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

Yuanming Gai1small orcid_display_4pp, Jingqi Chen1,2
small orcid_display_4pp, Shibin Zhang1,2small orcid_display_4pp, Beiwei Zhu3small orcid_display_4pp and Dawei Zhang1,2,3*small orcid_display_4pp

 


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




Article history:
Received: July 30, 2017
Accepted: January 22, 2018




Key words:
α-amylase, Bacillus subtilis, fermentation, site-directed mutagenesis, thermostability




Summary:
To improve the properties of α-amylase from Bacillus stearothermophilus (AmyS), a deletion mutant AmySΔR179-G180 was constructed by deleting arginine (Arg179) and glycine (Gly180) using site-directed mutagenesis. AmyS and AmySΔR179-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 AmySΔR179-G180 was enhanced and the half-life at 100 °C significantly increased from 24 to 33 min. In addition, AmySΔR179-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).




*Corresponding author:  tel3  +862224828749
                                                fax2  +862224828749
                                                 email3  zhang_dw@ tib.cas.cn



Paper was presented at the 7th International Forum on Industrial Bioprocessing - IFIBiop 2017, May 21-24, 2017, Wuxi, PR China

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