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. 1969 Dec;100(3):1355–1363. doi: 10.1128/jb.100.3.1355-1363.1969

Purification and Characterization of β-Glucosidase of Alcaligenes faecalis1

Y W Han a, V R Srinivasan a
PMCID: PMC250337  PMID: 5361217

Abstract

A cellobiose-utilizing bacterium isolated from sugar cane bagasse and identified as a strain of Alcaligenes faecalis (ATCC 21400) produced an inducible β-glucoside-splitting enzyme. The enzyme was purified by a series of streptomycin and ammonium sulfate fractionations and by Sephadex and diethylaminoethyl column chromatography. The final preparation was purified 130-fold, with a recovery of about 10% of the initial enzyme activity. The enzyme had a wide pH range, with optimal activity at pH 6.0 to 7.0. The enzyme was stable in solution at pH 6.5 to 7.8 when kept at 30 C for 2 hr, but it was destroyed by temperatures above 55 C. At 58 and 60 C, the time required to inactivate 90% of the initial activity was 16 and 6.5 min, respectively. An activation energy of 9,500 cal/mole and a Km of 1.25 × 10−4m were obtained by using p-nitrophenyl β-glucoside as a substrate. The Ki value and hydrolysis of cellobiose by the enzyme indicated a high affinity of the enzyme for the cellobiose. The enzyme had its specificity on β-glucosidic linkage and the rate of hydrolisis of glucosides depended upon the nature of the aglycon moiety. The inactivation studies showed the presence of sulfhydryl groups in the enzyme. The activity of the enzyme was easily destroyed by the Cu++ and Hg++ ions. The Michaelis-Menton relationship and the rate of heat inactivation indicated the presence of one type of noninteracting active site in the bacterial β-glucosidase. Molecular weight of the enzyme was estimated by gel filtration (Sephadex G-200) and sucrose density gradient, and a value of 120,000 to 160,000 was obtained.

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Selected References

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