Engineering Technological Center of Mushroom Industry, and School of Biological Science and Biotechnology, Minnan Normal University, 36 Xianqianzhi Street, Zhangzhou 363000, Fujian, People's Republic of China; Key Laboratory of Agro-products Processing Technology at Jilin Provincial Universities, Education Department of Jilin Provincial Government, Changchun University, 6543 Weixing Road, Changchun 130022, Jilin, People's Republic of China.
Key Laboratory of Agro-products Processing Technology at Jilin Provincial Universities, Education Department of Jilin Provincial Government, Changchun University, 6543 Weixing Road, Changchun 130022, Jilin, People's Republic of China.
Int J Biol Macromol. 2018 Jul 15;114:235-243. doi: 10.1016/j.ijbiomac.2018.03.073. Epub 2018 Mar 16.
The acidophilic and thermophilic pullulanases have many potential applications in the processes of starch liquefaction and saccharification. In this study, a gene encoding an amylopullulanase from Thermofilum pendens (TPApu) was heterologously expressed in Escherichia coli. Although TPApu possessed the same continuous GH57N_Apu domain and the succeeding α-helical region as other two amylopullulanases from Staphylothermus marinus (SMApu) and Caldivirga maquilingensis (CMApu), it only showed maximal amino acid identities of 25.7-28.7% with CMApu and SMApu. The purified TPApu appeared as a single band of SDS-PAGE with a molecular mass of 65.5kDa and exhibited the maximal activity at pH3.5 and 95-100°C. TPApu had the highest catalytic efficiency towards pullulan (kcat/km, 8.79smLmg) and α-cyclodextrin (kcat/km, 0.36smM). In the initial stages, the ring-opening reactions of γ-cyclodextrin, 6-O-glucosyl-β-cyclodextrin, 6-O-maltosyl-β-cyclodextrin and the debranching reactions of 6-O-maltooctaosyl-β-cyclodextrin were firstly catalyzed. In the subsequent reactions, a serial of maltooligosaccharides were produced. As the most acidophilic amylopullulanase among thermophilic pullulanases reported to date, TPApu preferred to debranch the DP6-12 side chains of amylopectin at pH4.5 and 100°C.
嗜酸性和嗜热 pullulanases 在淀粉液化和糖化过程中有许多潜在的应用。本研究中，来自 Thermofilum pendens 的基因编码的支链淀粉 pullulanase（TPApu）在大肠杆菌中异源表达。尽管 TPApu 具有与来自 Staphylothermus marinus（SMApu）和 Caldivirga maquilingensis（CMApu）的其他两种支链淀粉 pullulanase 相同的连续 GH57N_Apu 结构域和随后的α-螺旋区，但它与 CMApu 和 SMApu 的最大氨基酸同一性仅为 25.7-28.7%。纯化的 TPApu 在 SDS-PAGE 中呈现出单一条带，分子量为 65.5kDa，在 pH3.5 和 95-100°C 下表现出最大活性。TPApu 对 pullulan（kcat/km，8.79smLmg）和α-环糊精（kcat/km，0.36smM）具有最高的催化效率。在初始阶段，γ-环糊精、6-O-葡萄糖基-β-环糊精、6-O-麦芽糖基-β-环糊精的开环反应和 6-O-麦芽八糖基-β-环糊精的支链反应首先被催化。在随后的反应中，一系列麦芽低聚糖被产生。作为迄今为止报道的嗜热 pullulanases 中最嗜酸性的支链淀粉 pullulanase，TPApu 更喜欢在 pH4.5 和 100°C 下支化支链淀粉的 DP6-12 侧链。
