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Recombinant Chlorobenzene Dioxygenase from Pseudomonas sp. P51: A Biocatalyst for Regioselective Oxidation of Aromatic Nitriles
Author(s) -
Yildirim Selcuk,
Franko Telma T.,
Wohlgemuth Roland,
Kohler HansPeter E.,
Witholt Bernard,
Schmid Andreas
Publication year - 2005
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.200505075
Subject(s) - chemistry , biotransformation , pseudomonas putida , enantiomeric excess , dihydroxylation , biocatalysis , nitrilase , dioxygenase , escherichia coli , chlorobenzene , bioreactor , pseudomonas fluorescens , stereochemistry , organic chemistry , enzyme , biochemistry , catalysis , bacteria , enantioselective synthesis , gene , ionic liquid , genetics , biology
An efficient biocatalyst was developed for the cis ‐dihydroxylation of aromatic nitriles. The chlorobenzene dioxygenase (CDO) genes of Pseudomonas sp. strain P51 were cloned under the strict control of the P alk promoter of Pseudomonas putida GPo1. Escherichia coli JM101 cells carrying the resulting plasmid pTEZ30 were used for the biotransformation of benzonitrile in a 2‐L stirred tank bioreactor. Use of a stable expression system resulted in an average specific activity and an average volumetric productivity of 1.47 U/g cdw and 120 mg of product/h/L, respectively. The values represent a three‐fold increase compared to the results of the similar biotransformations with E. coli JM101 (pTCB144) where the genes of CDO were expressed under the control of lac promoter. The productivity of the cis ‐dihydroxylation process was limited by product toxicity. Removal of the products at toxic concentrations by means of an external charcoal column resulted in an additional increase in product concentration by 43%. E. coli JM101 (pTEZ30) was further used for the regio‐ and stereospecific dihydroxylations of various monosubstituted benzonitriles, benzyl cyanide, and cinnamonitrile. Biotransformations resulted in products with 42.9–97.1% enantiomeric excess. Initial enzymatic activities and isolated yields were obtained in the range of 1.7–4.7 U/g cdw and of 3–62%, respectively.