Overexpression of an endogenous raw starch digesting mesophilic α-amylase gene in Bacillus amyloliquefaciens Z3 by in vitro methylation protocol.

Abstract

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. 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. 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.