posted on 2021-06-08, 22:03authored byNilan
J. B. Kamathewatta, Tyler M. Nguyen, Rachel Lietz, Talisa Hughes, Banu Taktak Karaca, Dwight O. Deay, Mark L. Richter, Candan Tamerler, Cindy L. Berrie
Controlling
enzyme orientation and location on surfaces is a critical
step for their successful deployment in diverse applications from
biosensors to lab-on-a-chip devices. Functional activity of the enzymes
on the surface will largely depend on the spatial arrangement and
orientation. Solid binding peptides have been proven to offer versatility
for immobilization of biomolecules on inorganic materials including
metals, oxides, and minerals. Previously, we demonstrated the utility
of a gold binding peptide genetically incorporated into the enzyme
putrescine oxidase (PutOx–AuBP), enabling self-enzyme assembly
on gold substrates. PutOx is an attractive biocatalyst among flavin
oxidases, using molecular oxygen as an electron acceptor without requiring
a dissociable coenzyme. Here, we explore the selective self-assembly
of this enzyme on a range of surfaces using atomic force microscopy
(AFM) along with the assessment of functional activity. This work
probes the differences in surface coverage, distribution, size, shape,
and activity of PutOx–AuBP in comparison to those of native
putrescine oxidase (PutOx) on multiple surfaces to provide insight
for material-selective enzymatic assembly. Surfaces investigated include
metal (templated-stripped gold (TSG)), oxide (native SiO2 on Si(111)), minerals (mica and graphite), and self-assembled monolayers
(SAMs) with a range of hydrophobicity and charge. Supported by both
the coverage and the dimensions of immobilized enzymes, our results
indicate that of the surfaces investigated, material-selective binding
takes place with orientation control only for PutOx–AuBP onto
the TSG substrate. These differences are consistent with the measurements
of surface-bound enzymatic activities. Substrate-dependent differences
observed indicate significant variations in enzyme–surface
interactions ranging from peptide-directed self-assembly to enzyme
aggregation. The implications of this study provide insight for the
fabrication of enzymatic patterns directed by self-assembling peptide
tags onto localized surface regions. Enabling functional enzyme-based
nanoscale materials offers a fascinating path for utilization of sustainable
biocatalysts integrated into multiscale devices.