Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characterisation.
The nitrile hydratase (NHase, EC 4.2.1.84) genes (alpha and beta subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (alpha subunit, M(r) 23 kDa) and 218 amino acids (beta subunit, M(r) 24 kDa) and the NHase activator of 413 amino acids (M(r) 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5-17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30 degrees C and showed the highest stability at 4 degrees C in thermal inactivation studies between 4 degrees C and 50 degrees C.
Rzeznicka
Kamila
K
Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, 17487 Greifswald, Germany.
Schätzle
Sebastian
S
Böttcher
Dominique
D
Klein
Joachim
J
Bornscheuer
Uwe T
UT
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008689:Methacrylates / Q000378:metabolism; D009570:Nitriles / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016929:Rhodococcus equi / Q000201:enzymology* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008689:Methacrylates; D009570:Nitriles; D011994:Recombinant Proteins; C008385:methacrylonitrile; D006836:Hydro-Lyases; C048831:nitrile hydratase