Abstract:
Lipases are biocatalysts that possess remarkable abilities to hydrolyze triacylglycerols, breaking
them down into glycerol and fatty acids. These enzymes also catalyze reversible reactions, including
esterification, transesterification, and interesterification. The applications of lipases have greatly
expanded across various industries, notably in food, pharmaceuticals, cosmetics, detergents, textiles,
pulp and paper, leather, medical applications, and many others. Among all living organisms that
produce lipases, filamentous fungi are excellent extracellular lipase producers, and fungal lipases
have the ability to function at extreme temperatures and pH, are stable in organic solvents, chemo selective, and abundant. Many research studies have been conducted in this area; nevertheless, there
are many more lipolytic fungal species to be identified and characterized. Thus, the current study
was conducted to isolate and identify the novel lipolytic fungi from coconut oil-contaminated soil.
The soil samples were collected from four different areas (A: Normal soil sample from mini Sinharaja
area, Faculty of Science, University of Peradeniya, B: Compost sample from Gampaha area, C: Coconut
oil-contaminated soil sample from oil mill in Gampaha, D: Coconut oil-contaminated soil sample from
oil mill in Kandy). The collected soil samples were cultured in the PDA medium, and fungal species
were isolated using streak plate and point inoculation techniques. The fungal strains that were found
to be positive by phenol red and Tween 20 tests were subjected to para-nitrophenyl palmitate assay
to analyze the lipolytic activity of each species. The selected strains were identified using
morphological characteristics and molecular methods. Out of the 12 isolated species, 10 species gave
positive results for lipolytic activity, and those were identified based on colony morphology and
microscopic view (slide culture technique). Based on the results of the para-nitrophenyl palmitate
assay, DNA from three species with the highest lipolytic activity were extracted using the phenol chloroform method and heating method, and ITS regions were sequenced. Through BLAST search
and phylogenetic analysis, the most efficient fungal species capable of lipolysis were identified as
Rhizopus arrahizus (the most efficient), followed by Aspergillus niger (the second most efficient),
and Aspergillus nomius (the third most efficient). In order to establish these fungal species as viable
sources of lipases for industrial applications, further enzyme characterization and media
optimization studies are necessary.
