High throughput screening of monoamine oxidase (MAO-N-D5) substrate selectivity and rapid kinetic model generation

Abstract

a b s t r a c tFull 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.

Immobilisation and kinetics of monoamine oxidase (MAO-N-D5) enzyme in polyvinyl alcohol gels

“…The DOT was also monitored and remained close to 100% saturation, demonstrating that the combination of liquid fill volume, well geometry and shaking diameter/frequency used were sufficient to ensure that oxygen mass transfer into the microwell never became rate limiting. Similar results were found for MAO-N whole cell form in 96 microplates [10].…”

“…Previous studies have shown that the MAO-N-D5 kinetic profile has more resemblance to the human MAO-A, which follows a Ping-Pong bi-bi mechanism, rather than to the human MAO-B form, which follows a bi-bi ordered mechanism [12]. The mechanism and kinetic parameters of the whole cell MAO-N-D5 have previously been reported [10] based on high throughput, automated microwell experiments. In that work, a King-Altam scheme for the MAO-N-D5-mediated oxidation of amines was proposed which is shown in Fig.…”

“…The final values of those kinetic parameters for the immobilized MAO-N-D5 are summarized in Table 1. For comparison the table also shows the parameters previously determined for the whole cell form of the enzyme [10].…”

Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris

“…Monoamine oxidases (MAOs), enzymes catalysing the asymmetrical oxidation of a variety of amines, represent a relevant tool for biocatalysis. Wildtype or engineered MAOs have been expressed in Escherichia coli and characterised in terms of substrate specificity and regioselectivity; MAO from Aspergillus niger, in particular has been engineered to considerably extend its substrate specificity (Atkin et al 2008;Ramesh et al 2016;Rios-Solis et al 2015). Since the gene is originated from a filamentous fungus A. niger (Atkin et al 2008), the use of E. coli as a host for protein expression might have limited the overall yield of such catalysts.…”

“…Recently, a kinetic model for substrate specificity of MAO-N in different biocatalytic reactions was described (Rios-Solis et al 2015), while other groups analysed the influence of cultivation medium on enzyme activity (Ramesh et al 2016).…”