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http://dx.doi.org/10.4014/jmb.2101.01039

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)
Publication Information
Journal of Microbiology and Biotechnology / v.31, no.4, 2021 , pp. 570-583 More about this Journal
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
${\alpha}$-Amylase; Bacillus subtilis; chaperone; heat treatment; 3-L fermenter cultivation;
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