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首页> 外文期刊>Preparative biochemistry & biotechnology: An international journal for rapid communication >Purification, optimization of assay, and stability studies of dextransucrase isolated from weissella cibaria JAG8
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Purification, optimization of assay, and stability studies of dextransucrase isolated from weissella cibaria JAG8

机译：分离自Weissella cibaria JAG8的右旋糖核酸酶的纯化，优化测定和稳定性研究

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摘要

Dextransucrase-producing (Gen Bank accession no. KC110687) Weissella cibaria JAG8 was isolated from apple. The cell-free extract containing dextransucrase with specific activity of 1.0 U/mg was purified by polyethylene glycol (PEG). A concentration of 33% (v/v) PEG-400 fractionation gave a specific activity of 20.0 U/mg, whereas 15% (w/v) PEG-1500 resulted in a specific activity of 10.6 U/mg. The PEG-400-purified enzyme was further purified by chromatography using a Sephacryl S-300HR column, which resulted in 37-fold purification with 37 U/mg. The non-denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of column-purified enzyme showed a single homogenous band of 177 kDa by silver staining. The production of dextran was confirmed by in situ detection of the activity band using periodic acid-Schiff's base staining. The optimum assay conditions for dextransucrase were 35°C, pH 5.4, and 5.0% (w/v) sucrose concentration. The enzyme followed Michaelis-Menten kinetics with K_m of 13 mM and V_(max) 27.5 U/mg. The enzyme was stable in 10-500 mM sodium acetate buffer, pH 5.4. A 22% increase in enzyme activity was observed with 2 mM magnesium chloride; 64% loss in enzyme activity was observed with 10 mM ethylenediamine tetraacetic acid (EDTA), whereas a complete loss in activity was observed with 5 M urea. The dextransucrase was stable up to 35°C and pH of 5.4 for 1 hr.
机译：从苹果中分离出产生葡聚糖的酶（Gen Bank登录号：KC110687）魏氏杆菌Cibaria JAG8。用聚乙二醇（PEG）纯化比活性为1.0 U / mg的含右旋糖核酸酶的无细胞提取物。浓度为33％（v / v）的PEG-400分馏比活性为20.0 U / mg，而浓度为15％（w / v）的PEG-1500则比活性为10.6 U / mg。使用Sephacryl S-300HR色谱柱，通过色谱法进一步纯化PEG-400纯化的酶，从而得到37 U / mg的37倍纯化率。柱纯化的酶的非变性十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）通过银染显示177 kDa的单一均一带。通过使用高碘酸-希夫氏碱染色原位检测活性带证实了葡聚糖的产生。右旋糖核酸酶的最佳测定条件是35°C，pH 5.4和5.0％（w / v）蔗糖浓度。该酶遵循Michaelis-Menten动力学，K_m为13 mM，V_（max）27.5 U / mg。该酶在pH 5.4的10-500 mM乙酸钠缓冲液中稳定。用2 mM氯化镁观察到酶活性增加了22％;用10 mM乙二胺四乙酸（EDTA）观察到酶活性损失64％，而用5 M尿素观察到活性完全损失。右旋糖核酸酶在35℃和pH 5.4下稳定1小时。

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