High-level expression of a novel FMN-dependent heme-containing lyase, phenylacetaldoxime dehydratase of Bacillus sp. strain OxB-1, in heterologous hosts.
We examined the overexpression of a novel FMN-dependent heme-containing lyase, phenylacetaldoxime dehydratase (Oxd) of Bacillus sp. strain OxB-1, in Escherichia coli and Bacillus subtilis. Several plasmids were constructed to express the enzyme under the control of the lac promoter or its own promoter, together with or without nitrilase and a possible regulatory protein that is present in the wild-type genome. The enzyme was expressed using E. coli transfected with the plasmid pOxD-9OF. Expression was under the control of the lac promoter in the pUC18 vector and was much more effective when the start codon was changed from TTG to ATG. When the transfected cells were grown at 37 degrees C, the enzyme was produced mainly in inactive inclusion bodies, whereas the enzyme was largely soluble and active when the cells were grown at 30 degrees C. The production of active enzyme was markedly enhanced by increasing the volume of culture medium. This had the effect of slowing the rate of apoenzyme synthesis. A slow rate of synthesis allows for a more efficient incorporation of heme cofactor into the apoenzyme than a fast rate of synthesis. Under optimized conditions, the enzyme was produced in an active and soluble form at 15,000U/L of culture, which is about 1500-fold higher than the amount produced by the wild-type strain. Moreover, the enzyme comprised over 40% of total extractable cellular protein.
Kato
Yasuo
Y
Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, Kosugi, Toyama 939-0398, Japan.
Asano
Yasuhisa
Y
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005486:Flavin Mononucleotide / Q000378:metabolism*; D015870:Gene Expression; D006418:Heme / Q000378:metabolism*; D008190:Lyases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D006418:Heme; D005486:Flavin Mononucleotide; D008190:Lyases; C404242:oxd protein, Bacillus