Adsorption of amyloglucosidase from Aspergillus niger NRRL 3122 using ion exchange resin

Amyloglucosidase enzyme was produced by Aspergillus niger NRRL 3122 from solid-state fermentation, using deffated rice bran as substrate. The effects of process parameters (pH, temperature) in the equilibrium partition coefficient for the system amyloglucosidase - resin DEAE-cellulose were investigated, aiming at obtaining the optimum conditions for a subsequent purification process. The highest partition coefficients were obtained using 0.025M Tris-HCl buffer, pH 8.0 and 25ºC. The conditions that supplied the highest partition coefficient were specified, the isotherm that better described the amyloglucosidase process of adsorption obtained. It was observed that the adsorption could be well described by Langmuir equation and the values of Qm and Kd estimated at 133.0 U mL-1 and 15.4 U mL-1, respectively. From the adjustment of the kinetic curves using the fourth-order Runge-Kutta algorithm, the adsorption (k1) and desorption (k2) constants were obtained through optimization by the least square procedure, and the values calculated were 2.4x10-3 mL U-1 min-1 for k1 and 0.037 min-1 for k2 .

Sequential production of amylolytic and lipolytic enzymes by bacterium strain isolated from petroleum contaminated soil

Amylases and lipases are highly demanded industrial enzymes in various sectors such as food, pharmaceuticals, textiles, and detergents. Amylases are of ubiquitous occurrence and hold the maximum market share of enzyme sales. Lipases are the most versatile biocatalyst and bring about a range of bioconversion reactions such as hydrolysis, inter-esterification, esterification, alcoholysis, acidolysis, and aminolysis. The objective of this work was to study the feasibility for amylolitic and lipolytic production using a bacterium strain isolated from petroleum contaminated soil in the same submerged fermentation. This was a sequential process based on starch and vegetable oils feedstocks. Run were performed in batchwise using 2% starch supplemented with suitable nutrients and different vegetable oils as a lipase inducers. Fermentation conditions were pH 5.0; 30 degrees C, and stirred speed (200 rpm). Maxima activities for amyloglucosidase and lipase were, respectively, 0.18 and 1,150 U/ml. These results showed a promising methodology to obtain both enzymes using industrial waste resources containing vegetable oils.

Modificação enzimática da farinha de arroz visando a produção de amido resistente

The aim of this work was to study the enzymatic modification on rice flour using lipase pancreatic and amyloglucosidase to obtain resistant starch. For this, Response Surface Methodology (RSM) was used to determine the best operating conditions for each enzyme. For lypase pancreatic, the highest value for resistant starch (45%) was achieved within 2 h reaction at pH 7 using an enzyme/substrate ratio of 4% (w/w) and Dp= 100/200 tyler. For amyloglucosidase, optima conditions corresponded to an enzyme/substrate ratio of 0,006 mL/g and Dp= 100/200 tyler at 45 ºC, yielding 57% of resistant starch in 2 h reaction. These results show the potential of using both enzymes to modified rice flour, increasing the resistant starch in about 5.7 folds in relation to the flour without treatment (resistant starch=10.6%).

ENZYMATIC DETERMINATION OF STARCH IN DOCE DE LEITE USING DIALYSIS

The importance of starch for the food industry makes it necessary to develop new, fast, economic and accurate methodologies for its quantification. In the present paper starch hydrolysis using commercial enzymes of industrial grade are studied aiming to develop an easy and cheap analysis, available to a greater number of industries and technicians. The proposed method is simple, divided in a first step where soluble sugars are eliminated from the samples by using dialysis, followed by starch hydrolysis of the retained fraction with a thermoresistent bacterial alfa-amylase (Termamyl 120L®) and an amyloglucosidase (AMG 300L®). The hydrolysis conditions were those suggested by the enzyme producer. After the hydrolysis step the material was dialysed again for the extraction of glucose that was quantified by the glucose-oxidase colorimetric reactant. The results allowed the construction of calibration equations for starch determination on the analyzed samples. These samples were produced on a laboratory scale and native and acid-modified corn starches were added in known concentrations. By considering the final dilutions employed for glucose determination on the samples, it was possible to confirm that they were identical to that of the glucose-oxidase reactant calibration.

Concentração de enzimas amilolíticas na hidrólise do amido de gengibre

Uma possibilidade de incremento na cadeia produtiva do gengibre seria o uso de rizomas desclassificados para comercialização in natura, ou mesmo o resíduo da extração de óleos, como matérias-primas para a obtenção de bebidas destiladas. O amido não é diretamente fermentável, necessitando de uma hidrólise prévia de suas cadeias para a obtenção de glicose. Neste trabalho, objetivou-se avaliar o efeito das concentrações de amilases sobre o perfil de açúcares e rendimento no processo de hidrólise-sacarificação de gengibre. O processo seguiu o delineamento central composto rotacional para dois fatores, totalizando 11 tratamentos. Os resultados obtidos mostram o efeito das concentrações da α-amilase (Termamyl 2X) e da amiloglucosidase (AMG 300L) sobre o teor de glicose do hidrolisado e efeito da concentração de amiloglucosidase sobre o teor de dextrina. Os maiores rendimentos de hidrólise da suspensão de gengibre foram obtidos nas condições de elevada concentração de -amilase e amiloglucosidase.

Structural and functional characteristics of selected soft wheat starches

Starches from eight soft wheat samples (two parent lines and six offspring) were isolated; relationships between their structures and properties were examined. Branch chain-length distributions of amylopectins were determined by using high-performance anion exchange chromatography equipped with an amyloglucosidase reactor and a pulsed amperometric detector (HPAEC-ENZ-PAD). Results showed that the average chain length of the eight samples varied at DP 25.6-26.9. Starch samples of lines 02, 60, 63, 95, and 114 consisted of amylopectins with more long chains (DP greater than or equal to 37) and longer average chain length (DP 26.2-26.9) than that of other samples. These starch samples of longer branch chain length displayed higher gelatinization temperatures (55.3-56.5degreesC) than that of other samples (54.4-54.9degreesC) and higher peak viscosity (110-131 RVU) and lower pasting temperature (86.3-87.6degreesC) than others (83-100 RVU and 88.2-88.9degreesC, respectively). The M-w of amylopectins, determined by using high-performance size exclusion chromatography equipped with multiangle laser-light scattering and refractive index detectors (HPSEC-MALLS-RI), were similar for all samples (6.17 x 10(8) to 6.97 x 10(8)). There were no significant differences in amylose and phosphorus contents between samples. These results indicated that physical properties of wheat starch were affected by the branch-chain length of amylopectin.

Estrutura dos grânulos de amido de milho normal e ceroso

Visando obter informações a respeito da estrutura dos grânulos, amidos de milho normal e ceroso foram isolados e submetidos à ação da a-amilase e amiloglucosidase. Para elucidar a estrutura dos grânulos, os resíduos desta hidrólise foram submetidos à cromatografia de permeção em gel Sephadex G-50, diretamente e após sucessivas digestões enzimáticas com pululanase e b-amilase. Os resultados mostraram que existem diferenças nos resíduos dos amidos de milho ceroso e normal, tratados com a-amilase e amiloglucosidase. No resíduo do amido de milho ceroso, os perfis de eluição mostraram duas frações a 290 e 350 ml (picos I e II) respectivamente, que não eram suscetíveis ao ataque da a-amilase e amiloglucosidase, indicando que estas frações faziam parte das zonas cristalinas do amido. Estas frações também faziam parte das áreas cristalinas no amido normal. A presença do pico V à 390 ml na a-glucana do amido de milho normal sugeriu que além das duas frações não suscetíveis à hidrólise existia outra que também participava das zonas cristalinas deste amido como regiões não suscetíveis às enzimas formando, consequentemente, rede cristalina fortemente associada. A presença deste pico a 390 ml sugeriu arranjo cristalino distinto entre o amido de milho ceroso e o normal.

Aproveitamento do resíduo da produção de etanol a partir de farelo de mandioca, como fonte de fibras dietéticas

O presente trabalho teve por objetivo analisar resíduos do farelo de mandioca resultantes de processos de hidrólise enzimática para obtenção de etanol; visando o aproveitamento destes como fonte de fibras dietéticas. Foram realizados quatro ensaios enzimáticos utilizando as enzimas amilolíticas, a-amilase e amiloglucosidase, complementadas ou não com celulase e/ou pectinase. Os resíduos foram caracterizados quanto à composição centesimal, pH, acidez, perfil de açúcares e quanto às fibras (FDA, FDN, celulose, hemicelulose, lignina, açúcares neutros). Realizou-se também a análise microscópica dos resíduos. Pelos resultados obtidos na caracterização dos resíduos calculou-se a energia metabolizável aparente (EM). Observou-se que independente do ensaio enzimático todos os resíduos podem ser usados como fonte de fibras insolúveis. Os resíduos resultantes dos ensaios com pectinase apresentaram uma proporção aproximada de 1:1:1 de amido, fibras e açúcares, sendo a glicose o açúcar majoritário, e com energia metabolizável aparente de cerca de 2,6 kcal/g. Já os resíduos, onde não se utilizou a pectinase a proporção foi de 2:1:1 aproximadamente e a energia 3,1 kcal/g. A análise microscópica dos resíduos mostrou a presença de amido não hidrolisado preso às células em todos os ensaios enzimáticos sendo que, nos resíduos dos ensaios com pectinase a quantidade observada foi bem inferior aos demais. Uma possível alternativa para diminuir o valor calórico dos resíduos seria a lavagem com água após a prensagem para extração do hidrolisado para fermentação.

Preparation of silica with controlled pore sizes for enzyme immobilization

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

FACTORS THAT AFFECT THE ENZYMATIC DEGRADATION OF NATURAL STARCH GRANULES - EFFECT OF THE SIZE OF THE GRANULES

In this study the relationship between the enzymatic susceptibility and the size of the com and cassava starch granules was studied. The starch granules were separated by size and classified according to their average diameter in: a) larger than 16 mum; b) between 15 and 10 mum and c) smaller than 10 mum. The starch granules of various sizes were hydrolyzed by bacterial alpha-amylase and fungal amyloglucosidase. The results showed a relationship between the enzymatic susceptibility and the size of the starch granules; smaller size of the starch granules resulted in a higher percentage of hydrolysis. A basic difference in the mode of action of enzymes on small and large granules was observed. Enzymatic attack on the large granules was characterized by considerable surface corrosion, mainly at the radial axis. For small granules, the enzymatic action occurred on the surface of the granules and was characterized by an erosion with solubilization of the granules. Chemical and physical analysis of the starches suggested that hydrolysis should occur mainly at the amorphous areas of the granules.

The structure of waxy corn starch: Effect of granule size

The granules of waxy corn starch were isolated and various samples were separated by size and classified according to their average diameter in: non-separated granules (N), granules with diameter < 15 μm (S) and granules with diameter ≥ 15 μm (L). The samples were hydrolyzed by bacterial α-amylase and fungal amyloglucosidase. The starch granules remaining after enzymatic hydrolysis were analysed by X-ray diffraction and optical and scanning electron microscopy. Sephadex G-50 gel permeation chromatography of the dissolved residues from the hydrolysis of the N and S samples was performed directly and after successive enzymatic digestion with pullulanase and β-amylase. The results showed that the percentage of hydrolysis increased with a decrease in diameter. No apparent differences in waxy corn starch when observed under light and scanning electronic microscope were observed, regardless of diameter and enzyme action, although both large and small granules showed extensive surface corrosion after enzymatic attack. X-ray analysis suggested a decrease in the quantity of crystalline areas in the smaller granules, which would explain the high percentage of hydrolysis evidenced by these granules. The elution patterns of the α-glucans of both starches (N and S) were similar and reveled the presence of two fractions which were not susceptible to a-amylase and amyloglucosidase attack suggesting that these fractions were involved in the waxy corn starch crystalline regions. Debranching with pullulanase followed by gel-permeation chromatography showed that the amylopectins from the starch granules studied contained three groups of unit chains instead of the two reported in the literature.

Efeito da hidrólise enzimática seguida da moagem em moinho de bolas sobre as características estruturais e físico-químicas do amido de madioquinha-salsa

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo da hidrólise enzimática do amido de mandioquinha-salsa (Arracacia xanthorrhiza): efeito do tamanho dos grânulos

Pós-graduação em Engenharia e Ciência de Alimentos - IBILCE

Produção de destilado alcoólico a partir de mosto fermentado de batata-doce

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de frutose a partir de hidrolisado enzimático de amido de mandioca

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)