In the study, we investigated the contribution of Ca²⁺ to the thermostability of α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans , which has two calcium-binding sites (CaI and CaII), and β-CGTase from Bacillus circulans , which contains an additional calcium-binding site (CaIII), consisting of Ala315 and Asp577. It was found that the contribution of Ca²⁺ to the thermostability of two CGTases displayed a marked difference. Ca²⁺ affected β-CGTase thermostability significantly. After Ca²⁺ was added to β-CGTase solution to a final concentration of 5 mM followed by incubation for 120 min at 60 °C, residual activity of β-CGTase was 88.3%, which was much higher than that without Ca²⁺. However, Ca²⁺ had a small contribution to α-CGTase thermostability. Furthermore, A315D and D577K mutations at CaIII could significantly change the contribution of Ca²⁺ to β-CGTase thermostability. These results suggested that the contribution of Ca²⁺ to CGTase thermostability was closely related to CaIII.

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Highland barley (HB) is a nutritious crop with excellent health benefits, and shows promise as an economically important crop with diverse applications. Starch is the main component of HB and has great application potential owing to its unique structural and functional properties. This review details the latest status of research on the isolation, chemical composition, structure, properties, and applications of highland barley starch (HBS). Suggestions regarding how to better comprehend and utilize starches are proposed. The amylopectin content of HBS ranged from 74% to 78%, and can reach 100% in some varieties. Milling and air classification of barley, followed by wet extraction, can yield high-purity HBS. The surface of HBS granules is smooth, and most are oval and disc-shaped. Normal, waxy, and high-amylose HBS have an A-type crystalline. Due to its superb freeze-thaw stability, outstanding stability, and high solubility, HBS is widely used in the food and non-food industries. The digestibility of starch in different HB whole grain products varies widely. Therefore, the suitable HB variety can be selected to achieve the desired glycemic index. Further physicochemical modifications can be applied to expand the variability in starch structures and properties. The findings provide a thorough reference for future research on the utilization of HBS.

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Some thermophilic enzymes not only exhibit high thermostability at high temperatures but also have an activation effect by thermal incubation. However, the correlations between temperature-induced structural modulation and thermal activation are still unclear. In this study, we selected a thermophilic glycogen-debranching enzyme from Saccharolobus solfataricus STB09 (SsGDE), which was a promising starch-debranching enzyme with a thermal activation property at temperatures ranging from 50 to 70 °C, to explore the thermal activation mechanism. Molecular dynamics simulations were performed for SsGDE at 30, 50, or 70 °C to reveal the temperature dependence of structure modulation and catalytic function. The results revealed that four loops (loop1 313–337, loop2 399–418, loop3 481–513, and loop4 540–574) in SsGDE were reshaped, which made the catalytic cavity more open. The internal residues, including the catalytic triad Asp3631, Glu399, and Asp471, could be exposed, due to the structural modulation, to exert catalytic functions. We proposed that the thermal activation effect of SsGDE was closely associated with the temperature-induced modulation of the catalytic cavity, which paved the way for further engineering enzymes to achieve higher catalytic performance and stability.