Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in Escherichia coli.

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Expression

Background:

• Alkaline phytase application as a feed additive is limited by low specific activity.
• Escherichia coli is used for directed evolution but not ideal for food-grade production due to pathogenicity concerns.
• Combining expression systems can leverage advantages for improved protein production.

Purpose of the Study:

• To generate alkaline phytase mutants with improved activity and stability.
• To evaluate different expression hosts (E. coli, Bacillus subtilis, Pichia pastoris) for food-grade phytase production.
• To compare the biochemical properties of phytase mutants expressed in various systems.

Main Methods:

• Directed evolution of Bacillus subtilis 168 alkaline phytase (phy168) in E. coli.
• Transformation of phy168 mutants into food-grade hosts Bacillus subtilis and Pichia pastoris for secretory expression.
• Characterization of expressed phytase mutants, including specific activity, pH, and temperature profiles, and stability assays.

Main Results:

• The D24G/K70R/K111E/N121S phy168 mutant expressed in Bacillus subtilis showed the highest specific activity (30.4 U/mg) at 60°C and pH 7.0.
• This B. subtilis-expressed mutant retained approximately 70% of its activity after incubation at 80°C for 10 minutes, outperforming P. pastoris (25%) and E. coli (50%).
• Biochemical characterization revealed significant improvements in activity and thermostability for the B. subtilis-expressed mutant.

Conclusions:

• Bacillus subtilis is a suitable host for the secretory expression of engineered alkaline phytase mutants.
• The strategy of generating mutants in one host (E. coli) and expressing them in another (B. subtilis) offers a viable approach for industrial protein production.
• This method can overcome limitations of single expression systems to achieve desired enzyme properties for feed additives.