Industrial Applications of Whole-Cell Biocatalysis - Pharmaceutical Technology

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Industrial Applications of Whole-Cell Biocatalysis
Recombinant microbial whole-cell biocatalysis is a valuable approach for producing enantiomerically pure interemediates. The authors examine several groups of enzymes using this approach: dehydrogenases, hydantoinases, and acylases.


Pharmaceutical Technology


The hydantoinase platform


Figure 1 (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.)
The hydantoinase process was introduced in the 1970s for producing D-amino acids such as D-phenylglycine and p-OH-phenylglycine (7). Today, > 1000 tons of each of these amino acids are produced annually. They are used as side chains for the -lactame antibiotics ampicillin and amoxicillin.


Figure 2 (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.)
The D-hydantoinase process (see Figure 1) is an excellent example of a dynamic kinetic resolution process. As 5'-monosubstituted hydantoins racemize spontaneously or enzyme-catalyzed under conditions used for biotransformation, a 100% yield of optically pure D- or L-amino acid can be reached (8, 9). Another advantage of the hydantoinase route is that racemic 5'-monosubstituted hydantoins can be easily synthesized from cheap starting materials through the reactions shown in Figure 2. In addition, if the decarbamoylation step is done enzymatically, carbamoylases waste and by-product formation is extremely low (CO2 and NH4 are the only by-products), which is also advantageous in the product-isolation step. All these features make the hydantoinase route very attractive for the industrial production of optically pure artificial amino acids (10). To date, low space-time yields and high biocatalyst costs prevent the production of L-amino acids based on the hydantoinase process (11–13).

Therefore, Evonik expanded its hydantoinase platform for producing L-amino acids by focusing on strain development and process optimization by biochemical engineering (14, 15). Despite significant progress in reducing the biocatalyst production cost, increasing the activity of the biocatalyst and improving the space-time-yield process economics were still prohibitive for commercialization of this process.

To address these problems, Evonik developed a new generation of an L-hydantoinase process based on a tailor-made recombinant whole-cell biocatalyst. The biocatalyst costs have been reduced by designing recombinant Escherichia coli cells overexpressing a hydantoinase, carbamoylase, and hydantoin racemase from Arthrobacter sp. DSM 9771. Despite this progress, the D-selectivity of the hydantoinase for D,L-methylthioethylhydantoin was significantly limiting the space-time yield of the L-hydantoinase process (16, 17). As screening did not provide us with better hydantoinases, the authors intended to invert the enantioselectivity of the hydantoinase by directed evolution (18). The productivity of the process could be dramatically improved using the recombinant E. coli coexpressing the newly designed L-selective hydantoinase mutant with an L-carbamoylase and a hydantoin racemase. These improvements have been confirmed at industrial scale and resulted in a process for producing various natural and nonnatural L-amino acids.


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