Wangpaiboon Karan, Charoenwongpaiboon Thanapon, Klaewkla Methus, Field Robert A, Panpetch Pawinee
Center of Excellence in Structural and Computational Biology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
Department of Chemistry, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand.
Front Plant Sci. 2023 Jan 27;14:1114215. doi: 10.3389/fpls.2023.1114215. eCollection 2023.
Pullulanase (EC 3.2.1.41, PUL), a debranching enzyme belonging to glycoside hydrolase family 13 subfamily 13, catalyses the cleavage of α-1,6 linkages of pullulan and β-limit dextrin. The present work studied PUL from cassava Crantz (PUL) tubers, an important economic crop. The gene was successfully cloned and expressed in and rPUL was biochemically characterised. PUL was present as monomer and homodimer, as judged by apparent mass of ~ 84 - 197 kDa by gel permeation chromatography analysis. Optimal pH and temperature were at pH 6.0 and 50 °C, and enzyme activity was enhanced by the addition of Ca ions. Pullulan is the most favourable substrate for rPUL, followed by β-limit dextrin. Additionally, maltooligosaccharides were potential allosteric modulators of rPUL. Interestingly, short-chain maltooligosaccharides (DP 2 - 4) were significantly revealed at a higher level when rPUL was mixed with cassava isoamylase 3 (rISA3), compared to that of each single enzyme reaction. This suggests that PUL and ISA3 debranch β-limit dextrin in a synergistic manner, which represents a major starch catabolising process in dicots. Additionally, subcellular localisation suggested the involvement of PUL in starch catabolism, which normally takes place in plastids.
支链淀粉酶（EC 3.2.1.41，PUL）是一种属于糖苷水解酶家族13亚家族13的脱支酶，催化支链淀粉和β-极限糊精的α-1,6键的裂解。本研究以重要经济作物木薯（Crantz）块茎中的PUL为研究对象。该基因已成功克隆并在[具体表达系统]中表达，对重组PUL（rPUL）进行了生化特性分析。通过凝胶渗透色谱分析，rPUL的表观质量约为84 - 197 kDa，表明其以单体和同型二聚体形式存在。最适pH和温度分别为pH 6.0和50℃，添加钙离子可增强酶活性。支链淀粉是rPUL最有利的底物，其次是β-极限糊精。此外，麦芽寡糖是rPUL的潜在别构调节剂。有趣的是，与每个单一酶反应相比，当rPUL与木薯异淀粉酶3（rISA3）混合时，短链麦芽寡糖（聚合度2 - 4）的水平显著更高。这表明PUL和ISA3以协同方式对β-极限糊精进行脱支，这是双子叶植物中主要的淀粉分解过程。此外，亚细胞定位表明PUL参与淀粉分解代谢，淀粉分解代谢通常发生在质体中。
