Unlocking the molecular engineering of Geobacillus glycoside hydrolases as a source of industrial biocatalysts

This review examines Geobacillus sensu stricto as a source of thermostable glycoside hydrolases (GH) for biomass conversion, food processing, and enzyme engineering. Recent peer-reviewed literature was assessed with emphasis on taxonomy, genome-based Carbohydrate-Active Enzymes (CAZyme) prediction, biochemical validation, structural data, and engineering case studies. Taxonomic boundaries were interpreted using current Anoxybacillaceae frameworks, with Parageobacillus treated as a related comparator rather than as Geobacillus. The strongest evidence supports GH13 alpha-amylases, xylan-active systems, beta-xylosidases, and selected accessory enzymes. Recent studies also show that genome mining must be coupled with enzymatic assays and product profiling because CAZyme annotation alone does not prove industrial function. Molecular engineering has improved relevant traits, including the longer thermal half-life of engineered G. stearothermophilus alpha-amylase variants, the increased catalytic efficiency of oligo-alpha-1,6-glucosidase variants, and improved AmyS expression in Bacillus subtilis. Geobacillus glycoside hydrolases are best interpreted as process-specific, engineerable biocatalytic templates. Their translation requires reliable taxonomy, functional validation, structural interpretation, scalable expression and testing on realistic substrates. This synthesis also recognises current limitations: many predicted CAZymes still lack biochemical validation, complete cellulolytic systems remain less mature than xylan- and starch-active systems, and scale-up data remain scarce.