Karakterisering van die katalase van BCG : invloed van isoniasied op die gesuiwerde ensiem

Basson, Karen (1990-12)

Thesis (MSc)--Stellenbosch University, 1990.

Thesis

ENGLISH ABSTRACT: Catalase was extracted and isolated from BCG bacilli by the following precedures : sonication, treatment with deoxyribonuclease, treatment with octyl glucoside and sonification, ammonium sulphate precipitation, hydroxyapatite chromatography and chromatography on Sephacryl S-1000. A significant increase in the specific activity of the catalase and peroxidase functions was observed during purification and a final purity index (A405/A280) of 0.258 was obtained. Only one protein band, in addition to aggregates which could hardly penetrate the gel, was observed after non-denaturing polyacrylamide gel electrophoresis of the purified enzyme. The peroxidase activity (ABTS) assay) was consistent with the protein band. A.M. of 163.1 kDa was calculated from results obtained with SDS polyacrylamide gel electrophoresis, which correlates with a Mr of 144.54 kDa obtained by gelfiltration studies on Sephacryl S-1000. A characteristic Soret peak at 405 nm was observed on the UV spectrum of the purified enzyme. The optimum pH for catalase activity was 8,0. At pH 7,0 81% of the maximum activity at pH 6,0 and 56% at pH 9,0. This correlates with the fact that catalase must be able to survive over a broad pH range.

AFRIKAANSE OPSOMING: Katalase is geëkstraheer uit BCG basille en geïsoleer deur die volgende opeenvolgende stappe: sonikering, behandeling met deoksiribonuklease, behandeling met oktielglukosied en sonikering, ammoniumsulfaat presipitasie, hidroksi-apatiet chromatografie en chromatografie op Sephacryl S-1000. Die spesiefieke aktiwiteit van die katalatiese en peroksidatiewe funksies het beduidend toegeneem tydens suiwering en 'n finale RZ-waarde (A405/A280) van 0,258 is verkry. Analitiese poli-akrielanied gelelektroforese van die gesuiwerde ensien het hoofsaaklik een band getoon. Daar was ooreenstemming tussen die proteïenband en peroksidase aktiwiteit (ABTS bepaling). 'n Mr van 163,1 kDa is mbv SDS-PAGE bereken, wat ooreenstem met 'n Mr van 144,54 kDa wat bepaal is mbv gelfiltrasie studies op 'n Sephacryl S-1000 kolon. Die UV spektrum van die gesuiwerde ensiem het 'n kenmerkende Soret piek by 405nm getoon. Die optimum pH vir katalase aktiwiteit was 8,0. By neutrale pH was die aktiwiteit nog 81% van die maksimum waarde. By pH 6,0 het die ensiem nog 49% van die maksimum aktiwiteit getoon, terwyl 'n waarde van 56% by pH 9,0 gevind is. Hierdie bevindings is in ooreenstemming daarmee dat katalase oor 'n relatiewe breë pH gebied moet kan oorleef.

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