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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 computational-based 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. Conclusion This study can afford the new insights about the physicochemical and structural properties of the GH5-cellulase family from uncultured microorganisms. Furthermore, in silico analysis could be valuable in selecting a highly efficient cellulases for enhanced enzyme production.

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GbtXyl43A, a β-xylosidase that is isolated from Geobacillus thermoleovorans IT-08 and grouped in GH43 family. The substitution of 121Asp residue with Asn in GbtXyl43A caused decrease the enzyme activity. The aim of this study, determine the kinetic characteristics of wild-type GbtXyl43A and D121N variant using Vmax, KM, kcat, and kcat/KM. These parameters indicated catalytic mechanism of GbtXyl43A and its derivative. All of them were produced in Escherichia coli BL21 star. The purification of wild-type GbtXyl43A using affinity chromatography, but D121N variant also required anion-exchange chromatography. The specific activity of wild-type GbtXyl43A and D121N variant were 0.471 U mg-1 in purity level 55,44 and 0.012 U mg-1 in purity level 2,407, respectively. Both enzymes had same molecular weight, ~58 kDa. The kinetic parameters of wild-type GbtXyl43A were KM: 2.845 mM, kcat: 0.033 s-1, Vmax: 0.0033 mM min-1and kcat/KM: 0.0115 s-1mM-1. Furthermore, the KM, kcat, Vmax, and kcat/KM values of D121N variant were 4.565 mM, 1.01 × 10-4 mM min-1, 0.140 × 10-4 s-1, and 0.0307 s-1mM-1, respectively. The KM value of the D121N variant was higher than its wild type and showed the affinity of D121N variant was lower than GbtXyl43A