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Dubey, Nidhi Chandrama ; Tripathi, Bijay Prakash ; Müller, Martin ; Stamm, Manfred ; Ionov, Leonid:
Bienzymatic Sequential Reaction on Microgel Particles and Their Cofactor Dependent Applications.
In: Biomacromolecules.
Bd. 17
(2016)
Heft 5
.
- S. 1610-1620.
ISSN 1526-4602
DOI: https://doi.org/10.1021/acs.biomac.5b01745
Abstract
We report, the preparation and characterization of bioconjugates, wherein enzymes pyruvate kinase (Pk) and l-lactic dehydrogenase (Ldh) were covalently bound to poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgel support using glutaraldehyde (GA) as the cross-linker. The effects of different arrangements of enzymes on the microgels were investigated for the enzymatic behavior and to obtain maximum Pk-Ldh sequential reaction. The dual enzyme bioconjugates prepared by simultaneous addition of both the enzymes immobilized on the same microgel particles (PL), and PiLi, that is, dual enzyme bioconjugate obtained by combining single-enzyme bioconjugates (immobilized pyruvate kinase (Pi) and immobilized lactate dehydrogenase (Li)), were used to study the effect of the assembly of dual enzymes systems on the microgels. The kinetic parameters (Km, kcat), reaction parameters (temperature, pH), stability (thermal and storage), and cofactor dependent applications were studied for the dual enzymes conjugates. The kinetic results indicated an improved turn over number (kcat) for PL, while the kcat and catalytic efficiency was significantly decreased in case of PiLi. For cofactor dependent application, in which the ability of ADP monitoring and ATP synthesis by the conjugates were studied, the activity of PL was found to be nearly 2-fold better than that of PiLi. These results indicated that the influence of spacing between the enzymes is an important factor in optimization of multienzyme immobilization on the support.We report, the preparation and characterization of bioconjugates, wherein enzymes pyruvate kinase (Pk) and l-lactic dehydrogenase (Ldh) were covalently bound to poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgel support using glutaraldehyde (GA) as the cross-linker. The effects of different arrangements of enzymes on the microgels were investigated for the enzymatic behavior and to obtain maximum Pk-Ldh sequential reaction. The dual enzyme bioconjugates prepared by simultaneous addition of both the enzymes immobilized on the same microgel particles (PL), and PiLi, that is, dual enzyme bioconjugate obtained by combining single-enzyme bioconjugates (immobilized pyruvate kinase (Pi) and immobilized lactate dehydrogenase (Li)), were used to study the effect of the assembly of dual enzymes systems on the microgels. The kinetic parameters (Km, kcat), reaction parameters (temperature, pH), stability (thermal and storage), and cofactor dependent applications were studied for the dual enzymes conjugates. The kinetic results indicated an improved turn over number (kcat) for PL, while the kcat and catalytic efficiency was significantly decreased in case of PiLi. For cofactor dependent application, in which the ability of ADP monitoring and ATP synthesis by the conjugates were studied, the activity of PL was found to be nearly 2-fold better than that of PiLi. These results indicated that the influence of spacing between the enzymes is an important factor in optimization of multienzyme immobilization on the support.
Weitere Angaben
| Publikationsform: | Artikel in einer Zeitschrift |
|---|---|
| Begutachteter Beitrag: | Ja |
| Institutionen der Universität: | Fakultäten > Fakultät für Ingenieurwissenschaften > Professur Biofabrikation > Professur Biofabrikation - Univ.-Prof. Dr. Leonid Ionov Fakultäten Fakultäten > Fakultät für Ingenieurwissenschaften Fakultäten > Fakultät für Ingenieurwissenschaften > Professur Biofabrikation |
| Titel an der UBT entstanden: | Ja |
| Themengebiete aus DDC: | 500 Naturwissenschaften und Mathematik > 540 Chemie |
| Eingestellt am: | 21 Jun 2017 11:28 |
| Letzte Änderung: | 10 Mär 2022 10:19 |
| URI: | https://eref.uni-bayreuth.de/id/eprint/37712 |