Generation of phytase chimeras with low sequence identities and improved thermal stability

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Engineering

Background:

• Recombining multiple genes with numerous crossover points is a significant challenge in protein engineering.
• This limitation hinders the development of tailored enzymes for industrial use.

Purpose of the Study:

• To develop a method for efficient, sequence-independent recombination of multiple gene fragments.
• To engineer novel phytase variants with improved thermal stability and specific activity.

Main Methods:

• Computational analysis, including multiple sequence alignments and homology modeling, identified suitable gene fragments.
• Phosphorothioate-based cloning using the PTRec method facilitated combinatorial recombination of up to four phytase gene fragments.
• Functional phytase chimeras were generated and characterized for residual activity and specific activity.

Main Results:

• The PTRec method successfully recombined fragments from six different phytase genes.
• Two novel phytase variants, PTRec 74 and PTRec 77, demonstrated up to 32% improved residual activity at 90°C for 60 minutes.
• These variants retained high specific activities (> 1100 U/mg) and were composed of fragments from Citrobacter braakii, Hafnia alvei, and Yersinia mollaretii phytases.

Conclusions:

• Combinatorial DNA recombination strategies, like PTRec, are effective for generating enzymes with significantly altered properties.
• The identified phytase variants offer enhanced thermostability and specific activity, suitable for industrial applications.
• Generating novel enzymes with low sequence identity to parent enzymes presents opportunities for intellectual property claims.