Recombination of Thermo-Alkalistable, High Xylooligosaccharides Producing Endo-Xylanase from Thermobifida fusca and Expression in Pichia pastoris
| العنوان: | Recombination of Thermo-Alkalistable, High Xylooligosaccharides Producing Endo-Xylanase from Thermobifida fusca and Expression in Pichia pastoris |
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| المؤلفون: | Jiakun Wang, Ming-qi Liu, Jianxin Liu, Qian Wang, Du Wen, Xiao-Yan Weng |
| المصدر: | Applied Biochemistry and Biotechnology. 175:1318-1329 |
| بيانات النشر: | Springer Science and Business Media LLC, 2014. |
| سنة النشر: | 2014 |
| مصطلحات موضوعية: | Gene Dosage, Oligosaccharides, Glucuronates, Bioengineering, Applied Microbiology and Biotechnology, Biochemistry, Pichia, Pichia pastoris, chemistry.chemical_compound, Actinomycetales, Enzyme Stability, Protein purification, Xylobiose, Genetic Testing, Molecular Biology, Betula, Chromatography, High Pressure Liquid, Thermostability, Recombination, Genetic, Endo-1,4-beta Xylanases, biology, Temperature, General Medicine, Hydrogen-Ion Concentration, biology.organism_classification, Directed evolution, Enzyme Activation, Kinetics, chemistry, Genes, Bacterial, Structural Homology, Protein, Mutation, Xylanase, Xylans, Heterologous expression, Xylooligosaccharide, Half-Life, Biotechnology |
| الوصف: | For xylooligosaccharide (XO) production, endo-xylanase from Thermobifida fusca was modified by error-prone PCR and DNA shuffling. The G4SM1 mutant (S62T, S144C, N198D, and A217V) showed the most improved hydrolytic activity and was two copies expressed in Pichia pastoris under the control of GAP promoter. The maximum xylanase activity in culture supernatants was 165 ± 5.5 U/ml, and the secreted protein concentration reached 493 mg/l in a 2-l baffled shake flask. After 6× His-tagged protein purification, the specific activity of G4SM1 was 2036 ± 45.8 U/mg, 2.12 times greater than that of wild-type enzyme. Additionally, G4SM1 was stable over a wide pH range from 5.0 to 9.0. Meanwhile, half-life of G4SM1 thermal inactivation at 70 °C increased 8.5-fold. Three-dimensional structures suggest that two amino acid substitutions, S62T and S144C, located at catalytic domain may be responsible for the enhanced activity and thermostability of xylanase. Xylobiose was the dominant end product of xylan hydrolysis by G4SM1. Due to its attractive biochemical properties, G4SM1 has potential value in commercial XO production. |
| تدمد: | 1559-0291 0273-2289 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9b21a471fbeb6639b61ddd3d88a830abTest https://doi.org/10.1007/s12010-014-1355-7Test |
| حقوق: | CLOSED |
| رقم الانضمام: | edsair.doi.dedup.....9b21a471fbeb6639b61ddd3d88a830ab |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15590291 02732289 |
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