Optimized and automated protocols for high-throughput screening of amylosucrase libraries

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Engineering

Background:

• Amylosucrase is a valuable enzyme with applications in various industries.
• Improving enzyme stability, particularly thermostability and solvent resistance, is crucial for industrial viability.
• Existing methods for enzyme variant isolation can be time-consuming and low-throughput.

Purpose of the Study:

• To design and validate a general, high-throughput procedure for isolating amylosucrase variants with enhanced thermostability or organic solvent resistance.
• To optimize screening conditions for bacterial growth and recombinant protein production in miniaturized cultures.
• To identify improved amylosucrase mutants from a randomly generated variant library.

Main Methods:

• A two-step procedure involving in vivo selection and automated screening of enzyme variants.
• Utilizing an Escherichia coli expression vector for high-rate recombinant enzyme production in 96-well microplates.
• Validation of the screening assay by minimizing variability in bacterial growth and protein production, normalizing production to a coefficient of variance of 12.5%.

Main Results:

• The developed procedure successfully identified amylosucrase variants with enhanced thermostability and solvent resistance.
• Recombinant amylosucrase production was significantly normalized, reducing variability from 27% to 12.5%.
• A first-generation variant library yielded a mutant with a 25-fold increased stability at 50°C compared to the wild-type enzyme.

Conclusions:

• The designed high-throughput procedure is effective for isolating amylosucrase variants with improved functional properties.
• This method offers a robust platform for directed evolution of enzymes for enhanced stability.
• The identified mutant demonstrates significant potential for applications requiring thermostable or solvent-resistant amylosucrase.