Search

Carbohydrate binding modules (CBMs) are independent non-catalytic domains widely found in carbohydrate-active enzymes (CAZymes), and they play an essential role in the substrate binding process of CAZymes by guiding the appended catalytic modules to the target substrates. Owing to their precise recognition and selective affinity for different substrates, CBMs have received increasing research attention over the past few decades. To date, CBMs from different origins have formed a large number of families that show a variety of substrate types, structural features, and ligand recognition mechanisms. Moreover, through the modification of specific sites of CBMs and the fusion of heterologous CBMs with catalytic domains, improved enzymatic properties and catalytic patterns of numerous CAZymes have been achieved. Based on cutting-edge technologies in computational biology, gene editing, and protein engineering, CBMs as auxiliary components have become portable and efficient tools for the evolution and application of CAZymes. With the aim to provide a theoretical reference for the functional research, rational design, and targeted utilization of novel CBMs in the future, we systematically reviewed the function-related characteristics and potentials of CAZyme-derived CBMs in this review, including substrate recognition and binding mechanisms, non-catalytic contributions to enzyme performances, module modifications, and innovative applications in various fields. • Carbohydrate binding modules (CBMs) are essential for ligand recognition and specific binding. • The dominant interactions between CBMs and ligands are summarized. • CBMs are portable accessories to improve the properties of carbohydrate-active enzymes. • The novel processing methods and biochemical applications of CBMs are reviewed. [ABSTRACT FROM AUTHOR]

[Display omitted] • Corn starch is successively modified by two GBEs to rearrange glycosidic bonds. • Dual GBE-modified starch shows lower digestibility than single GBE-modified starch. • Synergy between Ro-GBE and Gt-GBE facilitates the rearrangement of glycosidic bonds. • Dual GBE modification offers an efficient approach to reducing starch digestibility. Enzymatically rearranging α-1,4 and α-1,6 glycosidic bonds in starch is a green approach to regulating its digestibility. A two-step modification process successively catalyzed by 1,4-α-glucan branching enzymes (GBEs) from Rhodothermus obamensi STB05 (Ro-GBE) and Geobacillus thermoglucosidans STB02 (Gt-GBE) was investigated as a strategy to reduce the digestibility of corn starch. This dual GBE modification process caused a reduction of 25.8 % in rapidly digestible starch fraction in corn starch, which were more effective than single GBE-catalyzed modification with the same duration. Structural analysis indicated that the dual GBE modified product contained higher branching density, more abundant short branches, and shorter external chains than those in single GBE-modified product. These results demonstrated that a moderate Ro-GBE treatment prior to starch gelatinization caused several suitable alterations in starch molecules, which promoted the transglycosylation efficiency of the following Gt-GBE treatment. This dual GBE-catalyzed modification process offered an efficient strategy for regulating starch digestibility. [ABSTRACT FROM AUTHOR]

Site-directed mutagenesis is a valuable strategy for modifying enzymes, but the lack of understanding of conserved residues regulating glycosidase function hinders enzyme design. We analyzed 1662 enzyme sequences to identify conserved amino acids in maltohexaose-forming amylase at both family and subfamily levels. Several conserved residues at the family level (G37, P45, R52, Y57, D101, V103, H106, G230, R232, D234, E264, H330, D331, and G360) were found, mutations of which resulted in reduced enzyme activity or inactivation. At the subfamily level, several conserved residues (L65, E67, F68, D111, E114, R126, R147, F154, W156, F161, G163, D165, W218H, V342, W345, and F346) were identified, which primarily facilitate substrate binding in the enzyme's active site, as shown by molecular dynamics and kinetic assays. Our findings provide critical insights into conserved residues essential for catalysis and can inform targeted enzyme design in protein engineering. [Display omitted] • The conserved residues at the family level comprise G37, P45, R52, Y57, D101, V103, H106, G230, R232, D234, E264, H330, D331, and G360. • Mutations conserved residues at the family level result in reduced enzyme activity or complete inactivation. • The subfamily level contained several conserved residues, such as L65, E67, F68, D111, E114, R126, R147, F154, W156, F161, G163, D165, W218H, V342, W345, and F346. • Conserved amino acids at the subfamily level primarily facilitate substrate binding in the enzyme's active site. [ABSTRACT FROM AUTHOR]

Enzymatic modification is a potential approach to effectively improve the stability of maltodextrin for food and industrial applications. In this study, a 1,4-α-glucan branching enzyme from Rhodothermus obamensis STB05 (Ro-GBE) was used to modify maltodextrin. The transparency of the Ro-GBE-modified maltodextrin prepared under the optimal modification conditions: enzyme dosage of 100 U/g, reacting time of 12 h at 55 °C, and pH of 7.0, was maintained above 90% for over 30 days. In addition, the viscosity stability, retrogradation property, and freeze–thaw stability were significantly improved. Because of the constant value of the dextrose equivalent (DE), increase in α-1,6-glycosidic linkage ratio, and decrease in weight-average molecular mass (Mw), we believed that the glycosidic bond rearrangement played a vital role in the Ro-GBE modification. Moreover, Ro-GBE exhibited high specificity in cleaving long chains with DP ≥ 15 and transferring the released chains as exterior chains to form a short-clustered structure. Pearson correlation analysis revealed that the transparency stability of maltodextrin mainly correlated with the branching degree, while the viscosity stability, retrogradation property, and freeze–thaw stability mainly contributed to the Mw of branched molecules. [Display omitted] • Improvement in maltodextrin stability by 1,4-α-glucan branching enzyme modification. • The transparency of Ro-GBE modified maltodextrin kept above 90% for over 30 days. • Improved viscosity stability, retrogradation property, and freeze–thaw stability. • Ro-GBE has chain length preference to form short-clustered structure of maltodextrin. • Correlations between molecular structure and storage properties of maltodextrin. [ABSTRACT FROM AUTHOR]