UNIVERSITA’ DEGLI STUDI DI PISA Scuola di dottorato “Galileo Galilei”
PhD IN CHEMICAL SCIENCE
XXIV cycle (2009-2011)
Development*and*Characterization*
of*Immobilized*Enzyme*Reactors*
(IMERs)*
Tommaso Martelli
Supervisor:
dr. Antonella Petri
External Supervisor:
prof. Irving W. Wainer
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! The! work! described! in! this! thesis! was! performed! at! the!
Dipartimento!di!Chimica!e!Chimica!Industriale,!(Università!di!Pisa,!
Pisa,!Italy)!and!at!the!Faculté!des!Sciences!et!Techniques,!pôle!St.!
Jerome! (Université! Paul! Cézanne,! AixKMarseille! III,! Marseille,!
France).!
This! thesis! is! a! result! of! a! collaboration! with! prof.! I.! Wainer!
(National! Institute! of! Health,! Baltimora,! USA),! prof! E.! Maser!
(Institute! of! Toxicology! and! Pharmacology! for! Natural! Scientists,!
University!Medical!School!SchleswigKHolstein,!Kiel,!Germany)!and!
prof.! A.! Archelas! (Université! Paul! Cézanne,! AixKMarseille! III! K!
Faculté! des! Sciences! et! Techniques,! pôle! St.! Jerome,! Marseille,!
France).!
Contract!grant!sponsor:!MIUR;!Contract!grant!number:!PRIN2007!
(Sintesi!e!Stereocontrollo!di!Molecole!Organiche!per!lo!Sviluppo!di!
Metodologie!InnoK!vative!di!Interesse!Applicativo).!
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Acknowledgements.
!
The! work! described! in! this! thesis! is! the! result! of! fruitful! collaborations! with!
various!professors,!researchers!and!colleagues!who!have!made!this!possible!and!I!would!
like!to!thank!them!all.!More!specifically!I!would!like!to!thank!
prof.! I.! W.! Wainer! (National! Institute! of! Health,! Baltimora,! USA),! who! gave! an! helpful!
support! to! the! project,! consisting! of! advices! and! discussion,! and! provided! some!
materials!necessary!for!our!research;!
prof.! A.! Archelas! (Université! Paul! Cézanne,! AixKMarseille! III! K! Faculté! des! Sciences! et!
Techniques,!pôle!St.!Jerome,!Marseille,!France),!who!allowed!me!to!attend!his!research!
lab! for! three! month,! in! order! to! improve! my! knowledge! about! enzyme! preparation,!
purification!and!use!and!for!the!materials!that!he!gently!furnished!us;!
prof.! E.! Maser! (Institute! of! Toxicology! and! Pharmacology! for! Natural! Scientists,!
University!Medical!School!SchleswigKHolstein,!Kiel,!Germany),!who!provided!samples!of!
enzyme;!
dr.!F.!Mazzini,!whose!reflections,!suggestion!and!ideas!were!very!helpful!and!fruitful!for!
my!work;!
dr.! A.! Cuzzola,! for! everything! concerning! mass! spectrometry! and! the! interesting!
discussions!about!chemistry!and!other;!
prof.! A.! Iuliano,! that! taught! me! how! to! pack! stainlessKsteal! columns! with! derivatized!
silica;!
prof.!R.!Menicagli,!for!her!help!and!advices;!
Sebastiano,! Maria! Rosa,! Sabriye,! Matteo,! Damiano,! Claudio,! Ravi,! Varsha,! Angela,!
Valentina,! Rosa,! Veronica,! Graziano,! Irene,! Virginia! and! all! the! other! with! whom! I’ve!
shared!at!least!a!part!of!this!journey.!
Last,!but!not!least,!a!great!thank!is!for!dr.!A.!Petri!who!guided!me!in!the!tortuous!
route!of!this!work!and!that!taught!me!how!to!appreciate!the!small!satisfaction!of!each!
days.!I!would!like!to!thank!her!also!for!her!suggestions,!comprehension,!presence!and!
above!all!for!her!friendship.!
Furthermore!I!would!like!to!thank!also!my!entire!family!and!all!my!friends;!also!
their!contributes!were!been!essential!for!me,!and!I!want!to!thank!all!of!them!for!their!
support!and!closeness.!
More! specifically! I! desire! to! tank! my! parents,! for! their! comprehension! and! their!
support!during!all!this!long!period!of!study;!Ilaria,!for!her!presence!in!my!life!and!for!her!
ability!to!show!me!the!positive!side!of!bad!things;!Peppino,!for!his!help!and!his!presence!
during!all!this!period.!
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The$most$exciting$phrase$to$hear$in$science,$
the$one$that$heralds$new$discoveries,$
is$not$'Eureka!'$(I've$found$it!),$but$"That's$funny..."$
Isaac$Asimov$
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....I$have$not$failed.$
I've$just$found$10,000$ways$that$won't$work.$$
Thomas$Edison$
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To!my!family,!
to!Ilaria!
and!to!everyone!
that!made!this!possible.!
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Index.
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INDEX* I!
LIST*OF*SYMBOLS*AND*ABBREVIATIONS* V!
ABSTRACT* VII!
CHAPTER*1:! INTRODUCTION* 1!
1.1*N! BIOCATALYSIS* 1!
1.1.1.! ENZYMES!IN!INDUSTRY! 1!
1.1.2.! ADVANTAGES!OF!BIOCATALYSTS! 3!
1.1.3.! DRAWBACKS!OF!BIOCATALYSTS! 4!
1.2*N! IMMOBILIZED*ENZYMES* 5!
1.2.1.! METHODS!OF!IMMOBILIZATION! 7!
1.3*N! IMMOBILIZED*ENZYME*REACTORS*(IMERS)* 8!
1.3.1.! SUPPORTS! 10!
1.3.2.! BIOSILICA!FOR!IMERS!PREPARATIONS! 12!
1.3.3.! ONKLINE!IMER!APPLICATIONS! 14!
1.3.3.1!K! PreKcolumn!assembly! 14!
1.3.3.2!K! PostKcolumn!assembly! 16!
1.3.3.3!K! Affinity!Chromatography! 17!
CHAPTER*2:! IMMOBILIZED*ENZYME*REACTORS*FOR*DRUG*DISCOVERY* 21!
2.1*N! INTRODUCTION* 21!
2.1.1.! IMMOBILIZED!ENZYME!ACTIVITY! 23!
2.2*N! IMMOBILIZED*ENZYME*REACTORS*BASED*ON*HUMAN*MONOAMINO*OXIDASE*TYPE*A*(MAONA)* 25!
2.2.1.! IMMOBILIZATION!OF!MAOKA! 28!
2.2.2.! KINETIC!STUDIES! 31!
2.2.3.! STABILITY! 32!
2.2.4.! EFFECT!OF!INHIBITORS!ON!MAOKA!ACTIVITY! 32!
2.2.5.! OXYGEN!ADSORPTION! 34!
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!II!
2.3*N! IMMOBILIZED*ENZYME*REACTORS*BASED*ON*HUMAN*11βNHYDROXYSTEROID*DEHYDROGENASE*
TYPE*1*(11βNHSD1)* 35!
2.3.1.! IMMOBILIZATION!STUDY! 38!
2.3.2.! KINETIC!STUDIES! 39!
2.3.3.! STABILITY! 40!
CHAPTER*3:! CMAC*COLUMN*AND*NLC*APPROACH* 43!
3.1*N! CELLULAR*MEMBRANE*AFFINITY*CHROMATOGRAPHY*(CMAC)* 43! 3.2*N! IMMOBILIZED*RECEPTORS*AND*MEMBRANE*PROTEINS* 44! 3.3*N! IMMOBILIZED*NICOTINIC*ACETYLCHOLINE*RECEPTORSNBASED*LIQUID*CHROMATOGRAPHIC*
STATIONARY*PHASE* 45!
3.3.1.! BINDING!STUDY! 47!
CHAPTER*4:! IMMOBILIZED*ENZYME*REACTORS*FOR*CHEMICAL*SYNTHESIS* 51!
4.1$%! INTRODUCTION* 51!
4.2*N! CHIRAL*EPOXIDES*AND*DIOLS* 55!
4.3$%! EPOXIDE*HYDROLASES*(EHS)* 57!
4.3.1.! EPOXIDE!HYDROLASE!FROM!ASPERGILLUS$NIGER$(ANEH)! 60! 4.3.2.! EPOXIDE!HYDROLASE!FROM!SOLANUM!TUBEROSUM!(STEH)! 63! 4.3.3.! ENANTIOCONVERGENT!PROCESS!WITH!COMPLEMENTARY!EHS! 64! 4.4*N! IMMOBILIZED*ENZYME*REACTORS*BASED*ON*ANEH*AND*STEH* 64!
4.4.1.1!K! Stability!of!immobilized!AnEH!and!StEH! 65!
4.4.1.2!K! Organic!solvent!stability! 68!
4.4.2.! PREPARATION!OF!STEH!AND!ANEHKBASED!IMERS! 70! 4.4.3.! ENANTIOCONVERGENT!SYNTHESIS!OF!PCSD!WITH!EH!IMERS! 71! 4.5*N! IMMOBILIZATION*OF*ANEH*AND*STEH*ON*BIOSILICA* 71!
CHAPTER*5:! EXPERIMENTAL*PART* 75!
5.1*N! CHEMICALS* 75!
5.2*N! APPARATUS* 76!
5.3*N! SILICA*GEL*DERIVATIZATION* 77!
5.4*N! BRADFORD*ASSAY* 78!
5.5*N! STAINLESSNSTEEL*COLUMN*PACKING* 78!
5.6*N! IMMOBILIZED*ENZYME*REACTORS*BASED*ON*MAONA* 79!
5.6.1.! CHROMATOGRAPHIC!METHODS! 79!
5.6.1.1!K! Analysis!of!kynuramine!/!4Khydroxyquinoline! 79!
5.6.1.2!K! Quantification!of!4Khydroxyquinoline!on!IMERs! 79!
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5.6.2.! IMMOBILIZATION!OF!MAOKA! 79!
5.6.2.1!K! IAM.PC!IMER!(IMER1)! 79!
5.6.2.2!K! EpoxySilica!IMER!(IMER2)! 80!
5.6.3.! KINETIC!STUDIES! 80!
5.6.3.1!K! Activity!Assay! 80!
5.6.3.2!K! Determination!of!the!kinetic!parameters! 81!
5.6.3.3!K! Optimization!of!the!IMER!activity!assay! 81!
5.6.3.4!K! Determination!of!the!kinetic!parameters!of!the!IMERs! 81!
5.6.4.! INHIBITION!STUDIES! 81!
5.6.4.1!K! “In!batch”!inhibitory!assay! 81!
5.6.4.2!K! “OnKline”!inhibitory!assay! 82!
5.6.5.! ANALYSIS!OF!THE!OXYGEN!ADSORPTION! 82!
5.6.6.! EXTRACTION!AND!PURIFICATION!OF!EUGENOL! 82!
5.7*N! IMMOBILIZED*ENZYME*REACTORS*BASED*ON*11βNHSD1* 83!
5.7.1.! CHROMATOGRAPHIC!METHODS! 83!
5.7.1.1!K! Analysis!of!Cortisone!/!Cortisol! 83!
5.7.2.! IMMOBILIZATION!OF!11βKHSD1! 83!
5.7.2.1!K! 11βKHSD1|EpoxySilica! 83!
5.7.2.2!K! 11βKHSD1|IAM! 84!
5.7.2.3!K! 11βKHSD1|BioSilica! 84!
5.7.3.! KINETIC!STUDIES! 84!
5.7.3.1!K! Activity!assay!for!11βKHSD1! 84!
5.7.3.2!K! Determination!of!the!kinetic!parameters! 85!
5.7.3.3!K! Activity!of!11βKHSD1|EpoxySilica! 85!
5.7.3.4!K! Activity!of!11βKHSD1|IAM! 85!
5.7.3.5!K! Activity!of!11βKHSD1|BioSilica! 86!
5.7.3.6!K! Determination!of!the!kinetic!parameters!of!11βKHSD1|BioSilica!samples! 86!
5.8*N! IMMOBILIZED*NACHRS* 86!
5.8.1.! NACHRSKIMER!PREPARATION! 86!
5.8.2.! NONKLINEAR!CHROMATOGRAPHIC!STUDIES! 87!
5.9*N! IMMOBILIZED*ENZYME*REACTORS*FOR*CHEMICAL*SYNTHESIS* 88!
5.9.1.! CHROMATOGRAPHIC!METHODS! 88!
5.9.1.1!K! Analysis!of!pCSO/pCSD! 88!
5.9.1.2!K! Analysis!of!(R)/(S)KpCSD! 88!
5.9.2.! IMMOBILIZATION!OF!EHS! 89!
5.9.2.1!K! EH|EpoxySilica! 89!
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!IV!
5.9.2.2!K! IMER3!and!IMER4! 89!
5.9.2.3!K! EH|BioSilica! 90!
5.9.3.! ENZYMATIC!ACTIVITY! 90!
5.9.3.1!K! Activity!assay! 90!
5.9.3.2!K! Activity!of!EH|EpoxySilica! 91!
5.9.3.3!K! Stability!test! 91!
5.9.3.4!K! Optimization!of!IMER3!and!IMER4!assay! 91!
5.9.3.5!K! Activity!of!EH|BioSilica! 91!
5.9.4.! ENANTIOCONVERGENT!PROCESS! 91!
5.9.4.1!K! “OnKline”!enantioconvergent!process! 91!
5.9.4.2!K! “In!batch”!enantioconvergent!proces! 92!
SUMMARY* 93!
APPENDIX*A:! MICHAELISNMENTEN*KINETICS* 95!
APPENDIX*B:! ENZYME*INHIBITION* 99!
APPENDIX*C:! NLC*CHROMATOGRAPHY* 103!
REFERENCES* 107!
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List.of.symbols.and.abbreviations.
Anab:! Anabasine!
AnEH:!! Epoxyde!Hydrolases!from!Aspergillus$niger$
BSA:!! Bovine!Serum!Albumine!
CMAC:! Cellular!Membrane!Affinity!Chromatography!
Cot:! Cotinine!
DMSO:! Dimethyl!sulphoxide!
DNA:! Deoxyribonucleic!acid!
E:!! Cortisone!
Ebt:! Epibatidine!
EHs:!! Epoxyde!Hydrolases!
F:!! Cortisol!
FAC:! Frontal!Affinity!Chromatography!
FAD:!! Flavin!Adenine!Dinucleotide!
FADH2:! Flavin!Adenine!Dinucleotide!hydroquinone!form!
HSA:! Human!Serum!Albumine!
HPLC:! High! Performance! Liquid! Chromatography! /! High! Pressure! Liquid!
Chromatography!
4HQ:!! 4KHydroxyKquinoline!
HTS:!! High!Throughput!Screening!
11βKHSD1:!! 11βKHydroxysteroid!dehydrogenases!type!1!
IAM:! Immobilized!Artificial!Membrane!
IC50:! Half!maximal!inhibitory!concentration!
IMERs:!! Immobilized!Enzyme!Reactors!
Ki:! Inhibition!constant!
KM:!! MichaelisKMenten!constant!
Kyn:!! Kynuramine!
MAOKA:!! Monoamino!oxidase!type!A!
MAOKB:!! Monoamino!oxidase!type!B!
MAOIs:!! Monoamino!oxidase’s!inhibitors!
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MS:! Mass!Spectrometer!
nAChRs:! Nicotinic!Acetylcholine!Receptors!
NADH/NAD+:!! Nicotinamide!adenine!dinucleotide!
NADPH/NADP+:!! Nicotinamide!adenine!dinucleotide!phosphate!
Nico:! Nicotine!
NLC:! NonKLinear!Chromatography!
NNico:! NorNicotine!
PBS:!! Phosphate!Buffer!Solutions!
pCSD:!! ParaKchloro!styrene!diol!
pCSO:!! ParaKchloro!styrene!oxide!
SD:!! Styrene!diol!
SO:!! Styrene!oxide!
StEH:!! Epoxide!Hydrolase!from!Solanum$tuberosum$
Tris:! Tris(hydroxymethyl)aminomethane!
VMAX:!! Maximum!velocity!(MichaelisKMenten!kinetics)!
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Abstract.
In! the! past! few! years,! the! applicability! and! versatility! of! immobilized! enzyme!
reactors! (IMERs)! has! been! extensively! demonstrated.! The! majority! of! IMERs! system!
relies!on!enzymes!immobilized!to!packed!matrices!within!flowKthrough!devices.!
This! approach! doesn’t! required! highly! purified! enzymes! or! a! great! amount! of!
protein,! but! principally! can! increase! the! enzymatic! stabilization! against! heat,! organic!
solvents! and! pH,! without! too! much! loss! of! catalytically! activity.! Thus,! in! IMERs! an!
immobilized!enzyme!becomes!a!catalyst!confined!or!localized!on!a!suitable!support!so!
that!it!can!be!used!repeatedly!for!as!long!as!it!remains!active,!thereby!minimizing!costs,!
time!of!analysis!and!make!it!economically!feasible!to!operate!in!a!continuous!mode.!A!
variety!of!matrices!have!been!investigated!over!the!years!for!enzyme!immobilization!or!
encapsulation;! in! particular! the! use! of! silica! based! IMERs! has! received! increasing!
attention! and! the! applications! have! grown! steadily.! Therefore! IMERs! now! find!
applications!in!scientific!fields!as!diverse!as!pharmacology,!chemistry,!materials!science!
and!chemical!engineering.!
This! thesis! focused! on! the! development! and! characterization! of! IMERs! based! on!
various!enzymes!immobilized!on!silica!based!matrices!by!using!different!immobilization!
techniques.!
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