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Yeast alcohol dehydrogenase immobilized in a glutaraldehyde–albumin matrix: Kinetics and cofactor diffusional effects *
Author(s) -
Millis James R.,
Wingard Lemuel B.
Publication year - 1981
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260230506
Subject(s) - chemistry , nad+ kinase , cofactor , michaelis–menten kinetics , alcohol dehydrogenase , glutaraldehyde , substrate (aquarium) , immobilized enzyme , reaction rate constant , kinetics , dehydrogenase , enzyme kinetics , nicotinamide adenine dinucleotide , chromatography , thiele modulus , oxidoreductase , membrane , ethanol , enzyme assay , enzyme , organic chemistry , biochemistry , active site , mass transfer , oceanography , physics , quantum mechanics , geology
This study was carried out to define how the overall rate of reaction would be influenced by different degrees of diffusional resistance to cofactor transport within an oxidoreductase membrane matrix. To accomplish this, 0.7–6.6μ M yeast alcohol dehydrogenase was immobilized in an albumin matrix crosslinked with 2.5 or 5.0% glutaraldehyde to give 102–1685 μM thick membranes. The enzyme half‐life was at least doubled at pH 7.5 or 8.8 on immobilization. Values of the kinetic constants for the soluble and immobilized enzyme were determined at 25°C and pH 8.8 over the range of 0.01–1.0 M bulk solution concentration of ethanol as substrate and 140–1000μ M bulk solution concentration of nicotinamide adenine dinucleotide (NAD + ) as cofactor, to give essentially single substrate kinetics in NAD + . Equilibrium partitioning of ethanol and NAD + between the solution and membrane was measured and used in the data analysis. The four kinetic constants for the soluble enzyme agreed with literature values; and all increased with immobilization of the enzyme. The Michaelis constants for NAD + and for ethanol were greater for the immobilized enzyme. The diffusional resistance to NAD + transport, presented in terms of the Thiele modulus, showed that the overall rate of reaction was decreased by about 50% even at values of the modulus as low as 2.0.