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-alpha-glucanotransferase from Thermoproteus uzoniensis (Tu alpha GT) 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 (SBDSt1 and SBDSt2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBDGA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBDGA 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-Tu alpha GT fusions increased 1.2-2.4-fold on amylose and decreased 3-9 fold on maltotriose compared with Tu alpha GT. 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 alpha-1,6/alpha-1,4-linkage ratio of the modified starch. Notably, SBDGA-Tu alpha GT and-SBDSt2-Tu alpha GT showed K-d of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas Tu alpha GT and SBDSt1-Tu alpha GT had 3-5-fold lower affinity. SBDSt2 contributed more than SBDSt1 to activity, substrate binding, and the stability of Tu alpha GT fusions.

Automated summary

In this study, a thermophilic 4-alpha-glucanotransferase was engineered by fusing starch binding domains (SBDs) to enhance its affinity for starch, leading to increased activity and stability.