Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: production, purification and novel properties
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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 Fe2+ and Mg2+ 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.
KeywordsAmyA isoform amylase Aspergillus oryzae Pichia pastoris Thermostability Glycosylation
This research was supported by the Ministry of Higher Education and Scientific Research of Tunisia under the contract program CBS-LMBEE/code: LR15CBS06 2015–2018.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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