title: Downsizing a pullulanase to a small molecule with improved soluble expression and secretion efficiency in Escherichia coli.
abstract: BACKGROUND
  Significant challenges, including low expression and extracellular secretion of soluble protein, are encountered in expressing and purifying Bacillus acidopullulyticus pullulanase (BaPul) in Escherichia coli.
  
  
  METHODS
  An N-terminal domain truncation was adopted to facilitate BaPul variant expression and/or secretion.
  
  
  RESULTS
  BaPul possesses a complex modular architecture that consists of CBM41-X45a-X25-X45b-CBM48-GH13. The activities of M1 (ΔCBM41) and M5 (ΔCBM41ΔX25) variants were 2.9- and 2.4-fold that of wild-type (WT) enzyme, respectively. The enhanced expression of soluble protein is the main reason for these improved activities. PelB-M1 and PelB-M5 were transported to the periplasmic space, where PelB is part of the PelB-pET28a(+) construct, and PelB-M3 (ΔX25) and PelB-WT variants were largely retained in the cytoplasm. After fermentation, about 56.6 and 93.4 % of the total activity of PelB-M1 and PelB-M5 were transferred to the periplasm, respectively, followed by cell lysis and leakage of the partial enzyme into the extracellular medium. The optimal temperature and pH for purified preparations of M1, M3, and M5 were similar to those of the WT enzyme. In a starch saccharification reaction, the dextrose equivalents of M1, M3, and M5 proteins were 94.7, 94.5, and 93.1 %, respectively, which were also essentially identical to that of WT (93.6 %).
  
  
  CONCLUSIONS
  The deletion of CBM41 and/or X25 domain did not affect the enzyme application, and the truncated variants were more highly expressed and secreted in E. coli. Thus, the truncated variants may be more suitable for industrial applications.
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China. ana_001_22@163.com.|School of Biochemical Engineering, Anhui Polytechnic University, Wuhu, 241000, China. ana_001_22@163.com.|The Key Laboratory of Industrial Biotechnology and The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. ana_001_22@163.com.
authors.forename: Ana
authors.initials: A
authors.lastname: Chen
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China. 506555937@qq.com.|The Key Laboratory of Industrial Biotechnology and The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. 506555937@qq.com.
authors.forename: Yang
authors.initials: Y
authors.lastname: Sun
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China. 1378206711@qq.com.|The Key Laboratory of Industrial Biotechnology and The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. 1378206711@qq.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China. 630906668@qq.com.|The Key Laboratory of Industrial Biotechnology and The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. 630906668@qq.com.
authors.forename: Feng
authors.initials: F
authors.lastname: Peng
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China. 1058274535@qq.com.|The Key Laboratory of Industrial Biotechnology and The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. 1058274535@qq.com.
authors.forename: Chunjun
authors.initials: C
authors.lastname: Zhan
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China. 1107988900@qq.com.|The Key Laboratory of Industrial Biotechnology and The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. 1107988900@qq.com.
authors.forename: Meng
authors.initials: M
authors.lastname: Liu
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China. yangyankun@jiangnan.edu.cn.|The Key Laboratory of Industrial Biotechnology and The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. yangyankun@jiangnan.edu.cn.
authors.forename: Yankun
authors.initials: Y
authors.lastname: Yang
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China. baizhonghu@jiangnan.edu.cn.|The Key Laboratory of Industrial Biotechnology and The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. baizhonghu@jiangnan.edu.cn.
authors.forename: Zhonghu
authors.initials: Z
authors.lastname: Bai
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005285:Fermentation / Q000502:physiology; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase