Lane 1: marker, Lane 2: crude cell extract, Lane 3, 60 ~ 80% A.S. sat., Lane 4: SP column at pH 6.9, Lane 5: Purified -glucosidase from Mono-S at pH 7.5.
Characteristics of -glucosidase No significant activity on hydrolysis of other glycosides. The estimated Mr. of the enzyme is 150 kDa by gel filtration and 78 kDa by SDS-PAGE. This dimeric enzyme has a pI= 9.0 and an optimal activity at pH 5.0 and temperature of 50 ℃.
The Bronsted plot & rate-limiting step For good substrates (phenol pKa<7) Deglucosylation For Poor substrates (pKa>7) Glucosylation Li, Y-K. *, Chir, J. and Chen, F-Y. 2001 ” Catalytic mechanism of a family 3 b- glucosidase and mutagenesis study on its Asp-247” Biochem. J. 355, 835-840.
Michaelis-Menten parameters of carboxyphenyl -D-glucosides and derivatives catalyzed by Fbgl and E473G NaN 3 KmKm k cat K cat /K m Aryl- -glucosides Enzyme(mM) (s -1 )(s -1 mM -1 ) WT0300.0120.0004 E473G00.20.0320.16 E473G2001.850.3500.19 WT00.120.0040.33 E473G00.130.00010.0008 E473G2001.890.00020.0001 WT02.20.10.045 E473G00.30.00030.001 E473G200ND WT00.721.171.63 E473G00.10.0030.03 E473G2004.50.0140.003
Summary A -glucosidase containing 726 amino acid from F. meningosepticum was cloned and identified as a new member of GH-3 enzyme GH-3 -glucosidase involves a two-step, double displacement mechanism. D247 and E473 function as the nucleophile and the general acid/base catalyst, respectively.