de atividade da enzima, permanecendo em 4,5. A imobilização resultou em um pequeno aumento da estabilidade térmica da ?-glicosidase. A ?-glicosidase imobilizada em lentes PVA - Lentikats® apresentou-se mais estável do que a enzima livre, após 3 h de tratamento na faixa de 45 a 55°C. O tempo de meia vida da ?-glicosidase imobilizada em sol-gel a 70°C foi 0,88 h. A ?-glicosidase imobilizada em sol-gel apresentou cerca de 10% de atividade residual após 3 h a 70°C, enquanto que a enzima livre foi inativada após 1 hora a 70°C. A ?-glicosidase imobilizada em sol-gel apresentou valores de KM na faixa de 1,0 a 1,25 mM de celobiose, a 60°C, estimados pelos métodos de Lineweaver - Burk, Hanes - Woolf e Solver, enquanto que a enzima livre apresentou valores de 0,92 a 1,69 mM de celobiose, sugerindo que não houve alteração da afinidade da enzima pelo substrato celobiose com a imobilização. A imobilização da enzima em lentes PVA - Lentikats® resultou em um aumento dos valores de KM estimados em 3,61; 2,7 e 3,03 mM de celobiose, a 50°C, respectivamente, pelos métodos de Lineweaver - Burk, Hanes - Woolf e Solver indicando que a imobilização resultou em diminuição da afinidade da enzima imobilizada pelo substrato. A taxa de conversão relativa da solução 1,5 mM de celobiose em tampão acetato 0,5 M pH 5,0 a 50°C, utilizando-se ?-glicosidase imobilizada em lentes PVA - Lentikats® em processo contínuo foi de 100% após 5 h, entretanto a porcentagem de conversão diminuiu para 40% após 148 h. A ?-glicosidase livre e imobilizada produzida pelo micro-organismo A. niger LBA 02 foi capaz de hidrolisar as isoflavonas glicosiladas de soja em suas formas agliconas. O teor da isoflavonas agliconas aumentou no extrato de isoflavonas de soja tratadas com ?-glicosidase livre e imobilizada em lentes PVA - Lentikats® e sol-gel, sendo que a daidzeína aumentou aproximadamente 2,6; 10,8 e 12,2 vezes e o teor de genisteína aumentou 11,7; 11,4 e 11,4 vezes quando aplicada a enzima ?-glicosidase livre e imobilizada em lentes PVA - Lentikats® e sol-gel, respectivamente, ao final de 24 hAbstract: This work aimed to study the production of ?-glucosidase by Aspergillus sp. strain using agricultural residues such as wheat bran, passion fruit peel and sugarcane bagasse, the immobilization of the enzyme in different support such as lens - shaped PVA - Lentikats®, sol-gel, Eupergit, Amberlite, gelatin and calcium alginate and the application of free and immobilized enzyme in the conversion of isoflavone glucosides in soy isoflavone aglycones. The fungus was identified as Aspergillus niger LBA 02. The crude enzyme extract obtained from A. niger LBA 02 showed activity of ?-glucosidase, CM-cellulase, amylase, polygalacturonase and pectinase. It was obtained higher ?-glucosidase activity (44.81 U / g) in the fermentation of strain A. niger LBA 02 in semisolid media composed of 25 g of wheat bran and 5.7 mL of distilled water, after 240 h of fermentation at 30 °C. The effects of the addition of yeast extract and salts KH2PO4, NH4NO3, MgSO4.7H2O in wheat bran medium for production of ?-glucosidase by A. niger LBA 02 were not significant in the levels studied. The passion fruit peel waste and sugarcane bagasse added in the culture media of wheat bran did not act as inducers of the production of ?-glucosidase levels studied. Among the methods of immobilization tested, the sol-gel technique and lens - shaped PVA - Lentikats® showed better results for ?-glucosidase immobilization. The free enzyme showed optimum activity at 65 °C and pH 4.5, while the enzyme immobilized in sol-gel showed optimum activity in the range of 60 - 65 °C. A temperature of 50 °C was set as the working optimum temperature for the enzyme immobilized in lens - shaped PVA - Lentikats® because above 60 °C occurred melting of the lenses. Immobilization of ?-glucosidase did not alter the optimum pH of the enzyme activity, remaining at pH 4.5. Immobilization resulted in a small increase in the thermal stability of ?-glucosidase. The ?-glucosidase immobilized in lens - shaped PVA - Lentikats® showed to be more stable than the free enzyme after 3 h of treatment in the range of 45 to 55 °C. The half-life of the ?-glucosidase immobilized in sol-gel at 70 °C was 0.88 h. The ?-glucosidase immobilized in sol-gel showed about 10% residual activity after 3 h at 70 °C while free enzyme was inactivated after 1 h at 70 °C. The ?-glucosidase immobilized in sol-gel showed KM in the range of 1.0 to 1.25 mM of cellobiose at 60 °C, estimated by the method of Lineweaver - Burk, Hanes - Woolf and Solver while free enzyme showed KM values in the range of 0.92 to 1.69 mM of cellobiose, suggesting no change in the affinity of the enzyme for the substrate cellobiose after immobilization. The immobilized enzyme in lens - shaped PVA - Lentikats® resulted in an increase in the KM values estimated 3.61, 2.7 and 3.03 mM of cellobiose at 50 °C, respectively, by methods Lineweaver - Burk, Hanes - Woolf and Solver indicating that immobilization resulted in decreasing affinity of the immobilized enzyme to the substrate. Relative conversion rate of 1.5 mM cellobiose solution in acetate buffer 0.5 M pH 5.0 at 50 °C, using ?-glucosidase immobilized in lens - shaped PVA - Lentikats® in continuous process was 100% after 5 h, but the conversion decreased to 40% after 148 h. The ?-glucosidase produced by the micro-organism A. niger LBA 02, free and immobilized was able to hydrolyze isoflavone glycosides from soy in their aglycone forms. The content of isoflavone aglycones increased in soy isoflavones extract treated with ?-glucosidase free and immobilized in lens - shaped PVA - Lentikats® and sol-gel, and the daidzein increased approximately 2.6, 10.8 and 12.2 times, and genistein increased content of 11.7, 11.4 and 11.4 fold when applied to ?-glucosidase enzyme free and immobilized in lens - shaped PVA - Lentikats ® and sol-gel, respectively, at the end of 24 hDoutoradoCiência de AlimentosDoutora em Ciência de Alimento

Sugarcane starch: quantitative determination and characterization

Starch is found in sugarcane as a storage polysaccharide. Starch concentrations vary widely depending on the country, variety, developmental stage, and growth conditions. The purpose of this study was to determine the starch content in different varieties of sugarcane, between May and November 2007, and some characteristics of sugarcane starch such as structure and granules size; gelatinization temperature; starch solution filterability; and susceptibility to glucoamylase, pullulanase, and commercial bacterial and fungal α-amylase enzymes. Susceptibility to debranching amylolytic isoamylase enzyme from Flavobacterium sp. was also tested. Sugarcane starch had spherical shape with a diameter of 1-3 µm. Sugarcane starch formed complexes with iodine, which showed greater absorption in the range of 540 to 620 nm. Sugarcane starch showed higher susceptibility to glucoamylase compared to that of waxy maize, cassava, and potato starch. Sugarcane starch also showed susceptibility to debranching amylolytic pullulanases similar to that of waxy rice starch. It also showed susceptibility to α-amylase from Bacillus subtilis, Bacillus licheniformis, and Aspergillus oryzae similar to that of the other tested starches producing glucose, maltose, maltotriose, maltotetraose, maltopentose and limit α- dextrin

Potential Applications of Carbohydrases Immobilization in the Food Industry

Carbohydrases find a wide application in industrial processes and products, mainly in the food industry. With these enzymes, it is possible to obtain different types of sugar syrups (viz. glucose, fructose and inverted sugar syrups), prebiotics (viz. galactooligossacharides and fructooligossacharides) and isomaltulose, which is an interesting sweetener substitute for sucrose to improve the sensory properties of juices and wines and to reduce lactose in milk. The most important carbohydrases to accomplish these goals are of microbial origin and include amylases (α-amylases and glucoamylases), invertases, inulinases, galactosidases, glucosidases, fructosyltransferases, pectinases and glucosyltransferases. Yet, for all these processes to be cost-effective for industrial application, a very efficient, simple and cheap immobilization technique is required. Immobilization techniques can involve adsorption, entrapment or covalent bonding of the enzyme into an insoluble support, or carrier-free methods, usually based on the formation of cross-linked enzyme aggregates (CLEAs). They include a broad variety of supports, such as magnetic materials, gums, gels, synthetic polymers and ionic resins. All these techniques present advantages and disadvantages and several parameters must be considered. In this work, the most recent and important studies on the immobilization of carbohydrases with potential application in the food industry are reviewed

Potential Applications Of Carbohydrases Immobilization In The Food Industry.

Carbohydrases find a wide application in industrial processes and products, mainly in the food industry. With these enzymes, it is possible to obtain different types of sugar syrups (viz. glucose, fructose and inverted sugar syrups), prebiotics (viz. galactooligossacharides and fructooligossacharides) and isomaltulose, which is an interesting sweetener substitute for sucrose to improve the sensory properties of juices and wines and to reduce lactose in milk. The most important carbohydrases to accomplish these goals are of microbial origin and include amylases (α-amylases and glucoamylases), invertases, inulinases, galactosidases, glucosidases, fructosyltransferases, pectinases and glucosyltransferases. Yet, for all these processes to be cost-effective for industrial application, a very efficient, simple and cheap immobilization technique is required. Immobilization techniques can involve adsorption, entrapment or covalent bonding of the enzyme into an insoluble support, or carrier-free methods, usually based on the formation of cross-linked enzyme aggregates (CLEAs). They include a broad variety of supports, such as magnetic materials, gums, gels, synthetic polymers and ionic resins. All these techniques present advantages and disadvantages and several parameters must be considered. In this work, the most recent and important studies on the immobilization of carbohydrases with potential application in the food industry are reviewed.141335-6

A multicomponent system based on a blend of agroindustrial wastes for the simultaneous production of industrially applicable enzymes by solid-state fermentation

<div><p>Abstract This study reports the use of statistical mixture design as a tool for the simultaneous production of lipase, CMCase, α-amylase, and β-glucosidase by Aspergillus niger under solid-state fermentation. Wheat bran, soybean meal, cottonseed meal, and orange peel were used as substrates, either individually or combined in different formulations, to study their synergistic or antagonistic effects on production of the enzymes. The highest lipase (323 U g-1) and CMCase (10 U g-1) activities were detected after 48 h, while the maximum activities of α-amylase (18 U g-1) and β-glucosidase (15 U g-1) occurred at 72 and 96 h, respectively. Considering the substrate formulation, the ternary mixture of wheat bran (1/3), soybean meal (1/3), and cottonseed meal (1/3) was the most versatile, showing production of CMCase (>5 U g-1) and α-amylase (>8 U g-1) at 24 h, lipase (>320 U g-1) at 72 h, and β-glucosidase (>10 U g-1) at 48 h.</p></div