Abstract
Activity tests of enzymes are often applied for determining their concentration. In the easiest case, just one product concentration is measured after a given time. This often leads to nonlinear dependences of the apparent activity with enzyme protein concentration. A general solution of this problem consists in using the balance equation of the assay system, which commonly represents a batch reactor. Here, the balance equation of the batch for a general Michaelis Menten-type reaction kinetics is used as the calibration function. The correlation of the apparent activity and enzyme concentration was established by capturing the enzyme by means of metal chelate interaction owing to a hexahistidine tag attached to the β-glucanase.
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Abbreviations
- A :
-
absorption (–)
- b :
-
regression coefficient (slope) (var. units)
- c :
-
molar concentration (mmol L−1)
- d :
-
path length of light (cm)
- E :
-
enzyme concentration (g L−1)
- f V :
-
dilution factor (–)
- k′2; k 2,app :
-
apparent rate constant (mmol L−1 min−1)
- K′M; K M,app :
-
apparent Michaelis constant (mmol L−1)
- Et :
-
normalized time of reaction (g min L−1)
- L :
-
productivity (mmol L−1 min−1)
- P :
-
product concentration (mmol L−1)
- r :
-
reaction rate (mmol L−1 min−1)
- S :
-
substrate concentration (mmol L−1)
- s′0M :
-
apparent saturation coefficient (–)
- t :
-
time of reaction (min)
- V :
-
volume (L)
- X :
-
substrate conversion (–)
- ε:
-
molar absorbance (L mol−1 cm−1)
- 0:
-
referring to initial conditions (t = 0; X = 0)
- app:
-
apparent
- cal:
-
referring to the calibration solution
- Glc:
-
glucose
- max:
-
maximal, maximum
- R:
-
reactor, referring to the assay conditions
- s:
-
sample
- rel:
-
relative (with respect to the maximum rate of reaction)
- V:
-
volume
References
Arrhenius S (1889) Ueber die Reaktionsgeschwindigkeit bei der Inversion von Rohrzucker durch Saeuren. Z Physik Chem 4:226–248
Dixon M, Webb EC (1964) Enzymes, 2nd edn. Longmans, Green and Co. Ltd., London, pp 145–166
Flaschel E (1992) Enzyme kinetics and reactor resign. In: Rozzell JD, Wagner F (eds) Biocatalytic production of amino acids and derivatives. Hanser Publishers, New York, pp 321–407
van Griethuysen E, Flaschel E, Renken A (1985) The influence of the ion content of whey on the pH-activity profile of the ß-galactosidase from Aspergillus oryzae. J Chem Tech Biotechnol 35B:129–133
Miksch G, Neitzel R, Fiedler R, Friehs K, Flaschel E (1997) Extracellular production of a hybrid β-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors. Appl Microbiol Biotechnol 47:120–126
Miksch G, Fiedler E, Dobrowolski P, Flaschel E (1997) Controlled secretion into the culture medium of a hybrid β-glucanase by Acetobacter methanolicus mediated by the kil gene of Escherichia coli located on a Tn5-derived transposon. Appl Microbiol Biotechnol 47:530–536
Beshay U, Miksch G, Friehs K, Flaschel E (2003) Production of a bacterial β-glucanase by expression in Escherichia coli and simultaneous adsorption on a metal chelate affinity resin. Arab J Biotechnol 6:187–194
Borris R, Olsen O, Thomsen KKK, von Wettstein D (1989) Hybrid Bacillus endo-(1-3,1-4)-β-glucanases: construction of recombinant genes and molecular properties of the gene product. Carlsberg Res Commun 54:41–54
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Zor T, Selinger Z (1996) Linearization of the Bradford protein assay increases its sensitivity. Theoretical and experimental studies. Anal Biochem 236:302–308
Walker AC, Schmidt CLA (1944) Studies on histidine. Archs Biochem 5:445–467
Foster RJ, Niemann C (1953) The evaluation of the kinetic constants of enzyme catalyzed reactions. Proc Natl Acad Sci USA 39:999–1003
Acknowledgments
One of the authors (U. Beshay) gratefully acknowledges the financial support from the Alexander von Humboldt-Foundation for a visiting fellowship to Bielefeld University, Germany.
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Beshay, U., Miksch, G. & Flaschel, E. Improvement of a β-glucanase activity test by taking into account the batch reactor balance of the test system. Bioprocess Biosyst Eng 30, 251–259 (2007). https://doi.org/10.1007/s00449-007-0121-4
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DOI: https://doi.org/10.1007/s00449-007-0121-4