Commercial biocatalysts

Although mammalian CYPs are attractive candidates for use as commercial biocatalysts, many functional characteristics limit the opportunities to exploit such a system. Association of the enzymes with membranes prevents easy extraction and purification and limits the opportunities to produce useful recombinant enzymes by cloning the relevant genes for expression in microbial systems. All P450s have a porphyrin-haem active site that requires a second protein to reduce the iron component, often cytochrome P450 reductase or... 

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

Cross-linked enzymes are commercial biocatalysts and can be reused in organic solvent and aqueous solution. They are purchased as crystals derived from a single cross-linked enzyme. 

Biotechnology has attracted enormous interest and high expectations over the past decade. However, the implementation of new technologies into industrial processes has been slower than initially predicted. Although biocatalytic methods hold great industrial potential, there are relatively few commercial applications of biocatalysts in organic chemical synthesis. The main factors that limit the application of biocatalysts are ... 

When compared to traditional chemical synthesis, processes based on biocatalysts are generally less reliable. This is due, in part, to the fact that biological systems are inherently complex. In bioprocesses involving whole cells, it is essential to use the same strain from the same culture collection to minimise problems of reproducibility. If cell free enzymes are used the reliability can depend on the purity of the enzyme preparation, for example iso-enzyme composition or the presence of other proteins. It is, therefore, important to consider the commercial source of the enzyme and the precise specifications of the biocatalyst employed. 

The industrial development of biotransfonmations is hampered currently by lack of commercial availability of biocatalysts at a reasonable price, insufficient operational stability of most biocatalysts and the practical problems associated with the exploitation of cofactor-dependent biocatalysts. 

Many procedures have been suggested to achieve efficient cofactor recycling, including enzymatic and non-enzymatic methods. However, the practical problems associated with the commercial application of coenzyme dependent biocatalysts have not yet been generally solved. Figure A8.18 illustrates the continuous production of L-amino adds in a multi-enzyme-membrane-reactor, where the enzymes together with NAD covalently bound to water soluble polyethylene glycol 20,000 (PEG-20,000-NAD) are retained by means of an ultrafiltration membrane. 

After discussing the biological capability to transform steroids, we briefly examine foe biotransformation of other terpenoids to ensure that the reader develops an awareness of the potential of biotechnology to modify or produce derivatives of a wide range of natural materials that are of tremendous potential, commercial value in the food and health care sectors. We also include a brief consideration of the use of biocatalysts to transform a range of other hydrocarbon compounds. 

This work deals with the preparation and study of a biocatalyst prepared by immobilising a commercial pectinase on a tailor made support, and its use in a... 

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. 

Nevertheless, development of processes for commercial purposes is still limited, particularly with interfacial effects the loss of activity of the biocatalyst, the slow coalescence, the biocatalyst aggregation, and accumulation of medium components at the interface. 

A prochiral bis(cyanomethyl) sulfoxide was converted into the corresponding mono-acid with enantiomeric excesses as high as 99% using a nitrilase-NHase biocatalyst. The whole-cell biocatalyst Rhodococcus erythropolis NCIMB 11540 and a series of commercially available nitrilases NIT-101 to NIT-107 were evaluated in this study. As outlined in Figure 8.18, the prochiral sulfoxide may be transformed into five different products (plus enantiomeric isoforms), of which, three are chiral (A, B, and C) and two achiral (D and E). Only products A, B, and E were observed with the biocatalysts employed in this investigation. Both enantiomerically enriched forms of both A and C could be obtained with one of the catalysts used. The best selectivities are as follows (S)-A 99% ee, (R)-A 33% ee, (S)-C 66% ee, and (R)-C 99% ee, using NIT-104, NIT-103, NIT-108, and NIT-107 respectively. Each of these catalysts produced more... 

One of the most important advantages of the bio-based processes is operation under mild conditions however, this also poses a problem for its integration into conventional refining processes. Another issue is raised by the water solubility of the biocatalysts and the biocatalyst miscibility in oil. The development of new reactor designs, product or by-product recovery schemes and oil-water separation systems is, therefore, quite important in enabling commercialization. Emulsification is thus a necessary step in the process however, it should be noted that highly emulsified oil can pose significant downstream separation problems. 

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. 

The limitations preventing commercialization are mainly related to biocatalyst activity and specificity. Currently, increasing rate with respect to the biocatalyst is the main objective of the biocatalytic refining processes being developed. New applications of by-products could contribute to improving economic parameters. 

The second important issue related to commercial use of desulfurization biocatalysts is their inhibition by sulfate. The sulfur repression mechanism in most Rhodococcus species limits their use or activity in presence of sulfate- and sulfur-containing amino-acids such as cysteine, methionine, etc. To alleviate this problem, expression of the dsz genes under the control of alternate promoters has been investigated. 

Thus, several improvements have been made in the Rhodococcus strains to make desulfurization application possible or attractive however, the sulfur removal rate still remains the biggest bottleneck and no biocatalysts capable of rates needed for commercialization exist as of yet. 

In addition to desulfurization activity, several other parameters are important in selecting the right biocatalyst for a commercial BDS application. These include solvent tolerance, substrate specificity, complete conversion to a desulfurized product (as opposed to initial consumption/removal of a sulfur substrate), catalyst stability, biosurfactant production, cell growth rate (for biocatalyst production), impact of final desulfurized oil product on separation, biocatalyst separation from oil phase (for recycle), and finally, ability to regenerate the biocatalyst. Very few studies have addressed these issues and their impact on a process in detail [155,160], even though these seem to be very important from a commercialization point of view. While parameters such as activity in solvent or oil phase and substrate specificity have been studied for biocatalysts, these have not been used as screening criteria for identifying better biocatalysts. 

Despite all the intellectual property generated in this field, the application has not reached commercial scale. It does not mean that there has not been any progress. In fact, the biocatalyst development has greatly advanced, much of it due to the advancements in the techniques, methods and tools related to MB and GE. MB techniques raised the understanding of the biocatalyst from the level of whole cells to clearly defined... 

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