Enzyme immobilization on nanomaterials integrated in microreactors

Mikroreaktorji, ki so po definiciji reaktorji z vsaj eno dimenzijo pod 1mm, so vedno bolj zanimivi industriji in vedno bolj uveljavljajo svojo pomembnost v biokatalitskih procesih. Njihove lastnosti, kot so dobro razmerje med površino in volumnom, lahka kontrola nad procesom, majhna poraba reagentov, omogočajo mikroreaktorjem učinkovite procese. Za dodatno povečanje razmerja med površino in volumnom v mikroreaktorjih, je v porastu integracija nanostruktur kot so nanovzmeti in nanotkanine, v mikroreaktorje. Omenjene nanostrukture so se v preteklosti izkazale kot izjemen nosilec za imobilizacijo encimov z označevalci. V okviru magistrskega dela sem razvila mikroreaktor v katerem sem želela izvajati imobilizacijo encimov in izvedbo biotransformacije. Opravila sem karakterizacijo nanovzmeti za določevanje hidrofobnosti. S ploščicami, ki so vsebovale različno gosto porazdeljene nanovzmeti, sem ugotovila, da z višjo gostoto porazdelitve nanovzmeti narašča tudi začetna hidrofobnost. V okviru karakterizacije sem opravila analizo z vrstičnim elektronskim mikroskopom (SEM). Ugotovila sem, da je površina nanovzmeti zgoščena in zlepljena. Predvidevam, da je to zaradi izpostavitve visoki temperaturi med njihovo pripravo. To je tudi lahko razlog za slabo učinkovitost nanovzmeti. ω-transaminazo z Zbasic2 označevalcem (N-SBM-ATA-wt) sem imobilizirala na silicijeve nanovzmeti preko ionskih interakcij med peptidnim označevalcem in nabito površino. Imobilizacija N-SBM-ATA-wt ni bila učinkovita, kar lahko pripišem zlepljenosti in zgoščenosti nanovzmeti in njihovi neustrezni hidrofilnosti. Karboksilno esterazo pridobljeno iz Archaeoglobus fulgidus (AF-Est2) s polihistidinskim označevalcem sem imobilizirala na nanotkanino z bakrovimi ioni na osnovi afinitetne imobilizacije med označevalcem in površino Ugotovila sem, da je bila imobilizacija AFEst2 na nanotkanine učinkovita, saj se je imobiliziralo tudi 100% encima, prav tako pa je bila uspešna tudi regeneracija nanotkanine z vnosom bakrovih ionov.Microreactors, which are by definition reactors with at least one dimension below 1 mm, are gaining more attention from industry and are more important in the field of biocatalysis. Their favorable characteristic, such as high surface-to-volume ration, easy process control, low reagent consumption, enable microreactors to perform highly efficient biocatalytic processes. For additional increase of surface-to-volume ratio nanomaterials, such as nanosprings and nanofibers, are becoming increasingly important. Such nanostructures are an efficient carrier for tagged enzyme immobilization. In this work a microreactor with immobilized enzymes for a desired biotransformation was developed. Characterization of nanosprings was performed. Nanosprings with different density were tested and it was concluded that nanosprings with higher density are more hydrophobic at the start. An analysis with scanning electron microscope (SEM) was performed as well. Study showed that the surface of nanosprings was melted and concentrated. It is presumed this is the consequence of the exposure to higher temperatures in the preparation of nanosprings. ω-transaminase with Zbasic2 (N-SBMATA-wt) tag was immobilized on silicon nanosprings via ionic interaction between Zbasic2 tag and surface. Immobilization yield was below expectation, which could be caused by melted nanosprings and consequently their unsuitable hydrophilic characteristic. Carboxyl esterase from Archaeoglobus fulgidus (AF-Est2) with polyhistidine tag was immobilized on nanofiber with functionalized Cu2+ via ionic interactions. Immobilization yield in this experiment even reached 100%. Regeneration of nanofibers was performed with attachment of Cu2+ particles on nanofibers and it was successful, because further immobilizations yields were high