Enhanced extracellular production of maltotetraose amylase from Pseudomonas saccharophila in Bacillus subtilis through regulatory element optimization

Abstract

Maltotetraose amylase (Mta) catalyzes the hydrolysis of amylaceous polysaccharides into maltotetraose, which is an important functional sugar used in the food industry. However, the lack of efficient expression systems for recombinant Mta has hindered its scale-up production and application. In this study, a codon-optimized mta gene from Pseudomonas saccharophila was efficiently produced in Bacillus subtilis by optimizing the regulatory elements. First, a plasmid library containing 173 different signal peptide sequences placed upstream of mta gene was constructed, and transformed into B. subtilis strain WB800N(amyEΔ1) for high-throughput screening. The signal peptide yhcR was found to significantly enhance the secretion of Mta, reaching an activity of 75.4 U/mL in the culture medium. After optimization of the promoters, the Mta activity was further increased to 100.3 U/mL using a dual-promoter P_HpaIIP_amyE. Finally, the carbon sources and nitrogen sources for recombinant Mta production were optimized, yielding a highest Mta activity of 288.9 U/mL under the optimal culture conditions. The crude enzyme solution containing recombinant Mta produced a highest maltotetraose yield of 70.3% with 200 g/L of maltodextrin as the substrate. Therefore, the present study have demonstrated a high yield of Mta produced in B. subtilis, laying the foundation for large-scale Mta production and application.