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-M

SUBMITTER: Chen A 

PROVIDER: S-EPMC4712565 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature