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Heterologous Expression of Xylanase xAor from Aspergillus oryzae in Komagataella phaffii T07 2022
Andrey Valentinovich Zadorozhny, and
Viktor Sergeevich Ushakov, and
Alexei Sergeevich Rozanov, and
Natalia Vladimirovna Bogacheva, and
Valeria Nikolayevna Shlyakhtun, and
Mikhail Evgenyevich Voskoboev, and
Anton Vladimirovich Korzhuk, and
Vladislav Anatolevich Romancev, and
Svetlana Valerevna Bannikova, and
Irina Anatolyevna Mescheryakova, and
Egor Vladimirovich Antonov, and
Asya Rifhatovna Vasilieva, and
Elena Iurevna Pavlova, and
Danil Olegovich Chesnokov, and
Elizaveta Dmitrievna Shedko, and
Alla Viktorovna Bryanskaya, and
Denis Vladimirovich Bochkov, and
Tatiana Nikolayevna Goryachkovskaya, and
Sergey Evgenyevich Peltek
Laboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, Russia.
Xylanases (EC 3.2.1.8) hydrolyze the hemicellulose of plant cell walls. Xylanases are used in the food and paper industries and for bioconversion of lignocellulose to biofuel. In this work, the producer-strain with four copies of the xAor xylanase gene was organized in two tandem copies for optimal expression in Komagataella phaffii T07 yeast. The secreted 35 kDa xylanase was purified from culture medium by gel filtration on Sephadex G-25 and anion exchange chromatography on DEAE-Sepharose 6HF. Tryptic peptides of the recombinant enzyme were analyzed by liquid chromatography-tandem mass spectrometry where the amino acid sequence corresponded to Protein Accession # O94163 for Endo-1,4-beta-xylanase from Aspergillus oryzae RIB40. The recombinant xylanase was produced in a bioreactor where the secreted enzyme hydrolyzed oat xylane with an activity of 258240 IU/mL. High activity in the culture medium suggested xylanase could be used for industrial applications without being purified or concentrated. The pH optimum for xylanase xAor was 7.5, though the enzyme was active from pH 2.5 to pH 10. Xylanase was active at temperatures from 35 °C to 85 °C with a maximum at 60 °C. In conclusion, this protocol yields soluble, secreted xylanase suitable for industrial scale production.
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