Co-Immobilization of Xylanase and Scaffolding Protein onto an Immobilized Metal Ion Affinity Membrane
Lignocellulosic biomass conversion technology seeks to convert agricultural waste to sugars through the use of various cellulases and hemicellulases. In practice, the application of free enzymes might increase the cost of the process due to difficulties with recovery of the enzymes and products. Imm...
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MDPI AG
2020-12-01
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| Series: | Catalysts |
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| Online Access: | https://www.mdpi.com/2073-4344/10/12/1408 |
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| author | Ho-Lam Wong Nien-Jen Hu Tzong-Yuan Juang Yung-Chuan Liu |
| author_facet | Ho-Lam Wong Nien-Jen Hu Tzong-Yuan Juang Yung-Chuan Liu |
| author_sort | Ho-Lam Wong |
| collection | DOAJ |
| description | Lignocellulosic biomass conversion technology seeks to convert agricultural waste to sugars through the use of various cellulases and hemicellulases. In practice, the application of free enzymes might increase the cost of the process due to difficulties with recovery of the enzymes and products. Immobilization might be an effective approach for recovering the hydrolysis products and improving the stability and reusability of the enzymes. In this study, we used a recombinant genetic engineering approach to construct a scaffold protein gene (CipA) and a xylanase gene (XynC) fused to a dockerin gene (DocT). After expressing CipA and XynC-DocT (XynCt) genes using <i>E. coli</i> hosts, the crude extracts were collected. An immobilized metal ion affinity membrane/Co<sup>2+</sup> ion (IMAM-Co<sup>2+</sup>) system was prepared to adsorb CipA in its crude extract, thereby allowing simultaneous purification and immobilization of CipA protein. A similar approach was applied for the adsorption of XynCt protein, exploiting the interaction between the cohesin units in IMAM-Co<sup>2+</sup>-CipA and the dockerin unit in XynCt. The activity of the xylanase unit was enhanced in the presence of Co<sup>2+</sup> for both the free XynCt enzymes and the immobilized CipA-XynCt. The heat resistance and stability over a wide range of values of pH of the immobilized CipA-XynCt were superior to those of the free XynCt. Furthermore, the immobilized CipA-XynCt retained approximately 80% of its initial activity after seven reaction cycles. The values of <i>K</i><sub>m</sub> and ν<sub>max</sub> of IMAM-Co<sup>2+</sup>-CipA-XynCt (1.513 mg/mL and 3.831 U/mg, respectively) were the best among those of the other tested forms of XynCt. |
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| issn | 2073-4344 |
| language | English |
| last_indexed | 2024-03-10T14:22:33Z |
| publishDate | 2020-12-01 |
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| record_format | Article |
| series | Catalysts |
| spelling | doaj.art-a0972ed31d854d4688c5aab66e23b4462023-11-20T23:17:01ZengMDPI AGCatalysts2073-43442020-12-011012140810.3390/catal10121408Co-Immobilization of Xylanase and Scaffolding Protein onto an Immobilized Metal Ion Affinity MembraneHo-Lam Wong0Nien-Jen Hu1Tzong-Yuan Juang2Yung-Chuan Liu3Department of Chemical Engineering, National Chung Hsing University, Taichung 402, TaiwanGraduate Institute of Biochemistry, National Chung Hsing University, Taichung 402, TaiwanDepartment of Cosmeceutics, China Medical University, Taichung 404, TaiwanDepartment of Chemical Engineering, National Chung Hsing University, Taichung 402, TaiwanLignocellulosic biomass conversion technology seeks to convert agricultural waste to sugars through the use of various cellulases and hemicellulases. In practice, the application of free enzymes might increase the cost of the process due to difficulties with recovery of the enzymes and products. Immobilization might be an effective approach for recovering the hydrolysis products and improving the stability and reusability of the enzymes. In this study, we used a recombinant genetic engineering approach to construct a scaffold protein gene (CipA) and a xylanase gene (XynC) fused to a dockerin gene (DocT). After expressing CipA and XynC-DocT (XynCt) genes using <i>E. coli</i> hosts, the crude extracts were collected. An immobilized metal ion affinity membrane/Co<sup>2+</sup> ion (IMAM-Co<sup>2+</sup>) system was prepared to adsorb CipA in its crude extract, thereby allowing simultaneous purification and immobilization of CipA protein. A similar approach was applied for the adsorption of XynCt protein, exploiting the interaction between the cohesin units in IMAM-Co<sup>2+</sup>-CipA and the dockerin unit in XynCt. The activity of the xylanase unit was enhanced in the presence of Co<sup>2+</sup> for both the free XynCt enzymes and the immobilized CipA-XynCt. The heat resistance and stability over a wide range of values of pH of the immobilized CipA-XynCt were superior to those of the free XynCt. Furthermore, the immobilized CipA-XynCt retained approximately 80% of its initial activity after seven reaction cycles. The values of <i>K</i><sub>m</sub> and ν<sub>max</sub> of IMAM-Co<sup>2+</sup>-CipA-XynCt (1.513 mg/mL and 3.831 U/mg, respectively) were the best among those of the other tested forms of XynCt.https://www.mdpi.com/2073-4344/10/12/1408immobilized metal ion affinity membranexylanaseenzyme immobilizationscaffolding proteinkinetic study |
| spellingShingle | Ho-Lam Wong Nien-Jen Hu Tzong-Yuan Juang Yung-Chuan Liu Co-Immobilization of Xylanase and Scaffolding Protein onto an Immobilized Metal Ion Affinity Membrane Catalysts immobilized metal ion affinity membrane xylanase enzyme immobilization scaffolding protein kinetic study |
| title | Co-Immobilization of Xylanase and Scaffolding Protein onto an Immobilized Metal Ion Affinity Membrane |
| title_full | Co-Immobilization of Xylanase and Scaffolding Protein onto an Immobilized Metal Ion Affinity Membrane |
| title_fullStr | Co-Immobilization of Xylanase and Scaffolding Protein onto an Immobilized Metal Ion Affinity Membrane |
| title_full_unstemmed | Co-Immobilization of Xylanase and Scaffolding Protein onto an Immobilized Metal Ion Affinity Membrane |
| title_short | Co-Immobilization of Xylanase and Scaffolding Protein onto an Immobilized Metal Ion Affinity Membrane |
| title_sort | co immobilization of xylanase and scaffolding protein onto an immobilized metal ion affinity membrane |
| topic | immobilized metal ion affinity membrane xylanase enzyme immobilization scaffolding protein kinetic study |
| url | https://www.mdpi.com/2073-4344/10/12/1408 |
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