Abstract
Full kinetic models provide insight into enzyme mechanism and kinetics and also support bioconversion process design and feasibility assessment. Previously we have established automated microwell methods for rapid data collection and hybrid kinetic modelling techniques for quantification of kinetic constants. In this work these methods are applied to explore the substrate selectivity and kinetics of monoamine oxidase, MAO-N-D5, from Aspergillus niger. In particular we examine the MAO-N-D5 variant Ile246Met/Asn336Ser/Met348Lys/Thr384Asn to allow the oxidation of secondary amines Initial screening showed that MAO-N-D5 enabled the selective oxidation of secondary amines in 8 and 9 carbon rings, as well as primary ethyl and propyl amines attached to secondary amines of indolines and pyrrolidines. Subsequently we developed a first kinetic model for the MAO-N-D5 enzyme based on the ping-pong bi-bi mechanism (similar to that for the human MAO-A enzyme). The full set of kinetic parameters were then established for three MAO-N-D5 substrates namely; 3-azabicyclo[3,3,0]octane, 1-(2 amino ethyl) pyrrolidine and 3-(2,3-dihydro-1H-indole-1-yl)propan-1-amine. The models for each amine substrate showed excellent agreement with experimentally determined progress curves over a range of operating conditions. They indicated that in each case amine inhibition was the main determinant of overall reaction rate rather than oxygen or imine (product) inhibition. From the perspective of larger scale bioconversion process design, the models indicated the need for fed-batch addition of the amine substrate and to increase the dissolved oxygen levels in order to maximize bioconversion process productivity.
Highlights
Biocatalytic processes are increasingly being considered in industrial organic syntheses since in certain situations they are considered ‘greener’ and can bring considerable reductions to overall process costs [1,2]
E. coli BL21 DE3 Gold was selected as the host strain for all further work due to the slightly higher cell concentrations produced during fermentation
The results here demonstrate the capacity of the enzyme to oxidize secondary amines in rings with 8 and 9 carbons, as well as primary ethyl and propyl amines attached to secondary amines of indolines and pyrrolidines
Summary
Biocatalytic processes are increasingly being considered in industrial organic syntheses since in certain situations they are considered ‘greener’ and can bring considerable reductions to overall process costs [1,2]. This is a consequence of the mild temperature, pressure and pH conditions under which enzymes function. Most reported bioconversion kinetic studies only report the maximum reaction rate, Vmax, and the Michaelis constant, Km, along with qualitative comments on the level of substrate and/or product inhibition This is because obtaining a full kinetic model along with values of all the inhibition constants involves multiple experiments and complex data processing, consuming valuable time and resources [8]
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