Downsizing a pullulanase to a small molecule with improved soluble expression and secretion efficiency in Escherichia coli.

backgroundSignificant challenges, including low expression and extracellular secretion of soluble protein, are encountered in expressing and purifying Bacillus acidopullulyticus pullulanase (BaPul) in Escherichia coli.

methodsAn N-terminal domain truncation was adopted to facilitate BaPul variant expression and/or secretion.

resultsBaPul 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 %).

conclusionThe 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.