Summary
An improved method for the production ofl-leucine dehydrogenase is described employing a mutant with a constitutive enzyme and a fed-batch cultivation technique yielding high cell concentrations. Purification ofl-leucine dehydrogenase to homogeneity was carried out starting with 30 kgBacillus cereus cells by heat treatment at 63°C, followed by two liquid-liquid extraction steps and three conventional column chromatographies. Crystals have been obtained from the 95-fold purified enzyme. The molecular weight of the native enzyme was determined by sedimentation equilibrium and gel filtration studies to be 310 000 containing eight identical subunits with a molecular weight of 39 000. The sedimentation coefficient was estimated to 11.65 S. Branched-chain amino acids likel-leucine,l-valine orl-isoleucine are deaminated by the NAD-dependent enzyme. In the reverse reaction a variety of 2-ketoacids, especially 2-ketoisocaproate, 2-ketoisovalerate and 2-keto-3-methyl-valerate, were reductive aminated to the correspondingl-amino acids in the presence of 0.9 M ammonia. The amino acid composition for the subunit ofl-leucine dehydrogenase is presented.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Andrews P (1965) The gel filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J 96:595–606
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Edelhoch H (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 7:1948–1954
Flossdorf J (1980) Erweiterte Meßmöglichkeiten in der analytischen Ultrazentrifugation durch die Verwendung eines neuartigen Kollimators. Makromolekulare Chemie 181:715–724
von der Haar F (1976) Purification of protein by fractional interfacial salting out on unsubstituted agarose gels. Biochem Biophys Res Commun 70:1009–1013
Habeeb A (1972) Reaction of protein sulfhydryl groups with Ellman's reagent. Methods Enzymol 25:457–464
Hirs C (1967) Performic acid oxidation. Methods Enzymol 11:197–199
Hummel W, Schütte H, Kula M-R (1981) Leucine dehydrogenase fromBacillus sphaericus. Optimized production conditions and an effective method for its large-scale purification. Eur J Appl Microbiol Biotechnol 12:22–27
Hummel W, Schütte H, Kula M-R (1984) New enzymes for the synthesis of chiral compounds; in: Enzyme Engineering 7; New York: Ann NY Acad Science 434:194–205
Hustedt H, Kroner KH, Kula M-R (1984) Continuous enzyme purification by crosscurrent extraction. In: Third European Congress on Biotechnology, Vol. I, Verlag Chemie Weinheim, pp 597–605
Jovin T, Chrambach A, Maughton MA (1964) An apparatus for preparative temperature-regulated polyacrylamide gel electrophoresis. Anal Biochem 9:351–369
Kula M-R, Kroner KH, Hustedt H (1982) Purification of enzymes by liquid-liquid extraction. Adv Biochem Eng 24:73–118
Kroner KH, Hustedt H, Kula M-R (1984) Extractive enzyme recovery — Economic considerations. Process Biochemistry 19:170–179
Obermeier N, Poralla K (1976) Some physiological functions of thel-leucine dehydrogenase inBacillus subtilis. Arch Microbiol 109:59–63
Ohshima T, Misono H, Soda K (1978) Properties of crystalline leucine dehydrogenase fromBacillus sphaericus. J Biol Chem 253:5719–5725
Poralla K (1971) The induction of a dehydrogenase activity for branched chain amino acids inBacillus subtilis. Arch Microbiol 77:339–343
Schütte H, Kroner KH, Hummel W, Kula M-R (1983) Recent developments in separation and purification of biomolecules. In: Biochemical Engineering III, Ann NY Acad Science 413:270–282
Shapiro AL, Vinuela E, Maizel JV (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun 28:815–820
Svedberg T, Pedersen KO (1959) In: The Ultracentrifuge. Claredon Press Oxford 1940, reprinted by Johnson, New York, p 48
Tsai H, Yang C-Y, Tsai JHJ (1974) The subunit structure ofNeurospora tryptophan synthase: A reappraisal. Biochem Biophys Res Commun 61:1332–1339
Wandrey C, Wichmann R, Kula M-R, Bückmann F (1984a) Enzym-Membran-Reaktor, Technologietransfer: Beispiel Produktion vonl-Aminosäuren. Die Umschau 3:88–91
Wandrey C, Fiolitakis E, Wichmann U, Kula M-R (1984b)l-Amino acids from a racemic mixture of α-hydroxy acids. In: Enzyme Engineering 7, New York: Ann NY Acad Science 434:91–94
Wandrey C, Wichmann R, Berke W, Morr M, Kula M-R (1984c) Continuous cofactor regeneration in membrane reactors. In: Third European Congress on Biotechnology, Vol. I. Verlag Chemie, Weinheim, pp 239–244
Yamane T, Shimizu S (1984) Fed-batch techniques in microbial processes. In: Adv Biochem Eng Biotechnol (Fichter A, ed) 30:147–194
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schütte, H., Hummel, W., Tsai, H. et al. l-leucine dehydrogenase fromBacillus cereus . Appl Microbiol Biotechnol 22, 306–317 (1985). https://doi.org/10.1007/BF00582413
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00582413