Thermostable Carbohydrate-Active Enzymes for High-Temperature Biomass Conversion: From Molecular Design to Process Integration.

High-temperature processing of carbohydrate-rich biomass, including starch liquefaction and lignocellulose saccharification, can improve sugar yields, reduce contamination risks, and facilitate integration with thermochemical pretreatments. However, the operational stability of native carbohydrate-active enzymes (CAZymes) at 60-90 °C remains a major bottleneck for efficient and economically viable biorefineries, and existing reviews rarely integrate multiscale molecular mechanisms with process-relevant engineering strategies. This review systematically examines the multiscale basis of thermostability in CAZymes-covering core hydrophobic packing, electrostatic and metal-mediated networks, disulfide bonds, conformational dynamics, and modular architectures involving catalytic domains, linkers, and carbohydrate-binding modules-and directly links these features to enzyme longevity under high-solids, inhibitor-rich conditions. We then critically compare key engineering strategies, including structure-guided rational design, semi-rational directed evolution, consensus design, ancestral sequence reconstruction, and machine learning-assisted workflows embedded in design-build-test-learn (DBTL) cycles, highlighting their respective strengths, limitations, and complementarity. Representative case studies demonstrate that engineered thermophilic α-amylases and cellulases achieving ΔTm improvements of approximately 10-20 °C can sustain >80-85% residual activity after prolonged exposure at process-relevant temperatures, translating into tangible benefits such as 15-30% reductions in enzyme dosing and measurable decreases in steam consumption, alongside higher sugar titers. By bridging molecular determinants, engineering paradigms, and quantitative process performance, this review provides a pragmatic roadmap for deploying thermostable CAZymes as robust biocatalysts in sustainable high-temperature biomass conversion.