Isolated Enzyme Processes

Cofactor regeneration is potentially possible via electrochemical, chemical, photochemical, and enzymatic methods. The enzymatic methods are of two types those that are enzyme-coupled (using two enzymes) and those that are substrate-coupled. Table 10.3 lists some of the regeneration options. The substrate-coupled approach is particularly attractive [32]. In such systems, it is common to use 2-propanol [isopropyl alcohol (IPA)] as the cosubstrate that leads to acetone as the coproduct. The complication with such a system is that a competitive equilibrium is established 

Whole cell Use cell respiration Cell respiration with added carbon source Supplement with second cell with regeneration enzyme Supplement with regeneration enzyme 

Isolated reductase Enzyme-coupled Substrate-coupled 

---

Disadvantages of Whole-Cell Process Compared with the Isolated Enzyme Process... 

The reaction was already being used in the 1930s by Knoll (Ludwigshafen, Germany) (later BASF, since 2001 Abbott) (German Patent DE 546459, 1932) to produce ephedrine via (R)-phenylacetylcarbinol, (R)-PAC. Recently, efforts have been renewed to obtain an isolated enzyme process or a whole-cell process based on recombinant DNA technology. It was found that pyruvate could be substituted by the less expensive acetaldehyde, which did not produce C02 as a side product. 

Not only the enzymes but also the cellular components such as coenzymes and carbohydrates are conserved in the cell, which makes the whole cell processes favorable. For example, the addition of an expensive coenzyme and an auxiliary enzyme for coenzyme regeneration is not necessary, which makes the system simple and economical when comparing with the equivalent isolated enzyme process. 

The hydrolysis of nitriles can be carried out with either isolated enzymes or immobilized cells. Eor example, resting cells of P. chlororaphis can accumulate up to 400 g/L of acrylamide in 8 h, provided acrylonitrile is added gradually to avoid nitrile hydratase inhibition (116). The degree of acrylonitrile conversion to acrylamide is 99% without any formation of acryUc acid. Because of its high efficiency the process has been commercialized and currentiy is used by Nitto Chemical Industry Co. on a multithousand ton scale. 

Here we will focus on the biochemical aspects. The techniques of isolating enzymes, the process of enzyme immobilisation and the behaviour of immobilised enzyme reactors are discussed in detail in the BIOTOL text Technological Applications of Biocatalysts", so will not deal with these aspects in detail here. In outline, however, once the desired enzyme is isolated, it is attached to a carrier material. In order to ascertain sufficient accessibility of the enzyme, a bifunctional spacer molecule is attached to the carrier ... 

The above two processes employ isolated enzymes - penicillin G acylase and thermolysin, respectively - and the key to their success was an efficient production of the enzyme. In the past this was often an insurmountable obstacle to commercialization, but the advent of recombinant DNA technology has changed this situation dramatically. Using this workhorse of modern biotechnology most enzymes can be expressed in a suitable microbial host, which enables their efficient production. As with chemical catalysts another key to success often is the development of a suitable immobilization method, which allows for efficient recovery and recycling of the biocatalyst. 

Goldberg, K., Schroer, K., Luetz, S. and Liese, A. (2007) Biocatalytic ketone reduction - a powerful tool for the production of chiral alcohols -part I processes with isolated enzymes. Applied Microbiology and Biotechnology, 76, 237-248. 

Hummel, W., Abokitse, K., Drauz, K. et al. (2003) Towards a large-scale asymmetric reduction process with isolated enzymes Expression of an (5)-alcohol dehydrogenase in E. coli and studies on the synthetic potential of this biocatalyst. Advanced Synthesis and Catalysis, 345 (1 + 2), 153-159. 

Whole-cell biotransformations frequently showed insufficient stereoselectivities and/or undesired side reactions because of competing enzymatic activities present in the cells. These side reactions can modify the substrates and/or products. Furthermore, whole-cell biotransformations are limited due to the intrinsic need to grow biomass, which generates its own metabolites that are not related to the biotransformation reactions and, therefore, which need to be removed during the downstream process. Both the cells themselves and the unrelated metabolites produced are impurities that need to be removed after the biotransformation reaction. With isolated enzymes, there are no organism and unrelated metabolites to remove after the biotransformation processes. 

In this section, the advantages of the use of whole cells over the use of isolated enzymes in biotransformation processes are presented. 

In the processes that require regeneration of cofactors such as nicotinamide adenine dinucleotide phosphate (NAD(P)H) and adenosine triphosphate (ATP), whole-cell biotransformations are more advantageous than enzymatic systems [12,15]. Whole cells also have a competitive edge over the isolated enzymes in complex conversions involving multiple enzymatic reactions [14]. 

Compared with isolated enzymes, application of whole cells as biocatalysts is usually more economical since there is no protein purification process involved. Whole cells can be used directly in chemical processes, thereby greatly minimizing formulation costs. Whole cells are cheap to produce and no prior knowledge of genetic details is required. Microorganisms have adapted to the natural environment and produce both simple and complex metabolic products from their nutrient sources through complex, integrated pathways. 

In vitro synthesis of polyesters using isolated enzymes as catalyst via non-biosynthetic pathways is reviewed. In most cases, lipase was used as catalyst and various monomer combinations, typically oxyacids or their esters, dicarboxylic acids or their derivatives/glycols, and lactones, afforded the polyesters. The enzymatic polymerization often proceeded under mild reaction conditions in comparison with chemical processes. By utilizing characteristic properties of lipases, regio- and enantioselective polymerizations proceeded to give functional polymers, most of which are difficult to synthesize by conventional methodologies. 

In vitro polyester syntheses using an isolated enzyme as catalyst via non-bio-synthetic pathways is reviewed. These enzymatic routes for production of biodegradable polyesters possess several advances in comparison with fermentation and chemical processes ... 

By means of genetic engineering, including cloning and site-directed mutagenesis, it has become possible for modern synthetic chemists to utilize a sufficient amount of isolated enzyme catalysts and to modify the reactivity, stability, or even specificity of enzymes. Therefore, polymerizations catalyzed by isolated enzyme are expected to create a new area of precision polymer syntheses. Furthermore, enzymatic polymerizations have great potential as an environmentally friendly synthetic process of polymeric materials. 

Toray (1) A large Japanese chemicals manufacturer, perhaps best known for its process for synthesizing /-lysine for use as a dietary supplement. The starting material is cyclohexene which is converted in five steps to racemic lysine. An enzymic process isolates the desired optical isomer, the other is recycled. 

The Diels-Alder reaction is one of the most powerful and versatile carbon-carbon bond-forming methods available to synthetic organic chemists attempts to isolate enzymes that catalyze such a process have been unsuccessful. Therefore, the acceleration of this reaction by an abzyme has been an important landmark in the field of catalytic antibodies and of considerable potential for chemical synthesis. 

---