Efficient Production and Characterization of Maltohexaose-Forming alpha-Amylase AmyM Secreted From the Methylotrophic Yeast Pichia pastoris

In the author's previous research, maltohexaose-forming alpha-amylase AmyM (KM114206), which belongs to the glycoside hydrolase family 13 (GH13), was identified from Corallococcus sp. strain EGB. In this study, the gene encoding the mature AmyM peptide is expressed under the pFLD1 promoter in Pichia pastoris GS115, and optimal conditions for the extracellular production of AmyM are determined through a combination of single-factor experiments and the response surface methodology. Waste molasses and industrial glucose are added in the experiments to increase protein expression. High-cell density fermentation of recombinant P. pastoris is performed using a fed-batch strategy over a period of approximately 60 h in a 50 L fermenter, and the maximum expression reached final values of 120 and 220 mg L-1 with a feeding strategy of 15 g L-1 waste molasses and 10 g L-1 industrial glucose, respectively. Thus, a highly efficient production of alpha-amylase AmyM in P. pastoris is observed, compared with a low expression level of 1.4 mg L-1 AmyM observe in Escherichia coli. Furthermore, the tolerance of the fermentative alpha-amylase AmyM toward salt and organic solvents shows that the enzyme may be highly efficient for the improvement of water-soluble total phenolic content and the antioxidant properties of wheat. These results demonstrate that AmyM hydrolysis is an efficient method for the improvement of the antioxidant potential of cereals. In the present study, alpha-amylase AmyM production in P. pastoris occurred at high levels, and the enzyme exhibited superior characteristics, which may enable its use in practical applications.