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

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Expression

Background:

• Bacillus acidopullulyticus pullulanase (BaPul) expression in E. coli faces challenges with low soluble protein yield and extracellular secretion.
• Understanding BaPul's modular structure is key to improving its production.

Purpose of the Study:

• To enhance the expression and secretion of soluble Bacillus acidopullulyticus pullulanase (BaPul) in Escherichia coli.
• To investigate the impact of N-terminal domain truncation on BaPul activity and application.

Main Methods:

• Engineered N-terminal domain truncations of BaPul, including removal of CBM41 and X25 domains.
• Utilized PelB signal peptide for periplasmic targeting of BaPul variants.
• Assessed enzyme activity, expression levels, secretion efficiency, and optimal conditions (pH, temperature).

Main Results:

• Truncated variants M1 (ΔCBM41) and M5 (ΔCBM41ΔX25) showed 2.9- and 2.4-fold higher activity than wild-type (WT) BaPul, attributed to enhanced soluble protein expression.
• PelB-tagged M1 and M5 variants were efficiently secreted into the periplasm (56.6% and 93.4% activity transfer).
• Truncated variants retained similar optimal temperature, pH, and starch saccharification efficiency (around 94%) compared to WT BaPul.

Conclusions:

• Deletion of CBM41 and/or X25 domains in BaPul does not compromise enzyme function or application.
• Truncated BaPul variants exhibit significantly improved expression and secretion in E. coli.
• These modified BaPul variants are potentially more suitable for industrial enzyme production and applications.