Microbial recombinant enzyme

This chapter is not intended to serve as a comprehensive review in drug metabolite biosynthesis rather, we will focus on practical considerations for metabolite synthesis at small to medium scale with three bioreactor systems mammalian bioreactors, microbial bioreactors and recombinant enzyme bioreactors. 

Metabolite biosynthesis has demonstrated its utility in drug metabolite preparation and characterization, and contributed to drag discovery and development. Although metabolite biosynthesis is a prerequisite step for metabolite structure elucidation in many cases, it is complementary to chemical synthesis in large-scale metabolite preparations. The merits for using these techniques should be determined on a case-by-case fashion. New techniques, such as recombinant enzyme and microbial glucuronidation systems, would have a great impact on the field. 

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... 

However, expression in a microbial cell is not always straightforward. For example, recombinant enzyme activity maybe different from that of the native enzyme. When incubated in a mixture of hydroperoxides, a HPL from green bell pepper (Capsicum annuum L.) that was expressed in Yarrowia lipolytica favours the production of hexanal although the native enzyme produces the unsaturated aldehyde ds-3-hexenal, both within the green bell pepper itself and when expressed in E. coli [22]. 

This microbial sensor system is based on the inhibitory action of the mutagens on the respiration of B subtilis Rec . B subtilis M45 (Rec") is genetically deficient in the DNA recombination enzyme system, whereas B subtilis H17 (Rec+) is a wild strain which has the ability to repair damaged DNA. The subsequent death of Rec bacteria is preceded by the decrease of respiration. As a result, the number of Rec cells on the surface of the oxygen electrode decreased and the current of the Rec electrode increased. On the other hand, the damaged DNA of Rec+ bacteria is repaired with the recombination system. Therefore, the number of Rec+ cells did not change and the current of the Rec electrode did not increase. 

Figure 5. Glycosylation pathway in insect cells and potential sites for metabolic engineering. Enzymes shown in bold type are those genes needed to be overexpressed to give recombinant proteins with more human-like glycans. The sialylation pathway is needed to supply galactose and sialic acid. These genes may be of microbial origin. Enzymes with an asterisk are those that may need to be inhibited or deleted.

Seeger M, M Gonzalez, B Camara, L Munoz, E Ponce, L Mejias, C Mascayano, Y Vasquez, S Sepulveda-Boza (2003) Biotransformation of natural and synthetic isoflavanoids by two recombinant microbial enzymes. Appl Environ Microbiol 69 5045-5050. 

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. 

Guoa, M., Hang, H. and Zhua, T. (2008) Effect of glycosylation on biochemical characterization of recombinant phytase expressed in Pichia pastoris. Enzyme and Microbial Technology, 42, 340-345. 

Rustler, S., Muller, A., Windeisen, V. et al. (2007) Conversion of mandelonitrile and phenylglycinenitrile by recombinant E. coli cells synthesizing a nitrilase from Pseudomonasfluorescens EBC191. Enzyme and Microbial Technology, 40, 598-606. 

Engelking, H., Pfaller, R., Wich, G. and Weuster-Botz, D. (2006) Reaction engineering studies on /3-ketoester reductions with whole cells of recombinant Saccharomyces cerevisiae. Enzyme and Microbial Technology, 38, 536-544. 

Microbial coagulants are now useful and are responsible for about one third of all the cheese produced worldwide, but suffer from the disadvantage of being too stable and so are threatened commercially by improved methods of produdng chymosin by recombinant DNA technology. The use of thermally destabilized microbial rennets results in residual enzyme levels in the milk product similar to or below those encountered when calf rennet is use (55). An unexpected benefit has been an increase on some occasions of the specificity of the microbial enzyme, making it virtually indistinguishable from the action of calf rennet. Also some microbial rennets help impart a flavor that is popular with consumers. 

---