Cloning, Expression, and Characterization of a D-Psicose 3-Epimerase from Clostridium cellulolyticum H10
Cloning, Expression, and Characterization of a D-Psicose 3-Epimerase from Clostridium cellulolyticum H10
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解纤维梭菌 H10 的 d-阿洛酮糖 3-差向异构酶的克隆、表达和表征
DOI：
10.1021/jf201356q
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发表时间：
2011-07-27
影响因子：
6.1
通讯作者：
Jiang, Bo
中科院分区：
文献类型：
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作者：
Mu, Wanmeng;Chu, Feifei;Jiang, Bo
克隆了原提出的木糖异构酶结构域TIM桶蛋白ACL75304，并在大肠杆菌中进行了表达。表达酶经镍亲和层析纯化，电泳均匀，鉴定为D-psicose 3- epimase。该酶严格依赖金属，在Co2+存在下表现出最大的活性。酶活性的最佳pH和温度为55℃，pH为8.0。在60℃条件下，该酶的半衰期分别为6.8 h和10 min，这表明该酶在有Co2+和无Co2+的条件下具有极强的热稳定性，但在没有金属离子的情况下极易失活。该酶对底物n-psicose的Michaelis-Menten常数（k -m）、周转数（k(cat)）和催化效率（k(cat)/ k -m）分别为17.4 mM、3243.4 min（-1）和186.4 nM min（-1）。在最佳条件下，该酶可将d -果糖外显异构化为D-psicose，转化率为32%，表明该酶是一种潜在的D-psicose生产者。
The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli. The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as D-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co2+. The optimum pH and temperature for enzyme activity were 55 degrees C and pH 8.0. The half-lives for the enzyme at 60 degrees C were 6.8 h and 10 min when incubated with and without Co2+, respectively, suggesting that this enzyme was extremely thermostable in the presence of Co2+ but readily inactivated without metal ion. The Michaelis-Menten constant (K-m), turnover number (k(cat)), and catalytic efficiency (k(cat)/K-m) values of the enzyme for substrate n-psicose were estimated to be 17.4 mM, 3243.4 min(-1,) and 186.4 nM min(-1), respectively. The enzyme carried out the epimerization of D-fructose to D-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential D-psicose producer.