Two-Step Biocatalytic Process

The biocatalytic process takes place in stirred tank reactors with sieves in the bottom and a working volume of several cubic meters (Fig. 7). The first biocatalytic step is a three-phase reaction with a solid biocatalyst, immobilized DAO on a spherical carrier, the liquid Ceph C solution and oxygen or air as the gaseous phase. The second step is a two-phase reaction with immobilized GA on a solid carrier and the glutaryl-7-ACA in solution. 

The two-stage biocatalytic reaction can be performed in a single reactor [14], but the separation of the two reactions is preferred because of different reaction parameters (e.g., pH value, temperature, oxygen) and stability of the enzymes used. With water as the solvent and enzymes fixed on a carrier, the process runs in a repeated batch mode at room temperature (20-30°C). Higher temperatures lead to increased reaction rates, but also to higher byproduct formation and reduced stability of the biocatalysts. A pH value between 7.0 and 8.5 is recommended with respect to thermodynamics, enzyme activities and stability and formation of byproducts. The use of cells is not recommended with respect to operational stability and possible product contamination. Therefore purified enzymes covalently immobilized on a polymeric carrier are chosen for the industrial process for both steps. The particle diameter of the spherical biocatalyst is about 100-300 pm, to allow for acceptable mass transfer and filtration times. 

The reuse of the expensive biocatalysts is a prerequisite for the economy of the biocatalytic process. On a lab-scale the carrier-fixed enzymes can be used for more than 100 cycles (DAO) and 180 cycles (GA), before reaching half of the starting activity [15]. Prolonging the reaction time can compensate for the decreasing activity. As claimed by reference [15] for the lab-scale preparation of 1 kg 7-ACA about 1.2 kU D-amino acid oxidase and 1.5 kU glutaryl-7-ACA acylase are consumed, but operational stability is dependent on scale. In production vessels gradients, e.g., pH value and shear stress, are different and could influence the operational stability of the biocatalysts, therefore a higher biocatalyst consumption is usually realistic. 

---

Various methods are known to produce 7-ACA from cephalosporin C (Ceph C) by removing the a-aminoadipyl side-chain. They can be classified into three types, the chemical process, a two-step enzymatic process and an enzymatic process in which the side-chain is directly removed from Ceph C. Today two processes are running commercially on an industrial scale, the classical chemical process and the modern two-step biocatalytic process (Fig. 2). Until now the favorable direct process is less effective, because of low conversion. 

Fig. 2 7 -ACA production starting from Ceph C (chemical and two-step biocatalytic process).

GlyC can be synthesized following a two-step biocatalytic process (Scheme 9.8). In the first step, one of two primary hydroxyl groups of... 

The same group developed a two-step biocatalytic process for e-caprolactone formation, starting from the cheap and easily available raw material cyclohexanol. The desired product was obtained in 94-97% conversion when operating at substrate concentrations in the range 20-60 mM. Additional aspects of the production of e-caprolactone were investigated by Bornscheuer and coworkers [23] by testing different enzyme ratios, coexpression of chaperone proteins... 

An alternative two-step biocatalytic route, first developed at Glaxo in the 1970s, utilized a D-amino acid oxidase and an amidase to provide 7-ACA under physiological conditions (Scheme 1.12). This process has since been established in several companies, with minor modifications. In fact, 7-ACA was manufactured by GSK at Ulverston (Cumbria, UK) using both the chemical and biocatalytic processes in parallel for a period of 2 years during which time the environmental benefits of the biocatalytic process were assessed (see Section 1.6). 

As mentioned above, pravastatin was produced by a two-step fermentation process the first step is production of ML-236B and the second is hydroxylation of ML-236B. This chapter surveys the biocatalytic production of pravastatin with a particular focus on the molecular mechanism of hydroxylation of ML-236B by cytochrome P450 (Cyt P450) from S. carbophilus. 

The discovery that the rate of reaction of the desulfurization of fossil fuels is enhanced by the addition of a flavoprotein to the biocatalyst was then claimed in the other two family patents. So, the patents are related to the use of a flavoprotein, particularly FMN reductase, in addition to the biocatalytic material for increasing the rate of desulfurization. In the World patent, ten more claims were allowed, compared to the US issued patent. The excess claims include a set of dependant claims in which the microorganism containing the recombinant DNA molecule is considered. However, in the invention a two-step process is stated, it is just the contact between the fossil fuel with an aqueous phase containing a biocatalyst and a rate-enhancing amount of a flavoprotein. There is no indication whatsoever on how much that amount could be. 

The integration of two unit operations lies at the heart of process engineering. More often in bioprocesses it is the integration of product formation with the following recovery steps that is critical.5 In the specific case of biocatalytic processes the product recovery is also critical, but in this chapter the focus will be on the integration of the surrounding chemical steps with the biocatalysis. 

Deracemization by DKR is in principle a kinetic resolution process in which the non-transformed enantiomer is racemized in situ. The conditions are that a chiral catalyst promotes the transformation of one enantiomer (Rj) into the product (Rp) while the other enantiomer is racemized at a comparable rate and the racemic mixture (R -i- S ) is restored. The product (Rp) is not racemized under the same conditions. While a simple kinetic resolution yields a maximum of 50% of the product, with this technique a 100% conversion can be reached. Although the majority of chiral molecules of industrial interest are stiU prepared by kinetic resolution, the continuous development of industrial enzymes and racemizing processes fosters new chemo- or biocatalytic systems for DKR to appear. A great impulse for deracemization methods based on the one-pot/two-steps resolution racemization process was brought about by BackvaU et al. over a 10-year period... 

The biocatalytic synthesis can be extended towards the production of L-alanine when using additionally an L-aspartate decarboxylase for a subsequent decarboxylation step. Such a process has been reported by Tanabe Seiyaku, and gave the desired L-alanine in 86% yield and with 99% ee [37]. A key feature of the decarboxylase process is the high substrate concentration of 2.5 M, and a space-time yield of 170 g/(L d) of L-alanine. Based on this two-step approach, L-alanine has been produced in annual amounts of ca. 60 tons [32b, 37]. 

Finally, a chemoenzymatic enantioconvergent procedure led to (S)-ibuprofen in four steps and 47% overall yield (Fig. 11.2-20). The latter compound is a widely used antiinflammatory drug and pain remedy and is one of the top ten drugs sold worldwide l,HH. In the key step, the conditions for the enantioconvergent hydrolysis of para-iso-butyl-a-methylstyrene oxide was optimized (elevated substrate concentration at +4 °C) to afford the non-reacted epoxide in >95 % ee[136l After separation from the epoxide, the formed diol (70% ee) was recycled via a two-step sequence via the corresponding bromohydrin, which was cyclized back to give ( )-epoxide. The latter material was subjected to repeated biocatalytic resolution in order to improve the economy of the process. 

Comparison of the two strategies for PCA synthesis from DHS depends upon the ultimate goal of die process. If synthesis of PCA as a final product is the goal, the two-step conversion that relies on thermal dehydration of DHS is superior. However, if PCA is needed only as an intermediate which will undergo further catalysis, biocatalytic synthesis using DHS dehydratase is superior, since the need for separate, sequential biocatalytic processes can be avoided. 

Deracemization via Biocatalytic Stereoinversion. Racemic secondary alcohols may be converted into a single enantiomer via stereoinversion which proceeds through a two-step redox sequence (Scheme 2.130) [38, 941, 942] In a first step, one enantiomer from the racemic mixture is selectively oxidized to the corresponding ketone while the other enantiomer remains unaffected. Then, the ketone is reduced in a second subsequent step by another redox-enzyme displaying opposite stereochemical preference. Overall, this process constitutes a deracemization technique, which leads to the formation of a single enantiomer in 100% theoretical yield... 

Bioprocesses incorporating more than one redox enzyme in an oxidative reaction system might involve, in the simplest case, two oxidizing enzymes coupled so that they act sequentially to effect two oxidation steps. A key issue in the development of such oxidative biocatalytic systems would be the determination of the values, for each enzyme involved, of the redox potentials. These can be determined by potentiometric titration using redox mediators (such as NADH) and techniques such as cyclic voltammetry or electrophoresis [44]. Knowledge of the redox potentials would facilitate the design and engineering of a process in which the two... 

Nevertheless, one process for synthesis of the low calorie sweetener, Aspartame, which is a methyl ester of a dipeptide, (Asp-Phe-OMe) involves a biocatalytic step. Aspartic acid amino protected by benzyloxycarboi rl group, is reacted with two moles of phenylalanine methylester catalysed by the protease thermolysin. The extra mole of ester makes the dipeptide precipitate and it is later recycled. For details see section 4.6. 

Several enzymes such as reductases and dehydrogenases utilize nicotinamide derivatives as reversible carriers of redox equivalents. The reduced dihydronicotinamide moiety NAD(P)H acts by donating a hydride equivalent to other molecules. In the corresponding two-electron oxidized NAD(P) form, the cofactor formally accepts a hydride ion from the substrate. Functional models of such reversible hydride transfer processes are of considerable interest for biomimetic chemistry, and the strategies to regenerate nicotinamide-type cofactors are crucial for the performance of many organic transformations involving biocatalytic key steps 139,140). 

Unfortunately the enzyme titer in the fermentation broth, even after classical mutation and several years of process development, was too low for the biocatalyt-ic process. With respect to the economics, the gene encoding for glutaryl-7-ACA acylase has been cloned, sequenced and expressed in E. coli to produce sufficient amounts. The enzyme titer could be increased by a factor of more than 100. Even the purification of the enzyme became much easier and resulted in higher yields with less side-activities, e.g., esterases. Two chromatographic purification steps were substituted by crystallization of the enzyme. The enzyme crystals could be stored long term without deactivation. To allow for reuse, the glutaryl-7-ACA acylase was immobilized on a polymeric carrier. 

Several approaches have been developed in the last two decades to combine KR with in situ racemization, allowing the 50% yield barrier associated with KR to be overcome. The process involves the typical KR in which one of the enantiomers is transformed quickly leaving the other enantiomer unreacted. As the faster reacting enantiomer is depleted, the equihbrium of (R)-/(S)- is constantly readjusted by racemization of the slow reacting enantiomer (Scheme 4.19). The process is nonstatic leading to the appHcation of the term dynamic kinetic resolution (DKR). In contrast to KR, DKR can provide an enantiomerically pure compound in 100% theoretical yield [50]. For the most effective process, the rate of racemization should equal or exceed the rate of the enantioselective transformation [51]. Chemical, thermal, biocatalytical or even spontaneous racemization processes can be involved — essentially those that can be performed in a single step under mild conditions are suitable. It is important that the conditions are adjusted not to promote racemization of the product. 

---