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Purification of Urease from Pleurotus sajor-caju.

Characterisation of GTPase Activity of UreG and

Biochemical Properties of the enzyme

Antik K. Bose

ABSTRACT

Biochemical properties of urease obtained from fruiting body extract of Pleurotus sajor-

caju have been described. The enzyme was purified 3176.623 fold by CM-cellulose, DEAE-

cellulose and Sephadex G-200 column chromatography. The molar mass estimated by

Sephadex G-200 was 510KD and saccharide content was 4%. 6% SDS-PAGE of purified

urease showed subunits UreA/B homodimer (28.8 KD ), UreC (150 KD), UreD (104 KD),

UreE (21.5 KD), UreF (102 KD) and UreG (103KD). GTPase activity of UreG was

characterized with KM, Vmax & Kcat to be 2.6mM, 1.1µ mol GTP hydrolyzed/ min and 0.27

min-1

. The enzyme hydrolyzed urea with KM and Vmax to be 1.85mM and 64.51 µ mol NH3

C and 7.4 respectively. The enzyme

activity was enhanced by Ca2+

(2.94%), 5-10 mM, Mn2+

( 2.67-52.406%), 20-50mM Ni2+

(2.6-6.93% ) and 5-10mM Mncl2 ( 0.58- 8.02% ). The enzyme showed product inhibition by

NH4+. Competitive inhibition by thiourea and uncompetitive inhibition by NaF were also

studied. Amino acids at the active site have been identified to be Histidine and Aspartic

acid using DEPC and Diazomethane with EDAC