Biomedical Engineering Reference
In-Depth Information
groups. permits. immobilization. without. surface. modiication. or. coupling. reagents.. In.
addition,.the.lat.surface.of.graphene.oxide.lends.itself.to.analysis.using.surface-imaging.
techniques.such.as.AFM..Enzyme.immobilization.was.performed.by.mixing.the.enzyme.
and.GO.together.in.phosphate.buffer.with.shaking.on.ice.for.30.min..After.centrifugation.
and.washing,.the.enzyme.activity.was.assayed..GO.is.negatively.charged.at.biologically.
relevant.pH.values,.while.HRP.with.a.PI.of.7.2.has.a.net.positive.charge.below.7.2.and.a.
net. negative. charge. above. that. pH.. Consequently,. the. enzyme. loading. for. HRP. varied.
inversely.with.pH..At.pH.7,.approximately.100.μg.enzyme/mg.of.GO.was.bound..In.con-
trast,.lysozyme.with.a.pI.of.10.3.exhibit.a.net.positive.charge.throughout.the.entire.biologi-
cally. relevant. pH. range. (∼4.8. to. 10). and. showed. a. much. higher. enzyme. loading. with. a.
maximum.loading.of.∼700.μg/mg.of.GO.at.pH.7..In.contrast.to.some.of.the.results.obtained.
with.nanotubes,.the.K
m
.of.the.immobilized.enzyme.was.similar.to.that.of.the.free.enzyme;.
however,.the.k
cat
.was.greatly.reduced,.leading.to.an.overall.decrease.in.catalytic.eficiency..
Based.on.their.AFM.observations,.the.authors.suggested.that.the.cause.of.the.decrease.in.
eficiency. was. changes. in. the. conformation. of. the. enzyme. due. to. multiple. interactions.
between.the.enzyme.and.the.graphene.oxide.
One. important. challenge. remaining. in. the. use. of. enzymes. in. industrial. processes. is.
the. loss. of. activity. in. organic. environments.. This. presents. a. signiicant. problem. when.
hydrophobic.substrates.are.desired.as.they.exhibit.low.solubility.in.aqueous.environments..
To.address.this.issue,.immobilization.has.been.extensively.employed..Two.recent.studies.
have.used.CNTs.as.the.immobilization.substrate.for.these.organic.environments..Das.and.
Das. (2009). placed. enzyme-CNT. hybrids. in. cationic. reverse. micelles.. As. was. previously.
observed.by.others,.the.enzymes.suffered.a.loss.of.structural.integrity.upon.immobilization.
to. the. CNTs. with. a. commensurate. loss. of. activity.. However,. upon. being. placed. in. the.
reverse. micelles. (cetyltrimethyl. ammonium. bromide/isooctane/
n
-hexanol/water),. the.
peroxidase-CNT. hybrids. showed. signiicant. activation. (seven-. to. ninefold). in. catalytic.
activity.compared.to.the.activity.in.aqueous.buffer..Moreover,.this.activity.was.∼1500.to.
3500.times.higher.than.observed.for.the.enzyme.in.macroscopic.aqueous-organic.biphasic.
mixtures.. Ji. et. al.. (2010). investigated. the. effects. of. MWNT. immobilization. on. lipases.
in. “pure”. organic. solvents. rather. than. micelles.. In. this. study,. the. lipase. was. covalently.
attached.using.carbodiimide.activation.of.the.MWNTs..As.seen.in.previous.studies,.the.
immobilized.lipase.retained.∼60%.of.its.α-helical.content.upon.immobilization..When.the.
lipase.was.used.for.the.resolution.of.(R,S)-1-phenyl.ethanol.in.the.organic.solvent.heptane,.
the. MWNT-immobilized. lipase. showed. nearly. 100%. of. the. possible. conversion. with. a.
modest. effect. of. temperature. between. 35°C. and. 60°C,. while. the. free. enzyme. showed. a.
maximum.conversion.of.∼70%.and.a.much.stronger.temperature.dependence.
15.3.1.2 Inorganic Materials
The. use. of. gold. nanoparticles. (AuNP). as. substrates. for. enzyme. immobilization. has.
been.studied.extensively.by.Rotello.and.coworkers.(Verma.et.al..2004;.Simard.et.al..2005;.
Bayir.et.al..2006;.Bayraktar.et.al..2006;.You.et.al..2006a,b;.Jordan.et.al..2009;.Samanta.et.al..
2009;. Jeong. et. al.. 2011).. In. their. earlier. studies,. they. examined. the. effects. of. amino-acid.
functionalized. AuNP. on. the. activity. and. selectivity. of. alpha-chymotrypsin. (ChT). (You.
et. al.. 2006a).. In. this. study,. the. amino. acids. are. covalently. linked. to. the. AuNP. using. a.
thiol-based.linker,.and.the.ChT.is.non-covalently.associated.with.the.amino.acids.on.the.
surface. of. the. particle.. Depending. on. the. charge. of. the. substrate. and. the. surface. amino.
acid,.signiicant.enhancement.or.inhibition.of.hydrolysis.could.be.achieved.with.cationic.
substrates.showing.increased.k
cat
/K
m
.in.the.presence.of.negatively.charged.amino.acids.and.
