Fungal Extracellular Lipases from Coffee Plantation Environments for the Sustainable Management of Agro-Industrial Coffee Biomass

Abstract

Coffee wastes have large amounts of by-products rich in phenolic compounds such as chlorogenic and caffeic acid, with potential applications for developing fine chemicals such as caffeic acid phenethyl ester (CAPE). A screening for microorganisms was undertaken in a coffee plantation environment to isolate native tropical species able to modify secondary metabolites present in this kind of biomass enzymatically. From the screening, 130 fungal strains could grow in lipase inducer media. Fungal strains were identified via ITS-based sequencing. Classification based on BLAST assigned 51 isolates to 12 different genera, including Absidia, Aspergillus, Cunninghamella, Fusarium, Metarhizium, Meyerozyma, Mucor, Neocosmospora, Papiliotrema, Penicillium, Rhizopus, and Trichoderma. DNA sequencing identified 14 putative extracellular lipases. According to the extracellular lipase activity, the most promising strain was identified as Fusarium sp. by DNA barcoding. Extracellular lipases from this strain exhibited maximal hydrolytic activity at a temperature of 45 °C, a pH of 7.00, and 200 ppm of NaCl, with an affinity towards substrates having carbon chain lengths of 8 or longer. Under these conditions, lipase instead of esterase activity is the main feature. The Km and Vmax values calculated using p-nitrophenyl palmitate (pNPP) as hydrolysis substrate were 0.003 mM and 299.8 μmol min−1 mg−1, respectively. Fusarium sp. lipases presented high stability during freeze–thawing, allowing the storage of enzyme solutions at −20 °C, but not as a lyophilized powder. According to our kinetic study, these lipases catalyzed CAPE hydrolysis, showing a progressive decrease in the concentration of the CAPE and a correspondent increase in the caffeic acid concentration as a product of this hydrolysis. Being able to carry out this type of reaction under mild conditions shows that Fusarium sp. lipases recognize CAPE as substrate and suggest CAPE synthesis (reverse reaction) and transformation can be engineered, using caffeic acid from coffee biomass, as a potential industrial application for these lipases.