Isomaltose Production by Modification of the Fructose-Binding Site on the Basis of the Predicted Structure of Sucrose Isomerase from “Protaminobacter rubrum”

“Protaminobacter rubrum” sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose and trehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration via a mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a ³²⁵RLDRD³²⁹ motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional active-site structure of SI was superimposed on and compared with those of other α-glucosidases in family 13. We identified two Arg residues that may play important roles in SI-substrate binding with weak ionic strength. Mutations at Arg³²⁵ and Arg³²⁸ in the fructose-binding site reduced isomaltulose production and slightly increased trehalulose production. In addition, the perturbed interactions between the mutated residues and fructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose could now bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designed based on structural analysis, the R³²⁵Q mutant enzyme exhibiting high relative activity for isomaltose production was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperature shift; the maximum isomaltose concentration and production yield were 57.9 g liter⁻¹ and 0.55 g of isomaltose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreased at a temperature of >35°C. Maximum isomaltose production (75.7 g liter⁻¹) was recorded at 35°C, and its yield for the consumed sucrose was 0.61 g g⁻¹ with the addition of 15% (wt/vol) glucose. The relative activity for isomaltose production increased progressively with temperature and reached 45.9% under the same conditions.     

Purification and characterization of an anionic isoperoxidase from scented-geranium callus

Secretory anionic isoperoxidase (EC 1.11.1.7), named PA1, was 68-fold purified from scented-geranium (Pelargonium graveolense) callus by using ion exchange chromatography and gel filtration. Isoperoxidase PA1 was a glycoprotein with an isoelectric point (pI) of 4.0. The molecular weight of PA1 was approximately 42.5 and 44 kDa, estimated by SDS-PAGE and Sephadex G-150 gel filtration, respectively. The optimum pH of the enzyme was 5.0 for guaiacol and H2O2, and the Km values for guaiacol and H2O2 were 1.96 and 8.5mM, respectively. Substrate studies in terms of optimum pHs and Km values with various synthetic and naturally occurring phenolic compounds were performed. In comparison with cationic isoperoxidase, PC3, which has been already characterized, anionic isoperoxidase PA1 had much lower Km values for synthetic phenolic compounds and much higher Km values for naturally occurring phenolic compounds than PC3. Moreover, anionic isoperoxidase PA1 could utilize ferulic acid as a substrate very well, while cationic isoperoxidase PC3 could not utilize ferulic acid as a substrate.     

Cytogenetic monitoring of farm animals under conditions of environmental pollution.

Cytogenetic examinations were carried out in 13 cattle farms, two herds of horses, one stag farm and 13 pig farms in areas with different levels of environmental contamination. The frequency of aberrant cells per 100 mitoses was 3.67 +/- 1.89 in pigs (n = 260) and 4.16 +/- 2.4 in herbivores (n = 497). This is a significant difference (p < 0.01). Ten times higher frequencies of chromatid exchanges were found in pigs. The examined herds were classified into three groups by the level of environmental contamination (satisfactory, impaired and severely impaired environment). Significant differences in aberrant cell counts were recorded between the groups of herbivorous animals. Significant differences in pigs were recorded only between herds in satisfactory and severely impaired environments. Significantly higher frequencies of aberrant cells were found in farms of herbivorous animals in the industrial area of Pardubice compared with findings in the South Moravian agricultural area (4.7% and 3.1% respectively). The effect of local contamination sources on farm environment was also investigated. A cattle farm located in the vicinity of a large chemical plant was examined five times at 6-month intervals. An autumn examination revealed significantly higher frequencies of aberrant cells compared with the spring examination.