Thermostable multifunctional GH74 xyloglucanase from Myceliophthora thermophila: high-level expression in Pichia pastoris and characterization of the recombinant protein
| Autor: | Liliya Sakhibgaraeva, Sergey V. Yarotsky, O. V. Berezina, Irina Krestyanova, Wolfgang Liebl, Wolfgang H. Schwarz, Sergey V. Rykov, Jonathan Herlet, Vladimir V. Zverlov, Artem Zavyalov, Petra Kornberger, Dmitriy Kozlov |
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| Rok vydání: | 2017 |
| Předmět: |
0106 biological sciences
0301 basic medicine beta-Glucans Glycoside Hydrolases Sordariales 01 natural sciences Applied Microbiology and Biotechnology Pichia Pichia pastoris law.invention Substrate Specificity 03 medical and health sciences chemistry.chemical_compound law 010608 biotechnology Enzyme Stability Cloning Molecular Glucans Thermostability chemistry.chemical_classification biology Thermophile Hydrolysis Temperature General Medicine Hydrogen-Ion Concentration biology.organism_classification Recombinant Proteins Xyloglucan Molecular Weight Kinetics 030104 developmental biology Enzyme chemistry Biochemistry Carboxymethylcellulose Sodium Recombinant DNA Fermentation Xylans Biotechnology Myceliophthora thermophila Half-Life |
| Zdroj: | Applied microbiology and biotechnology. 101(14) |
| ISSN: | 1432-0614 |
| Popis: | A xyloglucanase of the GH74 family was identified in the thermophilic fungus strain Myceliophthora thermophila VKPM F-244, and its gene sequence was optimized for cloning and expression in Pichia pastoris. The recombinant xyloglucanase MtXgh74 exhibited the highest activity toward tamarind seed xyloglucan with a K M value of 0.51 ± 0.06 mg/mL. The activities on barley β-glucan and carboxymethylcellulose were about 4 and 2%, respectively, compared to xyloglucan. Maximum xyloglucanase activity was observed at 70–75 °C and pH 6.5. After pre-incubation at 50 °C, pH 6.0 for 3 h, the enzyme retained 100% of its activity. The half-life of MtXgh74 at 60 °C, pH 6.0 was 40 min. In P. pastoris, MtXgh74 was produced in glycosylated form. The enzyme production in a 1 L bioreactor resulted in a yield of 118 U/mL or 5.3 g/L after 51 h fermentation. Kinetic studies of the hydrolysis product formation suggest that MtXgh74 has an endo-processive mode of action. The final products were the standard xyloglucan building blocks XXXG, XXLG, XLXG, and XLLG. Additionally, MtXgh74 hydrolyzed various linkages within the xyloglucan building blocks XXXG, XXLG, and XLXG (except XLLG) producing diverse low molecular weight oligosaccharides which may be identified by MALDI-TOF as XG, XX, XXG/GXX/XGX, XXX, LG, LX/XL, XLX/XXL, LLG, GXXXG, GXLLG, XLLGX. The unique combination of different activities within one enzyme along with its high thermostability and specificity toward xyloglucan makes MtXgh74 a promising candidate enzyme for industrial applications. |
| Databáze: | OpenAIRE |
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