Enzymatic acylation of a bifunctional molecule in 2-methyl-2-butanol: Kinetic modelling

The kinetics of enzymatic acylation of a bifunctional molecule exhibiting both an amino and a hydroxyl function, considered as a peptide-like model molecule, is studied. The acylation of 6-amino-1-hexanol by ethyl oleate in 2-methyl-2-butanol catalysed by Novozym 435® has been investigated and modelled. This acylation process generated two products issued respectively from the O-acylation of the 6-amino-1-hexanol, and from the N-acylation of the O-acyl product, which leads to the N,O-diacyl product. Experimental results confirmed the sequential reaction scheme. An experimental approach was adopted to identify the impact of several operating conditions on the reaction performances, such as initial substrates molar ratio and stirring rate. The kinetics of enzyme deactivation was also studied and included in the kinetic model. The establishment of the kinetic model was based on three major hypotheses: (i) a sequential bi bi ping-pong enzymatic mechanism, (ii) an interaction between acyl-acceptor and the acyl-enzyme formation steps, (iii) the absence of influence of the residual hydrolysis of ethyl oleate on the kinetics. Under this hypothesis, the kinetic parameters of the model were quantified using a genetic-evolutionary algorithm as an innovative identification method. The simplified model made it possible to describe the kinetics of the 6-amino-1-hexanol acylation for some ranges of 6-amino-1-hexanol/ethyl oleate molar ratios (2/1, 1/1, 1/2, 1/4) with a single kinetic parameter set. The case of a significant excess of 6-amino-1-hexanol (4/1) or ethyl oleate (1/8) constituted the limit of the kinetic model. However, these extreme molar conditions are not suitable to develop bioconversion processes involving bioactive peptides that are not available in large quantities.