Metagenomic analysis of Triacylglycerol Lipase enzyme in two soil samples

Rodrigo Ribeiro1*; Mateus Barbosa1; Jéssica Patrícia1; Maricy Bonfá1; Rodrigo Pereira1

1Faculdade de Ciências Biológicas e Ambientais – Universidade Federal da Grande Dourados. *Author’s e-

mail for correspondence: rodrigopereira@ufgd.edu.br

Introduction and objective

The enzyme Triacyglycerol lipase (EC 3.1.1.3) are

hydrolytic enzymes present in several organisms,
including animals, plants fungi and bacteria. In
their natural environment, these enzymes have a
function of catalyzing a hydrolysis of
triacylglycerols to the corresponding fatty acids
and glycerol. Lipase are classified as hydrolases
that can hydrolyse the bond of glycerol esters to
diacylglycerol, monoacylglycerol and free fatty
acids, and also act in the synthesis reaction of
esters through interesterification reaction. They
are enzymes commonly applied in the food,
chemical, pharmaceutical, oleochemistry, biodiesel
and detergents industries due to its bioconversion
functions. In the past, access to these
microorganism was restricted, but with the
emergence and improvement of metagenomics,
we were allowed to know and study in more
details all this unknown genomic material. The
current work aimed to analyze the occurrence of
genes that codify for the lipase enzyme through
bioinformatic programs to identify these
organisms, taxonomically classify those that are
found and which of the 2 different soil samples
provided by EMBRAPA company has more
enzymes.
Methods
The EMBRAPA company provided 5 different soil
samples, which two of them were used, the
integrated crop-livestock soil and the
conventionally planted soil sample. The genetic
material of the soil samples was sequenced
through Illumina next-generation sequencing by
the shotgun method. Data were pre-treated and
the Open Reads Frames (ORF’s) compared with a
local lipase database created from NCBI library
through the blastall command line on linux system.
The output of the local alignment was analyzed
using the Megan6 program to obtain the
cladogram and rarefaction graph, also see the
amount of organisms in each sample. The STAMP
program was used for statistical analyses in a
comparison two-by-two and obtaining extended
error bar graphs to see organisms present in
significant quantities.
Figure 1 – Flowchart conteining the process
steps
Results and discussion
In Megan6 program the analyze returned 73.614
reads and a total of 1.717 identified organisms
which 868 are from integrated farming livestock
soil sample and 849 are from conventionally
planted soil sample. Through Megan6 comparison
11 enzyme-producing phylum returned, which 4
them belong to the fungal kingdom, while the
others 7 vary between plants and microalgae.
Regarding the genus of the identified fungi, 41
different genus were obtained, with 713 organism
in the soil sample of integrated crop farming and
678 organisms in the sample of conventional
planting. Present in greater quantity, the family of
Aspergillaceae counts with 836 organisms,
representing more than half of the total identified
organisms. When the statistical comparison was
made between the two soil samples at phylum
level on STAMP program, organisms from
Streptophyte show greater statistical differences,
being significantly more present in the
conventionally planted soil sample and organisms
from Basidiomycota, Mucoromycota and
Labyrinthulomycetes was statistically more
significant in integrated livestock soil. At genus
level, organism from Stemphylium and Penicillium
was identified with a greater difference between
proportion in integrated livestock soil sample and
organisms from Pleosporaceae and Aspergillaceae
family – the STAMP program can’t find their genus
name – presents a statistically more significant
difference between proportion in conventionally
planted soil sample.

Conclusion

The Aspergillus nigger is the specie that was present in greater numbers in both soil samples. The integrated
farming livestock soil was the one that presented a more numerous mycrobiote and also proved to be a
carrier of greater biodiversity. Even so, there are a lot of organisms present in the conventional planting
sample. This show that both soils are viable for future studies involving lipase-producing organisms.