title: Optimal production of L-threo-2,3-dihydroxyphenylserine (L-threo-DOPS) on a large scale by diastereoselectivity-enhanced variant of L-threonine aldolase expressed in Escherichia coli.
abstract: This study examined the efficient production and optimal separation procedures for pure L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) from a mixture of diastereomers synthesized by whole-cell aldol condensation reaction, harboring diastereoselectivity-enhanced L-threonine aldolase in Escherichia coli JM109. The addition of the reducing agent sodium sulfite was found to stimulate the production of L-threo-DOPS without affecting the diastereoselectivity ratio, especially at the 50 mM concentration. The optimal pH for diastereoselective synthesis was 6.5. The addition of Triton X-100 also strongly affected the synthesis yield, showing the highest conversion yield at a 0.75% concentration; however, the diastereoselectivity of the L-threonine aldolase was not affected. Lowering the temperature to 10°C did not significantly affect the diastereoselectiviy without affecting the synthesis rate. At the optimized conditions, a mixture of L-threo-DOPS and L-erythro-DOPS was synthesized by diastereoselectivity-enhanced L-threonine aldolase from E. coli in a continuous process for 100 hr, yielding an average of 4.0 mg/mL of L-threo-DOPS and 60% diastereoselectivity (de), and was subjected to two steps of ion exchange chromatography. The optimum separation conditions for the resin and solvent were evaluated in which it was found that a two-step process with the ion-exchange resin Dowex 50 W × 8 and activated carbon by washing with 0.5 N acetic acid was sufficient to separate the L-threo-DOPS. By using two-step ion-exchange chromatography, synthesized high-purity L-threo-DOPS of up to 100% was purified with a yield of 71%. The remaining substrates, glycine and 3,4-dihydroxybenzaldehyde, were recovered successfully with a yield of 71.2%. Our results indicate this potential procedure as an economical purification process for the synthesis and purification of important L-threo-DOPS at the pharmaceutical level.
authors.affiliation: Department of Food Science and Human Nutrition, and Research Institute of Human Ecology, Chonbuk National University, Jeonju, Jeonbuk, Korea.
authors.forename: Hui-Jeong
authors.initials: HJ
authors.lastname: Gwon
authors.forename: Hideki
authors.initials: H
authors.lastname: Yoshioka
authors.forename: Nho-Eul
authors.initials: NE
authors.lastname: Song
authors.forename: Jong-Hui
authors.initials: JH
authors.lastname: Kim
authors.forename: Young-Ran
authors.initials: YR
authors.lastname: Song
authors.forename: Do-Youn
authors.initials: DY
authors.lastname: Jeong
authors.forename: Sang-Ho
authors.initials: SH
authors.lastname: Baik
mesh_terms: D000978:Antiparkinson Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019149:Bioreactors; D002852:Chromatography, Ion Exchange; D015103:Droxidopa / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics / Q000378:metabolism*; D007218:Industrial Microbiology / Q000191:economics / Q000379:methods*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D013237:Stereoisomerism
chemical_list: D000978:Antiparkinson Agents; D012696:Glycine Hydroxymethyltransferase; D015103:Droxidopa