Glucose thermostability

Monooxygenase Assays. Incubation media contained the following (final concentrations) 0.05M phosphate buffer, pH 7.A, glucose-6-phosphate (G-6-P, 2.3 mM), G-6-P dehydrogenase (3 units), NADP (0.23 mM), and KC1 (2.8 mM), and various tissue preparations. Substrates were added in small volumes (25 yl or less) of MeOH. Samples (1.1 ml) were shaken in a thermostated (usually at 22°C) water bath and reactions terminated by enzyme denaturation. Specific analytical procedures for aldrin epoxi-dation (13), 1 CH30-p-nitroanisole 0-demethylation (1A), and 3H-benzo(a)pyrene oxidation (15) have been described. 

The work to be summarized here is preliminary and will be published elsewhere (manuscripts in preparation). Thermoanaerobacter strain B6A produces a xylanase complex diat is inducible by xylose and catabolite repressed by glucose. Noticeably, when mixed with insoluble xylan, glucose-grown cells do not bind xylan whereas, xylose-grown cells bind very tightly. Canonized ferritin was used to identify xylanosome structures in xylose grown cells which are similar to cellulosomes observed in C. thermocellum, Thermoanaerobacter strain B6A produces thermostable cell-bound endoxylanase, which is also excreted. 

The kit includes necessary enzymes (thermostable a-amylase and amyloglucosidase), some reagents (buffer concentrate and glucose oxidase/peroxidase [GOPOD] reagent) and standards (glucose solution and com starch) to carry out 100 starch determinations. The other reagents required for this procedure may be obtained from any chemical supplier. 

I. Lasters, Enhancing the thermostability of glucose isomerase by protein engineering, Bio/Technology 1991, 9, 738-742. 

S. -Y. Liu, J. Wiegel, and F. C. Gherardini, Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489, J. Bacteriol. 1996, 178, 5938-5945. 

Glucose (Xylose) Isomerase (Gl) and Glycoamylase Enhancement of Thermostability 289... 

The by-products, such as isomaltose, have to be removed chromatographically, which is energy- and cost-intensive. Protein engineering focuses on increasing the glucose yield (affecting enzyme selectivity) and enzyme thermostability. 

Kenausis G, Chen Q, Heller A. Electrochemical glucose and lactate sensors based on wired thermostable soybean peroxidase operating continuously and stably at 37°C. 

D Auria S, DiCesare N, Staiano M, Gryczynski Z, Rossi M, Lakowicz JR. A novel fluorescence competitive assay for glucose determinations by using a thermostable glucokinase from the thermophilic microorganism Bacillus stearothermophilus. Analytical Biochemistry 2002, 303, 138-144. 

Index Entries Sol-gel glass chloroperoxidase glucose oxidase acetonitrile horseradish peroxidase thermostability. 

---