Exploring the enzymatic landscape of 4-α-glucanotransferases in carbohydrate bioprocessing

4-α-Glucanotransferases (4αGTs), also known as amylomaltases or disproportionating enzymes, catalyze α-1,4-glucan and maltooligosaccharide transfer in starch and glycogen metabolism of microorganisms, plants and animals. The present review covers their classification, reactions, structure-function relationships, engineering and applications. 4αGTs belong to glycoside hydrolase families GH13, GH57, and GH77, and catalyze four types of reactions: disproportionation, cyclization, coupling, and hydrolysis, of which the first two are particularly important for biotechnological applications. Insights into active site structures and substrate binding have facilitated the rational design of product specificity, modes of action, and increased product yields. Assays of the four reactions encompass monitoring amylose consumption by iodine staining, release of glucose in maltotriose disproportionation, chromatographic analysis of change in chain lengths, and release of reducing sugar by hydrolysis. Major reactions in transglycosylation of starch include formation of amylopectin with extended branch chains and cyclization to large-ring cyclodextrins (LR-CDs), also referred to as cycloamyloses (CAs). Product yields, chain length distribution, and size of LR-CDs depend on the enzyme, substrates and reaction conditions. 4αGT products are useful in the food, biomaterials and pharma sectors. Thus, chain length modification can elicit resistance of starch to digestion via structural reorganization and confer thermo-reversible gel formation, while LR-CDs can increase aqueous solubility of guest-molecules for controlled delivery and adjust rheological behavior of starches. Moreover, 4αGT can generate bioactive glycoconjugates and novel oligosaccharides by transglycosylation. Future development of 4αGT-catalyzed reactions includes optimization by rational enzyme engineering and high-throughput screening technologies. This review portrays the immense potential of 4αGTs in sustainable biomanufacturing.