Biocatalyst stability

Several scouting experiments were performed to find the best pH conditions. Figure 3 reports the ratio between the PG specific activity measured after the purification procedure (ASf) and the initial PG specific activity (ASi). At pH 3.5, the microspheres are able to remove from the broth the major part of the protein without PG activity, thus providing a four time increase of the enzyme specific activity. The purified PG from Kluyveromyces marxianus was immobilised following the above procedure. Batch reactions in the packed bed reactor were done to evaluate the biocatalyst stability. After an initial loss, due to enzyme release, the residual PG activity reaches a plateau value corresponding to about 40% of the initial activity. Probably, some broth component interfered during the immobilisation reaction weakening the protein-carrier interactions. 

On the other hand, biocatalyst stability can be affected by the presence of organic-aqueous interface. In our previous work [25], we studied the effect of interface with octane on the lipoxygenase stability in an octane-buffer pH 9.6 biphasic system. This loss in activity is more pronounced than that observed in the aqueous system. During lipoxygena-tion, the active enzyme concentration [E] in the aqueous phase of our biphasic bioreactor was ... 

The liquid-liquid interface has been identified as the major factor responsible for papain deactivation in a biphasic system [66]. If the interfacial tension can be decreased to a small value using surfactant, the biocatalyst stability will be expected to increase. 

Organic solvent Log P Biphasic medium Biocatalyst Reaction Biocatalyst stability Activity Reference... 

The volumetric ratio of the two liquid phases (j6 = Forg/ Faq) can affect the efficiency of substrate conversion in biphasic media. The biocatalyst stability and the reaction equilibrium shift are dependent on the volume ratio of the two phases [29]. In our previous work [37], we studied the importance of the nonpolar phase in a biphasic system (octane-buffer pH 9) by varying the volume of solvent. The ratio /I = 2/10 has been the most appropriate for an improvement of the yield of the two-enzyme (lipase-lipoxygenase) system. We found that a larger volume of organic phase decreases the total yield of conversion. Nevertheless, Antonini et al. [61] affirmed that changes in the ratios of phases in water-organic two-phase system have little effect upon biotransformation rate. 

In a study looking at solvent stability [424], various aqueous-miscible solvents (tetrahydrofuran, acetonitrile, isopropanol, methanol, and A,A-dimethylformamide) were used with pinacyanol chloride as substrate. Although a PAH was not used as a substrate, the results may be extrapolated to PAH reactions. The greater impact of peroxide as compared to the solvent on biocatalyst stability was reported in this study and the need to control peroxide concentration was noted. 

The patents, however, protected the microorganisms (biocatalysts/biocatalytic systems) [86,87] as well as process to use the microorganism [87], So far, there are no records of any other international protection. The patent reports new cultures of Rhodococcus strains, and a method to improve biocatalyst stability, which allows recycling. 

The use of immobilized cell reactors have shown improved biocatalyst stability, however, the specific rates of desulfurization have been much lower than for suspended cell (stirred) reactors. Mass transfer limitations have been significant resulting in lower rates. Thus, the activity is sacrificed to achieve stability. Further work in this area and improved immobilization matrices can help improve the activity along with the stability. 

Immobilization onto a solid support, either by surface attachment or lattice entrapment, is the more widely used approach to overcome enzyme inactivation, particularly interfacial inactivation. The support provides a protective microenvironment which often increases biocatalyst stability, although a decrease in biocata-lytic activity may occur, particularly when immobilization is by covalent bonding. Nevertheless, this approach presents drawbacks, since the complexity (and cost) of the system is increased, and mass transfer resistances and partition effects are enhanced [24]. For those applications where enzyme immobilization is not an option, wrapping up the enzyme with a protective cover has proved promising [21]. 

Quite common in applied biocatalysis, where the purity of biocatalyst often is not known, is the expression of biocatalyst stability as an the enzyme consumption number (e.c.n.) [Eq. (2.26)]. 

The threshold value for sufficient biocatalyst stability depends on the application, as does the value for product yield. For any application in synthesis the TTN should exceed 10 000, and for large-scale processing a value of > 1 000 000 is preferred. 

On the basis of the biocatalyst stability results it was obvious that there were indirect effects that influenced the reaction, for example the density-dependent physical properties were affected by the change in pressure. An increase in pressure led to an enhanced solvent density with improvement in its solvating power in the reaction bulk. The solubility in the liquid reaction mixture increased with pressure as well. In the liquid subcritical region rich in CO2, at pressures below... 

Adercruetz P and Mattiasson B. Aspects of Biocatalyst Stability in Organic Solvents. Biocatalysis 1987 1 99-108. 

Thermodynamic effects on biocatalysts working in the presence of non-conventional media have an impact on two levels i) phase and reaction equilibria and ii) biocatalyst stability and activity [34]. The thermodynamic effects on the first level are by now relatively well understood. It is probably safe to say that a certain scientific foundation for rational phase and reaction equilibrium engineering exists. Based on this knowledge, it is possible to conceive, if not to design, bio catalytic systems with tailored selectivities and/or improved product yields due to low water activity, the presence of non-aqueous non-conventional solvents [33], or characterized by a very high solid content [35, 36]. It has been shown for particular cases that this type of engineering may be based directly on standard thermodynamic tools such... 

Further bottlenecks of the whole-cell biocatalytic process are to be addressed such as substrate and product inhibition, pH and temperature intolerance (Doig et al. 2003), as well as oxygen limitations. In the case of the oxidation of, e.g., bicyclo[3.3.0]-hep-2-en-6-one, the process limitations are the oxygen supply which limits the rate of the reaction, the product inhibition which limits the final yield, and the biocatalyst stability which limits the total reaction time (Baldwin and Woodley 2006). Here, with increasing cell concentrations, the dissolved oxygen tension drops to zero, and the initial specific reaction rate is reduced dramatically. 

Brocklebank S, Woodley JM, Lilly MD. Immobilised trans-ketolase for carbon-carbon bond synthesis biocatalyst stability. J. Mol. Catal B Enzym. 1999 7 223-231. 

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