LAUSR

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, due to the existence of the restriction-modification system (RMS), 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 CFUs per microgram 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 flask and 386.03 ± 16.25 U mL-1 in 5-L stirred-tank fermenter, respectively. The B. 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. Besides, exceptional ability to hydrolyze starch makes α-amylase AmyZ3 and strain BAZ3-16 valuable in the starch industry. This article is protected by copyright. All rights reserved.