Biomedical Engineering Reference
In-Depth Information
inal.degradation.of.the.guar.gum.(as.measured.by.solution.viscosity),.indicating.promise.
for.this.technique.
15.3.1.4 Cofactor Immobilization
A.inal.area.of.enzymatic.biocatalysis.that.may.beneit.from.the.introduction.of.nanotech-
nology.is.for.reactions.that.require.cofactor.addition..Cofactors.are.non-protein.chemical.
compounds.that.are.required.for.the.biological.activity.of.the.protein..The.most.common.
cofactor.compounds.desired.for.industrial.biocatalysis.are.the.electron.carriers.required.
for.redox.reactions,.most.commonly.NAD(H)..These.cofactors,.which.are.quite.expensive,.
are.consumed.in.stoichiometric.quantities.for.each.enzymatic.reaction..However,.they.can.
be. regenerated. by. an. additional. redox. reaction. and. then. reused. if. they. can. be. retained.
near.the.original.enzyme..Liu.et.al..immobilized.glutamate.dehydrogenase.(GLDH),.lac-
tate.dehydrogenase.(LDH),.and.NAD(H).separately.to.silica.nanoparticles.by.functional-
izing.the.nanoparticles.with.epoxides.(Liu.et.al..2009).and.then.reacting.each.enzyme.or.
the.cofactor.with.the.functionalized.particles..The.particles.were.then.combined.to.create.
a.reactor.system..In.this.combined.system,.glutamate.was.oxidized.to.α-ketoglutarate.with.
a.commensurate.reduction.of.NAD
+
.to.NADH..The.NADH.was.then.reoxidized.to.NAD
+
.
with.a.commensurate.oxidation.of.pyruvate.to.lactate..At.low.cofactor.concentrations,.the.
reaction. rates. were. similar. to. solution. rates,. but. as. the. cofactor. concentration. increased,.
the. V
max
. for. the. immobilized. reactions. was.∼50%. of. the. free. solution. reaction,. and. the.
immobilized. system. reached. cofactor. saturation. at. a. much. lower. cofactor. concentration.
than.the.free.solution..In.their.efforts.to.optimize.the.immobilized.system,.they.found.that.
the.buffer.system.had.a.dramatic.effect.on.the.turnover.number.as.did.the.salt.concentra-
tion..The.liquid.to.solid.ratio.also.had.a.dramatic.effect.with.higher.particle.concentrations.
giving.substantially.reduced.turnover.numbers..They.hypothesized.that.this.was.due.to.
aggregation,.which.reduced.particle.mobility,.reducing.collision.frequency.
15.3.2 Separations
A. wide. range. of. modalities. are. applied. for. separating. biological. materials,. depending.
on.the.size,.charge,.density,.surface.activity,.biological.afinity,.etc..of.the.materials.being.
separated..The.majority.of.the.applications.are.in.the.separations.of.proteins.(occasion-
ally. nucleic. acids). to. obtain. a. material. of. the. desired. purity. for. industrial,. diagnostic,.
or. therapeutic. use.. Another. important. application. is. peptide. separations. for. the. grow-
ing.ield.of.proteomics..In.proteomic.research,.proteins.are.digested.with.proteases.into.
peptides.that.are.then.separated.(generally.by.chromatography).before.analysis.by.mass.
spectrometry..In.macroscale.separations,.two.of.the.most.commonly.used.techniques.are.
membrane.separations,.in.which.compounds.are.primarily.separated.by.size,.with.large.
compounds.being.retained.by.the.membrane.and.smaller.particles.lowing.through.the.
membrane. pores,. and. chromatography. (or. ixed. bed. adsorption). in. which. compounds.
preferentially. partition. between. the. solid. and. mobile. phases. depending. on. charge,.
size,.hydrophobicity,.and.biological.afinity..Both.of.these.separation.techniques.can.be.
extended.into.the.nanoscale.using.either.nanoparticles.or.nanostructured.materials..As.
in. the. case. of. enzyme. immobilization,. nanoscale. materials. provide. very. high. surface.
area.to.volume.ratios.and.substantially.reduced.mass-transfer.resistance.compared.with.
micrometer.sized.particles..In.addition.to.these.nanoscale.materials,.microluidic.devices.
(while.not.truly.nanoscale),.often.made.using.the.same.lithographic.techniques.used.in.
the.semiconductor.industry,.are.increasingly.employed.for.bioseparations.or.development.
