Establishment of High-Throughput Screening Protocol Based on Isomerase Using Geobacillus sp. L-Rhamnose Isomerase

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Engineering

Background:

• Directed evolution generates enzyme variants but requires efficient high-throughput screening (HTS) for identifying improved functionalities.
• L-rhamnose isomerase (L-RI) is crucial for isomerizing D-allulose to D-allose, with activity detectable via colorimetric assays.
• Existing screening methods may lack the efficiency and accuracy needed for large mutant libraries.

Purpose of the Study:

• To establish a robust and reliable high-throughput screening (HTS) protocol for selecting high-activity L-rhamnose isomerase (L-RI) variants.
• To optimize and validate a colorimetric assay for quantifying L-RI activity based on D-allulose depletion.
• To adapt the protocol for a 96-well plate format suitable for large-scale screening in protein engineering.

Main Methods:

• Optimization of reaction conditions in a single-tube format to accurately measure D-allulose depletion using Seliwanoff's reaction.
• Validation of the single-tube assay against high-performance liquid chromatography (HPLC) for precise quantification.
• Adaptation of the assay to a 96-well plate format, including steps for cell harvest, supernatant removal, and filtration to minimize interference.

Main Results:

• The optimized protocol accurately quantifies D-allulose depletion, showing excellent agreement with HPLC measurements.
• The 96-well plate format demonstrated high analytical quality with a Z'-factor of 0.449, signal window (SW) of 5.288, and assay variability ratio (AVR) of 0.551.
• These statistical metrics meet the acceptance criteria for a high-quality HTS assay.

Conclusions:

• A reliable and efficient HTS protocol for screening L-RI activity has been successfully established.
• This protocol is suitable for identifying high-activity isomerase variants in protein engineering efforts.
• The developed HTS method has broad applicability in industrial and research settings for enzyme discovery and optimization.