Racemase: Dynamic Kinetic Resolution

Use of Hydrolases in amino acid manufacture

Aminoacylase biocatalysts specific for the hydrolysis of N-acetyl-L-amino acids have been extensively developed in resolution processes for large-scale industrial production of optically pure L-amino acids. L-Methionine, for example is currently produced at multi-thousand ton scale from N-acetyl DL-methionine using immobilized L-aminoacylase. The acylase based process has also been demonstrated for bulk production of a number of unnatural L-amino acids (Figure 1.) in response to increasing demand for non-proteinogenic L-amino acids and D-amino acids as components of single isomer and peptide based pharmaceuticals.

Figure 1. Non-proteinogenic L-amino acids produced by amino acylase resolution.

This process remains the most widespread enzymatic method for manufacture of L-amino acids. Although, in specific cases, this enzymatic approach is economically less attractive than other methods, such as fermentation, the breadth of current application and the potential for future expansion of the aminoacylase route is far greater than that of fermentative routes to optically pure amino acids. This is due to the general inability to produce non-proteinogenic amino acids by fermentation, in contrast to the relaxed substrate range of aminoacylase and the ability to generally prepare N-acetyl amino acid substrates at industrial scale.

Figure 2. Aminoacylase-catalyzed resolution of N-acetyl amino acids.

The single greatest impediment to wider commercialisation of the aminoacylase approach has been the limited reaction yield. Since these reactions are based on the resolution of the corresponding racemate, the maximum yield in the best case scenario is only 50% (Figure 2.). Increased yield requires recycling of the unreacted isomer using often costly and severe chemical conditions, usually including isolation and separate processing of the chiral ballast.

To overcome this limitation, Lybradyn has developed and implemented a genetic screening process to identify efficient N-acetyl amino acid racemase biocatalysts which are compatible with L- or D- aminoacylases and can be used in a wholly enzymatic process to produce either L- or D-amino acids at high optical purity from corresponding racemates. The substrate specific in situ racemization afforded by the racemase enables the process to achieve yields significantly exceeding 50% in a single pass.

The development and implementation of this dynamic resolution is of high relevance to the growing demand for non-proteinogenic L- and D-amino acids as components of peptidomimetic pharmaceuticals in areas such as HIV protease inhibition and oncology (Figure 3).

Figure 3. Examples of unnatural amino acids in peptidomimetic drugs in development.

Technology Summary of Lybradyn Racemases

Lybradyn's technology has combined earlier work which identified a limited number of inefficient N-acetyl amino acid racemase enzymes with new high-throughput screens and a microbial genomics approach to significantly improve existing racemase enzymes and identity new racemases which are sufficiently active and robust for industrial application.

The screen is based upon the laboratory design and construction of specific bacterial mutants which require N -acetyl amino acid racemase activity for growth on a selective growth medium. This method enables the enormous selective power of natural microbial evolution to be applied to the isolation of efficient new biocatalysts. The screen is capable of analyzing millions of independent enzyme variants in a single experiment and can be adapted to a wide variety of amino acid targets.

Lybradyn's expertise in molecular biology and directed evolution enables us to rapidly isolate and manipulate genes encoding racemases and racemase progenitor enzymes. Mutagenesis, screening and characterization of improved variants have been optimized with novel biocatalysts readily assessed against a variety of targets and subjected to repeated rounds of improvement. In this way optimal biocatalytic properties can be introduced, including substrate specificity, stability and resistance to product inhibition.

Lybradyn's expertise in fermentation process development enables design of microbial systems capable of producing racemase enzymes on manufacturing scale cost effectively. Expression and fermentation systems are optimized to include, high cell densities, maximized protein yields, cost effective culture media formulations, efficient culture processes and downstream protein processing methods.

Opportunities for Commercialization

Lybradyn is an enabling technology discovery and development biotechnology company specializing in integrating protein expression and fermentation to create new products. The racemase program represents a unique opportunity to introduce new enzyme products to a number of applications including pharamceutical, agricultural and fine chemical products.

Lybradyn is interested in developing relationships with companies that can assist in commercializing the racemase/aminoacylase process for the manufacture of unnatural amino acid targets for pharmaceutical, agrochemical and fine chemical applications. We believe that the partnership of a small biotech and a respected manufacturing firm is a force that can effectively commercialize products such as the dynamic kinetic resolution of chiral compounds. This also presents an effective pairing to participate in government contracts, global foundations, as well as commercial sales.