Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(-)-mandelic acid production.
A nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli BL21 (DE3) in a soluble form. The encoded protein with a His₆-tag was purified to nearly homogeneity as revealed by SDS-PAGE with a molecular weight of approximately 38.5 kDa, and the holoenzyme was estimated to be composed of 10 subunits of identical size by size exclusion chromatography. The V(max) and K(m) parameters were determined to be 27.9 μmol min⁻¹ mg⁻¹ protein and 21.8 mM, respectively, with mandelonitrile as the substrate. The purified enzyme was highly thermostable with a half life of 155 h at 30 °C and 94 h at 40 °C. Racemic mandelonitrile (50 mM) could be enantioselectively hydrolyzed to (R)-(-)-mandelic acid by the purified nitrilase with an enantiomeric excess of 97%. The extreme stability, high activity and enantioselectivity of this nitrilase provide a solid base for its practical application in the production of (R)-(-)-mandelic acid.
Zhang
Zhi-Jun
ZJ
Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
Xu
Jian-He
JH
He
Yu-Cai
YC
Ouyang
Li-Ming
LM
Liu
You-Yan
YY
D000097:Acetonitriles / Q000737:chemistry; D000421:Alcaligenes / Q000201:enzymology* / Q000378:metabolism; D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002413:Cations, Divalent / Q000737:chemistry; D019976:Cloning, Organism / Q000379:methods; D004492:Edetic Acid / Q000737:chemistry; D004795:Enzyme Stability / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008333:Mandelic Acids / Q000138:chemical synthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature
D000097:Acetonitriles; D002413:Cations, Divalent; D008333:Mandelic Acids; D011994:Recombinant Proteins; C045516:mandelonitrile; D004492:Edetic Acid; D000619:Aminohydrolases; C021867:nitrilase; C037938:mandelic acid