Overexpression of an endogenous raw starch digesting mesophilic α‐amylase gene in Bacillus amyloliquefaciens Z3 by in vitro methylation protocol. (5th March 2020)

• Record Type: Journal Article
• Authors: Tang, Shizhe; Xu, Tingliang; Peng, Jing; Zhou, Kaiyan; Zhu, Yuling; Zhou, Wenbo; Cheng, Haina; Zhou, Hongbo
• Is Part Of: Journal of the science of food and agriculture. Volume 100:Number 7(2020)
• Page Start: 3013
• Page End: 3023
• Publication Date: 2020-03-05
• Subjects: mesophilic α‐amylase -- Bacillus amyloliquefaciens -- restriction‐modification system -- genetic engineering -- industrial strain -- raw starch hydrolysis; Food -- Periodicals; Agriculture -- Periodicals; 664
• DOI: 10.1002/jsfa.10332
• Languages: English
• ISSNs: 0022-5142

Abstract

Abstract: 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 × 10 2 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