Purification and properties of a heat stable inulin fructotransferase (DFA III-producing) from Arthrobacter pascens T13-2

A strain of thermophilic bacteria, Bacillus stearothermophilus, with pectolytic activity has been isolated. It produced an endo-polygalacturonic acid trans-eliminase (endo-PATE, EC 4.2.2.1) extracellularly when grown at 65 °C on a pectic acid medium. The PATE was purified 62-fold by the rapid affinity chromatographic method on a Sepharose–polygalacturonamide linked matrix. The absorbed PATE was eluted from the column with a continuous gradient of 0–10−3 M ethylenediaminetetraacetic acid (EDTA) in phosphate buffer at pH 7.6.The endo-PATE of this organism was much more heat stable than similar enzymes from the mesophilic Bacillus polymyxa and the thermotolerant Bacillus pumilus. The maximum activity of the enzyme occurred at 70 °C. With pectic acid as the substrate, the endo-PATE had an optimal pH of 9.0, the highest optimal pH compared with those of similar enzymes from other species of the genus.The molecular weight of the endo-PATE, as determined by chromatography on a Sephadex G-100 gel column, was 24 000.

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Purification and properties of Penicillium glucose 6-phosphate dehydrogenase

Biochemical Journal ◽

10.1042/bj1280817 ◽

1972 ◽

Vol 128 (4) ◽

pp. 817-831 ◽

Cited By ~ 11

Author(s):

A. Anne Malcolm ◽

M. G. Shepherd

Keyword(s):

Molecular Weight ◽

Thermal Inactivation ◽

Gradient Centrifugation ◽

Sucrose Density Gradient ◽

Thermophilic Fungus ◽

Sucrose Density Gradient Centrifugation ◽

Coenzyme Specificity ◽

Heat Stable ◽

Purification And Properties ◽

Glucose 6 Phosphate Dehydrogenase

1. Glucose 6-phosphate dehydrogenase was isolated and partially purified from a thermophilic fungus, Penicillium duponti, and a mesophilic fungus, Penicillium notatum. 2. The molecular weight of the P. duponti enzyme was found to be 120000±10000 by gelfiltration and sucrose-density-gradient-centrifugation techniques. No NADP+- or glucose 6-phosphate-induced change in molecular weight could be demonstrated. 3. Glucose 6-phosphate dehydrogenase from the thermophilic fungus was more heat-stable than that from the mesophile. Glucose 6-phosphate, but not NADP+, protected the enzyme from both the thermophile and the mesophile from thermal inactivation. 4. The Km values determined for glucose 6-phosphate dehydrogenase from the thermophile P. duponti were 4.3×10−5m-NADP+ and 1.6×10−4m-glucose 6-phosphate; for the enzyme from the mesophile P. notatum the values were 6.2×10−5m-NADP+ and 2.5×10−4m-glucose 6-phosphate. 5. Inhibition by NADPH was competitive with respect to both NADP+ and glucose 6-phosphate for both the P. duponti and P. notatum enzymes. The inhibition pattern indicated a rapid-equilibrium random mechanism, which may or may not involve a dead-end enzyme–NADP+–6-phosphogluconolactone complex; however, a compulsory-order mechanism that is consistent with all the results is proposed. 6. The activation energies for the P. duponti and P. notatum glucose 6-phosphate dehydrogenases were 40.2 and 41.4kJ·mol−1 (9.6 and 9.9kcal·mol−1) respectively. 7. Palmitoyl-CoA inhibited P. duponti glucose 6-phosphate dehydrogenase and gave an inhibition constant of 5×10−6m. 8. Penicillium glucose 6-phosphate dehydrogenase had a high degree of substrate and coenzyme specificity.

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