Cell-free enzyme synthesis

As stated earlier (see p. 342), study of the cell-free, enzymic synthesis of dextran was initiated by Hehre and Sugg They demonstrated the ability of a cell-free extract of Leuconostoc mesenteroidea to catalyze the formation of dextran. The over-all reaction was represented by equation (1). In addi-... 

Fig. 4. The time dependence of cell-free enzyme synthesis uninhibited and inhibited at varying times by actinomycin or rifampicin. Synthesis was carried out as described in Table 1 except for the following modifications. In the control experiment, samples were removed from the synthesis system and assayed at the times indicated on the abscissa. In the actinomycin and rifampicin experiments, the inhibitor (2 jitg of actinomycin or 1 ju,g of rifampicin per milliliter) was added at the time indicated on the abscissa, and the incubation was continued for another 60 minutes before assay. All values have been normalized to an assay time of 200 minutes. The S-30 used was prepared from strain 514 which contains a deletion of the entire lac region including the repressor gene.

This review will summarize data and ideas that have, in any way, been collected and promoted by cell-free enzyme synthesis (Chapter I). The second part will deal in some detail with the characteristics of cell-free enzyme synthesis and the principles of available systems (Chapter II). We feel that these details will make the potential of the method apparent. 

Some of these genes have now been detected and characterized, either by cell-free enzyme synthesis (Herrlich and Schweiger, 1970 Schweiger et ah, 1971 ScHERZiNGER et ah, 1972a) (A), by measurement of UV sensitivity (ScHERZiNGER et ah, 1972a Herrlich et ah, 1974) (B) or by the isolation of deletion mutants (Studier, 1972 Studier, 1973) (C). 

By cell-free enzyme synthesis, the host shut-off was analysed and two mechanisms distinguished Shut-off of host translation and inhibition of host messenger RNA synthesis. 

Again, the discussion of the lac operon will have to be limited to advances made in conjunction with cell-free enzyme synthesis. 

The in vitro synthesis of trp enzymes is another good example of the faithfulness of cell-free enzyme synthesis. The appearance of tryptophan synthetase [trp and trpK) and of anthranilate synthetase [trp and trpT>) in vitro parallels the kinetics in vivo and both enzymes are S5mthesized in equimolar amounts, as they are in vivo (Pouwels and van Rotterdam, 1972). 

Wetekam, W. Identification of template strand in heteroduplex DNA directing cell-free enzyme synthesis. Molec. gen. Genet. 118, 57-60 (1972). 

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. 

GM Whitesides. Applications of cell-free enzymes in organic synthesis. In Enzymes in Organic Synthesis. London Pitman Publishing, 1985, pp 76-96. 

Finally, some authors propose the use of enzyme mixtures for the biosynthesis of complex molecules. A cell free protein synthesis (CFPS) system is a novel approach that was successfully used for the production of complex mammalian proteins with multiple disulfide bonds (Bhatta-charya, 2004). However, even if the technical potential of this method is proved, its economical feasibility has still to be proven. 

In spite of the progress that has been made, several difficulties limit the use of cell-free enzymes for the synthesis of polysaccharides. The major problem is the complexity of many polysaccharide-synthesizing systems. Isolation, purification, and stabilization of the required enzymes is often difficult, as many enzymes lose activity when they are no longer membrane-associated. Enzyme isolation from eukaryotic sources is tedious, because of low cellular enzyme concentration. It is unlikely that cell-free enzymatic synthesis will provide better routes to most natural polysaccharides than do fermentation and isolation. The use of genetic engineering,... 

Whitesides, G.M. Large-Scale Organic Synthesis Using Cell-free Enzymes. In Applications of Biochemical Systems in Organic Chemistry. Jones, J.B., Sih, C.J., and Perlman, D. (eds) Wiley Interscience New York, 1976, pp. 901-927. 

Synthesis and Chemical Features of Disaccharides and their Derivatives.—For several successful syntheses of disaccharides from 1-thioglycosides see Section 2 of this Chapter. An extensive review has appeared (in Czech) on the chemistry of sucrose, and its synthesis and that of trehalose, by cell-free enzymic methods using appropriate, coupled synthetase systems, have been described. These afford practical routes to millimolar quantities of the disaccharides. Injection of [l- C]-D-glucose into crickets allowed the synthesis of 1,1 -labelled trehalose in 27% yield. ... 

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