Directed evolution of D-2-keto-3-deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of D- and L-sugars
Directed evolution of D-2-keto-3-deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of D- and L-sugars
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DOI：
10.1016/s1074-5521(00)00035-1
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发表时间：
2000-11-01
影响因子：
--
通讯作者：
Wong, CH
中科院分区：
文献类型：
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作者：
Fong, S;Machajewski, TD;Wong, CH
背景：开发和改进酶作为有机合成的催化剂是当前生物催化研究的热点。研究的代表性酶是大肠杆菌D-2-酮基-3-脱氧-6-磷酸葡萄糖酸（KDPG）醛缩酶，其使用D-构型的KDPG作为substrate.Results催化高度特异性的可逆aldol反应，使用体外进化，醛缩酶已被转化为具有改进的催化效率、改变的底物特异性和立体选择性的醛缩酶。特别是，一些进化的醛缩酶能够接受在非磷酸化形式的D-和L-甘油醛作为可逆的羟醛缩合反应的底物已经获得，提供了一个新的方向的酶促合成的D-和L-suggestions.Conclusions：这项研究已经证明了使用体外进化的有效性，迅速改变的醛缩酶的性质，以提高其效用在不对称合成。进化的醛缩酶不同于高度磷酸盐和D-糖依赖性的天然酶，其催化从非磷酸化醛和丙酮酸有效合成D-糖和L-糖。本研究中所描述的原理和策略应适用于其他醛缩酶，以进一步扩大其合成用途的范围。
Background: Exploitation and improvement of enzymes as catalysts for organic synthesis is of current interest in biocatalysis. A representative enzyme for investigation is the Escherichia coli D-2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes the highly specific reversible aldol reaction using the D-configurated KDPG as substrate.Results: Using in vitro evolution, the aldolase has been converted into aldolases with improved catalytic efficiency, altered substrate specificity and stereoselectivity. In particular, some evolved aldolases capable of accepting both D- and L- glyceraldehyde in the non-phosphorylated form as substrates for reversible aldol reaction have been obtained, providing a new direction to the enzymatic synthesis of both D- and L-sugars.Conclusions: This research has demonstrated the effectiveness of using in vitro evolution to rapidly alter the properties of an aldolase to improve its utility in asymmetric synthesis. The evolved aldolases, differing from the native enzyme which is highly phosphate- and D-sugar-dependent, catalyze the efficient synthesis of both D- and L-sugars from non-phosphorylated aldehydes and pyruvate. The principles and strategies described in this study should be applicable to other aldolases to further expand the scope of their synthetic utility.