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Enhanced Production of Soluble Pyrococcus furiosus α-Amylase in Bacillus subtilis through Chaperone Co-Expression, Heat Treatment and Fermentation Optimization

  • Zhang, Kang (State Key Laboratory of Food Science and Technology, Jiangnan University) ;
  • Tan, Ruiting (State Key Laboratory of Food Science and Technology, Jiangnan University) ;
  • Yao, Dongbang (State Key Laboratory of Food Science and Technology, Jiangnan University) ;
  • Su, Lingqia (State Key Laboratory of Food Science and Technology, Jiangnan University) ;
  • Xia, Yongmei (State Key Laboratory of Food Science and Technology, Jiangnan University) ;
  • Wu, Jing (State Key Laboratory of Food Science and Technology, Jiangnan University)
  • Received : 2021.02.01
  • Accepted : 2021.03.22
  • Published : 2021.04.28

Abstract

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

Keywords

References

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