Temperature isomerase

The ultimate objective of an X-ray cryoenzymological study is the mapping of the structures of all kinetically significant species along the reaction pathway. In the case of ribonuclease A this has been largely achieved, as described above. Other enzymatic reactions now await application of the same techniques. Unfortunately, not all crystalline enzymes lend themselves to study by this method. In some cases it may be impossible to find a suitable cryoprotective mother liquor in others, the reaction may occur too rapidly at ordinary temperature. A reaction with Acat of 10 seconds and an activation enthalpy of —6 kcal mol will not be quenched even at — 75°C. The approach we have described in this article can be applied to only a small number of enzymes. Two likely candidates for successors to ribonuclease are the enzymes yeast triosephosphate isomerase and porcine pancreatic elastase. 

Figure 3(A). Comparison of temperature optima for activities of glucose isomerase, amylase, and >galactosidase. Enzymes were assayed with cell extract from xylose-grown cells. A 100% activity value corresponds to 0.60, 0.58, and 0.46 U/mg for glucose isomerase, amylase, and -galactosidase, respectively. Cell extracts in 50 mM sodium phosphate buffer (pH 7.0), 100 mM sodium acetate buffer (pH 5.5), and 100 mM sodium phosphate buffer (pH 6.0) for glucose isomerase, amylase, and -galactosidase, respectively, were preincubatcd at the indicated temperatures, prior to the assay for residual enzyme activities. Reprinted with permission from ref. 20. Copyright 1990 American Society for Microbiology.

Figure 3(B). Comparison of temperature optima for stabilities of glucose isomerase, amylase, and d-galactosidase. Reprinted with permission from ref. 20. Copyright 1990 American Society for Microbiology.

Xylose isomerases with higher thermostability were found in the strains of Streptomyces and relaxed Actinoplanaceae (which includes the generdLAmpullariella and Actinopianes). High thermo-tolerance is desirable for production of HFCS because at equilibrium, as the temperature of the enzyme reaction is increased, the ketose/aldose ratio increases proportionately 30). In addition, reactors running at higher temperatures have less risk of microbial contamination, allowing for less frequent and less costly enzyme replacement. 

The isolation of mutants in S. typhimurium by Rick and Osborn (11,12) and mutants in E. coli by Nishijima and Raetz (44) that accumulate the Lipid A precursor indicate that KDO synthesis and Lipid A synthesis are not coordinately controlled. The initial steps in the synthesis of the Lipid A precursor are totally unknown. The temperature sensitive mutants of E. coli isolated by Nishijima and Raetz (44) that are defective in phosphaditylglyc-erol phosphate synthesis at 42°C and accumulate the Lipid A precursors indicate that there is some relationship between the synthesis of phosphatidylglycerol and LPS. The reasons for the acu-cumulation of the Lipid A precursors in this E. coli mutant are not obvious. We have shown that CDP-diglyceride, one of the substrates for phosphatidylglycerol phosphate synthesis, is an inhibitor of D-arabinose-5-phosphate isomerase with an 1 value... 

J. A. Roels and R. van Tilberg, Temperature dependence of the stability and the activity of immobilized glucose isomerase,... 

High-Fructose Corn Syrup (HFCS) occurs as a water white to light yellow, somewhat viscous liquid that darkens at high temperatures. It is a saccharide mixture prepared as a clear, aqueous solution from high-dextrose-equivalent corn starch hydrolysate by the partial enzymatic conversion of glucose (dextrose) to fructose, using an insoluble glucose isomerase preparation that complies with 21 CFR 184.1372 and that has been obtained from a pure culture fermentation that produces no antibiotics. It is miscible in all proportions with water. 

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