82890-22-0Relevant articles and documents
Directed Evolution Strategies for Enantiocomplementary Haloalkane Dehalogenases: From Chemical Waste to Enantiopure Building Blocks
van Leeuwen, Jan G.E.,Wijma, Hein J.,Floor, Robert J.,Van der Laan, Jan-Metske,Janssen, Dick B.
, p. 137 - 148 (2012)
We used directed evolution to obtain enantiocomplementary haloalkane dehalogenase variants that convert the toxic waste compound 1,2,3-trichloropropane (TCP) into highly enantioenriched (R)- or (S)-2,3-dichloropropan-1-ol, which can easily be converted into optically active epichlorohydrins-attractive intermediates for the synthesis of enantiopure fine chemicals. A dehalogenase with improved catalytic activity but very low enantioselectivity was used as the starting point. A strategy that made optimal use of the limited capacity of the screening assay, which was based on chiral gas chromatography, was developed. We used pair-wise site-saturation mutagenesis (SSM) of all 16 noncatalytic active-site residues during the initial two rounds of evolution. The resulting best R- and S-enantioselective variants were further improved in two rounds of site-restricted mutagenesis (SRM), with incorporation of carefully selected sets of amino acids at a larger number of positions, including sites that are more distant from the active site. Finally, the most promising mutations and positions were promoted to a combinatorial library by using a multi-site mutagenesis protocol with restricted codon sets. To guide the design of partly undefined (ambiguous) codon sets for these restricted libraries we employed structural information, the results of multiple sequence alignments, and knowledge from earlier rounds. After five rounds of evolution with screening of only 5500 clones, we obtained two strongly diverged haloalkane dehalogenase variants that give access to (R)-epichlorohydrin with 90% ee and to (S)-epichlorohydrin with 97% ee, containing 13 and 17 mutations, respectively, around their active sites.
Industrialization of the Microbial Resolution of Chiral C3 and C4 Synthetic Units: From a Small Beginning to a Major Operation, a Personal Account
Kasai, Naoya,Suzuki, Toshio
, p. 437 - 455 (2007/10/03)
This account describes the research and development of the microbial resolution of chiral C3 and C4 synthetic units through to the production stage. These chiral C3 and C4 synthetic units are mainly used for the production of various pharmaceuticals, new materials such as liquid crystals, chiral polymers, and natural compounds as well as in basic chemical research. The research started in 1983 and the industrial plant was built in 1994. The development is still ongoing and is being broadened to include C4 chiral units, chiral propylene glycol, and so on. This project started as simple research on the activated sludge from an epichlorohydrin plant and evolved through many events and much research to an industrial production. We describe the various implications and the flow of events in the research and development through to the production of these chiral C3 and C4 synthetic units.
Degradation of 2,3-Dichloro-1-propanol by a Pseudomonas sp.
Kasai, Naoya,Tsujimura, Kazuya,Unoura, Kinya,Suzuki, Toshio
, p. 3185 - 3190 (2007/10/02)
A bacterium that assimilates 2,3-dichloro-1-propanol was isolated from soil by enrichment culture.The strain was identified as Pseudomonas sp. by the taxonomic studies.The strain converted 2,3-dichloro-1-propanol to 3-chloro 1,2-propanediol, releasing chloride ions.The conversion was stereospecific because the residual 2,3-dichloro-1-propanol and formed 3-chloro-1,2-propanediol gave optical rotation.The resting cells converted various halohydrins to the dehalogenated alcohols, and cell-free extracts had strong epoxyhydrolase activity.These results indicated that the strain assimilated 2,3-dichloro-1-propanol via 3-chloro-1,2-propanediol, glycidol and glycerol.The possibility to manufacture optically active 2,3-dichloro-1-propanol is discussed.
