TY - JOUR
T1 - Immobilisation and kinetics of monoamine oxidase (MAO-N-D5) enzyme in polyvinyl alcohol gels
AU - Markošová, Kristína
AU - Dolejš, Igor
AU - Stloukal, Radek
AU - Rios-Solis, Leonardo
AU - Rosenberg, Michal
AU - Micheletti, Martina
AU - Lye, Gary J.
AU - Turner, Nicholas
AU - Rebroš, Martin
PY - 2016/7/1
Y1 - 2016/7/1
N2 - This study focused on the production and immobilisation of the crude enzyme extract of recombinant monoamine oxidase (EC 1.4.3.4), originating from Aspergillus Niger (MAO-N-D5) and expressed in Escherichia coli, in PVA gel using the LentiKats® technique. MAO-Ns are important enzymes in the chemical industry due to their stereoselectivity and they are often used for the deracemisation of non-optically pure mixtures of amines. Biomass production, enzyme preparation, enzyme immobilisation, process parameters for the immobilised enzyme and characterisation of the enzyme are described in detail here. The biomass was prepared in laboratory bioreactors, and two different disruption techniques were compared. The activity of the enzyme was determined by biotransformation with secondary amine 3-azabicyclo [3,3,0] octane as a substrate. The crude enzyme extract showed 61.5% of the whole cell activity and the immobilised enzyme showed a wider optimum pH and temperature ranges than the free enzyme. The initial specific activity of the immobilised monoamine oxidase crude enzyme extract remained at 80% after 12 repeated biotransformations. For the first time, the full kinetic parameters of an immobilised MAO-N-D5 were obtained based on a ping-pong bi-bi reaction mechanism. The specific activity was 0.29 U g-1 (Lentikats) and the Km was 7.31 mM, which were similar in comparison to whole cell MAO-N-D5. Characterisation of immobilised MAO-N-D5 showed particular benefits in terms of activity and stability in comparison with free and whole cell MAO-N-D5; therefore, the immobilisation of this enzyme is very suitable for industrial applications.
AB - This study focused on the production and immobilisation of the crude enzyme extract of recombinant monoamine oxidase (EC 1.4.3.4), originating from Aspergillus Niger (MAO-N-D5) and expressed in Escherichia coli, in PVA gel using the LentiKats® technique. MAO-Ns are important enzymes in the chemical industry due to their stereoselectivity and they are often used for the deracemisation of non-optically pure mixtures of amines. Biomass production, enzyme preparation, enzyme immobilisation, process parameters for the immobilised enzyme and characterisation of the enzyme are described in detail here. The biomass was prepared in laboratory bioreactors, and two different disruption techniques were compared. The activity of the enzyme was determined by biotransformation with secondary amine 3-azabicyclo [3,3,0] octane as a substrate. The crude enzyme extract showed 61.5% of the whole cell activity and the immobilised enzyme showed a wider optimum pH and temperature ranges than the free enzyme. The initial specific activity of the immobilised monoamine oxidase crude enzyme extract remained at 80% after 12 repeated biotransformations. For the first time, the full kinetic parameters of an immobilised MAO-N-D5 were obtained based on a ping-pong bi-bi reaction mechanism. The specific activity was 0.29 U g-1 (Lentikats) and the Km was 7.31 mM, which were similar in comparison to whole cell MAO-N-D5. Characterisation of immobilised MAO-N-D5 showed particular benefits in terms of activity and stability in comparison with free and whole cell MAO-N-D5; therefore, the immobilisation of this enzyme is very suitable for industrial applications.
KW - Immobilisation
KW - Kinetic parameters
KW - Monoamine oxidase
KW - Polyvinyl alcohol
UR - http://www.scopus.com/inward/record.url?scp=84964598902&partnerID=8YFLogxK
U2 - 10.1016/j.molcatb.2016.04.009
DO - 10.1016/j.molcatb.2016.04.009
M3 - Article
AN - SCOPUS:84964598902
SN - 1381-1177
VL - 129
SP - 69
EP - 74
JO - Journal of Molecular Catalysis - B Enzymatic
JF - Journal of Molecular Catalysis - B Enzymatic
ER -