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EziG in the Literature

A few weeks ago, Professor Nick Turner’s research group at the Manchester Institute of Biotechnology in collaboration with Matthew Truppo and Paul Devine from Merck put EziG to the test in their article published in Tetrahedron. They set out to test how “general” EziG really is as an immobilization platform by testing how well six major classes of biocatalysts work on EziG. We are proud to say that EziG passed their tests with flying colors.

 

The Turner group immobilized a broad panel of biocatalysts including an alcohol oxidase, alcohol dehydrogenase, amine dehydrogenase, carboxylic acid reductase, P450 oxidase, and reductive aminase on EziG. Each of these enzymes has distinct properties and needs different cofactors and reagents so the team tested out each EziG type to find the best support for each process. They got the most use out of the semi-hydrophilic Amber line, using it for 3 of the 4 chemical processes demonstrated.

 

The team first tested how well a reductive amination could be performed with EziG. Reductive aminations are ubiquitous reactions in chemical synthesis because they have excellent atom economy and provide a mild way of converting an aldehyde or ketone to an amine. They also offer a great opportunity to couple two smaller molecules together.

 

The group set up two different processes for reductive amination. The first used a formate dehydrogenase co-immobilized with an amine dehydrogenase to reduce 4-fluorophenyl acetone into (R)-4-fluoroamphetamine, oxidizing the formate ion to form carbon dioxide as the only byproduct.

 

Using just 150 mg of EziG in a packed bed running in a continuous flow of aqueous buffer, gave an impressive productivity of over 300 gL-1day-1. This performance could be attributed to the high loading and retained activity that EziG provided. The team found that the amine dehydrogenase could be loaded at 32% of the dry weight of the immobilized formulation and retained >99% of its original activity once bound.

 

The second reductive amination process the team tested used a reductive aminase enzyme recently discovered in Aspergillus fungi to catalyse the NADPH-dependent reductive coupling of cyclohexanone and allylamine. The team co-immobilized their reductive aminase with glucose dehydrogenase to provide the reducing power needed to regenerate NADPH for the reductive aminase. Stability has been a serious challenge for reductive aminases, so the success of this process depended entirely on the ability of EziG to stabilize the enzyme.

 

In a side-by-side comparison of non-immobilized enzyme mixture to the co-immobilized formula on EziG Amber, the team saw longer lasting catalysis with the immobilized enzyme. The added stability of the EziG-immobilized formulation drove the total conversion of cyclohexanone up from 50% to over 90% and allowed them to recycle the enzyme, achieving a total turnover number 6-fold higher than the equivalent non-immobilized enzyme mixture.

 

Building on this, the team tried using EziG-immobilized enzymes to make aldehydes and ketones that could feed into the reductive amination process. They tested three different methods. The first method used an alcohol dehydrogenase to oxidized 1-(4-fluorophenyl)propan-2-ol to form 4-fluorophenyl acetone. Alcohol dehydrogenase depends on NAD+ as a cofactor, so this process was immediately coupled to the amine dehydrogenase reductive amination process (which uses NADH) by co-immobilizing alcohol dehydrogenase with amine dehydrogenase on EziG. The two enzymes working together were able to rapidly exchange the alcohol for an amine, working through the ketone intermediate, generating water as the only byproduct. Such an elegant process is a fantastic demonstration of green chemistry principles and the power of enzyme immobilization.

 

The second method they ran took advantage of a carboxylic acid reductase (CAR). CAR depends on an NADPH cofactor and a steady stream of ATP to reduce carboxylic acids to aldehydes. Because of this need for NADPH and ATP, CAR-dependent processes are usually run with whole cells. However, living cells can also drive unwanted side reactions, making product cleanup expensive. The team envisioned that co-immobilizing three enzymes might make it possible to run this process cleanly and efficiently in flow. They immobilized CAR on EziG Opal alongside AMP phosphotransferase and adenylate kinase which work together to regenerate expensive ATP from inexpensive polyphosphate. Using this regenerative co-immobilized system, the team saw a 98% yield in the conversion of benzoic acid to benzaldehyde needing as little as 1 molecule of ATP for every 500 molecules of product. This result is a clear demonstration of the ability of EziG to improve the economics of biocatalysis.

 

The third method they tried used an alcohol oxidase enzyme. Alcohol oxidase uses a copper cofactor and the oxidizing power of dissolved oxygen to convert primary alcohols into aldehydes. If an aldehyde is the desired product (as is often the case in the perfume industry), the use of oxygen in place of NAD+ makes this process far more atom efficient.

 

However, this is a challenging process to run. Aldehydes can be quite reactive, damaging the biocatalyst or becoming over-oxidized into carboxylic acids. Furthermore, the reaction with molecular oxygen generates reactive hydrogen peroxide as a byproduct which usually needs to be removed using a significant amount of added catalase. By taking advantage of the flow chemistry enabled by EziG, the team was able to optimize their in-flow mixture and continuously remove reactive products from the process.

 

The Turner group’s thoughtful exploration of process conditions using EziG led them to a remarkable result. They found that the ideal conditions for this oxidation used 1-hexanol dissolved in cyclohexane pumped through an EziG column loaded with alcohol oxidase. The enzyme showed remarkable tolerance for the solvent, giving a consistent output of hexanal over at least 120 column volumes of the effluent. The use of cyclohexane also eliminated over-oxidation and made product recovery easy.

 

Overall, we are very excited to see EziG enabling and enhancing biocatalysis. Our congratulations and our thanks go out to Prof. Nick Turner and the rest of the team for putting together a great paper and for putting EziG to the test and showing what it can do. If you have a synthetic challenge that you would like to discuss with us, reach out at the address below.

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