Summary
To develop a large-scale isolation of leucine dehydrogenase (E.C. 1.4.1.9) as industrial catalyst we carried out a limited screening for microorganisms with high leucine dehydrogenase activity.
Conditions for the growth and enzyme formation of Bacillus sphaericus (DSM 396) which proved to be the best enzyme producer were optimized. The highest yield in volume and specific activity were obtained using glucose and yeast-extract in the medium.
The highest specific enzyme activity was found at the end of the exponential growth phase. Cultivation of Bacillus sphaericus under optimal conditions increased the yield to about 3 U mg−1. The heat stability of the enzyme was exploited to develop a simple large-scale purification. Together with an ultrafiltration step, the enzyme could be enriched 9fold in a short time. After further purification using DE-cellulose an enzyme preparation (25fold enriched) was obtained; suitable as a technical catalyst in amino acid production.
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References
Aharonowitz Y, Friedrich CG (1980) Alanine dehydrogenase of the β-lactam antibiotic producer Streptomyces clavuligerus. Arch Microbiol 125:137–142
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
Bridson EY, Brecker A (1970) Design and formulation of microbial culture media. In: Norris JR, Ribbsons DW (eds) Methods in microbiology, vol 3A. Academic Press, London New York, pp 229–295
Lebeault JM, Zevaco C, Hermier J (1970) Purification and properties of the NAD-linked dehydrogenase involved in Bacillus subtilis spore germination. L-Alanine: NAD oxidoreductase (desaminating). Bull Soc Chim Biol 52:1073–1088
Ohshima T, Misono H, Soda K (1978) Properties of crystalline leucine dehydrogenase from Bacillus sphaericus. J Biol Chem 253:5719–5725
Samejima H, Kimura K, Ado Y (1980) Recent development and future directions of enzyme technology in Japan. Biochimie 62:299–315
Sanwal BD, Zink MW (1961) L-leucine dehydrogenase of Bacillus cereus. Arch Biochem Biophys 94:430–435
Soda K, Misono H, Mori K, Sakato H (1971) Crystalline L-leucine dehydrogenase. Biochem Biophys Res Commun 44:931–935
Wandrey C, Wichmann R, Bückmann AF, Kula M-R (1978) Immobilization of biocatalysts using ultrafiltration techniques. In: First European Congress on Biotechnology, Interlaken, pp 44–47
Wandrey C, Wichmann R, Bückmann AF, Kula M-R (1980) Immobilization of biocatalysts using ultrafiltration techniques. In: Weetall H, Royer G (eds) Enzyme engineering, vol V. Plenum Press, New York, pp 453–456
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Hummel, W., Schütte, H. & Kula, MR. Leucine dehydrogenase from Bacillus sphaericus. Optimized production conditions and an efficient method for its large-scale purification. European J. Appl. Microbiol. Biotechnol. 12, 22–27 (1981). https://doi.org/10.1007/BF00508114
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DOI: https://doi.org/10.1007/BF00508114