Biomedical Engineering Reference
In-Depth Information
have.been.used.for.decades,.there.have.been.a.number.of.recent.advances.in.immobiliza-
tion.and.stabilization,.many.of.them.due.to.the.incorporation.of.nanotechnology,.both.car-
bon-based.and.inorganic.nanomaterials,.to.create.novel.enzyme-nanomaterial.complexes..
These. materials. comprise. a. variety. of. nanostructures. including. nanoporous. materials,.
nanoibers,. and. nanoparticles.. While. some. of. these. advances. have. been. reviewed. rela-
tively.recently.(Kim.et.al..2008),.the.ield.continues.to.advance.rapidly..These.advances.and.
their.applications.will.be.discussed.next.
15.3.1.1 Carbon-Based Materials
As. described. in. Section. 15.2.2,. carbon. can. be. found. in. a. number. of. nanoscale. forms..
For. enzyme. applications,. carbon. nanotubes. are. the. most. common. form,. with. enzymes.
immobilized.to.both.SWNT.and.multiwall.nanotubes.(MWNTs).although.C
60
.fullerenes.
have.also.been.reported.(Asuri.et.al..2007b)..In.addition,.graphene.oxide.has.been.used.as.
a.substrate.for.enzyme.immobilization.(Zhang.et.al..2010b).
CNTs. with. their. extremely. high. length-to-diameter. ratios. (up. to. ∼1.×.10
8
),. provide.
a. unique. substrate. for. immobilization,. with. very. high. surface. area. but. none. of. the.
diffusional. limitations. observed. in. typical. microporous. substrates.. In. addition,. their.
aspect.ratio.makes.it.possible.to.recover.the.nanotubes.by.iltration,.something.that.is.not.
possible.with.other.nanoscale.materials..Asuri.and.colleagues.immobilized.a.number.of.
different. enzymes. to. MWNTs. (Asuri. et. al.. 2006b).. Prior. to. the. enzyme. immobilization,.
they. acid-oxidized. commercially. available. nanotubes. and. then. fractionated. them. by.
repeatedly.sonicating.and.iltering.until.a.water-soluble.fraction.was.obtained..Enzymes.
were.then.attached.to.the.nanotubes.using.carbodiimide.chemistry..Soybean.peroxidase.
(SBP).immobilized.in.this.manner.showed.substantially.increased.thermal.stability.and.
thermal. activation. when. compared. with. the. soluble. enzyme. as. well. as. reusability. after.
recovery.by.iltration.and.improved.storage.stability..The.authors.hypothesized.that.two.
mechanisms. might. lead. to. the. improved. thermal. stability,. multi-point. attachment. of.
the. enzyme. to. the. support,. preventing. denaturation. and/or. decreased. protein-protein.
interactions. on. the. highly. curved. surface. of. carbon. nanotubes. (Asuri. et. al.. 2006a).. Of.
particular. note. was. that. this. increased. thermostability. occurred. while. the. enzyme.
was. in. a. soluble. form,. in. contrast. to. most. immobilization. strategies. that. are. insoluble..
In. a. subsequent. study,. these. authors. attached. several. enzymes. to. water-soluble. SWNTs.
prepared.in.a.similar.manner.as.the.MWNTs.(Asuri.et.al..2007a)..In.this.study,.in.addition.
to.demonstrating.increased.enzyme.stability.in.the.presence.of.guanidine.hydrochloride.
(GdnHCl),.a.strong.chemical.denaturant,.they.were.able.to.perform.spectroscopic.analysis.
of. the. immobilized. protein. structure,. something. they. were. unable. to. do. for. enzymes.
immobilized.to.the.MWNTs.due.to.interference.from.the.nanotubes..SWNT.also.provided.
much.higher.loading.due.to.the.surface.accessibility.of.every.single.atom..Typical.loadings.
for.several.different.enzymes.were.∼1.2.mg.enzyme/mg.SWNT..They.found.∼50%.activity.
was.retained.after.immobilization.and.using.far-UV.circular.dichroism.(CD).spectroscopy,.
they. observed. 60%-80%. of. the. native. solution. secondary. structure. was. maintained..
Using. CD. spectroscopy. and. luorescence. measurements,. they. were. able. to. calculate. an.
increase.in.the.free.energy.of.unfolding.between.horseradish.peroxidase.free.in.solution.
and.immobilized.onto.SWNTs..They.found.ΔG.of.unfolding.increased.∼6.6.kJ/mol.upon.
immobilization,.accounting.for.the.increased.stability.
Cang-Rong.and.Pastorin.analyzed.the.effects. of.different.immobilization.methods.for.
amyloglucosidase.(AMG).attached.to.both.SWNTs.and.MWNTs.(Cang-Rong.and.Pastorin.
2009).. They. explored. both. physical. adsorption. and. chemical. ligation. using. oxidized.
