Alves-Prado Heloiza Ferreira, Gomes Eleni, da Silva Roberto
Biochemistry and Applied Microbiology Laboratory, UNESP-State University of São Paulo, Rua Cristóvão Colombo n 2265, 15054-000, São José do Rio Preto, SP, Brazil.
Appl Biochem Biotechnol. 2007 Apr;137-140(1-12):41-55. doi: 10.1007/s12010-007-9038-2.
A cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19) from a newly isolated alkalophilic and moderately thermophilic Paenibacillus campinasensis strain H69-3 was purified as a homogeneous protein from culture supernatant. Cyclomaltodextrin glucanotransferase was produced during submerged fermentation at 45 degrees C and purified by gel filtration on Sephadex G50 ion exchange using a Q-Sepharose column and ion exchange using a Mono-Q column. The molecular weight of the purified enzyme was 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the pI was 5.3. The optimum pH for enzyme activity was 6.5, and it was stable in the pH range 6.0-11.5. The optimum temperature was 65 degrees C at pH 6.5, and it was thermally stable up to 60 degrees C without substrate during 1 h in the presence of 10 mM CaCl(2). The enzyme activity increased in the presence of Co(2+), Ba(2+), and Mn(2+). Using maltodextrin as substrate, the K(m) and K(cat) were 1.65 mg/mL and 347.9 micromol/mg x min, respectively.
从新分离的嗜碱中度嗜热的坎皮纳斯芽孢杆菌菌株H69 - 3中提取的环麦芽糊精葡糖基转移酶（E.C. 2.4.1.19），作为一种纯蛋白从培养上清液中纯化得到。环麦芽糊精葡糖基转移酶在45℃的深层发酵过程中产生，并通过在Sephadex G50上进行凝胶过滤、使用Q - Sepharose柱进行离子交换以及使用Mono - Q柱进行离子交换来纯化。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测得纯化酶的分子量为70 kDa，其等电点为5.3。酶活性的最适pH为6.5，在pH 6.0 - 11.5范围内稳定。在pH 6.5时最适温度为65℃，在10 mM CaCl₂存在下，无底物时在60℃下热稳定1小时。在Co²⁺、Ba²⁺和Mn²⁺存在下酶活性增强。以麦芽糊精为底物时，Kₘ和Kₑₜ分别为1.65 mg/mL和347.9 μmol/mg·min。
