Novel CaLB-like Lipase Found Using ProspectBIO, a Software for Genome-Based Bioprospection

Area of Science:

• Biotechnology
• Bioinformatics
• Enzyme Engineering

Background:

• Enzymes are crucial in food, pharmaceutical, and industrial fuel sectors.
• In silico bioprospecting offers an efficient strategy for discovering new enzyme candidates.
• Limitations exist in conventional similarity-based searches for novel enzymes.

Purpose of the Study:

• To introduce and validate the ProspectBIO software for in silico enzyme discovery.
• To identify novel lipase candidates using sequence homology and conserved motifs.
• To experimentally validate a newly discovered lipase for biotechnological applications.

Main Methods:

• Development of ProspectBIO software for non-annotated sequence identification via genome-based homolog searching.
• Prospecting for lipases using sequence homology to *Candida antarctica* lipase B (CaLB) and conserved motifs.
• Experimental validation including gene expression in *Pichia pastoris*, enzyme activity assays, and structural analysis.

Main Results:

• ProspectBIO identified 1672 lipase sequences, significantly more than the 733 found by conventional database searches.
• A novel lipase gene from *Ustilago hordei* (UhL) was selected based on homology and lack of patent protection.
• Recombinant UhL exhibited tributyrin hydrolysis activity, with activity enhanced 4-fold under optimized induction conditions.
• Structural analysis revealed high similarity to CaLB but with distinct active pocket entrance features, suggesting altered substrate specificity.

Conclusions:

• ProspectBIO is an effective tool for discovering novel enzyme candidates, including lipases, from genomic data.
• The identified *Ustilago hordei* lipase (UhL) shows promise for biotechnological applications.
• Optimized expression and structural insights provide a foundation for further enzyme engineering and application development.