State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, NO.1 West Beichen Road, Chaoyang District, Beijing 100101, China.
Appl Microbiol Biotechnol. 2010 Jun;87(1):225-33. doi: 10.1007/s00253-010-2439-0. Epub 2010 Feb 13.
A thermostable glucoamylase (TtcGA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli. The full-length gene (2112 bp) encodes a 703-amino acid polypeptide including a predicted signal peptide of 21 residues. The recombinant mature protein was partially purified to 30-fold homogeneity by heat treatment and gel filtration chromatography. The mature protein is a monomer with the molecular weight of 77 kD. The recombinant enzyme showed maximum activity at 75 degrees C and pH 5.0. It is the most thermostable bacterial glucoamylase described to date with nearly no activity loss after incubation at 75 degrees C for 6 h. TtcGA can hydrolyze both alpha-1, 4- and alpha-1, 6-glycosidic linkages in various alpha-glucans. It showed preference for maltooligosaccharides over polysaccharides with specific activity of 80 U/mg towards maltose. Kinetic studies revealed that TtcGA had the highest activity on maltooligosaccharide with four monosaccharide units. The cations Ca2+, Mn2+, Co2+, Mg2+, and reducing agent DTT showed no obvious effects on the action of TtcGA. In contrast, the enzyme was inactivated by Zn2+, Pb2+, Cu2+, and EDTA.
一株嗜热厌氧菌（Thermoanaerobacter tengcongensis MB4）来源的热稳定糖化酶（TtcGA）在大肠杆菌中成功表达。全长基因（2112 bp）编码一个 703 个氨基酸的多肽，包括一个预测的 21 个氨基酸的信号肽。重组成熟蛋白经热处理和凝胶过滤层析部分纯化至 30 倍纯度。成熟蛋白是一个单体，分子量为 77 kD。重组酶在 75°C 和 pH 5.0 时表现出最大活性。它是迄今为止描述的最耐热的细菌糖化酶，在 75°C 孵育 6 小时后几乎没有活性损失。TtcGA 可以水解各种α-葡聚糖中的α-1,4-和α-1,6-糖苷键。它对麦芽寡糖表现出偏好，对麦芽四糖的比活性为 80 U/mg。动力学研究表明，TtcGA 在四糖单位的麦芽寡糖上具有最高的活性。阳离子 Ca2+、Mn2+、Co2+、Mg2+和还原剂 DTT 对 TtcGA 的作用没有明显影响。相比之下，Zn2+、Pb2+、Cu2+和 EDTA 使酶失活。
