Recycling considerations enzyme production

Improved sensitivity and scope can be achieved by coupling two (or more) enzymatic reactions in a chain, cycling, or catalytic mechanism (9). For example, a considerable enhancement of the sensitivity of enzyme electrodes can be achieved by enzymatic recycling of the analyte in two-enzyme systems. Such an amplification scheme generates more than a stoichiometric amount of product and hence large analytical signals for low levels of the analyte. In addition, a second enzyme can be used to generate a detectable (electroactive) species, from a nonelectroactive product of the first reaction. 

As early as 1984 the porcine pancreas lipase-catalysed enantioselective synthesis of (R)-glycidol was described. At pH 7.8 and ambient temperatures the reaction was allowed to proceed to 60% conversion (Scheme 6.9). This means that the enzyme was not extremely enantioselective, otherwise it would have stopped at 50% conversion. Nonetheless, after workup the (R)-glycidol was obtained in a yield of 45% with an ee of 92% [42]. This was a remarkable achievement and the process was developed into an industrial multi-ton synthesis by Andeno-DSM [34, 43]. While on the one hand a success story, it also demonstrated the shortcomings of a kinetic resolution. Most enzymes are not enantiospecific but enantioselective and thus conversions do not always stop at 50%, reactions need to be fine-tuned to get optimal ees for the desired product [28]. As mentioned above kinetic resolutions only yield 50% of the product, the other enantiomer needs to be recycled. As a result of all these considerations this reaction is a big step forward but many steps remain to be done. 

The cost of enzyme preparations has been decreasing in recent years however, it continues to affect considerably the price of ethanol obtained from cellulosic raw materials. Increased enzymatic hydrolysis efficiency is one way to reduce the enz)me cost in bioethanol production. Another method is enzyme recycle and reuse. Immobilization of biocatalysts allows for their economic reuse and development of continuous bioprocess. Although immobilization poses problems of substrate accessibility and binding for most endo- and exocellulases, P-glucosidase exhibits characteristics amenable to immobilization, such as activity on soluble substrates and the lack of a carbohydrate-binding module. Among the possible approaches, immobilization of (J-glucosidase is one prospective solution to the problem. 

The selection of a bioreactor and operation mode starts with the type of biocatalysts (enzymes or whole cells) that will be used to produce the desired product, and if the biocalalysts will be suspended or immobilized with a recycle option. Each of these possibihties should be examined while taking into consideration the reactions stoichiometry and kinetics, mixing needs, gas exchange, heat removal and capital and operating costs (Merchuk and Asenjo, 1995). 

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