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Preparative synthesis of dTDP‐ L ‐rhamnose through combined enzymatic pathways
Author(s) -
Kang YoungBok,
Yang YungHun,
Lee KwangWon,
Lee SunGu,
Sohng Jae Kyung,
Lee Hei Chan,
Liou Kwangkyoung,
Kim ByungGee
Publication year - 2005
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/bit.20648
Subject(s) - rhamnose , enzyme , biochemistry , chemistry , chromatography , polysaccharide
dTDP‐ L ‐rhamnose, an important precursor of O‐antigen, was prepared on a large scale from dTMP by executing an one‐pot reaction in which six enzymes are involved. Two enzymes, dTDP‐4‐keto‐6‐deoxy‐ D ‐glucose 3,5‐epimerase and dTDP‐4‐keto‐rhamnose reductase, responsible for the conversion of dTDP‐4‐keto‐6‐deoxy‐ D ‐glucose to dTDP‐ L ‐rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti , functionally expressed in Escherichia coli , and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP‐4‐keto‐6‐deoxy‐ D ‐glucose involving TMP kinase, acetate kinase, dTDP‐glucose synthase, and dTDP‐glucose 4,6‐dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP‐ L ‐rhamnose using dTMP and glucose‐1‐phosphate as starting materials. About 82% yield of dTDP‐ L ‐rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose‐1‐phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP‐ L ‐rhamnose was obtained in an overall yield of 60% after two‐step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identified by HPLC, ESI‐MS, and NMR, showing about 95% purity. © 2005 Wiley Periodicals, Inc.