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Bacterial Luciferase of Vibrio harveyi MAV: Purification, Characterization, and Application
Lang, Dietmar Erdmann, Helmut Schmid, Rolf D.
Lang, Dietmar
Erdmann, Helmut
Schmid, Rolf D.
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Issue Date
1992
Submitted date
2024-04-17
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Abstract
In the medical and biotechnological industry is an increasing interest for bioluminescent systems for example as analytical tools and genetic markers. We introduced a new procedurefora large scale purification of luciferase from Vibrio harveyi MAV in view of subsequentcrystallization of the enzyme. In four steps the luciferase was obtained in greater than 99 % purity using the chromatography equipment (FPLC-System)from Pharmacia/LKB, Freiburg, Germany. Homogeneity of the luciferase was proved by SDS-PAGE andsilver staining. The enzyme was purified 34fold to a yield of 29 % and a specific activity of 1.8*10'' [LU/mg]. The optimum activity of luciferase was detected at pH 6.8 and 30°C.Theisoelectric point proved to be at pH 4.7. Treatment with the inhibitors diethylcarbonate, phenylmethylsulfonyifluoride, and diethyl-p-nitrophenylphosphate until unknownasluciferase inhibitors, affected the activity with the same extent and nearly the samevelocity as other inhibitors, reacting with histidine and cysteine residues at the active center of luciferase. The enzymecrystallized depending upondifferent precipitation agentsin various forms. We obtained twotypesof crystals, needles in ammonium sulfite and rhombic-like in ammonium sulfate.
Citation
Biosensors : fundamentals, technologies and applications, 239 - 244
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Book chapter
conference paper
conference paper
Language
en
Description
Series/Report no.
GBF monographs ; Volume 17
ISSN
0930-4320
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ISBN
3527284370
1560812206
1560812206
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Attribution-NonCommercial-ShareAlike 4.0 International