RESUMO
In this study, the behavior of enzyme activity as a function of pH and temperature is modeled on the basis of fundamental considerations. A formulation is developed that includes the activation of enzymes with increasing temperatures and the deactivation of enzymes at higher temperature, together with the effect of protonation and hydroxylation on activity at various constant pH levels. The model is calibrated and validated against an extensive set of experimental data on phytases from seven different origins. The percentage variance accounted for (R(2)(adj)), obtained by statistical nonlinear regression analysis on all data sets, was shown to range from 97.6% to 99.5%. The equilibrium constant of protonation and hydroxylation proved to be independent of temperature.
Assuntos
6-Fitase/metabolismo , Modelos Biológicos , 6-Fitase/biossíntese , Bactérias/enzimologia , Ativação Enzimática , Concentração de Íons de Hidrogênio , Hidroxilação , Plantas/enzimologia , Prótons , Análise de Regressão , Reprodutibilidade dos Testes , TemperaturaRESUMO
To elucidate if there is any possibility to identify highly processed foods as produced through genetic engineering, beer, soya bean oil, processed tomato (ketch-up, paste, pizza tomatoes, peeled tomatoes, soup) and potato (french fries, crisps, mashed potatoes, flour, starch, fried potatoes) products as well as an enzyme preparation (Natuphos) were investigated by PCR. In pizza tomatoes, peeled tomatoes, french fries, fried potatoes, potato flour and potato crisps DNA suitable for PCR was found. Therefore, it is possible to identify these products as produced through genetic engineering. Such an identification is impossible in certain beers (pilsener, export, Nutfield lyte), soya bean oil, tomato soup, potato starch, mashed potatoes and Natuphos since PCR-analysis gave no indication of the presence of DNA in these products. As it was shown by adding Escherichia coli DNA the used method is, in principle, capable of detecting specifically small amounts of DNA in such products.
Assuntos
DNA Bacteriano/análise , Análise de Alimentos/métodos , Manipulação de Alimentos , Engenharia Genética , Reação em Cadeia da Polimerase/métodos , Primers do DNA , Enzimas/genética , Escherichia coli , Alimentos Fortificados , Solanum lycopersicum/genética , Óleos , Sensibilidade e Especificidade , Solanum tuberosum/genética , Glycine max/genéticaRESUMO
A cytoplasmatic phytase was purified about 410-fold to apparent homogeneity with a recovery of 28%. The enzyme is induceable under carbon limitation in the presence of phytate. It behaves as a monomeric protein of a molecular mass of about 40 kDa. The phytase is rather specific for phytate and exhibits optimal conditions for phytate degradation at pH 5.0 and 58 degrees C. Kinetic parameters for the hydrolysis of Na phytate are KM 300 microM and kcat 180 s-1 at 35 degrees C and pH 5.0. Phytate is hydrolyzed in a stepwise manner; the penta- and tetrakisphosphate were identified as I(1,2,4,5,6)P5 and I(1,2,5,6)P4. Consequently, this enzyme is a 3-phytase (EC 3.1.3.8).
Assuntos
6-Fitase/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Klebsiella/enzimologia , 6-Fitase/metabolismo , Proteínas de Bactérias/metabolismo , Cátions Bivalentes/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Ácido Fítico/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
Escherichia coli phytase was covalently immobilized on NHS-activated Sepharose High Performance. The pH dependence of the phytase activity was not influenced by immobilization, whereas stability against heat treatment was enhanced as a consequence of immobilization. Compared to the free phytase the immobilized enzyme exhibits the same excellent substrate specifity, but showed an increased Km-value. Using the immobilized phytase in a packed-bed bioreactor makes special isomers of the lower myo-inositol phosphate esters available. The major isomers formed were identified as I(1,2,3,4,5)P5, I(2,3,4,5)P4, I(2,4,5)P3 and I(2,5)P2.
Assuntos
6-Fitase/metabolismo , Reatores Biológicos , Ácido Fítico/metabolismo , Biotecnologia , Estabilidade Enzimática , Enzimas Imobilizadas , Escherichia coli/enzimologia , Concentração de Íons de Hidrogênio , Fosfatos de Inositol/metabolismo , Cinética , TemperaturaRESUMO
Two periplasmatic phytases, called P1 and P2, were purified about 16,500-fold to an apparent homogeneity with a recovery of 7 and 18%, respectively. The enzymes behave as monomeric proteins with molecular masses of about 42 kDa. Because of the limited amounts recovered, the amino terminal sequence of only one of the phytases was determined. Both enzymes are very specific for phytate and have little or no activity on other phosphate esters tested. The kinetic parameters for the hydrolysis of Na-phytate and p-nitrophenyl phosphate are kcat/KM 478 x 10(5) s-1 M-1 and 0.6 x 10(5) s-1 M-1 at pH 4.5. The hydrolysis pathway for phytate was elucidated for P2; consequently, this enzyme is a 6-phytase. The chemical and kinetic properties of the purified phytase P2 points to an identity with an enzyme described by Dassa et al. (1982, J. Biol. Chem. 257, 6669-6676) as a pH 2.5 acid phosphatase. Because of the kinetic parameters it would be better to denote this enzyme as a phytase.
