title: A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization.
abstract: In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V max and K m toward 3-cyanopyridine were 77.5 μmol min(-1) mg(-1) and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
authors.forename: Hualei
authors.initials: H
authors.lastname: Wang
authors.forename: Guinan
authors.initials: G
authors.lastname: Li
authors.forename: Mingyang
authors.initials: M
authors.lastname: Li
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
authors.forename: Xuedong
authors.initials: X
authors.lastname: Wang
mesh_terms: D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009570:Nitriles / Q000378:metabolism; D016366:Open Reading Frames; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D009570:Nitriles; D021122:Protein Subunits; D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase
references.citation: Protein Expr Purif. 2006 Jun;47(2):672-81
references.pmid: 16495079
references.citation: BMC Biotechnol. 2013 Feb 18;13:14
references.pmid: 23414071
references.citation: Eur J Biochem. 1990 Dec 27;194(3):765-72
references.pmid: 2269298
references.citation: J Appl Microbiol. 2003;95(6):1161-74
references.pmid: 14632988
references.citation: Microbiology (Reading). 2005 Nov;151(Pt 11):3639-3648
references.pmid: 16272385
references.citation: Bioprocess Biosyst Eng. 2011 Mar;34(3):315-22
references.pmid: 20960011
references.citation: Appl Microbiol Biotechnol. 2009 May;83(2):273-83
references.pmid: 19153727
references.citation: Appl Environ Microbiol. 2004 Apr;70(4):2429-36
references.pmid: 15066841
references.citation: Appl Microbiol Biotechnol. 2003 Apr;61(2):118-22
references.pmid: 12655453
references.citation: J Am Chem Soc. 2003 Sep 24;125(38):11476-7
references.pmid: 13129332
references.citation: J Biol Chem. 1992 Oct 15;267(29):20746-51
references.pmid: 1400390
references.citation: Arch Microbiol. 2006 Feb;184(6):407-18
references.pmid: 16341723
references.citation: J Biotechnol. 2008 Feb 1;133(3):327-33
references.pmid: 18061298
references.citation: Appl Microbiol Biotechnol. 2006 Dec;73(3):567-75
references.pmid: 17061133
references.citation: J Biotechnol. 2009 Aug 10;143(1):17-26
references.pmid: 19539670
references.citation: J Biotechnol. 2007 May 10;129(4):645-50
references.pmid: 17350705
references.citation: Appl Environ Microbiol. 2003 Aug;69(8):4359-66
references.pmid: 12902216
references.citation: Appl Microbiol Biotechnol. 2011 Feb;89(3):665-72
references.pmid: 20862583
references.citation: Appl Biochem Biotechnol. 2011 Dec;165(7-8):1556-67
references.pmid: 21938420
references.citation: Planta. 2001 Mar;212(4):508-16
references.pmid: 11525507
references.citation: Chem Biodivers. 2006 Dec;3(12):1279-87
references.pmid: 17193242
references.citation: J Bacteriol. 1990 Sep;172(9):4807-15
references.pmid: 2394676