Impact of Starch Binding Domain Fusion on Activities and Starch Product Structure of 4-α-Glucanotransferase.

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBD St1 and SBD St2 ) from Solanum tuberosum disproportionating enzyme 2 ( St DPE2) and the C-terminal domain (SBD GA ) of glucoamylase from Aspergillus niger ( An GA). In silico analysis of CBM20s revealed that SBD GA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2-2.4-fold on amylose and decreased 3-9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3-1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBD GA -TuαGT and-SBD St2 -TuαGT showed K d of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBD St1 -TuαGT had 3-5-fold lower affinity. SBD St2 contributed more than SBD St1 to activity, substrate binding, and the stability of TuαGT fusions.