Abstract
Heat conditioning of cell homogenates of B. cereus and a recombinant E. coli was studied for the isolation of leucine dehydrogenase and alanine racemase, respectively. The strain of E. coli carried the gene of the thermostable alanine racemase from B. stearothermophilus. Activity loss can be minimized (<5%) and aggregation and flocculation of soluble proteins (70–80%) and other cell components can be achieved, depending on temperature, biomass concentration and pH-value.
Thereby a 3–6 fold increase in specific activity was obtained. The resulting extract after solid-liquid separation showed lower viscosity and less turbidity than unheated controls, making it more suitable for chromatographic separations.
Similar content being viewed by others
Abbreviations
- ADH :
-
alcohol dehydrogenase
- AlaR :
-
alanine racemase
- BM %:
-
biomass concentration
- D 1/s:
-
Shear rate
- η mPas:
-
viscosity
- EF :
-
enrichment factor
- LeuDH :
-
leucine dehydrogenase
- \(\dot Q\) KW:
-
heat rate
- RA %:
-
remaining activity
- RCF :
-
relative centrifugal force
- RP %:
-
remaining protein
- ϱ kg/dm3 :
-
density of homogenate
- T F°C:
-
temperature of feed
- T HI°C:
-
temperature of heating fluid (inlet)
- T HO°C:
-
temperature of heating fluid (outlet)
- K Pdm3/h:
-
volumetric flow rate of the feed
References
Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 (1976) 248–254
Brandts, J. F.: Heat effects on proteins and enzymes. In: Rose, A. H. (ed.): Thermobiology, pp. 25–72. London, New York: Academic Press 1967
Klibanov, A. M.: Stabilization of enzymes against thermal inactivation. Adv. Appl. Microbiol. 29 (1983) 1–28
Kroner, K. H.; Hustedt, H.; Kula, M.-R.: Purification of enzymes by liquid-liquid extraction. In: Fiechter, A. (ed.): Advances in Biochemical Engineering, vol. 24, pp. 73–118. Berlin, Heidelberg, New York: Springer 1982
Ohshima, T.; Wandrey, C.; et al.: Screening of thermostable leucine and alanine dehydrogenases in thermophilic Bacillus strains. Biotechnol. Lett. 7 (1985) 871–876
Ohshima, T.; Nagata, S.; Soda, S.: Purification and characterization of thermostable leucine dehydrogenase from B. stearothermophilus. Arch. Microbiol. 141 (1985) 407–411
Sanwal, B. D.; Zink, M. W.: l-leucin dehydrogenase from Bacillus cereus. Arch. Biochem. Biophys. 94 (1961) 430–435
Singleton, R.; Amelunxen, R. E.: Proteins from thermophilic microorganisms. Bacteriol. Rev. 37 (1973) 320–342
Schnell, J.; Kula, M.-R.: Investigations of heat treatment to improve the isolation of intracellular enzymes. Bioprocess Engineering 4 (1989) 129–138
Schütte, H.; Hummel, W.; Tsai, H.; Kula, M.-R.: l-leucin dehydrogenase from Bacillus cereus. Appl. Microbiol. Biotechnol. 22 (1985) 306–317
Schütte, H.; Kula, M.-R.: Purification of proteins and the disruption of microbial cells. Biotech. Progress 3 (1987) 31–42
Soda, K., et al.: Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization. Biochem. 25 (1986) 3268–3274
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kula, MR., Schnell, J. Investigations of heat treatment to improve the isolation of intracellular enzymes. Bioprocess Eng. 5, 31–38 (1990). https://doi.org/10.1007/BF00369644
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00369644