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Production of recombinant L‐leucine dehydrogenase from Bacillus cereus in pilot scale using the runaway replication system E. coli [piet98]
Author(s) -
Ansorge Marion Bettina,
Kula MariaRegina
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(20000605)68:5<557::aid-bit10>3.0.co;2-j
Subject(s) - dehydrogenase , escherichia coli , chromatography , biochemistry , enzyme assay , bacillus cereus , enzyme , biology , recombinant dna , chemistry , bacteria , genetics , gene
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°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. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 557–562, 2000.