Abstract
Maltotetraose amylase, which catalyzes the hydrolysis of amylaceous polysaccharides into maltooligosaccharides with maltotetraose as the main product, is extensively used in the food industry. However, the lack of efficient expression system for maltotetraose amylase has hampered its production and application. In this study, high-level production of a maltotetraose amylase mutant (referred to as Pp-Mta∆CBM) from Pseudomonas saccharophila was achieved in Pichia pastoris X-33. First, the gene of maltotetraose amylase with the carbohydrate-binding module (CBM) removed was codon-optimized and cloned into the pPICZαA vector, followed by transformation into P. pastoris X-33 for expression. Using the promoter PAOX1 and signal peptide α-factor, high-level production of Pp-Mta∆CBM with minimal extracellular impurity proteins was achieved, resulting in an extracellular activity of 367.9 U/mL after 7 days of cultivation in shake flasks. Next, the expressed Pp-Mta∆CBM was purified and characterized. This recombinant enzyme was glycosylated and has maximum activity at 55 ℃ and pH 7.0. Its Km for soluble starch was 4.1 g/L, and its kcat was 3237.6 s−1. Finally, the Pp-Mta∆CBM was found to produce a maximum maltotetraose yield of 57.1% in the presence of 200 g/L of substrate. The findings presented in this study demonstrate the efficient production of Pp-Mta∆CBM in P. pastoris, providing a new expression system for maltotetraose amylase and laying the foundation for its scale-up production and industrial application.
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The data that support the findings of the present study are available from the corresponding author on reasonable request.
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This research was funded by Scientific Research Fund of Liaoning Provincial Education Department (grant number LJKFZ20220213).
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Investigation, data curation, writing—original draft, Xinyu Wang; investigation, data curation, Ming Xu, Xiaopeng Ren, and Mingyu Li; investigation, data curation, supervision, conceptualization, writing—review and editing, funding acquisition, Conggang Wang; writing—review and editing, Fan Yang and Xianzhen Li. All authors have read and agreed to the published version of the manuscript.
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Wang, X., Xu, M., Ren, X. et al. High-Level Expression and Biochemical Characterization of a Maltotetraose Amylase in Pichia pastoris X-33 for Maltotetraose Production. Appl Biochem Biotechnol (2024). https://doi.org/10.1007/s12010-024-04871-0
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DOI: https://doi.org/10.1007/s12010-024-04871-0