Efficient expression and secretion of endo-1,4-β-xylanase from Penicillium citrinum in non-conventional yeast Yarrowia lipolytica directed by the native and the preproLIP2 signal peptides

Autor: Sophie Bozonnet, Chanika Ouephanit, Warawut Chulalaksananukul, Monnat Theerachat, Nassapat Boonvitthya
Přispěvatelé: Program in Biotechnology, Faculty of Science, Chulalongkorn University [Bangkok], Department of Botany, Faculty of Science, Biofuels by Biocatalysts Research Unit, Department of Botany, Faculty of Science, Innovation Institute, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Thailand Research Fund through the RGJ (Royal Golden Jubilee Ph.D. Program, Thailand) [PHD/0073/2552], Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)
Jazyk: angličtina
Rok vydání: 2019
Předmět:
Zdroj: Protein Expression and Purification
Protein Expression and Purification, Elsevier, 2019, 160, pp.1-6. ⟨10.1016/j.pep.2019.03.012⟩
Protein Expression and Purification, 2019, 160, pp.1-6. ⟨10.1016/j.pep.2019.03.012⟩
ISSN: 1046-5928
1096-0279
DOI: 10.1016/j.pep.2019.03.012⟩
Popis: Filamentous fungi are the most common industrial xylanase producers. In this study, the xynA gene encoding xylanase A of Penicilium citrinum was successfully synthesized and expressed in Yarrowia lipolytica under the control of the strong constitutive TEF promoter. Native and preproLIP2 secretion signals were used for comparison of the expression and secretion level. The recombinant xylanase was produced as a soluble protein, and the total activity production reached 11 and 52 times higher than the level of activity produced by the fungus P. citrinum native strain, respectively. Maximum activity was observed with the preproLIP2 secretion signal at 180 U/mL. Post translational glycosylation affected the molecular mass of the recombinant xylanase, resulting in an apparent molecular weight larger than 60 kDa, whereas after deglycosylation, the recombinant XynA displayed a molecular mass of 20 kDa. The deglycosylated xylanase was purified by ion exchange chromatography and reached 185-fold of purification. The enzyme was optimally active at 55 degrees C and pH 5 and stable over a broad pH range (3-9). It retained more than 80% of the original activity after 24 h. It conserved around 80% of the original activity after pre-incubation at 40 degrees C for 6 h. With birchwood xylan as substrate, the enzyme showed a K-m of 5.2 mg/mL, and K-cat of 245 per s. The high level of secretion and the stability over a wide range of pH and at moderate temperatures of the re-XynA could be useful for variety of biotechnological applications.
Databáze: OpenAIRE
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