Enhanced Enzyme Immobilization Using a Novel Agarose‐binding Tag Leads to Improved Flow Reactor Performance

Continuous enzymatic synthesis has gained increasing prom-
inence, extending its applicability to the chemical industry. A
practical approach for integrating enzymes into continuously
operated reactors is their entrapment within polymer-based
hydrogels. In this study, we present a novel approach to
enhance enzyme retention within agarose hydrogels, employ-
ing a carbohydrate-binding module sourced from the Micro-
bulbifer thermotolerans thermostable β-agarase I (MtCBM) as a
versatile agarose-binding tag. Among the four tested carbohy-
drate-binding modules, MtCBM exhibited superior binding
affinity to solid agarose and fusion proteins incorporating
MtCBM demonstrated significantly enhanced retention within
agarose hydrogels. Employing a newly developed fluidic flat-
bed agarose reactor, we demonstrate that, due to increased
retention in agarose hydrogels, a phenacrylate decarboxylase
tagged with MtCBM yielded nearly six times more product
compared to a similarly sized fusion enzyme lacking the
MtCBM-tag. The higher leaching rate of the unlabeled enzyme
could not be compensated even by tripling the hydrogel layer
thickness, clearly documenting the effectiveness of MtCBM
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labeling for retention in the gel matrix. In addition, we
performed theoretical modelling of the reactor, which led to a
deeper understanding of the retention kinetics. The results
suggest that this innovative approach is promising for improv-
ing stability and reusability of enzymes in agarose-based
carriers.