Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 88, Issue 6, Pages 1277-1285Publisher
SPRINGER
DOI: 10.1007/s00253-010-2836-4
Keywords
4-Chloro-3-oxobutanoate ethyl ester; (S)-4-Chloro-3-hydroxybutanoate ethyl ester; Crude enzyme catalysis; Whole-cell catalysis
Categories
Funding
- Major Basic Research Program of China [2009CB724700]
- National Key Technology RD Program [2008BAI63B07]
- Innovation Fund for Doctoral Dissertation of Nanjing University of Technology [BSCX200911, KJ103736]
Ask authors/readers for more resources
A cofactor regeneration system for enzymatic biosynthesis was constructed by coexpressing a carbonyl reductase from Pichia stipitis and a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3) PlySs. Transformants containing the polycistronic plasmid pET-PII-SD2-AS1-B exhibited an activity of 13.5 U/mg protein with 4-chloro-3-oxobutanoate ethyl ester (COBE) as the substrate and an activity of 14.4 U/mg protein with glucose as the substrate; NAD(H) was the coenzyme in both cases. Asymmetric reduction of COBE to (S)-4-chloro-3-hydroxybutanoate ethyl ester [(S)-CHBE] with more than 99% enantiomeric excess was demonstrated by transformants. Furthermore, the paper made a comparison of crude enzyme catalysis and whole-cell catalysis in an aqueous monophasic system and a water/organic solvent biphasic system. In the water/n-butyl acetate system, the coexpression system produced 1,398 mM CHBE in the organic phase, which is the highest yield ever reported for CHBE production by NADH-dependent reductases from yeasts. In this case, the molar yield of CHBE was 90.7%, and the total turnover number, defined as moles (S)-CHBE formed per mole NAD+, was 13,980.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available