The authors have no conflict of interest to declare.
Quantitative analysis of enzymatic fractionation of multiple substrate mixtures†
Article first published online: 8 AUG 2012
Copyright © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 110, Issue 1, pages 78–86, January 2013
How to Cite
Kaki, S. S. and Adlercreutz, P. (2013), Quantitative analysis of enzymatic fractionation of multiple substrate mixtures. Biotechnol. Bioeng., 110: 78–86. doi: 10.1002/bit.24613
- Issue published online: 20 NOV 2012
- Article first published online: 8 AUG 2012
- Accepted manuscript online: 18 JUL 2012 10:52AM EST
- Manuscript Accepted: 9 JUL 2012
- Manuscript Revised: 6 JUL 2012
- Manuscript Received: 10 MAY 2012
- enzyme catalysis;
- competing substrates;
- enzymatic enrichment;
The enzymatic conversion of mixtures of multiple substrates was studied quantitatively, based on established methodology used for the enzymatic kinetic resolution of racemic mixtures, involving the use of competitive factors: ratios of specificity constants (kcat/KM) of substrate pairs. The competitive factors of the substrates were defined in relation to a reference substrate. These competitive factors were used to predict the composition of the reaction mixture as a function of the degree of conversion of the reaction. The methodology was evaluated using three different lipases to hydrolyze a model mixture of four fatty acid methyl esters and for the esterification of a mixture of the same fatty acids in free form with ethanol. In most cases, the competitive factors determined from the initial phase of the reactions predicted the product composition during the rest of the reaction very well. The slowest reacting fatty acid was erucic acid (both in free form and as methyl ester), which was thus enriched in the remaining substrate fraction, while the other fatty acids: lauric acid, palmitic acid and oleic acid were converted faster. Simulations of the compositions of reaction mixtures with different values of the competitive factors were carried out to provide an overview of what could be achieved using enzymatic enrichment. Possible applications include reactions involving homologous substrates and mixtures of multiple isomers. The analysis presented provides guidelines that can be useful in the screening and development of enzymes for enzymatic enrichment applications. Biotechnol. Bioeng. 2013; 110: 78–86. © 2012 Wiley Periodicals, Inc.