Integral kinetics of alpha-galactosidase purified from Glycine max for simultaneous hydrolysis of stachyose and raffinose

Biotechnology and Bioengineering
J E PorterM R Ladisch


alpha-Galactosidase from soybean (Glycine max) was purified by a five-step procedure. The enzyme's natural substrates, raffinose and stachyose, have K(m)'s of 3. 0 mM and 4. 79 mM, respectively. The products, galactose and sucrose, were measured after separation by liquid chromatography. Galactose is a competitive product inhibitor of stachyose and raffinose hydrolysis with a K(i) of 0. 12 mM. We determined these parameters by an integral kinetic approach. Stachyose hydrolysis gives a nearly constant level of raffinose shortly after hydrolysis begins. Thus, cleavage of the first alpha-(1,6)-bond in the tetrasaccharide is the rate-limiting step. Since the stachyose hydrolysis yields raffinose, soybean alpha-galactosidase simultaneously hydrolyzes two substrates. We present a novel approach for analyzing simultaneous substrate hydrolysis with competitive product inhibition by a modified integral rate expression. The experimentally found kinetic parameters are confirmed by solving the simultaneous equations which describe the hydrolysis. This technique may be applicable to other hydrolytic enzymes with multiple substrates.


Mar 25, 1992·Biotechnology and Bioengineering·J E Porter, M R Ladisch
Aug 1, 1992·Enzyme and Microbial Technology·J E PorterM R Ladisch
Jan 29, 2000·The Plant Journal : for Cell and Molecular Biology·T PeterbauerA Richter
Feb 27, 2003·Artificial Cells, Blood Substitutes, and Immobilization Biotechnology·Seçil Onal, Azmi Telefoncu


Apr 1, 1988·Analytical Biochemistry·S Y HwangC Gilvarg
Oct 1, 1969·The Biochemical Journal·P M Dey, J B Pridham
Nov 15, 1981·Analytical Biochemistry·W WrayR Hancock
Mar 1, 1982·Plant Physiology·E Del Campillo, L M Shannon

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