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Cloning and Expression of D-Threonine Aldolase from Ensifer arboris NBRC100383
Authors: Sang-Ho Baik
Abstract:D-erythro-cyclohexylserine (D chiral unnatural β-hydroxy amino acid expected for the synthesis of drug for AIDS treatment. To develop a continuous bioconversion system with whole cell biocatalyst of D-threonine aldolase (D genes for the D-erythro-CHS production, D-threonine aldolase gene was amplified from Ensifer arboris 100383 by direct PCR amplication using two degenerated oligonucleotide primers designed based on genomic sequence of Shinorhizobium meliloti Sequence analysis of the cloned DNA fragment revealed one open-reading frame of 1059 bp and 386 amino acids. This putative D-TA gene was cloned into NdeI and EcoRI (pEnsi His-tag sequence or BamHI (pEnsi-DTA) sequence of the pET21(a) vector. The expression level of the cloned gene was extremely overexpressed by E. coli BL21(DE3) transformed with pEnsi-DTA compared to E. coli BL21(DE3) transformed with pEnsi-DTA. When the cells expressing the wild used for D-TA enzyme activity, 12 mM glycine was successfully detected in HPLC analysis. Moreover, the whole cells harbouring the recombinant D-TA was able to synthesize D-erythro of 0.6 mg/ml in a batch reaction.
Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1330655Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1435
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