Lipases present biotechnological applications in various industrial sectors due to their ability to perform multiple biochemical reactions. However, the high cost sometimes discourages their potential uses, besides the extensive number of patents involving them. One of the most utilized and researched lipases is Candida antarctica lipase B (CALB), known for its versatility, encompassing enantioselectivity, thermostability, and a wide range of substrates. Therefore, finding new CALB-like lipases is an interesting strategy to enable the implementation of biocatalysts, especially if intellectual property analysis is included. The present study identified and produced six CALB-like enzymes without patent protection, with differences in pocket amino acids and substrate specificity. We conducted genomic searches in almost 7000 Fungal genomes, identifying over 1500 unique CALB homolog candidates. The phylogenetic and intellectual property analysis filtered those results into a few sequences without protection that were very similar to CALB. One cloned lipase had a lower hydrophobicity at the pocket entrance and preferred the C4 p-nitrophenyl ester as substrate. Another had a wider opening and more polar pocket, showing no preference. These results identified new patent-free lipases with conserved essential catalytic elements and diverse substrate specificity due to variations in the catalytic pocket. These enzymes can be the starting point for biocatalyst innovation with potential applications in diverse biotechnological areas.
Keywords: CALB; Enzyme activity; Lipase; Patent protection; Pichia pastoris; Prospection.
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