title: Clostridium thermocellum Nitrilase Expression and Surface Display on Bacillus subtilis Spores.
abstract: Nitrilases are an important class of industrial enzymes. They require mild reaction conditions and are highly efficient and environmentally friendly, so they are used to catalyze the synthesis of carboxylic acid from nitrile, a process considered superior to conventional chemical syntheses. Nitrilases should be immobilized to overcome difficulties in recovery after the reaction and to stabilize the free enzyme. The nitrilase from Clostridium thermocellum was expressed, identified and displayed on the surface of Bacillus subtilis spores by using the spore coat protein G of B. subtilis as an anchoring motif. In a free state, the recombinant nitrilase catalyzed the conversion of 3-cyanopyridine to niacin and displayed maximum catalytic activity (8.22 units/mg protein) at 40 °C and pH 7.4. SDS-PAGE and Western blot were used to confirm nitrilase display. Compared with the free enzyme, the spore-immobilized nitrilase showed a higher tolerance for adverse environmental conditions. After the reaction, recombinant spores were recovered via centrifugation and reused 3 times to catalyze the conversion of 3-cyanopyridine with 75.3% nitrilase activity. This study demonstrates an effective means of nitrilase immobilization via spore surface display, which can be applied in biological processes or conversion.
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, China.
authors.forename: Huayou
authors.initials: H
authors.lastname: Chen
authors.forename: Tianxi
authors.initials: T
authors.lastname: Zhang
authors.forename: Tengyun
authors.initials: T
authors.lastname: Sun
authors.forename: Zhong
authors.initials: Z
authors.lastname: Ni
authors.forename: Yilin
authors.initials: Y
authors.lastname: Le
authors.forename: Rui
authors.initials: R
authors.lastname: Tian
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chen
authors.forename: Chunxia
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000619:Aminohydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D009525:Niacin / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013171:Spores, Bacterial / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D011725:Pyridines; D011993:Recombinant Fusion Proteins; D009525:Niacin; D000619:Aminohydrolases; C021867:nitrilase; C020729:3-cyanopyridine