Scale biocatalytic processes

The p-lactams, mainly penicillins and cephalosporins, are by production volume the most important class of antibiotics worldwide, enjoying wide applicability towards a range of infectious bacteria. Most of the key molecules are semi-synthetic products produced by chemical modification of fermentation products. Production of these molecules has contributed significantly to the development of large-scale microbial fermentation technology, and also of large-scale biocatalytic processing. 

Even a few years ago, biocatalytic processes on an industrial scale were few. As with so many novel technologies, however, the time lag between research, development, and large-scale application just had to pass before we could witness a range of such processes in industrial practice today. In a review summarizing the status of biocatalytic processing in industry, Straathof (2002) records that the number of industrial-scale biocatalytic processes has more than doubled over the last 10 years. 

Performance data for biocatalytic reactions and processes will be discussed throughout this book, especially in Chapters 2, 7, 18, and 19. Table 1.3 lists some of the largest-scale biocatalytic processes, categorized by size stated as of tons per year... 

The isomerization of glucose to fructose, catalyzed by the enzyme xylose isomerase, is by far the largest-scale biocatalytic process. Already known for several decades,... 

In summary, enzyme immobilization is extremely important in the scale-up of many biocatalytic processes. The preferred method for pharmaceutical production involves covalent binding through cross-linking or attachment to a support. Noncovalent attachment is less attractive, but it is heavily utihzed owing to the commercial availabihty of industrial quantities of some enzymes immobilized using this technique. 

The biocatalytic reduction of carboxylic acids to their respective aldehydes or alcohols is a relatively new biocatalytic process with the potential to replace conventional chemical processes that use toxic metal catalysts and noxious reagents. An enzyme known as carboxylic acid reductase (Car) from Nocardia sp. NRRL 5646 was cloned into Escherichia coli BL21(DE3). This E. coli based biocatalyst grows faster, expresses Car, and produces fewer side products than Nocardia. Although the enzyme itself can be used in small-scale reactions, whole E. coli cells containing Car and the natural cofactors ATP and NADPH, are easily used to reduce a wide range of carboxylic acids, conceivably at any scale. The biocatalytic reduction of vanillic acid to the commercially valuable product vanillin is used to illustrate the ease and efficiency of the recombinant Car E. coli reduction system." A comprehensive overview is given in Reference 6, and experimental details below are taken primarily from Reference 7. 

Cmcial to the development of a biocatalytic route is furthermore the selection of a readily available biocatalyst with sufficient activity, selectivity and stability, which preferably should be commercially available. Other factors that play an important role are the scale at which the process should be ran and the time it takes to develop such a biocatalytical process. 

Biocatalytic processes increasingly penetrate the chemical industry. In a recent study, 134 industrial-scale biotransformations, on a scale of > 100 kg with whole cells or enzymes starting from a precursor other than a C-source, were analyzed. Hydrolases (44%), followed by oxido-reductases (30%), dominate industrial biocatalytic applications. Average performance data for fine chemicals (not pharmaceuticals) applications are 78% yield, a final product concentration of 108 g I.1, and a volumetric productivity of 372 g (L d) 1. 

The enzyme membrane reactor (EMR) is an established mode for running continuous biocatalytic processes, ranging from laboratory units of 3 mL volume via pilot-scale units (0.5-500 L) to full-scale industrial units of several cubic meters volume and production capacities of hundreds of tons per year (Woltinger, 2001 Bommarius, 1996). The analogous chemzyme membrane reactor (CMR) concept, discussed in Chapter 18, Section 18.4.5, is based on the same principles as the EMR but is far less developed yet. 

As the use of lipase for industrial chemical synthesis becomes easier, several chemical companies have begun to increase significantly their biocatalytic process used in synthetic application. Among these companies, BASF, in which enantiomerically pure alcohols and amines are produced on industrial scale.98... 

The seventh and final paper, "Development of a Fermentation-Based Process for 1,3-Propanediol Highlights of a Successful Path from Corn to Textile Fiber," by Tyler Ames of DuPont, reviewed the multiyear effort by DuPont and its development partners (Genecor International and Tate Lyle) to commercialize a new biocatalytic process for the production of 1,3-propanediol (PDO), a key ingredient in DuPont s new Sorona advanced polymer platform. PDO is currently being produced at pilot scale at Tate Lyle s Decatur, IL, site, and construction of a commercial-scale facility is expected to begin soon. 

As with another class of compounds, the scale of synthesis and time required at the research stage before product can be made influence which method is finally used. At small scale, a plethora of methods exist to prepare amino acids, in addition to isolation of the common ones from natural sources. The majority of these small-scale reactions rely on the use of a chiral auxiliary or template. At larger scale, asymmetric hydrogenation and biocatalytic processes come into their own. For the amino acids approaching commodity chemical scales, biological approaches, either as biocatalytic or total fermentation, provide the most cost-efficient processes. 

In general, a set of conditions are required for the successful development and scale up of a novel biocatalytic process the availability of a suitable biocatalyst, methods... 

A biocatalytic enantioselective addition of ammonia to a C=C bond of an a,)9-unsaturated compound, namely fumaric acid, makes the manufacture of L-aspartic acid possible on an industrial scale. This process, which is applied by, e. g., Kyowa Hakko Kogyo and Tanabe Seiyaku, is based on the use of an aspartate ammonia lyase as a biocatalyst [119]. Another comparable reaction is the asymmetric biocatalytic addition of ammonia to trans-cinnamic acid, which represents a technically feasible process for the production of L-phenyl-alanine [120]. 

Biocatalysis covers a broad range of scientific and technical disciplines, which are geared to develop biocatalysts and biocatalytic processes for practical purposes. The natural pool of biocatalysts is extremely diverse and includes whole cells of microbial, plant or animal origin, as well as cell-free extracts and enz3rmes derived from these sources. The wide range of catalytic power offered by nature remains, however, largely imexplored. Currently, only a very small fraction of the known biocatalysts are actually being applied on a commercial scale. For example, of the approximately 4,000 known enzymes, about 400 are available commercially, but only about 40 are actually used for industrial applications. 

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. 

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. 

A convenient biocatalytic process has been developed using a novel whole-cell biocatalyst for the preparation of (R)-l,3-butanediol (BDO) by stereo-specific oxidoreduction on an industrial scale. (R)-l,3-BDO is an important chiral synthon for the synthesis of various optically active compounds, such as azetidinone derivatives, which are used to prepare penem and carbapenem antibiotics for industrial usage. 

A cost effective and easily scaled-up process has been developed for the synthesis of (S)-3-[2- (methylsulfonyl)oxy ethoxy]-4-(triphenylmethoxy)-1 -butanol methanesulfonate, a key intermediate used in the synthesis of a protein kinase C inhibitor drug through a combination of hetero-Diels-Alder and biocatalytic reactions. The Diels-Alder reaction between ethyl glyoxylate and butadiene was used to make racemic 2-ethoxycarbonyl-3,6-dihydro-2H-pyran. Treatment of the racemic ester with Bacillus lentus protease resulted in the selective hydrolysis of the (R)-enantiomer and yielded (S)-2-ethoxycarbonyl-3,6-dihydro-2H-pyran in excellent optical purity, which was reduced to (S)-3,6-dihydro-2H-pyran-2-yl methanol. Tritylation of this alcohol, followed by reductive ozonolysis and mesylation afforded the product in 10-15% overall yield with excellent optical and chemical purity. Details of the process development work done on each step are given. 

Attempts to resolve the racemic acid of 3 and its ester through classic resolution failed. In the early stages of development, a process based on SchoUkopf s asymmetric synthesis was developed (see Section 9.3). Large-scale development work was aimed at finding a biocatalytic process to resolve the amino acids. Racemic a,a-disubstituted a-amino esters were synthesized by standard chemistry through alkylation of the Schiff s bases formed from the amino esters (Scheme 9.5), or through formation of hydantoins. ... 

Enzymes have many potential advantages when used as catalysts for chemical synthesis. The unique properties offered by these biocatalysts are, first of all, their often outstanding chemo-, regio-, and, in particular, stereoselectivity. Furthermore, enzymes are highly efEcient catalysts working under very mild conditions. However, enzymes do also have some drawbacks that may limit their potential use, such as ability to accept a limited substrate pool only, and a moderate operational stability. Ways of overcoming most of these potential limitations exist and they pose in most cases more of a perceived than a real problem. Well over 100 different biocatalytic processes have been implemented on an industrial scale [1]. A few processes are... 

Even though ILs may offer some imique opportunities to the development of new biocatalytic processes they also carry several inherent problems that may limit their use, especially in processes to be performed on an industrial scale. The lack of vapor pressure may be a potential advantage in certain applications, but it also poses a problem in many others. Thus, isolation of polar, nonvolatile products may be problematic and new technologies to enable it must be developed. The lack of volatility may also make it very difficult to remove ILs completely from the products. 

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