title: Bienzymatic Sequential Reaction on Microgel Particles and Their Cofactor Dependent Applications.
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.
authors.affiliation: Department of Chemistry, Technische Universität Dresden , 01069 Dresden, Germany.
authors.forename: Nidhi C
authors.initials: NC
authors.lastname: Dubey
authors.forename: Bijay P
authors.initials: BP
authors.lastname: Tripathi
authors.affiliation: Department of Chemistry, Technische Universität Dresden , 01069 Dresden, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Müller
authors.affiliation: Department of Chemistry, Technische Universität Dresden , 01069 Dresden, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Stamm
authors.affiliation: College of Engineering, College of Family & Consumer Science, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Leonid
authors.initials: L
authors.lastname: Ionov
mesh_terms: D000178:Acrylamides / Q000737:chemistry; D000180:Acrylic Resins / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D005782:Gels / Q000737:chemistry*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology; D011108:Polymers / Q000737:chemistry*; D011770:Pyruvate Kinase / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits
chemical_list: D000178:Acrylamides; D000180:Acrylic Resins; D004800:Enzymes, Immobilized; D005782:Gels; D011108:Polymers; C052970:poly-N-isopropylacrylamide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C067295:N-isopropylacrylamide; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase