Exploring hot-spring metagenomes for the repertoire of carbohydrate-active enzymes and potential bacterial strains for enzyme production

This study employs a targeted metagenome mining approach to identify carbohydrate-active enzymes (CAZymes) from thermal aquatic environments in the Sikkim Himalaya, followed by functional validation through enzyme screening of bacterial isolates. Metagenomic approach highlighted an abundance of CAZymes in the metagenomic data of Yumthang (3,934), followed by Borong (1,606), Lingdem (1,557), Polok (1096), and Reshi (831). Over 50% of the predicted repertoire was found to be primarily constituted by two classes of CAZyme: glycosyl transferases (GT) (45%) and glycoside hydrolases (GH) (28%). Bacterial isolates were screened to produce high-value biomass transforming enzymes such as xylanase, pectinase, amylase, and β-glucosidase based on the metagenomic data. The enzymes produced from the selected bacterial isolates of Bacillus velezensis PBW5, Bacillus subtilis PBW7, Bacillus haynesii ZNS4, and Bacillus licheniformis PAS3 showed activity at high temperature. B. velezensis PBW5 and strains of B. subtilis were identified as potential producers of xylanase apart from ability to produce other CAZymes. At the same time, B. licheniformis PAS3 and B. haynesii ZNS4 were recorded to be the effective pectinase and amylase producers. The process was developed for submerged and solid-state fermentation (using industrial orange pomace) for simultaneous production of xylanase and pectinase from B. velezensis PBW5 and B. licheniformis PAS3 having activity at high temperature range (50-80°). This study has been pivotal in exploring the enzymatic repertoire of microorganisms from untapped ecological niches of Sikkim and identifying novel strains for the biomass transformation into value-added products.