Abstract:
Background: Hydrolysis of cellulose-based biomass by cellulases produce fermented sugar for making biofuels,
such as bioethanol. Cellulases hydrolyze the β-1,4-glycosidic linkage of cellulose and can be obtained from cultured
and uncultured microorganisms. Uncultured microorganisms are a source for exploring novel cellulase genes
through the metagenomic approach. Metagenomics concerns the extraction, cloning, and analysis of the entire
genetic complement of a habitat without cultivating microbes. The glycoside hydrolase 5 family (GH5) is a cellulase
family, as the largest group of glycoside hydrolases. Numerous variants of GH5-cellulase family have been identified
through the metagenomic approach, including CelGH5 in this study. University-CoE-Research Center for
Biomolecule Engineering, Universitas Airlangga successfully isolated CelGH5 from waste decomposition of oil palm
empty fruit bunches (OPEFB) soil by metagenomics approach. The properties and structural characteristics of GH5-
cellulases from uncultured microorganisms can be studied using computational tools and software.
Results: The GH5-cellulase family from uncultured microorganisms was characterized using standard computationalbased tools. The amino acid sequences and 3D-protein structures were retrieved from the GenBank Database and
Protein Data Bank. The physicochemical analysis revealed the sequence length was roughly 332–751 amino acids, with
the molecular weight range around 37–83 kDa, dominantly negative charges with pI values below 7. Alanine was the
most abundant amino acid making up the GH5-cellulase family and the percentage of hydrophobic amino acids was
more than hydrophilic. Interestingly, ten endopeptidases with the highest average number of cleavage sites were
found. Another uniqueness demonstrated that there was also a difference in stability between in silico and wet lab.
The II values indicated CelGH5 and ACA61162.1 as unstable enzymes, while the wet lab showed they were stable at
broad pH range. The program of SOPMA, PDBsum, ProSA, and SAVES provided the secondary and tertiary structure
analysis. The predominant secondary structure was the random coil, and tertiary structure has fulfilled the structure
quality of QMEAN4, ERRAT, Ramachandran plot, and Z score.
