preprints.org > biology and life sciences > biology and biotechnology > doi: 10.20944/preprints202308.0633.v1

Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Version 1 : Received: 4 August 2023 / Approved: 7 August 2023 / Online: 8 August 2023 (08:30:15 CEST)

Lipases have tremendous potential for industrial use, particularly, those mostly active against water-insoluble substrates, such as triglycerides composed of long-chain fatty acids. However, in most cases, mutants often need to be constructed to achieve optimal performance for such substrates. Protein engineering techniques have been reported as strategies for improving lipase characteristics by introducing specific mutations in the cap domain of esterases or in the lid domain of lipases, or through lid domain swapping. Here, we have improved the lipase character of a lipase, retrieved from the Marine Metagenomics MarRef Database and assigned to the Actinoalloteichus genus (WP_075743487.1), through site-directed mutagenesis and by substituting its lid domain (FRGTEITQIKDWLTDA) by the one of Rhizopus delemar (previously Rhizopus oryzae) lipase (FRGTNSFRSAITDIVF). Results demonstrated that the redesigned mutants gain activity against bulkier triglycerides such as glyceryl tridecanoate and tridodecanoate, olive oil, coconut oil, and palm oil. The absence of a residue in the new lid, present in the original lid, appears to be a key aspect to the increase in lipase character, although full activity recovery is only obtained with lid swapping.

lipase; lid domain; protein engineering; rational design

Biology and Life Sciences, Biology and Biotechnology

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