Theoretical and Practical Aspects of Immobilized Multi-Step Enzyme Systems

Mosbach, Klaus; Mattìasson, Bo; Gestrelius, Stina; Srere, Paul A.

doi:10.1007/978-1-4615-8897-9_20

Cited by 5 publications

(6 citation statements)

References 9 publications

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“…One of the potential advantages of immobilized enzymes is their increased stability (8). To compare the stability of free and immobilized BCCPluciferase and BCCP-oxidoreductase, the enzymes were stored at 4°C with and without DTT, and their activities were measured periodically ( Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…As part of the development of these diagnostic assays, luciferase and oxidoreductase have been expressed in E. coli and immobilized on solid materials in several labs (2,3,6,8,10,15). The immobilization of oxidoreductase and luciferase by the methods described in these reports generally resulted in low binding efficiency, with significant loss of enzyme activity (3,10,11).…”

Section: Discussionmentioning

confidence: 97%

“…This suggests that sensitive biosensors with a wide useful detection range can be developed using the BCCPluciferase and -oxidoreductase system. For application in biosensors, coimmobilization of oxidoreductase and luciferase is also of interest because of the increased coupling efficiency between the enzymes (3,8). The increased efficiency is due to a decrease in nonenzymatic oxidation of FMNH 2 , which competes with production of light through enzymatic oxidation of FMNH 2 .…”

Section: Figmentioning

confidence: 98%

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Specific Immobilization ofin VivoBiotinylated Bacterial Luciferase and FMN:NAD(P)H Oxidoreductase

Min

Andrade

Stewart

1999

Analytical Biochemistry

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 97%

Section: Figmentioning

confidence: 98%

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Specific Immobilization ofin VivoBiotinylated Bacterial Luciferase and FMN:NAD(P)H Oxidoreductase

Min

Andrade

Stewart

1999

Analytical Biochemistry

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“…For instance water-insoluble bifunctional aggregates can be useful as an alternative to matrix-bound two-step and multi-step enzyme systems [16].…”

Section: Omentioning

confidence: 99%

Preparation of a Soluble, Bifunctional Enzyme Aggregate and Studies on Its IGnetic Behaviour in Polymer Media

Mattlasson

Johansson

Mosbach

1974

European Journal of Biochemistry

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Soluble, bifunctional enzyme aggregates have been prepared by crosslinking sequentially acting enzymes with glutaraldehyde. Aggregates of j3-glucosidase and glucose oxidase with molecular weights in the range 200 000-300 000 have been characterized from such a preparation. The sequentially working enzymes malate dehydrogenase and citrate synthase have also been aggregated using the same method. The kinetic behaviour of a system comprising two sequentiaily acting enzymes, either separately or as a n aggregate, was studied in media containing increasing concentrations of poly(ethyleneglyco1). A plot of transition time against polymer concentrations was bell-shaped in the case of /3-glucosidase and glucose oxidase whereas in the system malate dehydrogenase-citrate synthase almost no lagphase was observed and comequently no transition time was registered. I n both enzyme systems the steady-state rate in the overall reaction was stimulated up to 30 O/ , , a t lower polymer concentrations, whereas reduced rates were obtained in more concentrated polymer solutions. These effects are interpreted in terms of (a) an exclusion effect leading to a relative enrichment of the rate-limiting intermediate and an apparently higher concentration of the enzymes in the sequence and (b) a decreased diffusion rate which lowers the apparent V.

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“…There have been many studies on the properties of enzymes immobilized to various supports (26,31). In many cases, the enzymes are stabilized and shdw some altered properties with regard to pH optima or kinetic properties.…”

mentioning

confidence: 99%

Immobilization of bacterial luciferase and FMN reductase on glass rods.

Jablonski¹,

DeLuca²

1976

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial luciferase and NAD(P)H: FMN oxidoreductase isolated from Beneckea harveyi were covalently linked via diazotization to arylamine porous glass beads which had been cemented onto plain glass rods. These immobilized enzymes are individually active and also function to produce light via a coupled reaction utilizing NADH or NADPH. These enzymes have properties similar to the soluble forms with regard to pH and substrate optima and also exhibit linearity in peak intensity of the initial flash of light emitted as a function of NADH or NADPH concentration. Linearity with NADH is obtained in the range of 1 pmol to 50 nmol, and between 10 pmol to 200 nmol for NADPH. The bound enzymes are stable and reusable. This immobilized system offers a rapid and inexpensive method of assaying low concentrations of NADH and NADPH, and also serves as a model to study the association of these two enzymes. Bacterial luciferase catalyzes the oxidation of FMNH2 in the presence of a long chain aldehyde with the emission of light (1-4). The aldehyde is converted to the corresponding acid (5-7) and the emitted light has an emission peak at 490 nm. Because of the rapid autoxidation of FMNH2 (8) in an Aminco Chem-Glo photometer and recorded on an Aminco recorder. The initial peak light intensity was linear with respect to added luciferase in the range of 8 ng to 8 ,ug/ml using this instrument. Immobilized luciferase was assayed with the same concentrations of substrates. The rod containing the glass beads was placed in a test tube in the photometer and FMNH2 was injected.Soluble reductase was assayed by measuring the rate of disappearance of absorption at 340 nm in a Cary model 14 recording spectrophotometer. The reaction was initiated by adding 0.1 ml of NADH to 1 ml of 0.015 M phosphate buffer at pH 7.0, containing 70 ,AM EDTA, 0.4 mg of reductase, and FMN. Final concentrations were 0.2 mM NADH, 0.13 mM FMN. When the immobilized enzyme was assayed, the rod containing the enzyme (see below) was dipped into the cuvet which was mixed for 1-min intervals, then removed and the absorbance at 340 measured. This assay gave data that when plotted showed linearity for at least 3 min.The coupled assay was measured by peak light intensity obtained following injection of NADH into 0.5 ml of 0.1 M phosphate buffer at pH 7.0 containing 7.5 Mug of reductase, 5 ,ug luciferase, 2.3 MuM FMN, and 0.0005% decanal. When the immobilized enzyme was being assayed, the rod was immersed in the solution containing FMN and aldehyde, and NADH was injected directly into the solution.Beneckea harveyi strain no. 392 (19) was obtained from K. Nealson of the Scripps Institution of Oceanography. Large quantities of the bacteria were-grown in complete media essentially as described by Farghaly (20) The reductase was separated from the luciferase during chromatography on DEAE-Sephadex, the last step in the purification (22). The peak of reductase activity eluted between 1000 and 1325 ml of column eluant, before the luciferase was eluted. The peak tubes were com...

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Theoretical and Practical Aspects of Immobilized Multi-Step Enzyme Systems

Cited by 5 publications

References 9 publications

Specific Immobilization ofin VivoBiotinylated Bacterial Luciferase and FMN:NAD(P)H Oxidoreductase

Specific Immobilization ofin VivoBiotinylated Bacterial Luciferase and FMN:NAD(P)H Oxidoreductase

Preparation of a Soluble, Bifunctional Enzyme Aggregate and Studies on Its IGnetic Behaviour in Polymer Media

Immobilization of bacterial luciferase and FMN reductase on glass rods.

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