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From Gene to Function: Rapid Identification of a Robust Biocatalyst for Preparing Optically Pure Alcohols

Case Study

 

Figure 1. Chain A of Tcalid SDR with NADP and cofactor.
Figure 1. Chain A of Tcalid SDR with NADP and cofactor.

The production of optically pure alcohols, key chiral building blocks in pharmaceuticals, is often costly and inefficient. Kinetic resolution (KR) selectively converts one enantiomer in a racemic mixture, but it is limited to 50% yield (1,2). Despite this, KR remains valuable for high-purity chiral compound synthesis.


Challenge


GECCO Biotech, in collaboration with Boehringer Ingelheim, Zymvol Biomodeling, and Kiel University, aimed to develop an eco-friendly method for producing optically pure alcohols (3). The focus was on biocatalytic kinetic resolution of racemic alcohol by efficient and cost-effective identification and characterisation of ketoreductases (KREDs).



Multi-faceted strategy to address the challenge


In Silico Design: Computational methods to design and select promising enzyme candidates.


Cloning, Expression and Purification: We cloned optimized synthetic genes into an E. coli expression vector for efficient protein production. Our optimized screening protocol was employed to identify optimal strains and expression conditions, maximizing expression yield. The enzymes were produced, and purified in an MTP format.


In Vitro Testing: Parallelized processing enabled us to efficiently test mutants for stability, activity, and enantioselectivity under diverse conditions. This phase often includes reaction optimization to identify optimal conditions, including catalytic efficiency and stability across pH and temperatures.


Efficient Screening: We measured conversion and enantioselectivity via chiral gas chromatography (GC) to evaluate enzyme performance and identify lead candidates rapidly.


Rational Enzyme Design & Validation: Structural analysis to determine structure-function relationship.


Outcomes


This research identified Tcalid SDR, a novel short-chain dehydrogenase/reductase (SDR), as a promising enzyme due to its exceptional thermal and pH stability. The enzyme retained activity at temperatures up to 90°C and across a pH range of 5-8. Furthermore, Tcalid SDR exhibited high enantioselectivity and a strong affinity for NADH, making it a suitable candidate for enzyme engineering and optimization. The research successfully demonstrated the biocatalytic kinetic resolution of racemic substrate, a key step for potential industrial applications.


Figure 2. A. Target kinetic resolution of racemic ketone. B. Performance of selected enzyme variants in production of enantiopure alcohols.
Figure 2. A. Target kinetic resolution of racemic ketone. B. Performance of selected enzyme variants in production of enantiopure alcohols.


Key benefits for GECCO’s customers


  1. Fast and Efficient: We use E. coli for rapid growth and easy genetic manipulation, leading to fast turnaround times for your projects.

  2. Customization Options: We can accommodate different host organisms and purification tags, tailoring the process to your specific enzyme.

  3. Optimized for Performance: We balance scoring of different properties to produce enzymes with improved substrate affinity and stability.

  4. Comprehensive Analysis: We offer diverse analytical techniques and detecion capabilities, including chiral (U)HPLC, LC-MS and plate reader assays.

  5. Efficient Screening: Our in-house methods allow us to screen many enzyme variants quickly, to find the best candidate.



Notes


  1. Hall et al. - General overview of enzymatic approaches for asymetric synthesis.

  2. Hollmann et al.; Qin et al. - ADHs in organic synthesis, and more specific, in kinetic resolution.

  3. Song et al. - Original work, whose parts were used to prepare this case study.


If you would like to find out more or would like to contract any of our services, please contact us.





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