Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: production, purification and novel properties

• Published in: Molecular biology reports 2019-02, Vol.46 (1), p.921-932
• Main Authors: Trabelsi, Sahar, Sahnoun, Mouna, Elgharbi, Fatma, Ameri, Rihab, Ben Mabrouk, Sameh, Mezghani, Monia, Hmida-Sayari, Aïda, Bejar, Samir
• Format: Article
• Language: English
• Subjects: Animal Anatomy; Animal Biochemistry; Biomedical and Life Sciences; Bread; Dough; Enzymes; Glycosylation; Histology; Iron; Life Sciences; Magnesium; Methanol; Morphology; Original Article; Pichia pastoris; Purification; Thermal stability
• ISSN: 0301-4851; 1573-4978
• DOI: 10.1007/s11033-018-4548-2

A synthetic cDNA-AmyA gene was cloned and successfully expressed in Pichia pastoris as a His-tagged enzyme under the methanol inducible AOX1 promoter. High level of extracellular amylase production of 72 U/mL was obtained after a 72 h induction by methanol. As expected, the recombinant strain produced only the AmyA isoform since the host is a protease deficient strain. Besides, the purified r-AmyA showed a molecular mass of 54 kDa, the same pH optimum equal to 5.6 but a higher thermoactivity of 60 °C against 50 °C for the native enzyme. Unlike AmyA which maintained 50% of its activity after a 10-min incubation at 60 °C, r-AmyA reached 45 min. The higher thermoactivity and thermostability could be related to the N -glycosylation. The r-AmyA activity was enhanced by 46% and 45% respectively in the presence of 4 mM Fe 2+ and Mg 2+ ions. This enzyme was more efficient in bread-making since such ions were reported to have a positive impact on the nutriment quality and the rheological characteristics of the wheat flour dough. The thermoactivity/thermostability as well as the iron and magnesium activations could also be ascribed to the presence of an additional C-terminal loop containing the His tag.