Thermostable Transketolase from Geobacillus stearothermophilus: Characterization and Catalytic Properties
Thermostable Transketolase from Geobacillus stearothermophilus: Characterization and Catalytic Properties
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DOI：
10.1002/adsc.201200590
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发表时间：
2013-01-01
影响因子：
5.4
通讯作者：
Hecquet, Laurence
中科院分区：
文献类型：
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作者：
Abdoul-Zabar, Juliane;Sorel, Isabelle;Hecquet, Laurence
在这里，我们鉴定了第一个来自嗜热微生物——嗜热脂肪地杆菌的转酮酶（TK），该酶是由大肠杆菌合成基因表达的。G. stearotherophilus TK （mTKgst）在50？在摄氏65度的高温下工作3天。温度范围在6070℃左右。这将有助于制备应用和未来的生物催化剂的发展。mTKgst的热稳定性使我们能够在65℃的温度下对粗细胞提取物进行热休克处理，进行简单的一步纯化。温度45分钟，从1l培养物中直接产生132 mg纯mTKgst。在70℃时，mTKgst与乙醇醛的反应速率提高了14倍。比20摄氏度高吗？摄氏50度是摄氏4倍？与相同条件下的大肠杆菌TK相比，50岁时测试？以其他醛为受体时，mTKgst活性约为20℃时的3倍。以羟基丙酮酸为供体，三种醛为受体，分别制备了l -赤己糖1、d -甘油醛和丁醛，得到了相应的产物l -赤己糖1、d -木酮糖2和1,3-二羟基己酮-2- 1 - 3。产物1和2的旋光性表明，mTKgst的立体特异性与其他TK源相同，导致（3S）构型。对于非羟基化底物丁醛，ee值为85% (3S)，比大肠杆菌TK （75% ee, 3S）具有更高的对映选择性。高温下的工艺可以通过有利于疏水醛受体底物的溶解度和对非常规介质的耐受性，为扩展TK生物催化的应用提供机会。
Here we have characterized the first transketolase (TK) from a thermophilic microorganism, Geobacillus stearothermophilus, which was expressed from a synthetic gene in Escherichia coli. The G. stearothermophilus TK (mTKgst) retained 100% activity for one week at 50?degrees C and for 3 days at 65?degrees C, and has an optimum temperature range around 6070?degrees C, which will be useful for preparative applications and for future biocatalyst development. The thermostability of the mTKgst allowed us to carry out an easy, one-step purification by heat shock treatment of crude cell extracts at 65?degrees C for 45 min, directly yielding 132 mg of pure mTKgst from 1 L of culture. The reaction rate of mTKgst with glycolaldehyde was 14 times higher at 70?degrees C than at 20?degrees C, and 4 times higher at 50?degrees C when compared to E. coli TK under identical conditions. When tested at 50?degrees C with other aldehydes as acceptors, mTKgst activity was approximately 3 times higher than those obtained at 20?degrees C. Applications of this new TK in biocatalysis were performed with hydroxypyruvate as donor and three different aldehydes as acceptors glycolaldehyde, D-glyceraldehyde and butyraldehyde from which the corresponding products L-erythrulose 1, D-xylulose 2 and 1,3-dihydroxyhexan-2-one 3 were obtained, respectively. The optical rotations for products 1 and 2 indicate that the stereospecificity of mTKgst is identical to that of other TK sources, leading to a (3S) configuration. With the non-hydroxylated substrate, butanal, the ee value was 85% (3S), showing higher enantioselectivity than the E. coli TK (75% ee, 3S). Processes at elevated temperatures could offer opportunities to extend the applications of TK biocatalysis, by favoring hydrophobic aldehyde acceptor substrate solubility and tolerance towards non-conventional media.