Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark.
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Molecules. 2023 Jan 30;28(3):1320. doi: 10.3390/molecules28031320.
A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from (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 and SBD) from disproportionating enzyme 2 (DPE2) and the C-terminal domain (SBD) of glucoamylase from (GA). In silico analysis of CBM20s revealed that SBD 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-TuαGT and-SBD-TuαGT showed of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBD-TuαGT had 3-5-fold lower affinity. SBD contributed more than SBD to activity, substrate binding, and the stability of TuαGT fusions.
广泛的酶被用于修饰淀粉以用于各种应用。在这里，一种来自嗜热菌的 4-α-葡聚糖转移酶（TuαGT）通过在其 N 端融合碳水化合物结合模块家族 20（CBM20）的淀粉结合结构域（SBD）来进行工程改造，以增强其对颗粒状淀粉的亲和力。这些 SBD 是来自歧化酶 2（DPE2）的 N 端串联结构域（SBD 和 SBD）和来自 （GA）的葡萄糖淀粉酶的 C 端结构域（SBD）。对 CBM20 的计算机分析表明，SBD 和 GH77 DPE2 的 2 个拷贝属于进化树上分隔良好的簇；第二个拷贝与非 CAZyme CBM20 更为接近。SBD-TuαGT 融合蛋白在直链淀粉上的活性比 TuαGT 提高了 1.2-2.4 倍，而在麦芽三糖上的活性则降低了 3-9 倍。融合蛋白在糊化的普通玉米淀粉（NMS）上显示出相似的歧化活性。值得注意的是，融合蛋白的水解活性提高了 1.3-1.7 倍，导致修饰淀粉的分子量降低，α-1,6/α-1,4 键的比例升高。值得注意的是，SBD-TuαGT 和-SBD-TuαGT 对蜡质玉米淀粉（WMS）颗粒的结合常数（Kd）分别为 0.7 和 1.5mg/mL，而 TuαGT 和 SBD-TuαGT 的亲和力则低 3-5 倍。SBD 对融合蛋白的活性、底物结合和稳定性的贡献大于 SBD。
