SINTEZA ZAMREŽENIH ENCIMSKIH SKUPKOV IZ ENCIMA AMILAZE

Kac, Andreja

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Title:	SINTEZA ZAMREŽENIH ENCIMSKIH SKUPKOV IZ ENCIMA AMILAZE
Authors:	ID Kac, Andreja (Author) ID Leitgeb, Maja (Mentor) More about this mentor... ID Primožič, Mateja (Comentor)
Files:	UNI_Kac_Andreja_2009.pdf (17,82 MB) MD5: 765EB78EEED9EAD9D5C0397D51EE9BFC PID: 20.500.12556/dkum/af27642d-ab0c-40b5-b3fe-c66aeb980f22
Language:	Slovenian
Work type:	Undergraduate thesis
Organization:	FKKT - Faculty of Chemistry and Chemical Engineering
Abstract:	Delo opisuje sintezo aktivnih encimskih skupkov ali agregatov iz encima α — amilaze in postopek zamreženja le — teh z glutaraldehidom, z namenom priprave končne oblike stabilnih zamreženih encimskih skupkov ali na kratko CLEAs. V splošni praksi je postopek priprave zamreženih encimskih skupkov razdeljen na dva ključna dela, in sicer, na precipitacijo topnega oziroma nativnega encima s pomočjo ustreznega organskega topila ali nasičene anorganske soli ter na nadaljnjo zamreženje tako izoborjenega encima s pomočjo zamrežitvenega agensa. Iz literature je znano, da je glutaraldehid, kot homobifunkcionalna organska molekula, najbolj pogosto uporabljen zamrežitveni agens, ker je relativno poceni in je njegova uporaba zelo enostavna. Pri postopku denaturacije oziroma obarjanja nativnega encima je potrebno preizkusiti čim več različnih denaturantov, z namenom, da se ugotovi, kateri uporabljeni denaturant ne povzroči ireverzibilne denaturacije encima, oziroma zagotovi, da je po ponovni resuspenziji izoborjenega encima v vodni raztopini aktivnost encima ponovno blizu 100 %. V našem primeru smo uspešno sintetizirali zamrežene encimske skupke iz α — amilaze. Aktivnost CLEAs smo določili z reakcijo hidrolize vodotopnega škroba. Optimalni reakcijski pogoji sinteze zamreženih encimskih skupkov so bili doseženi pri koncentraciji encima α — amilaze Î³α-amilaza = 8 mg/mL in koncentraciji encima albumina (BSA) Î³BSA = 8 mg/mL. Za obarjanje smo uporabili φ = 90 % (v/v) delež različnih topil in φ = 10 % (v/v) delež raztopljenega encima. Obarjalna agensa, metanol in etanol, v katerih je encim obdržal najvišjo aktivnost, sta bila izbrana za naslednji korak — zamreženje. Reakcija zamreženja je potekala t = 3 h pri sobni temperaturi. Ugotovljeno je bilo, da je bila aktivnost zamreženih skupkov iz encima α — amilaze najvišja pri koncentraciji glutaraldehida φ = 1 % (v/v) in metanolu kot obarjalnemu reagentu. Končni videz raztopine zamreženih encimskih agregatov je podoben motni suspenziji, v kateri so jasno vidni skupki encimov značilne sferične oblike in premera okrog 1 µm. Tehnika za sintezo zamreženih encimskih agregatov je zelo enostavna in nasploh zelo obetavna metoda na področju imobilizacij različnih encimov.
Keywords:	zamreženi encimski skupki, CLEAs, α – amilaza, mrežni povezovalec, glutaraldehid
Place of publishing:	Maribor
Publisher:	[A. Kac]
Year of publishing:	2009
PID:	20.500.12556/DKUM-11161
UDC:	577.15:663.03(043.2)
COBISS.SI-ID:	13325334
NUK URN:	URN:SI:UM:DK:YLUDKQS4
Publication date in DKUM:	22.12.2009
Views:	4369
Downloads:	310
Metadata:
Categories:	KTFMB - FKKT
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Secondary language

Language:	English
Title:	SYNTHESIS CROSSLINKED ENZYMES AGGREGATES OF ENZYME AMYLASA
Abstract:	This work describe synthesis active enzyme aggregates of α – amylase, further cross – linked with glutaraldehyde in order to obtain the final form of stable cross – linked enzyme aggregates or CLEAs. In practise, the procedure to prepare cross – linked enzyme aggregates generally includes two major steps, that involves first the precipitation of the soluble enzyme with a suitable precipitant such as different organic solvents or inorganic salts and second the crosslinking step with an appropriate cross – linker. Generally, glutaraldehyde is the most common used crosslinking agent, since it is inexpensive and easily applicable. The precipitant of choise to make highly stable cross – linked enzyme aggregates is the one which gives the highest, preferably 100 %, activity yield after the re – suspension of the aggregated enzyme under investigation. In the present study, the cross – linked enzyme aggregates of α – amylase were successfully produced. The activity of CLEAs was determined by the reaction hydrolsis of starch soluble in water. The optimal conditions for the synthesis of cross – linked enzymes aggregates were predicted as following: α – amylase concentration γα-amilaza = 8 mg/mL and enzyme bovine serum albumin (BSA) concentration γBSA = 8 mg/mL. The reaction of precipitation was conducted with up to φ = 90 % (v/v) of different precipitants and φ = 10 % (v/v) of dissolved enzyme. The precipitant agents, metanol and etanol, were further selected or optimization of the reaction of crosslinking. Reaction of crosslinking lasted for t = 3 h at room temperature. The highest activity recovery of CLEAs was obtained at glutaraldehyde concentration φ = 1 % (v/v) and methanol as a precipitant of choice. The final suspension of CLEAs obtained is moderately turbid and enzyme particles can be normally observed with an average diameter of 1 m. Moreover, CLEAs, as such, represent a promising and relatively facile technique for the preparation of any immobilized enzyme, respectively.
Keywords:	cross – linked enzyme aggregates, CLEAs, α – amylase, cross-linker, glutaraldehyde

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