Recombinant enzymes isolation

Cyclic dithioketals and acetals represent another important class of sulfur containing chiral auxiliaries, which are available in chiral form by biooxidation. Biotransformations were performed on a preparative scale using whole-cells (wild type and recombinant) and isolated enzyme. Again, enantiocomplementary oxidation of unsubstituted dithianes (linear and cyclic, R = H) was observed when using and CPMOcomo (Scheme 9.28) [211,212]. Oxygenation of functionalized substrates (R = substituted alkyl) with gave preferably trans... 

D-Aminoacid oxidase has been isolated from a nnmber of yeasts, and the nucleotide sequence of the enzyme from Rhodotorula gracilis ATCC 26217 has been established (Alonso et al. 1998). The gene could be overexpressed in Escherichia coli, and levels of the enzyme were greater under conditions of low aeration the enzyme isolated from the recombinant organisms was apparently the apoenzyme since maximum activity required the presence of FAD. 

ANDERSEN, M.D., M0LLER, B.L., Cytochromes P450 from Cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin cloning, functional expression in Pichia pastoris and substrate specificity of the isolated recombinant enzymes, J. Biol. Chem., 2000,275, 1966-1975. 

Wilson, M.P. Majerus, P.W. Isolation of inositol 1,3,4-trisphosphate 5/6-ki-nase, cDNA cloning, and expression of the recombinant enzyme. J. Biol. Chem., 271, 11904-11910 (1996)... 

Using site-directed mutagenesis techniques, you isolate a series of recombinant enzymes in which specific lysine residues are replaced with aspartate residues. The enzymatic assay results are shown in the table. 

S. typhimurium enzyme (S94), which appears to be essentially identical to the E. coli sulfite reductase, are in agreement with the above results. Thus mutants lacking the flavoprotein or the hemoprotein component of the enzyme and containing only the appropriate partial activities have been obtained and the respective partial enzymes isolated. The absorption spectra of sulfite reductase preparations from the wild type and from these mutants are shown in Fig. 46, and the proposed structure for the two components of the wild-type enzyme is shown in Fig. 47. Reconstitution of NADPH-sulfite reductase by recombination of the flavoprotein... 

This red species has also been shown to form in the reaction of reduced enzyme with DMSO (Adams et at., 1999), and appears to represent a kinetically viable DMSO Michaelis complex, which rapidly achieves equilibrium with the eorresponding Eox DMS species in the enzyme active site. Consistent with this, recent kinetic work with DMSO reductase has demonstrated that the enzyme is kinetically competent to oxidized DMS as well as reduce DMSO (Adams et al., 1999). Interestingly, the former reaction is sensitive to extended preincubation of enzyme with DMS under aerobic (but not anaerobic), but the latter reaction is not. The spectrum of the DMS-modified enzyme resembles that exhibited by recombinant as-isolated enzyme heterologously expressed in E. coli, which can be converted to native enzyme by reduction and reoxidation (Hilton et al.,... 

Using an enzyme isolated from human milk,key polar groups on the acceptor and donor substrates have been decoded [48,92]. The availability of larger quantities of the recombinant form of fucosyltransferase III (Lewis-enzyme, EMBL accession no. X 53578) [93] allowed evaluation of the enzyme for preparative use with non-natural substrates [56,89,94,95]. 

Ex ample 1 The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary alcohol dehydrogenase was overexpressed in Escherichia coli to form more than 10% to total protein11361. The recombinant enzyme was purified by heat treatment and precipitation with aqueous (NH4)2S04 and isolated in 67% yield. Enzymes with mutation(s) around the active site residues were also created to examine the catalytically important zinc binding motif in the proteins. 

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