Cloning, purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1
Cloning, purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1
复制标题
柯达热球菌 KOD1 有机溶剂、去垢剂和热稳定淀粉支链淀粉酶的克隆、纯化和生化表征
DOI：
10.1016/j.procbio.2013.04.007
复制
发表时间：
2013-05-01
影响因子：
4.4
通讯作者：
Jia, Baolei
中科院分区：
文献类型：
--
作者：
Guan, Qingtian;Guo, Xiaohan;Jia, Baolei
耐热性淀粉葡聚糖酶可以催化α -1,4和α -1,6糖苷键的水解，在淀粉糖化工业中具有相当大的意义。本研究从极嗜热的柯达热球菌（Thermococcus kodakarensis）的厌氧古菌KOD1中克隆了编码胞外淀粉样葡聚糖酶的基因Apu-Tk。Apu-Tk编码一个含有27个残基的信号肽的1100个氨基酸的蛋白，该蛋白在信号肽切割后的预测质量为125 kDa。序列比对显示Apu-Tk包含GH57家族蛋白中保守的5个区域。全长Apu-Tk在大肠杆菌中表达并纯化至均匀性。纯化后的酶具有普鲁兰酶和淀粉酶活性。Apu-Tk水解普鲁兰和可溶性淀粉的最适温度为150℃~ 100℃。Apu-Tk在较宽的pH范围内（4 ~ 7）也有活性，最适pH为5.0 ~ 5.5。Apu-Tk在8%的SDS或10%的β -巯基乙醇的存在下仍保持了约30%的活性和部分折叠的球状结构。Apu-Tk具有产率高、pH范围宽、稳定性好等特点，是一种具有工业应用前景的酶。(C) 2013 Elsevier Ltd.版权所有。
Thermostable amylopullulanases can catalyse the hydrolysis of both alpha-1,4 and alpha-1,6 glucosidic bonds and are of considerable interest in the starch saccharification industry. In this study, the gene Apu-Tk encoding an extracellular amylopullulanase was cloned from an extremely thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1. Apu-Tk encodes an 1100-amino acid protein with a 27-residue signal peptide, which has a predicted mass of 125 kDa after signal peptide cleavage. Sequence alignments showed that Apu-Tk contains the five regions conserved in all GH57 family proteins. Full-length Apu-Tk was expressed in Escherichia coli and purified to homogeneity. The purified enzyme displayed both pullulanase and amylase activity. The optimal temperature for Apu-Tk to hydrolyse pullulan and soluble starch was >100 degrees C. Apu-Tk was also active at a broad range of pH (4-7), with an optimum pH of similar to 5.0-5.5. Apu-Tk also retained >30% of its original activity and partially folded globular structure in the presence of 8% SDS or 10% beta-mercaptoethanol. The high yield, broad pH range, and stability of Apu-Tk implicate it as a potential enzyme for industrial applications. (C) 2013 Elsevier Ltd. All rights reserved.