Cloning and Secretory Expression of Aspergillus niger α-amylase with a Novel Synthetic Promoter in Pichia pastoris and Its Application in Apple Juice

Amylase enzyme catalyzes the breakdown of starch by acting on the α-1,4 glycosidic bond. The use of amylases is common in areas such as baking and fruit juice production in the food industry, as well as in the detergent, textile, and paper industries. Due to their broad industrial applicability, the recombinant production of amylases has received increasing attention in recent years. In this study, the production of Aspergillus niger α-amylase enzyme was investigated for the first time in Pichia pastoris under the control of the ethanol-inducible synthetic ADH2 (SNT5) promoter. A codon-optimized A. niger α-amylase gene was expressed extracellularly in the P. pastoris MK115-PDI strain. Optimal production conditions were 24 °C and pH 6.0. In a 5 L bioreactor, total secreted protein reached 2.2 g/L and enzyme activity reached 44,062 U/mL. The recombinant enzyme was characterized and showed optimal activity at 60 °C and pH 7. In apple juice assays, the enzyme hydrolyzed starch and demonstrated suitability for juice clarification, although performance depended on enzyme concentration. Overall, these results indicate that the SNT5 synthetic promoter enables efficient recombinant α-amylase production in P. pastoris and represents a promising alternative to conventional promoter systems for industrial enzyme manufacturing.