Currently, the catalytic residue of the highly prolific fungal β-glucosidase (BGL) of Trichoderma asperellum UC1 remains unvalidated. The study used the alanine scanning method to confirm the catalytic residues of the BGL as Glu165, Asp226, and Glu423. This method cancels out all intermolecular hydrogen bonds with substrates, lignin, hemicellulose, and cellulose. Results revealed an overall decline in the stability of the energy-minimized mutant enzymes' compared to the wild-type BGL. The mutant enzyme registered lower PROCHECK (91.0%), ERRAT (93.09%), and Verify-3D (98.92%) values, in comparison to 90.2%, 92.09%, 98.06%, in the wild-type BGL, respectively. The mutant BGL UC1-substrate complexes were less stable than the wild-type enzyme, in which the mutant exhibited higher binding energies for docked lignin (−7.4% kcal mol-1), cellulose (−7.2 kcal mol-1), and hemicellulose (−7.2 kcal mol-1). Binding energies of the wild-type BGL with the corresponding substrates were lower at −7.9 kcal mol-1, −8.1 kcal mol-1, and −7.8 kcal mol-1. An interesting observation was that the alanine scanning changed the substrate preference order based on the calculated binding energies. The mutant BGL bound preferentially to lignin>cellulose=hemicellulose, while the wild-type BGL was selective to cellulose>lignin>hemicellulose. Hence, the findings convey the high likelihood of Glu165, Asp 256, and Glu423 are the catalytic residues of the BGL of T. asperellum UC1.
Digestive gland Achatina fulica is source of bioprospecting of glycoside hydrolases enzymes for many biotechnological and industrial processes. Nevertheless, there is limitation to discover novel enzymes. The functional-based approach analyzes the metagenomic library based on the genomic function of an organism used to look for new enzyme-producing genes. The aim of this study was to determine the chances of obtaining the novel genes for glycoside hydrolases from enzymes reservoir of digestive gland A. fulica through functional metagenomic method. The results showed that the total RNA concentration isolated from the digestive gland of A. fulica was 2,343.2 ng/uL. A total of 2 uL of total RNA has been used to construct the metagenomic library, so there are 4.69. 1010 - 1.87. 1011 transcribed molecules from 1.17. 109 - 4.68. 109 genes. This is a great number of chances to acquire the novel glycoside hydrolase genes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.