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Fang Zhong – One of the best experts on this subject based on the ideXlab platform.

Charles F. Shoemaker – One of the best experts on this subject based on the ideXlab platform.

  • effects of Alcalase protease n treatments on rice starch isolation and their effects on its properties
    Food Chemistry, 2009
    Co-Authors: Charles F. Shoemaker, Changrong Luo, Fang Zhong
    Abstract:

    Abstract The effects of different protease treatments on rice starches and their properties were studied. The rice starches produced from protease N exhibited higher pasting viscosities than those produced from Alcalase. The hot pastes of the starches produced from protease N also showed higher elastic moduli, zero-order Newtonian viscosities and yield stresses than those produced from Alcalase. No differences were found in the crystalline pattern, thermal properties, granules appearance, and average molecular weight ( M w ) of the rice starches between the two protease treatments. But the M w of the pasted starch produced from protease N was higher than that produced from Alcalase. When additional protease was added to the isolated starches and the mixture pasted, the M w of the starches pasted with added Alcalase was significant lower than that of the starches pasted with added protease N. The reduction in molecular weight suggested that Alcalase had modified the starch molecules during pasting.

  • Effects of Alcalase/Protease N treatments on rice starch isolation and their effects on its properties
    Food Chemistry, 2009
    Co-Authors: Charles F. Shoemaker, Changrong Luo, Fang Zhong
    Abstract:

    Abstract The effects of different protease treatments on rice starches and their properties were studied. The rice starches produced from protease N exhibited higher pasting viscosities than those produced from Alcalase. The hot pastes of the starches produced from protease N also showed higher elastic moduli, zero-order Newtonian viscosities and yield stresses than those produced from Alcalase. No differences were found in the crystalline pattern, thermal properties, granules appearance, and average molecular weight ( M w ) of the rice starches between the two protease treatments. But the M w of the pasted starch produced from protease N was higher than that produced from Alcalase. When additional protease was added to the isolated starches and the mixture pasted, the M w of the starches pasted with added Alcalase was significant lower than that of the starches pasted with added protease N. The reduction in molecular weight suggested that Alcalase had modified the starch molecules during pasting.

Paulo Waldir Tardioli – One of the best experts on this subject based on the ideXlab platform.

  • Hydrolysis of Proteins by Immobilized‐Stabilized Alcalase‐Glyoxyl Agarose
    Biotechnology progress, 2003
    Co-Authors: Paulo Waldir Tardioli, Justo Pedroche, Roberto Fernandez-lafuente, Raquel L. C. Giordano, Jose M. Guisan
    Abstract:

    This paper presents stable Alcalase-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced by immobilizing-stabilizing Alcalase on cross-linked 10% agarose beads, using low and high activation grades of the support and different immobilization times. The Alcalase glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde and CNBr as activation reactants. The performance of derivatives in the hydrolysis of casein was also tested. At pH 8.0 and 50 degrees C, Alcalase derivatives produced with 1 h of immobilization time on agarose activated with glutaraldehyde, CNBr, and low and high glyoxyl groups concentration presented half-lives of ca. 10, 29, 60, and 164 h, respectively. More extensive immobilization monotonically led to higher stabilization. The most stabilized Alcalase-glyoxyl derivative was produced using 96 h of immobilization time and high activation grade of the support. It presented half-life of ca. 23 h, at pH 8.0 and 63 degrees C and was ca. 500-fold more stable than the soluble enzyme. Thermal inacinactivation of all derivatives followed a single-step non-first-order kinetics. The most stable derivative presented ca. 54% of the activity of the soluble enzyme for the hydrolysis of casein and of the small substrate Boc-Ala-ONp. This behavior suggests that the decrease in activity was due to enzyme distortion but not to wrong orientation. The hydrolysis degree of casein at 80 degrees C with the most stabilized enzyme was 2-fold higher than that achieved using soluble enzyme, as a result of the thermal inacinactivation of the latter. Therefore, the high stability of the new Alcalase-glyoxyl derivative allows the design of continuous processes to hydrolyze proteins at temperatures that avoid microbial growth.

  • hydrolysis of proteins by immobilized stabilized Alcalase glyoxyl agarose
    Biotechnology Progress, 2003
    Co-Authors: Paulo Waldir Tardioli, Justo Pedroche, Raquel L. C. Giordano, Roberto Fernandezlafuente, Jose M. Guisan
    Abstract:

    This paper presents stable Alcalase-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced by immobilizing-stabilizing Alcalase on cross-linked 10% agarose beads, using low and high activation grades of the support and different immobilization times. The Alcalase glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde and CNBr as activation reactants. The performance of derivatives in the hydrolysis of casein was also tested. At pH 8.0 and 50 degrees C, Alcalase derivatives produced with 1 h of immobilization time on agarose activated with glutaraldehyde, CNBr, and low and high glyoxyl groups concentration presented half-lives of ca. 10, 29, 60, and 164 h, respectively. More extensive immobilization monotonically led to higher stabilization. The most stabilized Alcalase-glyoxyl derivative was produced using 96 h of immobilization time and high activation grade of the support. It presented half-life of ca. 23 h, at pH 8.0 and 63 degrees C and was ca. 500-fold more stable than the soluble enzyme. Thermal inacinactivation of all derivatives followed a single-step non-first-order kinetics. The most stable derivative presented ca. 54% of the activity of the soluble enzyme for the hydrolysis of casein and of the small substrate Boc-Ala-ONp. This behavior suggests that the decrease in activity was due to enzyme distortion but not to wrong orientation. The hydrolysis degree of casein at 80 degrees C with the most stabilized enzyme was 2-fold higher than that achieved using soluble enzyme, as a result of the thermal inacinactivation of the latter. Therefore, the high stability of the new Alcalase-glyoxyl derivative allows the design of continuous processes to hydrolyze proteins at temperatures that avoid microbial growth.

  • Hidrólise controlada de proteínas do soro de queijo usando carboxipeptidase A e Alcalase imobilizadas multipontualmente em agarose.
    Universidade Federal de São Carlos, 2003
    Co-Authors: Paulo Waldir Tardioli
    Abstract:

    Hidrolisados protéicos de alto valor agregado podem ser obtidos através da hidrólise seqüencial de proteínas com tripsina, quimotripsina, carboxipeptidase A (CPA) e Alcalase (preparação comercial de subtilisina). A viabilidade econômica do processo requer a utilização de enzimas imobilizadas e estabilizadas e o conhecimento da cinética das reações catalisadas com esse tipo de biocatalisador. Visando contribuir para o desenvolvimento de tal processo, os objetivos deste trabalho foram preparar derivados estáveis de CPA e Alcalase e estudar a cinética da hidrólise de polipeptídios. Esses polipeptídios foram produzidos por hidrólise seqüencial de proteínas do soro de queijo com tripsina e quimotripsina. Utilizandose agarose entrecruzada (6% p/p para CPA e 10% p/p para Alcalase) como suporte de imobilização, foram estudados diferentes métodos de ativação e condições de imobilização. Suportes glioxil-agarose altamente ativados (75 e 210 eqv de grupos aldeídos por mililitro de suporte, respectivamente para CPA e Alcalase) 25oC, pH 10,05 e tempo prolongado de contato (48 horas para CPA e 96 horas para Alcalase) produziram os melhores derivados. Os derivados CPA-glioxil agarose-6% e Alcalase-glioxil agarose-10% eram aproximadamente 213 e 515 vezes mais estáveis que as respectivas enzimas na forma solúvel. Esses derivados estabilizados retiveram 42% (para CPA-glioxil agarose-6%) e 54% (para Alcalase-glioxil agarose-10%) da atividade imobilizada, medidas com substratos de menor massa molecular (hipuril-L-Phe para CPA, e Boc-Ala-ONp para Alcalase) e substratos de maior massa molecular (polipeptídios com Phe carboxi-terminal para CPA, e caseína para Alcalase). Esses resultados mostraram que toda a perda de atividade estava associada à distorção da molécula de enzima imobilizada, devido a multi-interação enzima-suporte. Derivados preparados em glutaraldeído-agarose-6% apresentaram impedimentos estéricos na hidrólise de substratos macromoleculares. A análise de aminoácidos de hidrolisados ácidos das enzimas solúveis e imobilizadas (para os derivados mais estáveis) mostrou que aproximadamente 30 e 40%, para CPA e Alcalase, dos resíduos de lisina ligaram-se no suporte, sugerindo a existência de uma intensa ligação covalente multipontual entre a enzima e o suporte. As temperaturas de máximas taxas de hidrólise, usando respectivamente os derivados estabilizados de CPA e Alcalase, foram 20oC e 10oC mais elevadas que aquelas obtidas para as respectivas enzimas solúveis. O derivado CPA-glioxil mais estável pôde ser eficientemente utilizado na hidrólise de polipeptídios (proteínas do soro de queijo hidrolisadas com tripsina e quimotripsina) a altas temperaturas (por exemplo, 60oC), liberando duas vezes mais aminoácidos aromáticos (Tyr, Phe e Trp) do que a enzima solúvel, sob as mesmas condições operacionais. O grau de hidrólise de caseína, a 80oC, obtido com o derivado Alcalase-glioxil mais estável, foi duas vezes maior que aquele obtido com a enzima solúvel. Assim, os derivados produzidos permitem o projeto de um processo contínuo para a produção de hidrolisados protéicos, compostos de pequenos peptídios e com uma baixa concentração de aminoácidos aromáticos. Esse processo pode ser conduzido a alta temperatura, evitando-se assim problemas de contaminação microbiana do meio reacional. A hidrólise de resíduos carboxi-terminais a 45oC (pH 7,0), catalisada pelo derivado CPA-glioxil, pôde ser adequadamente representada por cinética de Michaelis-Menten, com inibição pelo substrato e produto. O modelo cinético foi representado em termos de ligações peptídicas carboxiterminais hidrolisáveis pela CPA, sem considerar-se a natureza do resíduo a ser liberado. A concentração de cada aminoácido liberado em função do tempo de hidrólise pôde ser ajustada por modelos empíricos (hiperbólico e decaimento exponencial). Assim, a partir da cinética de hidrólise total, é possível estimar-se a concentração de cada aminoácido em função do tempo de hidrólise. A hidrólise catalisada pelo derivado CPA-glioxil agarose-6%, com alta carga enzimática imobilizada, não foi limitada pela resistência difusional intrapartícula. A hidrólise de peptídios (bateladas de longa duração) a 50oC (pH 9,5), catalisada pelo derivado Alcalase-glioxil agarose-10%, pôde ser adequadamente representada por cinética de Michaelis-Menten com inibição pelo produto, e os parâmetros cinéticos Vmax, KM e KI foram correlacionados com o grau de hidrólise inicial do substrato (grau de hidrólise total obtido pela prévia ação de tripsina e quimotripsina sobre as proteínas do soro de queijo). Hidrólises em batelada de longa duração, catalisadas por Alcalase-glioxil agarose-10% com alta carga enzimática imobilizada, apresentaram efeitos de difusão, com um fator de efetividade, ηI, de aproximadamente 0,5.High value food protein hydrolysates can be obtained by sequential hydrolysis of proteins with trypsin, chymotrypsin, carboxypeptidase A (CPA) and Alcalase (commercial preparation of subtilisin). For the process to be economically feasible, immobilized and stabilized enzymes should be used, and the kinetics of the reactions with this kind of biocatalyst must be known. To contribute to the development of such a process, this work focused on preparing stable CPA and Alcalase derivatives, and on studying the kinetics of hydrolysis of polypeptides. These polypeptides were produced after the sequential hydrolysis of cheese wheywhey proteins with trypsin and chymotrypsin. Cross-linked agarose beads (6% w/w for CPA, and 10% w/w for Alcalase) were used as immobilization support, and different methods of activation and immobilization conditions were studied. A highly activated glyoxyl-agarose support (75 and 210 eqv of aldehyde groups per milliliter of support, respectively for CPA and Alcalase), 25oC, pH 10.05, and longer contact time (48 hours for CPA and 96 hours for Alcalase), provided the best derivatives. CPA-glyoxyl agarose-6% and Alcalase-glyoxyl agarose-10% derivatives were ca. 213- and 515-fold more stable than the soluble enzymes. These stabilized derivatives retained 42% (for CPA-glyoxyl agarose– 6%) and 54% (for Alcalase-glyoxyl agarose-10%) of the immobilized activity, assessed with small substrates (hippuryl-L-Phe for CPA, and Boc-Ala-ONp for Alcalase) and large substrates (Phe carboxy-terminal polypeptides for CPA, and casein for Alcalase). These results showed that all activity losses were caused by the distortion of the immobilized enzyme molecule, due to the enzyme-support multi-interaction. Derivatives prepared using glutaraldehyde-agarose presented spatial hindrances when hydrolysis of macromolecular substrates was taking place. The amino acid analysis of acid hydrolysates of the soluble and immobilized enzymes (for the more stable derivatives) showed that ca. 30 and 40%, for CPA and Alcalase, of the lysine residues were linked to the support, suggesting that there is intense multi-point interactions between enzyme and support, through covalent linkages. The temperatures for maximum hydrolysis rates, using respectively stabilized CPA and Alcalase derivatives, were 20oC and 10oC higher than the ones obtained using soluble enzymes. The most stable CPA-glyoxyl derivative could efficiently be used for polypeptides (cheese wheywhey proteins hydrolyzed with trypsin and chymotrypsin) hydrolysis at high temperatures (e.g., 60oC), releasing ca. 2-fold more aromatic amino acids (Tyr, Phe and Trp) than the soluble enzyme, under the same operational conditions. The casein degree of hydrolysis, at 80oC, obtained using the most stable Alcalase-glyoxyl derivative, was 2-fold higher than the one obtained with the soluble enzyme. Hence, the produced derivatives allow the design of a continuous process for the production of protein hydrolysates, which are composed of small peptides and have a low concentration of aromatic amino acids. This process can use higher temperature, avoiding microbial growth in the reaction medium. The C-terminal residues hydrolysis at 45oC (pH 7.0), catalyzed by CPA-glyoxyl, could be adequately represented by Michaelis-Menten kinetics, with substrate and product inhibition. The kinetic model was expressed in terms of C-terminal peptide bonds that can be hydrolyzed by CPA, regardless of the amino acid released. The concentration of each released amino acid as a function of the time of reaction could be well fitted by empirical models (hyperbolic or exponential decay). Hence, from the kinetics of total hydrolysis, it is possible to estimate the concentration of each amino acid as function of time. The hydrolysis catalyzed by the highly-loaded CPA-glyoxyl agarose-6% derivative was not limited by intra-particle diffdiffusion resistance. The hydrolysis of peptides (long-time batch) at 50oC (pH 9.5), catalyzed by Alcalase-glyoxyl agarose-10% derivative, could be adequately represented by Michaelis-Menten kinetics with product inhibition, and the kinetic parameters Vmax, KM e KI were correlated against the substrate initial degree of hydrolysis (total degree of hydrolysis obtained by previous action of trypsin and chymotrypsin on cheese wheywhey proteins). Long-time batch hydrolyses, catalyzed by highly-loaded Alcalase-glyoxyl agarose-10% derivative, presented diffusion effects, with effectiveness coefficient, ηI, of ca. 0.5

Jose M. Guisan – One of the best experts on this subject based on the ideXlab platform.

  • Hydrolysis of Proteins by Immobilized‐Stabilized Alcalase‐Glyoxyl Agarose
    Biotechnology progress, 2003
    Co-Authors: Paulo Waldir Tardioli, Justo Pedroche, Roberto Fernandez-lafuente, Raquel L. C. Giordano, Jose M. Guisan
    Abstract:

    This paper presents stable Alcalase-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced by immobilizing-stabilizing Alcalase on cross-linked 10% agarose beads, using low and high activation grades of the support and different immobilization times. The Alcalase glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde and CNBr as activation reactants. The performance of derivatives in the hydrolysis of casein was also tested. At pH 8.0 and 50 degrees C, Alcalase derivatives produced with 1 h of immobilization time on agarose activated with glutaraldehyde, CNBr, and low and high glyoxyl groups concentration presented half-lives of ca. 10, 29, 60, and 164 h, respectively. More extensive immobilization monotonically led to higher stabilization. The most stabilized Alcalase-glyoxyl derivative was produced using 96 h of immobilization time and high activation grade of the support. It presented half-life of ca. 23 h, at pH 8.0 and 63 degrees C and was ca. 500-fold more stable than the soluble enzyme. Thermal inactivation of all derivatives followed a single-step non-first-order kinetics. The most stable derivative presented ca. 54% of the activity of the soluble enzyme for the hydrolysis of casein and of the small substrate Boc-Ala-ONp. This behavior suggests that the decrease in activity was due to enzyme distortion but not to wrong orientation. The hydrolysis degree of casein at 80 degrees C with the most stabilized enzyme was 2-fold higher than that achieved using soluble enzyme, as a result of the thermal inactivation of the latter. Therefore, the high stability of the new Alcalase-glyoxyl derivative allows the design of continuous processes to hydrolyze proteins at temperatures that avoid microbial growth.

  • hydrolysis of proteins by immobilized stabilized Alcalase glyoxyl agarose
    Biotechnology Progress, 2003
    Co-Authors: Paulo Waldir Tardioli, Justo Pedroche, Raquel L. C. Giordano, Roberto Fernandezlafuente, Jose M. Guisan
    Abstract:

    This paper presents stable Alcalase-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced by immobilizing-stabilizing Alcalase on cross-linked 10% agarose beads, using low and high activation grades of the support and different immobilization times. The Alcalase glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde and CNBr as activation reactants. The performance of derivatives in the hydrolysis of casein was also tested. At pH 8.0 and 50 degrees C, Alcalase derivatives produced with 1 h of immobilization time on agarose activated with glutaraldehyde, CNBr, and low and high glyoxyl groups concentration presented half-lives of ca. 10, 29, 60, and 164 h, respectively. More extensive immobilization monotonically led to higher stabilization. The most stabilized Alcalase-glyoxyl derivative was produced using 96 h of immobilization time and high activation grade of the support. It presented half-life of ca. 23 h, at pH 8.0 and 63 degrees C and was ca. 500-fold more stable than the soluble enzyme. Thermal inactivation of all derivatives followed a single-step non-first-order kinetics. The most stable derivative presented ca. 54% of the activity of the soluble enzyme for the hydrolysis of casein and of the small substrate Boc-Ala-ONp. This behavior suggests that the decrease in activity was due to enzyme distortion but not to wrong orientation. The hydrolysis degree of casein at 80 degrees C with the most stabilized enzyme was 2-fold higher than that achieved using soluble enzyme, as a result of the thermal inactivation of the latter. Therefore, the high stability of the new Alcalase-glyoxyl derivative allows the design of continuous processes to hydrolyze proteins at temperatures that avoid microbial growth.

Changrong Luo – One of the best experts on this subject based on the ideXlab platform.

  • effects of Alcalase protease n treatments on rice starch isolation and their effects on its properties
    Food Chemistry, 2009
    Co-Authors: Charles F. Shoemaker, Changrong Luo, Fang Zhong
    Abstract:

    Abstract The effects of different protease treatments on rice starches and their properties were studied. The rice starches produced from protease N exhibited higher pasting viscosities than those produced from Alcalase. The hot pastes of the starches produced from protease N also showed higher elastic moduli, zero-order Newtonian viscosities and yield stresses than those produced from Alcalase. No differences were found in the crystalline pattern, thermal properties, granules appearance, and average molecular weight ( M w ) of the rice starches between the two protease treatments. But the M w of the pasted starch produced from protease N was higher than that produced from Alcalase. When additional protease was added to the isolated starches and the mixture pasted, the M w of the starches pasted with added Alcalase was significant lower than that of the starches pasted with added protease N. The reduction in molecular weight suggested that Alcalase had modified the starch molecules during pasting.

  • Effects of Alcalase/Protease N treatments on rice starch isolation and their effects on its properties
    Food Chemistry, 2009
    Co-Authors: Charles F. Shoemaker, Changrong Luo, Fang Zhong
    Abstract:

    Abstract The effects of different protease treatments on rice starches and their properties were studied. The rice starches produced from protease N exhibited higher pasting viscosities than those produced from Alcalase. The hot pastes of the starches produced from protease N also showed higher elastic moduli, zero-order Newtonian viscosities and yield stresses than those produced from Alcalase. No differences were found in the crystalline pattern, thermal properties, granules appearance, and average molecular weight ( M w ) of the rice starches between the two protease treatments. But the M w of the pasted starch produced from protease N was higher than that produced from Alcalase. When additional protease was added to the isolated starches and the mixture pasted, the M w of the starches pasted with added Alcalase was significant lower than that of the starches pasted with added protease N. The reduction in molecular weight suggested that Alcalase had modified the starch molecules during pasting.