Enzyme recombinant

The key enzyme in this sequence, isopenicillin N synthase (IPNS), has been purified from E. coli (59) and the recombinant enzyme shown to be a single polypeptide of 336 amino acids containing two cysteines, numbers 106 and 255 from the /V-teiminus, and probably a ferrous ion in a nonheme environment. The enzyme has been crystallized and studies undertaken to obtain suitably sized crystals for diffraction studies. 

Figure 18.4 The hanging-drop method of protein crystallization, (a) About 10 pi of a 10 mg/ml protein solution in a buffer with added precipitant—such as ammonium sulfate, at a concentration below that at which it causes the protein to precipitate—is put on a thin glass plate that is sealed upside down on the top of a small container. In the container there is about 1 ml of concentrated precipitant solution. Equilibrium between the drop and the container is slowly reached through vapor diffusion, the precipitant concentration in the drop is increased by loss of water to the reservoir, and once the saturation point is reached the protein slowly comes out of solution. If other conditions such as pH and temperature are right, protein crystals will occur in the drop, (b) Crystals of recombinant enzyme RuBisCo from Anacystis nidulans formed by the hanging-drop method. (Courtesy of Janet Newman, Uppsala, who produced these crystals.)...

Molecular characteristics of luciferase. A molecule of the luciferase of G. polyedra comprises three homologous domains (Li et al., 1997 Li and Hastings, 1998). The full-length luciferase (135 kDa) and each of the individual domains are most active at pH 6.3, and they show very little activity at pH 8.0. Morishita et al. (2002) prepared a recombinant Pyrocystis lunula luciferase consisting of mainly the third domain. This recombinant enzyme catalyzed the light emission of luciferin (luminescence A.max 474 nm) and the enzyme was active at pH 8.0. The recombinant enzyme of the third domain of G. polyedra luciferase was crystallized and its X-ray structure was determined (Schultz et al., 2005). A -barrel pocket putatively for substrate binding and catalysis was identified in the structure, and... 

Ratnatilleke A, JW Vrijbloed, JA Robinson (1999) Cloning and sequencing of the coenzyme B,2-binding domain of isobutyryl-CoA mutase from Streptomyces cinnamonensis. Reconstitution of mutase activity and characterization of the recombinant enzyme produced in Escherichia coli. J Biol Chem 274 31679-31685. 

Thus recombinant enzyme constructs for use in activity assays should be designed to faithfully reflect the physiological state of the enzyme to the extent that is practical in vitro. 

Effenberger and coworkers have utilized the tolerance of methyl ketones by the recombinant enzyme to develop an alternative synthesis of tetronic acids and their amino derivatives, as shown in Figure 5.18. Treatment of O-acyl cyanohydrins with lithium disilazide resulted in base-induced ring closure to amino tetronic acid derivatives. Alternatively, the cyanohydrins could be converted to a-hydroxy esters prior to acylation, and the same base-induced cyclization then led to tetronic acid derivatives [89]. 

Varela, E., Guillen, F., Martinez, A.T. and Martinez, M.J. (2001) Expression of Pleurotus eryngii aryl-alcohol oxidase in Aspergillus nidulans purification and characterization of the recombinant enzyme. Biochimica et Biophysica Acta, Protein Structure and Molecular, Enzymology, 1546 (1), 107-113. 

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. 

Kendrew, S.G., Hopwood, D.A. and Marsh, E.N.G. (1997) Identification of a monooxygenase from Streptomyces coelicolor A3(2) involved in biosynthesis of actinorhodin purification and characterization of the recombinant enzyme. Journal of Bacteriology, 179, 4305—4310. 

Most of the known microorganisms to be active for BDS (by the date that patent [408] was introduced), were considered in this invention. Furthermore, their mutational or engineered derivatives, enzymes, cell-free extracts, recombinant enzymes, recombinant DNA, plasmids, vectors, and fragments were also incorporated in the intellectual property document. The mentioned operating conditions regard ambient temperature, mechanical agitation and a 1 9 biocatalyst/petroleum ratio. 

Inhibitors of several enzymes in the glycolytic pathway, upon which survival of T. brucei is dependent, have been described. Lonidamine (29) has been shown to inhibit T. brucei hexokinase (IC50 = 850 pM) and be toxic to the parasite (T.b. LD50 = 50 pM) in culture [31]. A series of mannitol derivatives have been discovered, which inhibit T. brucei phos-phofructokinase (TbPFK) [32]. The most potent compound (30) within this series exhibits an IC50 = 23 pM in a recombinant enzyme assay and inhibits parasite growth in vitro (IC50 = 30 pM). 

Characterization of the exact nature of the post-translational modifications the mammary system is capable of undertaking. For example, the carbohydrate composition of tPA produced in this system differs from the recombinant enzyme produced in murine cell culture systems. 

Laronidase is yet an additional recombinant enzyme now approved for general medical use. The product, used to treat mucopolysaccharidosis, is overviewed in Box 12.2. 

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