Halophilic enzymes in tandem flow reactions

化学反应通常是多步骤的过程，其中起始材料转化为第一中间体，然后是另一个中间体，依此类推，直到合成最终分子。传统上，这些反应在单独的烧瓶中顺序进行，每个步骤都需要后处理，通常需要纯化。当合成在工业环境中按比例放大时，每个步骤都必须优化，成本成为关键因素。在可能的情况下，催化反应以最小化反应时间，并且如果特定反应中产生的杂质可以继续进行而不会在下一步中引起问题，则避免纯化。为了简化生产并最大化产量，流动化学的发展正在迅速扩大，其中反应是在反应器中连续流动而不是批量进行的。在这种装置中，催化剂可以固定在固体基质上，试剂以受控的流量通过，从而使产品能够连续合成。近年来，使用酶来取代化学催化剂也有了显着的发展，主要制药公司现在正在寻找生物技术，以更环保的方式生产药物。此外，生物催化工艺减少了产品被有毒杂质如（过渡）金属污染，这是其他催化工艺中的主要成本和时间因素。因此，生物催化在流动化学中的应用也是一个很好的目标生物技术。此外，多个酶可以按顺序固定到连续的生物反应器中，以进行级联反应，有效地组装人工生物合成序列，其中酶识别由前一个酶产生的产物作为底物，等等。这可以在无细胞系统中实现，或者人工途径可以在生物宿主内构建，该生物宿主本身成为生物反应器。在这个项目中，我们正在开发一个级联反应系统，利用从死海分离的生物体的酶。Haloferax volcanii生长在非常高的盐环境中，其酶不仅可以在高盐存在下催化反应，而且更有趣的是，它们可以在有机溶剂存在下催化反应。酶通常在非水介质中不稳定，这使得它们通常与许多分子不溶于水的化学反应不相容。因此，Haloferax酶是化学合成的理想生物催化剂。我们以前已经研究了来自Haloferax的醇脱氢酶（HvADH 1）的特性，该酶能够将非常广泛的醇转化为醛或酮。我们还知道，我们可以很容易地将HvADH 1不可逆地固定在市售的珠子上，进一步提高其稳定性和活性。我们现在想要将联合收割机HvADH 1与同样来自Haloferax的第二种酶结合，以将第一步中产生的醛和酮转化为胺，从而实现从醇开始的总体官能团相互转化以生成胺。胺是药物和农用化学品合成中非常常见的中间体，我们将从容易获得的醇中生产它们。我们将测试两个不同的系统，其中一个系统中的酶单独产生，分离，固定化，并优化底物转化的流量，和第二个系统中的酶将在Haloferax细胞内同时过量生产，该细胞本身将被固定化，并用作一个独立的反应器，其中两个步骤都将发生。

Chemical reactions are often multi step processes in which a starting material is converted into a first intermediate, then another and so on till the final molecule is synthesized. Traditionally these reactions are done sequentially, in separate flasks and each step requires a work-up and often purification. When the syntheses are scaled up for an industrial setting, each step must be optimized and costs become a critical factor. Where possible, reactions are catalyzed to minimize reaction times, and purification is avoided if impurities generated in a particular reaction can be carried forward without causing issues in the next step. In an effort to streamline production and maximize throughput, the development of flow-chemistry, where a reaction is carried out in a continuous flow through a reactor rather than in a batch, is rapidly expanding. In this set up a catalyst can be immobilized on a solid matrix and the reagents are passed through in a controlled flow allowing for the product to be synthesized continuously.In recent years the use of enzymes to replace chemical catalysts has also evolved significantly, with major pharmaceuticals now looking at biotechnology for the production of drugs with a more environmentally friendly approach. Moreover, biocatalytic processes reduce the contamination of products with toxic impurities such as (transition) metals, a major cost and time factor in other catalytic processes. The application of biocatalysis to flow chemistry is therefore an excellent target biotechnology also. Furthermore, multiple enzymes can be immobilized in sequence into contiguous bioreactors for cascade reactions to be developed, effectively assembling an artificial biosynthetic sequence where an enzyme recognizes as substrate the product generated by the previous enzyme and so on. This can be achieved either in a cell free system, or the artificial pathway can be constructed inside a biological host which becomes itself a bioreactor. In this project we are looking at developing a cascade reaction system utilizing enzymes from an organism isolated from the Dead Sea. Haloferax volcanii grows in very high salt environments and its enzymes can catalyze reactions not only in the presence of high salts, but more interestingly they can do this in the presence of organic solvents. Enzymes are normally unstable in non-aqueous media and this makes them often incompatible with chemical reactions where many molecules are insoluble in water. Haloferax enzymes are therefore ideal biocatalysts for chemical synthesis. We have previously investigated the characteristics of an alcohol dehydrogenase from Haloferax (HvADH1) and this enzyme is capable of transforming a very broad range of alcohols into aldehydes or ketones. We also know that we can easily immobilize HvADH1 irreversibly on commercially available beads further increasing its stability and activity. We now want to combine HvADH1 with a second enzyme, also from Haloferax, to transform the aldehydes and ketones produced in the first step to amines, achieving overall a functional group inter-conversion starting from alcohols to generate amines. Amines are very common intermediates in the synthesis of pharmaceuticals and agrochemicals and we will generate them from readily available alcohols. We will test two different systems, one in which the enzymes are individually produced, isolated, immobilized, and the flow optimized for substrate conversion, and a second system in which the enzymes will be over-produced simultaneously inside Haloferax cells which will be itself immobilized and used as a self contained reactor where both steps will take place.