The β- D-glucosidase system of an Actinoplanes sp.

Abstract

Actinoplanes sp. No. 1700, a sporangium-forming, filamentous, soil bacterium possesses a β- D-glucosidase (β- D-glucoside glucohydrolase, E.C. 3.2.1.21). The enzyme was induced to higher concentrations by addition of methyl or phenyl β- D-glucopyranoside, gentiobiose, or salicin to growing cultures. Addition of D-glucose, lactate, or acetate repressed enzyme induction back to the constitutive level, but never below it. The properties of this inducible system place it in the semi-constitutive category. Both the constitutive and the inducible enzyme were purified 60-fold; their properties were compared and found to be identical. Their pH optima lay between 5.8 and 6.0; the enzymes were stable for 2 h at 30° at pH 5.5 to 7.3. Rapid inactivation occurred at temperatures above 50°. The enzymes were inactivated by 100μM CU 2+, Hg 2+, Pb 2+, and Ag +. Each of these β- D-glucosidases was inhibited by p-chloromercuribenzoate (100 μ/M); this effect was overcome by cysteine or 2-mercaptoethanol, indicating that the β- D-glucosidase is a sulfhydryl enzyme. Kinetic determinations with chromogenic p-nitrophenyl β- D-glucopyranoside established a K m. of 2.5 x 10 -4 and an Arrhenius activation-energy of 8.5 kcal.mole -1. The molecular weight of the induced enzyme was 165,000 as determined by elution from Sephadex G-200. Chromatographic studies showed the enzyme to be a hydrolase, not a transferase.