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Stereospecific Synthesis of ( R )‐2‐Hydroxy Carboxylic Acids Using Recombinant E. coli BL21 Overexpressing YiaE from Escherichia coli K12 and Glucose Dehydrogenase from Bacillus subtilis
Author(s) -
Yun Hyungdon,
Choi HyeonLok,
Fadnavis Nitin W.,
Kim ByungGee
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp049694w
Subject(s) - escherichia coli , bacillus subtilis , stereospecificity , recombinant dna , dehydrogenase , enzyme , lac operon , chemistry , biochemistry , carboxylic acid , stereoselectivity , bacteria , stereochemistry , biology , catalysis , gene , genetics
The yiaE gene from Escherichia coli K12 was functionally expressed in E. coli BL21 using an IPTG inducible pET expression system (2.1 U/mg), and YiaE was purified to a specific activity of 18 U/mg. The purified enzyme catalyzes reduction of various aromatic and aliphatic 2‐oxo carboxylic acids to the corresponding ( R )‐2‐hydoxy carboxylic acids using NADPH. For practical applications, the problem of NADPH recycle was effectively solved by using recombinant E. coli overexpressing YiaE and glucose dehydrogenase from Bacillus subtilis in the same cell. The recombinant E. coli was used to prepare ( R )‐phenyllactic acid and ( R )‐2‐hydroxy‐4‐phenylbutanoic acid from the corresponding 2‐oxo carboxylic acids (98% ee) while the α‐carbonyl group of 2,4‐dioxo‐4‐phenylbutyric acid was reduced regio‐ and stereospecifically to give ( R )‐2‐hydroxy‐4‐oxo‐4‐phenylbutyric acid (97% ee) in quantitative yields. The cells could be recycled for 3 days at room temperature in 100 mM phosphate buffer (pH 7.0) without loss of activity, which reduced to 70% after 1 week.