Biocatalyst improvements

In an in depth comparison of the cumulative knowledge discussed in Chapter 3, with what one could extract from the technological results reported in this Chapter, perhaps the first observation that one can make is the difference between the content of the biocatalyst development vs. process development results. The results on biocatalyst improvements constitute the majority of the open literature reports. The most important bottleneck holding advancement of the biodesulfurization technology is the ability to break the second C-S bond, releasing the sulfur from the organosulfur molecules. The IP portfolio does not provide a real solution for that problem. 

Any organization which does not attempt to innovate is moribund and consequently, industry is a constantly seeking new or improved biocatalysts. Improvements will be sought in response to commercial needs, some fundamental, such as the requirement for a totally new catalyst, some more detailed, such as a desire to change specific parameters in an existing process. 

In order to increase productivity, thereby reducing the cost of ethanol fermentation, various alternatives to traditional fermentation techniques, including biocatalyst improvement, have been studied. 

Today s tools of evolutionary engineering certainly fulfill these requirements, and directed evolution has in fact emerged as the method of choice for biocatalyst improvement. However, we are only beginning to explore the power of evolutionary design. 

Adsorption on solid matrices, which improves (at optimal protein/support ratios) enzyme dispersion, reduces diffusion limitations and favors substrate access to individual enzyme molecules. Immobilized lipases with excellent activity and stability were obtained by entrapping the enzymes in hydrophobic sol-gel materials [20]. Finally, in order to minimize substrate diffusion limitations and maximize enzyme dispersion, various approaches have been attempted to solubilize the biocatalysts in organic solvents. The most widespread method is the one based on the covalent linking of the amphiphilic polymer polyethylene glycol (PEG) to enzyme molecules [21]. 

Alcohol oxidoreductases capable of oxidizing short chain polyols are useful biocatalysts in industrial production of chiral hydroxy esters, hydroxy adds, amino adds, and alcohols [83]. In a metagenomic study without enrichment, a total of 24 positive clones were obtained and tested for their substrate specifidty. To improve the detedion frequency, enrichment was performed using glycerol or 1,2-propanediol and further 24 positive clones were deteded in this study. 

Since stereoselectivities of biocatalytic reductions are not always satisfactory, modification of biocatalysis are necessary for practical use. This section explains how to find, prepare, and modify the suitable biocatalysts, how to recycle the coenzyme, and how to improve productivity and enantioselectivity of the reactions. 

The change of the anion results in alteration of enzymatic activity and also allows improvement in the enantioselectivity from an unacceptable level (1.1) to a synthetically useful value of 24. The cationic component of the IL also affects the activity and selectivity of the biocatalyst. Scheme 5.16 presents the study on the kinetic resolution of adrenaline-type aminoethanol in ILs [64]. 

In one of the above-mentioned cases [86], a significant improvement of reaction rates was observed when compared to the reactions carried out by uncoupled biocatalysts. This fact suggests that owing to the close proximity of the enzymes the local concentration of the intermediate is higher around the fused biocatalyst. 

The use of molecular biology methods, described in Section 5.3 seems to be especially worthwhile as it offers novel possibilities of optimization on process adjustment. Directed evolution leads to the formation of new biocatalysts with improved characteristics (selectivity, activity, stability, etc.). Incorporation ofnon-proteinogenic amino acids makes it possible to reach beyond the repertoire of building blocks used by nature. The prospect of bioconjugate preparation offers the possibility to form functional clusters of enzymes and to perform multiple synthetic steps in one pot. 

A large amount of biocatalyst is usually required to reduce a considerable amount of substrate (the b/s for baker s yeast is about 50-350). On the contrary, a low b/s ratio (2.6-0.5) could be achieved using the cyanobacteria. The improvement in the b s ratio is caused by the fact that the cyanobacterium can utilize the power of light effectively to reduce the substrate. 

Metabolic and enzyme engineering have received a lot of attention in academic institutions and are now being applied for the optimization of biocatalysts used in the production of a diverse range of products. Engineered microorganisms, even with non-native enzyme activities, are being used for novel products and process improvements for the production of precursors, intermediates and complete compounds, required in the pharmaceutical industry (Chartrain et ai, 2000). 

The biotransformation process has been improved by significant advances in biochemical engineering advances in genetic and protein engineering, microbiological manipulations for the production of enzymes, and the use of biocatalysts in immobilized form and large-scale purification methods. 

Several kinds of states in which enzymes may be used for various reactions in aqueous-organic biphasic systems have been developed in previous work (Table 2). In biphasic media, the biocatalyst is easily recovered after the reaction then it is not always necessary to be immobilized. Nevertheless, the immobilization can confer important properties, such as improved stability of biocatalyst. Furthermore, protection of the biocatalyst against a damaging turbulent environment can also play a role. 

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. 

Woodyer, R., van der Donk, W.A. and Zhao, H.M. (2006) Optimizing a biocatalyst for improved NAD(P)H regeneration directed evolution of phosphite dehydrogenase. Combinatorial Chemistry High Throughput Screening, 9, 237-245. 

The application of biocatalytic technologies in the refining industry will be possible only if it can improve product yields and produce cleaner fuels economically. The hurdle to commercialization of the biodesulfurization process is still the activity of the biocatalyst. The reasons for this will be evident from the discussion in Chapter 3. 

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