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Evaluation of the operational conditions of the glucose oxidase and catalase multienzymatic system through enzyme co-immobilization on amino hierarchical porous silica
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| DOI | 10.1016/J.CARRES.2024.109096 | ||||
| Año | 2024 | ||||
| Tipo | artículo de investigación |
Citas Totales
Autores Afiliación Chile
Instituciones Chile
% Participación
Internacional
Autores
Afiliación Extranjera
Instituciones
Extranjeras
Abstract
Hexaric acids have attracted attention lately because they are platform chemicals for synthesizing pharmaceuticals. In particular, gluconic acid is one of the most studied because it is readily available in nature. In this work, operational conditions like temperature and pH were evaluated for the enzymatic production of gluconic acid. For this purpose, glucose oxidase (GOx) and catalase (CAT) were individually immobilized and co -immobilized using amino -silica as support. The catalytic performance of the enzymes both as separate biocatalysts (GOx or CAT) and as an enzymatic complex (GOx-CAT) was assessed in terms of enzymatic activity and stability at temperatures 45 degrees C and 50 degrees C and pH 6 to 8. The results show that CAT is a key enzyme for gluconic acid production as it prevents GOx from being inhibited by H 2 O 2 . However, CAT was found to be less stable than GOx. Therefore, different GOx to CAT enzymatic ratios were studied, and a ratio of 1 - 3 was determined to be the best. The highest glucose conversion conditions were 45 degrees C and pH 7.0 for 24 h. Regarding the biocatalyst reuse, GOxCAT retained more than 70% of its activity after 6 reaction cycles. These results contribute to further knowledge and application of oxidases for hexaric acid production and shed greater light on the role of the glucose oxidase/ catalase pair in better catalytic performance. Both enzymes were immobilized in one pot, which is relevant for their potential use in industry; an enzyme system was obtained in a single step.
| Ord. | Autor | Género | Institución - País |
|---|---|---|---|
| 1 | Galaz, Tamara | - |
Pontificia Universidad Católica de Valparaíso - Chile
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| 2 | Ottone, Carminna | - |
Pontificia Universidad Católica de Valparaíso - Chile
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| 3 | RODRIGUEZ-NUNEZ, KAREN | Mujer |
Universidad de la Serena - Chile
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| 4 | BERNAL-ZULUAGA, CLAUDIA PATRICIA | Mujer |
Universidad de la Serena - Chile
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| Fuente |
|---|
| Fondo Nacional de Desarrollo Científico y Tecnológico |
| Pontificia Universidad Católica de Valparaíso |
| VINCI |
| Agencia Nacional de Investigación y Desarrollo |
| ANID Chile through FONDECyT |
| VINCI-DI PUCV |
| DI PUCV |
| APTES |
| Agradecimiento |
|---|
| ANID Chile funded this research through FONDECYT 1220134 and granted it to C.B. |
| The silica support was synthesized from a reaction mixture with the following molar ratio of reagents: SiO2: Na2O: CTAB: EtAc: H2O = 1: 0.3: 0.24: 7.2: 193 (SiO2:Na2O refers to Na2SiO3). The mixture was first incubated at 80 °C for 48 h. Then, the solid was dried at 60 °C and calcined at 540 °C for 6 h. The synthesized silica was chemically modified with aminopropyl groups, now called silica amino (SA). The synthesized silica was silylated with APTES to obtain the SA support using the protocol that Bernal et al. reported with some modifications [29]: 30 mL of toluene was added to 5% APTES per g of silica under magnetic stirring. After refluxing at 105 °C for 5 h, the material was filtered and washed with acetone, water, and pH 7 phosphate buffer.The co-immobilization of GOx and CAT was accomplished by contacting 1 g of SA support and 30 mg of total protein at a 1:1 GOx:CAT protein mass ratio for 48 h. The reactions were conducted at 45 °C, pH 7, and the glucose concentration was 8 g L−1, 16 U of GOx, and 58 U of CAT. The reactions were carried out in bottles with screw caps and shaken in a shaker with temperature control for a final volume of 20 mL. The immobilization yield of activity (YE) and immobilization yield of protein (YP) were calculated according to Eq. (1) and Eq. (2), respectively [35].Thermal stability is an essential parameter for preparing novel biocatalysts, especially when aiming for industrial applications like gluconic acid (GA) synthesis. Fig. 3 shows the thermal stability of the GOx and CAT derivates in SA supports. The inactivation curves were modeled according to the theory of enzymatic inactivation [36]. Both free and immobilized enzymes followed a first-order inactivation model at 45 °C and 50 °C (Eq. (3)). It should be noted that this temperature range was chosen based on the optimal temperatures of GOx and CAT from Fig. 2. In all cases, a decrease in activity over time was observed. The stabilizing effect of immobilization is evident for GOx (Fig. 3a), where GOX-SA kept higher residual activity than the free enzymes at 45 °C and 50 °C. This agrees with the reported stabilities for GOx in other works, where free and immobilized enzymes maintain their activity at elevated temperatures, and the immobilized enzyme improves greatly over the free enzyme [46,52].ANID Chile funded this research through FONDECYT 1220134 and granted it to C.B.The authors gratefully acknowledge Luigi Tavernini for providing help with the language and Catalina Morales and Mariela Muñoz for HPLC measurements. C. Ottone thanks the financial support from VINCI-DI PUCV (DI regular 039.316/2023). C. Bernal acknowledges the financial support from ANID Chile through FONDECYT 1220134. T. Galaz thanks the ANID scholarship N° 22200791 and PUCV for the fee waiver scholarship. |
| The silica support was synthesized from a reaction mixture with the following molar ratio of reagents: SiO2: Na2O: CTAB: EtAc: H2O = 1: 0.3: 0.24: 7.2: 193 (SiO2:Na2O refers to Na2SiO3). The mixture was first incubated at 80 °C for 48 h. Then, the solid was dried at 60 °C and calcined at 540 °C for 6 h. The synthesized silica was chemically modified with aminopropyl groups, now called silica amino (SA). The synthesized silica was silylated with APTES to obtain the SA support using the protocol that Bernal et al. reported with some modifications [29]: 30 mL of toluene was added to 5% APTES per g of silica under magnetic stirring. After refluxing at 105 °C for 5 h, the material was filtered and washed with acetone, water, and pH 7 phosphate buffer.The co-immobilization of GOx and CAT was accomplished by contacting 1 g of SA support and 30 mg of total protein at a 1:1 GOx:CAT protein mass ratio for 48 h. The reactions were conducted at 45 °C, pH 7, and the glucose concentration was 8 g L−1, 16 U of GOx, and 58 U of CAT. The reactions were carried out in bottles with screw caps and shaken in a shaker with temperature control for a final volume of 20 mL. The immobilization yield of activity (YE) and immobilization yield of protein (YP) were calculated according to Eq. (1) and Eq. (2), respectively [35].Thermal stability is an essential parameter for preparing novel biocatalysts, especially when aiming for industrial applications like gluconic acid (GA) synthesis. Fig. 3 shows the thermal stability of the GOx and CAT derivates in SA supports. The inactivation curves were modeled according to the theory of enzymatic inactivation [36]. Both free and immobilized enzymes followed a first-order inactivation model at 45 °C and 50 °C (Eq. (3)). It should be noted that this temperature range was chosen based on the optimal temperatures of GOx and CAT from Fig. 2. In all cases, a decrease in activity over time was observed. The stabilizing effect of immobilization is evident for GOx (Fig. 3a), where GOX-SA kept higher residual activity than the free enzymes at 45 °C and 50 °C. This agrees with the reported stabilities for GOx in other works, where free and immobilized enzymes maintain their activity at elevated temperatures, and the immobilized enzyme improves greatly over the free enzyme [46,52].ANID Chile funded this research through FONDECYT 1220134 and granted it to C.B.The authors gratefully acknowledge Luigi Tavernini for providing help with the language and Catalina Morales and Mariela Muñoz for HPLC measurements. C. Ottone thanks the financial support from VINCI-DI PUCV (DI regular 039.316/2023). C. Bernal acknowledges the financial support from ANID Chile through FONDECYT 1220134. T. Galaz thanks the ANID scholarship N° 22200791 and PUCV for the fee waiver scholarship. |
