Production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale using the runaway replication system E. coli[pIET98].
A method for the production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale is described employing the temperature induced runaway replication vector pIET98 and the Escherichia coli host strain BL21. Fed-batch cultivation using a semi-synthetic high-cell densitiy medium was adjusted in 5-L scale to yield a constant growth rate of 0,17 h(-1) and a final cell concentration of 27 g dry weight/L by exponentially increasing the nutrient supply. Runaway replication and thus, LeuDH expression was induced during the feeding phase by increasing the cultivation temperature to 41 degrees C yielding a specific enzyme activity of 110 U/mg, which corresponds to 30% of the soluble cell protein. The cultivation was terminated when the dissolved oxygen content fell below 10% saturation. The final volume activity was 600,000 U/L cultivation. No change in growth, cell density, or expression activity was observed scaling up the cultivation volume to 200 L. Thus, 120,000,000 units L-leucine dehydrogenase were obtained from one cultivation. The purification of L-leucine dehydrogenase to homogeneity was carried out by heat denaturation, liquid-liquid extraction, gel filtration, and anion-exchange chromatography to give pure enzyme in 65% yield. The integrity of the recombinant enzyme was tested measuring the molecular weight and determining the N-terminal amino acid sequence.
Ansorge
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MB
Institut für Enzymtechnologie der Heinrich-Heine-Universität Düsseldorf, D-52426 Jülich, Germany.
Kula
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MR
D000085:Acetates / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000302:isolation & purification; D001409:Bacillus cereus / Q000201:enzymology*; D002455:Cell Division; D003470:Culture Media; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics*; D050803:Leucine Dehydrogenase; D008970:Molecular Weight; D010100:Oxygen / Q000378:metabolism; D010865:Pilot Projects; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D013997:Time Factors
D000085:Acetates; D003470:Culture Media; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase; D010100:Oxygen