Izolovanje i delimično karakterisanje proteaze iz Pseudomonas aeruginosa ATCC 27853

Enzymatic characteristics of a protease from a medically important, referent strain of Pseudomonas aeruginosa ATCC 27853 were determined. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE, and gel filtration, it was estimated that the molecular mass of the purified enzyme was about 15 kDa. Other enzymatic properties were found to be: pH optimum 7.1, pH stability between 6.5 and 10; temperature optimum around 60 °C while the enzyme was stable at 60°C for 30 min. Inhibition of the enzyme was observed with metal chelators, such as EDTA and 1,10-phenanthroline, suggesting that the protease is a metalloenzyme. Furthermore, the enzyme contains one mole of zinc ion per mole of enzyme. The protease was stable in the presence of different organic solvents, which enables its potential use for the synthesis of peptides.U ovom radu je okarakterisana ekstracelularna proteaza medicinski značajnog, referentnog soja Pseudomonas aeruginosa ATCC 27853. Molekulska masa prečišćenog enzima određena SDS PAGE i gel filtracijom iznosi oko 15 kDa. Određeni su sledeći enzimski parametri: pH optimum 7,1; pH stabilnost u opsegu 6,5-10; temperaturni optimum 60°C, a enzim je stabilan na 60°C 30 min. Na osnovu inhibicije enzima pomoću EDTA i 1,10-fenantrolina, utvrđeno je da proteaza predstavlja metaloenzim. Pokazano da proteaza sadrži 1 mol jona cinka po molu enzima. Proteaza je stabilna u prisustvu različitih organskih rastvarača, što omogućava upotrebu za sintezu peptida

Adsorption of Candida rugosa lipase onto alumina: Effect of surface charge

The impact of the surface charge of alumina supports on the adsorption of Candida rugosa lipase was investigated in terms of the zeta potentials of the adsorption partners. The lipase adhered onto alumina with similar efficiency under both repulsive and attractive electrostatic conditions, shifting the zeta potential of the support towards that of the enzyme. The behavior was explained by a heterogeneous distribution of the surface charge of the lipase molecule. Special emphasis in this study was placed on the effect of immobilization on the enzyme kinetics and principal reasons for enzyme immobilization: improvement in stability and potential for reuse. The enzyme affinity was not altered by its adsorption onto alumina, while the Vmax value of the lipase decreased. The thermostability of the adsorbed lipase was improved. A significant potential for reuse was found

Immobilization of Candida rugosa lipase by adsorption onto biosafe meso/macroporous silica and zirconia

Lipase from Candida rugosa was immobilized by adsorption onto laboratory prepared supports, silica SBA-15 and zirconia. The adsorption process was studied as a function of pH in terms of percent of adsorbed lipase, enzyme activity and zeta potential of support and enzyme. Several analytical approaches such as laser Doppler electrophoresis, Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) showed that the lipase was successfully immobilized onto both supports. The zeta-potential data suggest that the adsorption efficiency does not depends on the sign but on the magnitude of the surface charge of adsorption partners, and therefore underline the importance of their dispersion stability. Adsorption to material surface altered enzyme characteristics. nu(max), for the lipase immobilized onto silica and zirconia were 4.8-fold and 3.6-fold lower than that of the free lipase, respectively. The Km showed no alteration of enzyme-substrate affinity on zirconia support, whereas the enzyme immobilized on silica had 3.6 times lower affinity. Thermostability at 60 degrees C of the lipase was improved 12-fold on zirconia and 4-fold on silica. Finally, in examining reusability, the immobilized lipase retained more than 90% of initial activity after eight reuses on both supports. (C) 2014 Elsevier B.V. All rights reserved