Biomedical Engineering Reference
In-Depth Information
aldehyde.group.of.the.GA-MNPs.to.form.NTA-MNPs,.and.inally,.Ni
2+
.was.loaded.on.the.
chelating.NTA.arms.to.produce.Ni-NTA-MNPs..These.Ni-NTA-MNPs.were.then.used.in.a.
one-step.puriication.and.immobilization.of.
Solanum tuberosum
.epoxide.hydrolase.(stEH1).
produced.in.
Pichia pastoris
..They.found.that.the.conjugated.MNPs.reached.a.speciic.loading.
capacity.of.146.mg.protein/mg.MNPs,.corresponding.to.82%.of.the.maximum.theoretical.
monolayer.loading..The.process.showed.high.speciicity.for.the.His-tagged.stEH1,.with.no.
other.proteins.from.the.cell.lysate.immobilized..The.nanobiocatalyst.showed.∼80%.of.the.
speciic.activity.of.the.free.enzyme.in.solution.with.identical.enantioselectivity,.suggesting.
no. signiicant. change. in. enzyme. conformation. upon. immobilization.. In. addition,. the.
immobilized.catalyst.retained.∼80%.activity.after.eight.cycles.of.recycle.and.reuse.using.a.
magnetic.ield.for.separation.
A.novel.approach.to.immobilization.of.lipases.on.MNPs.without.loss.of.catalytic.activity.
was. proposed. by. Ren. et. al.. (2011).. In. this. study,. the. MNP. surfaces. were. coated. with.
polydopamine,. an. in. situ. formed. coating. inspired. by. the. adhesive. proteins. secreted. by.
marine. mussels.. Iron-oxide. MNPs. were. incubated. in. an. alkaline. dopamine. solution. to.
create.an.adherent.polydopamine.ilm.on.the.MNPs..The.enzyme.was.then.immobilized.
by.exposure.of.the.particles.to.a.lipase-containing.solution.(
Candida rugosa
.lipase.type.VII)..
After. optimization. of. reaction. conditions,. they. achieved. a. lipase. loading. of. 429.mg/g. of.
material.(85.8%.of.the.amount.of.added.lipase).with.8.78.U/mg.speciic.activity.(73.9%.of.
free.lipase.speciic.activity),.signiicantly.higher.than.in.previous.reports..The.immobilized.
enzyme.showed.improved.pH.and.thermal.stability,.and.maintained.>70%.activity.after.21.
cycles.of.isolation.and.reuse.
Increasing.interest.in.the.use.of.cellulosic.materials.as.feedstocks.for.biofuels.or.bio-based.
chemical.synthesis.has.focused.attention.on.the.immobilization.of.cellulases..Two.recent.
reports.detail.the.immobilization.of.cellulase.on.MNPs.(Jordan.et.al..2011;.Khoshnevisan.
et.al..2011)..Khoshnevisan.et.al..(2011).immobilized.cellulase.from.
Trichoderma viride
.to.MNPs.
coated.with.an.ionic.coating,.presumably.by.electrostatic.interactions..Approximately.95%.
of.the.cellulase.activity.was.bound.to.the.MNPs;.the.resulting.cellulase/MNP.complexes.
were.quite.large.(∼1.5.μm),.regardless.of.the.size.of.the.starting.MNPs..In.their.experiments,.
the.free.enzyme.showed.greater.activity.at.all.temperatures.between.37°C.and.80°C.and.
both. the. free. and. immobilized. enzymes. showed. comparable. stability.. Interestingly,. the.
immobilized. enzyme. showed. little. pH. sensitivity,. whereas. the. free. enzyme. exhibited. a.
sharp.decrease.in.activity.at.pH.greater.than.7..This.pH.insensitivity.may.be.due.shielding.
of.the.active.site.by.the.ionic.surface.coating..In.contrast.to.the.large.particles.generated.
by.Khoshnevisan.et.al.,.Jordan.et.al..(2011).deliberately.sought.to.develop.cellulase/MNP.
complexes.of.nanometer.scales..Using.carbodiimide.chemistry,.they.generated.cellulase/
MNP. complexes. with. a. mean. diameter. of. approximately. 14.nm.. They. found. that. at. a.
ratio.of.1.mg.cellulase:.50.mg.of.MNPs,.the.maximum.fractional.binding.occurred.(∼78%);.
however,.the.maximum.loading.of.0.159.mg.enzyme/mg.of.nanoparticles.occurred.at.ratio.
of.25.mg.cellulase:.50.mg.of.MNPs..Unfortunately,.the.relative.activity.dropped.sharply.at.
a.loading.above.0.021.mg.enzyme/mg.of.nanoparticles,.most.likely.due.to.steric.crowding..
In.addition,.the.activity.loss.was.fairly.high.on.subsequent.uses.with.the.majority.(47.5%).
being.lost.after.the.irst.reuse.of.the.enzyme/MNP.complexes.
15.3.1.3 Polymeric Materials
Polymeric. materials. have. been. used. both. as. solid. supports. and. for. encapsulation. of.
enzymes,.to.create.nanoscale.immobilized.enzyme.complexes..In.the.case.of.solid.supports,.
the.approaches.and.results.are.similar.to.those.used.for.immobilization.of.enzymes.to.
