Whole limited substrate

Industrial applications of P450s have so far been restricted to whole-cell systems, which mostly solve the problem of cofactor delivery and regeneration. In such instances, however, physiological effects such as limited substrate uptake and reduced efflux of products out of cells, substrate or product toxicity, product degradation, as well as elaborate downstream processing are additional limiting factors that must be taken into account and often require optimization [34]. 

Limited substrate and product transport into and out of whole-cell biocatalysts. A third problem, which also concerns those reductions carried out in whole-cell format, is that the cell membrane can act as a barrier for diffusion of substrate into the cell and product out of the cell, leading to low specific reaction rates (rate per mass of cells). Both cell permeabilization and use of transporters or alternatively the use of isolated enzymes are potential strategies to overcome this challenge. 

The sensor usually consists of a coil of wire made from the material that is wound on a former and the whole sealed to prevent oxidization, although a film of the metal deposited on a ceramic substrate can also be used. The resistor is connected in a Wheatstone bridge network (Figure 17.17), using fixed resistors in the other three arms. The instrument connected across the bridge is calibrated directly in terms of temperature. The range is limited by the linearity of the device and the upper temperature, which can be measured, must be well below the melting point of the material. 

Several of the problems associated with whole cell bioprocesses are related to the highly effective metabolic control of microbial cells. Because cells are so well regulated, substrate or product inhibition often limits the concentration of desired product that can be achieved. This problem is often difficult to solve because of a poor understanding of the kinetic characteristics of the metabolic pathway leading to the desired product. 

Like enzymes, whole cells are sometime immobilized by attachment to a surface or by entrapment within a carrier material. One motivation for this is similar to the motivation for using biomass recycle in a continuous process. The cells are grown under optimal conditions for cell growth but are used at conditions optimized for transformation of substrate. A great variety of reactor types have been proposed including packed beds, fluidized and spouted beds, and air-lift reactors. A semicommercial process for beer used an air-lift reactor to achieve reaction times of 1 day compared with 5-7 days for the normal batch process. Unfortunately, the beer suffered from a mismatched flavour profile that was attributed to mass transfer limitations. 

The high affinity of the decarboxylase enzyme for its substrate (10 pM in the brain) makes it unlikely that this stage could ever become rate-limiting for the pathway as a whole. Nevertheless, the for this enzyme is considerably higher than tissue concentrations of 5-hydroxytryptophan and so, again, supply of this substrate is likely to be a crucial factor. 

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. 

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is widely used in foods, beverages, perfumes and the pharmaceuticals industries. Biotransformation of isoeugenol from essential oil to vanillin represents an economic route for the supply of vanillin, which has a limited supply due to the availability of vanilli pod plants. The conversion yield of isoeugenol to vanillin by the whole-cell biotransformation process of Bacillus fusiformis was low due to the product inhibition effect. Adding resin HD-8 to the whole-cell biotransformation eliminated the product inhibition effect, yielding 8 gL 1 of vanillin in the final reaction mixture [27]. The resin HD-8 also facilitated the separation of vanillin from the used substrate. The recovered isoeugenol can be used for the subsequent biotransformation reaction. 

The specificity of several of the enzymes identified in the 4S pathway of different organisms has been studied. In case of the DBT desulfurizing enzymes, little difference is expected in the specificity of the enzymes, say DszA, from different Rhodococcus strains found to date. This is essentially because the DNA sequence for the enzymes investigated so far has been the same. The difference in the specificity observed with whole cell assays is essentially due to the differences in substrate intake via the cell membrane and not necessarily due to a difference in the intrinsic enzyme specificity. It has been found that while isolated enzyme DszC (from KA2-5-1) can desulfurize up to 4,6 dipropyl DBT, whole cells cannot, indicating substrate transport as limiting factor. 

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