Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-β-1,4-xylanase for applications in pulp bleaching
| Autor: | Verawat Champreda, Aritsara Jaruwat, Katewadee Boonyapakron, Thidarat Nimchua, Benjamas Liwnaree, Penchit Chitnumsub |
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| Rok vydání: | 2017 |
| Předmět: |
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Molecular Paper 0106 biological sciences 0301 basic medicine Hot Temperature Bioengineering Protein Engineering 01 natural sciences Applied Microbiology and Biotechnology 03 medical and health sciences Hydrolysis 010608 biotechnology Hydrolase Escherichia coli Biomass Enzyme kinetics Thermostability chemistry.chemical_classification Endo-1 4-beta Xylanases Chemistry General Medicine Protein engineering Hydrogen-Ion Concentration Combinatorial chemistry Recombinant Proteins 030104 developmental biology Enzyme Biochemistry Xylanase Bagasse Biotechnology |
| Zdroj: | Journal of Biotechnology. 259:95-102 |
| ISSN: | 0168-1656 |
| DOI: | 10.1016/j.jbiotec.2017.07.035 |
| Popis: | In the pulp bleaching industry, enzymes with robust activity at high pH and temperatures are desirable for facilitating the pre-bleaching process with simplified processing and minimal use of chlorinated compounds. To engineer an enzyme for this purpose, we determined the crystal structure of the Xyn12.2 xylanase, a xylan-hydrolyzing enzyme derived from the termite gut symbiont metagenome, as the basis for structure-based protein engineering to improve Xyn12.2 stability in high heat and alkaline conditions. Engineered cysteine pairs that generated exterior disulfide bonds increased the kcat of Xyn12.2 variants and melting temperature at all tested conditions. These improvements led to up to 4.2-fold increases in catalytic efficiency at pH 9.0, 50°C for 1h and up to 3-fold increases at 60°C. The most effective variants, XynTT and XynTTTE, exhibited 2-3-fold increases in bagasse hydrolysis at pH 9.0 and 60°C compared to the wild-type enzyme. Overall, engineering arginines and phenylalanines for increased pKa and hydrogen bonding improved enzyme catalytic efficiency at high stringency conditions. These modifications were the keys to enhancing thermostability and alkaliphilicity in our enzyme variants, with XynTT and XynTTTE being especially promising for their application to the pulp and paper industry. |
| Databáze: | OpenAIRE |
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