Industrial biotransformations biocatalysts

For an industrial biotransformation, it is often necessary to further optimize an appropriate biocatalyst. This includes the elimination of the follow-up enzymes in the wild-type strain by mutations and the improvement of other characteristics by additional mutations and the selection of improved strains. Alternatively, the genetic information for a desired enzyme might be introduced in a host that has many of the preceding characteristics, and that has no enzymes that could modify or degrade the desired product. 

The quest for microorganisms capable of performing the desired biotransformation of indene led to the isolation of several strains of the genus Rhodococcus from soil samples contaminated with aromatic compounds that are able to oxidize indene to 1,2-indandiols of different chirality, and various other oxygenated derivatives [8]. Induction studies indicated that several oxygenases were present and differentially induced by naphthalene, toluene, and indene. The stereospecific nature of the enzymes expressed in Rhodococcus as well as their abihty to tolerate indene as a substrate makes these microorganisms promising candidates for development as an industrial-scale biocatalyst for the production of (21 )-indandiol. 

Taylor SJC, Holt KE, Brown RC, Keene PA, Taylor IN (2000) Choice of biocatalyst in the development of industrial biotransformations. In Patel RN (ed) Stereoselective biocatalysis. Dekker, New York, p 397... 

In industrial biotransformations, hydrolytic reactions occupy a prominent position for the production of optically active amines, alcohols, and carboxylic acids. Compared with other reactions, hydrolytic reactions are feasible to scale up because they are cofactor-free, relatively simple, and chemically tunable systems. In addition to home-made whole-cell biocatalysts, which are considered to be more cost-effective for specific syntheses, some commercially available hydrolases, including lipases/esterases, epoxide hydrolases, nitrilases, and glycosidases, are also employed for the enantioselective production of chiral chemicals. 

Though use of isolated purified enzymes is advantageous in that undesirable byproduct formation mediated by contaminating enzymes is avoided [37], in many industrial biotransformation processes for greater cost effectiveness the biocatalyst used is in the form of whole cells. For this reason baker s yeast, which is readily available, has attracted substantial attention from organic chemists as a catalyst for biotransformation processes. One of the first commercialized microbial biotransformation processes was baker s yeast-mediated production of (R)-phenylacetyl carbinol, where yeast pyruvate decarboxylase catalyzes acyloin formation during metabolism of sugars or pyruvate in the presence of benzaldehyde [38]. 

Some of the industrial biocatalysts are nitrile hydralase (Nitto Chemicals), which has a productivity of 50 g acrylamide per litre per hour penicillin G amidase (Smith Kline Beechem and others), which has a productivity of 1 - 2 tonnes 6-APA per kg of the immobilized enzyme glucose isomerase (Novo Nordisk, etc.), which has a productivity of 20 tonnes of high fmctose syrup per kg of immobilized enzyme (Cheetham, 1998). Wandrey et al. (2000) have given an account of industrial biocatalysis past, present, and future. It appears that more than 100 different biotransformations are carried out in industry. In the case of isolated enzymes the cost of enzyme is expected to drop due to an efficient production with genetically engineered microorganisms or higher cells. Rozzell (1999) has discussed myths and realities... 

Since cinnamyl aldehyde is the main component of cassia oil (approximately 90%) and Sri Lanka cinnamon bark oil (approximately 75%) [49], it is industrially more important to generate cinnamyl alcohol, which is less abundantly available from nature but is important as cinnamon flavour, by biotransformation of natural cinnamyl aldehyde than vice versa. Recently, a whole-cell reduction of cinnamyl aldehyde with a conversion yield of 98% at very high precursor concentrations of up to 166 g L was described [136]. Escherichia coli DSM 14459 expressing a NADPH-dependent R alcohol dehydrogenase from Lactobacillus kefir and a glucose dehydrogenase from Thermoplasma acidophilum for intracellular cofactor regeneration was applied as the biocatalyst (Scheme 23.8). 

Inactivation of the biocatalyst owing to these effects can be a significant limitation for industrial application of enzymatic and whole-cell biotransformation. For more than 20 years, many attempts have been made to associate the toxicity of different solvents with some of their physicochemical properties and to explain the influence of the two-phase system composition on bioconversion efficiency. 

One of the reactions catalyzed by esterases and lipases is the reversible hydrolysis of esters (Figure 19.1, Reaction 2). These enzymes also catalyze transesterilications and the desymmetrization of mew-substrates (vide infra). Many esterases and lipases are commercially available, making them easy to use for screening desired biotransformations without the need for culture collections and/or fermentation capabilities.160 In addition, they have enhanced stability in organic solvents, require no co-factors, and have a broad substrate specificity, which make them some of the most ideal industrial biocatalysts. Alteration of reaction conditions with additives has enabled enhancement and control of enantioselectivity and reactivity with a wide variety of substrate structures.159161164... 

As an example, it has been estimated that for the industrial production of fine chemicals, biotransformations should accomplish a minimum space-time yield of 0.1 g l-1 h-1 and a minimum final product concentration of 1 g l-1, while for pharmaceuticals, the minimum requirements are 0.001 g l-1 h-1 and 0.1 g l1 for volumetric productivity and product concentration, respectively [6]. Analysis of enzymes with recognized industrial potential, such as cytochrome P450, showed that some of the parameters are already within industrially relevant ranges [7]. The improvements achieved with these biocatalysts through protein, cell, and process engineering are based on the understanding of their molecular arrangement and catalytic mechanisms. 

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