In order to assess if the activity displayed by the developed immobilized TAs can only be attributed to heterogeneous catalysis, hot-filtration-tests were performed on both optimized TsRTA_HPAN3b and TsRTA_PP3 biocatalysts (Figure ). The TA-loaded membrane was removed from the reaction media after a short reaction time (15 minutes), and the activity was monitored and compared to a classical catalytic test (i.e. in which the membrane was not removed). Some residual activity could be detected after the removal of TsRTA_HPAN3b (Figure ), which highlights the fact that a small fraction of leached TsRTA contributed to the observed activity. Such leaching fraction was either estimated to 5.1 % (in the form of immobilized TsRTA) or 1.8 % (in the form of soluble TsRTA), based on the slope between 15and 60-minutes activity points. This can be explained by a slow hydrolysis of the imine bonds between GA and TsRTA or PEI. On the other hand, the hot-filtration test exhibited a completely flat profile after the removal of TsRTA_PP3 biocatalyst (Figure ), demonstrating that, in this case, only heterogeneous catalysis is involved in the observed activity. The two selected catalysts were also tested in 4 successive catalytic cycles to assess their recyclability. At the end of each cycle, the membrane-immobilized TAs were washed twice with 5 mL of buffer solution (i.e. HEPES 0.1 M pH 8 containing PLP 1 mM, pyruvate 10 mM), and then immersed into a fresh reaction medium. The obtained reaction profiles (Figure )