Property Improvement of α-Amylase from Bacillus stearothermophilus by Deletion of Amino Acid Residues Arginine 179 and Glycine 180

Yuanming Gai; Jingqi Chen; Shibin Zhang; Beiwei Zhu; Dawei Zhang

doi:10.17113/ftb.56.01.18.5448

Property Improvement of α-Amylase from Bacillus stearothermophilus by Deletion of Amino Acid Residues Arginine 179 and Glycine 180

Food Technol Biotechnol. 2018 Mar;56(1):58-64. doi: 10.17113/ftb.56.01.18.5448.

Authors

Yuanming Gai¹, Jingqi Chen^{1

2}, Shibin Zhang^{1

2}, Beiwei Zhu³, Dawei Zhang^{1

2

3}

Affiliations

¹ Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, 300308 Tianjin, PR China.
² Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, 300308 Tianjin, PR China.
³ School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, No. 1st Qinggongyuan, Ganjingzi, 116034 Dalian, PR China.

Abstract

To improve the properties of α-amylase from Bacillus stearothermophilus (AmyS), a deletion mutant AmyS∆R179-G180 was constructed by deleting arginine (Arg179) and glycine (Gly180) using site-directed mutagenesis. AmyS and AmyS∆R179-G180 were expressed in Bacillus subtilis and purified by ammonium sulfate precipitation, after which the enzymatic properties were characterized and compared. By deleting amino acids Arg179 and Gly180, the thermostability of α-amylase AmyS∆R179-G180 was enhanced and the half-life at 100 °C significantly increased from 24 to 33 min. In addition, AmyS∆R179-G180 exhibited greater acid resistance and lower calcium requirements to maintain α-amylase activity. The secretory capacity of the recombinant strain was evaluated by fed-batch fermentation in a 7.5-litre fermentor in which high α-amylase activity was obtained. The highest activity reached 3300 U/mL with a high productivity of 45.8 U/(mL·h).

Keywords: Bacillus subtilis; fermentation; site-directed mutagenesis; thermostability; α-amylase.