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.

In the current study, an ethyl acetate extract from the endophytic fungus Aspergillus ochraceus SPH2 isolated from the stem parts of the endemic plant Bethencourtia palmensis was screened for its biocontrol properties against plant pathogens (Fusarium moniliforme, Alternaria alternata and Botrytis cinerea), insect pests (Spodoptera littoralis, Myzus persicae, Rhopalosiphum padi), plant parasites (Meloidogyne javanica) and ticks (Hyalomma lusitanicum). SPH2 gave extracts with strong fungicidal and ixodicidal effects at different fermentation times. The bioguided isolation of these extracts gave compounds 1-3. Mellein (1) showed strong ixodicidal effects and was also fungicidal. This is the first report on the ixodicidal effects of 1. Neoaspergillic acid (2) showed potent antifungal effects. Compound 2 appeared during the exponential phase of the fungal growth while neohydroxyaspergillic acid (3) appeared during the stationary phase, suggesting that 2 is the biosynthetic precursor of 3. Additional molecular ions compatible with pyrazynes that were detected during the exponential phase included flavacol, and aspergilliamide while ochramide A was mostly detected during the stationary phase of the fermentation. Moreover, polyketids were also detected during the sationaty phase of the fermentation curve (dihydroaspyrone, aspyrone, asperlactone) and the alkaloid circumdatin H. Ochratoxin A was not detected. Therefore, SPH2 could be a potential biotechnological tool for the production of ixodicidal mellein.