title: Protein engineering of a nitrilase from Burkholderia cenocepacia J2315 for efficient and enantioselective production of (R)-o-chloromandelic acid.
abstract: The nitrilase-mediated pathway has significant advantages in the production of optically pure aromatic α-hydroxy carboxylic acids. However, low enantioselectivity and activity are observed on hydrolyzing o-chloromandelonitrile to produce optically pure (R)-o-chloromandelic acid. In the present study, a protein engineering approach was successfully used to enhance the performance of nitrilase obtained from Burkholderia cenocepacia strain J2315 (BCJ2315) in hydrolyzing o-chloromandelonitrile. Four hot spots (T49, I113, Y199, and T310) responsible for the enantioselectivity and activity of BCJ2315 were identified by random mutagenesis. An effective double mutant (I113M/Y199G [encoding the replacement of I with M at position 113 and Y with G at position 199]), which demonstrated remarkably enhanced enantioselectivity (99.1% enantiomeric excess [ee] compared to 89.2% ee for the wild type) and relative activity (360% of the wild type), was created by two rounds of site saturation mutagenesis, first at each of the four hot spots and subsequently at position 199 for combination with the selected beneficial mutation I113M. Notably, this mutant also demonstrated dramatically enhanced enantioselectivity and activity toward other mandelonitrile derivatives and, thus, broadened the substrate scope of this nitrilase. Using an ethyl acetate-water (1:9) biphasic system, o-chloromandelonitrile (500 mM) was completely hydrolyzed in 3 h by this mutant with a small amount of biocatalyst (10 g/liter wet cells), resulting in a high concentration of (R)-o-chloromandelic acid with 98.7% ee, to our knowledge the highest ever reported. This result highlights a promising method for industrial production of optically pure (R)-o-chloromandelic acid. Insight into the source of enantioselectivity and activity was gained by homology modeling and molecular docking experiments.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Hualei
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Wenyuan
authors.initials: W
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Huihui
authors.initials: H
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Lifeng
authors.initials: L
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Lujia
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, People's Republic of China xdwang@ecust.edu.cn dzhwei@ecust.edu.cn.
authors.forename: Xuedong
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, People's Republic of China xdwang@ecust.edu.cn dzhwei@ecust.edu.cn.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000619:Aminohydrolases / Q000235:genetics* / Q000378:metabolism; D019149:Bioreactors / Q000382:microbiology*; D057508:Burkholderia cenocepacia / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D002626:Chemistry, Pharmaceutical / Q000379:methods; D008333:Mandelic Acids / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
chemical_list: C541344:2-chloromandelic acid; D008333:Mandelic Acids; D000619:Aminohydrolases; C021867:nitrilase