BACKGROUND Mesophilic α‐amylases function effectively at low temperatures with high rates of catalysis and require less energy for starch hydrolysis. Bacillus amyloliquefaciens is an essential producer of mesophilic α‐amylases. However, because of the existence of the restriction‐modification system, introducing exogenous DNAs into wild‐type B. amyloliquefaciens is especially tricky. RESULTS α‐Amylase producer B. amyloliquefaciens strain Z3 was screened and used as host for endogenous α‐amylase gene expression. In vitro methylation was performed in recombinant plasmid pWB980‐amyZ3. With the in vitro methylation, the transformation efficiency was increased to 0.96 × 102 colony‐forming units μg–1 plasmid DNA. A positive transformant BAZ3‐16 with the highest α‐amylase secreting capacity was chosen for further experiments. The α‐amylase activity of strain BAZ3‐16 reached 288.70 ± 16.15 U mL−1 in the flask and 386.03 ± 16.25 U mL−1 in the 5‐L stirred‐tank fermenter, respectively. The Bacillus amyloliquefaciens Z3 expression system shows excellent genetic stability and high‐level extracellular production of the target protein. Moreover, the synergistic interaction of AmyZ3 with amyloglucosidase was determined during the hydrolysis of raw starch. The hydrolysis degree reached 92.34 ± 3.41% for 100 g L−1 raw corn starch and 81.30 ± 2.92% for 100 g L−1 raw cassava starch after 24 h, respectively. CONCLUSION Methylation of the plasmid DNA removes a substantial barrier for transformation of B. amyloliquefaciens strain Z3. Furthermore, the exceptional ability to hydrolyze starch makes α‐amylase AmyZ3 and strain BAZ3‐16 valuable in the starch industry. © 2020 Society of Chemical Industry

α-Amylase is the most extensively applied enzyme in industry. There is an urgent need for improvement on the yield of α-amylases currently. Herein, a strategy which combined Atmospheric and Room Temperature Plasma (ARTP) mutagenesis tool for construction of mutant library of Bacillus amyloliquefaciens with a 24-well plates screening technique was adopted to improve the yield of recombinant Bacillus amyloliquefaciens α-amylases (BAA). A mutant strain named B. amyloliquefaciens ZN mut-7# was obtained, and the activity of BAA produced by this mutant strain was 86.92% higher than that of the original strain. B. amyloliquefaciens ZN mut-7# has an unchanged BAA gene and genetic stability. This successful application proved that ARTP can be applied to the genetically engineering strains that contain recombinant plasmid. Furthermore, response surface methodology offers an achievable and efficient strategy to optimize the composition of medium used to generate BAA in B. amyloliquefaciens ZN mut-7#. A 1.28-fold increase had been obtained compared to the production of non-optimized fermentation medium. This study demonstrates that ARTP mutagenesis and medium optimization are efficient and feasible methods for increasing recombinant enzyme production in the genetically engineering strains.