Purification And Characterization Of Nitrile Hydratase From Nocardia Sp. And Amidase From Bacillus Cereus

Organonitriles are widespread in plants,fungi,bacteria,arthropods and insect.In biosystems,three kinds of nitrile-converting enzymes transform nitriles to the corresponding carboxylic acids via two metabolic pathways.Due to the potentially extensive applications of amides and carboxylic acids in fine chemical and pharmaceutical synthesis, organonitrile biotransformation and biocatalysis,including unsaturated and saturated aliphatic nitriles,aromatic and heterocyclic nitriles,is of critical industrial importance.In the present dissertation,the purification methods and the properties of nitrile hydratase(NHase)and amidase were summarized. Nitrile hydratase from Nocardia sp.108 and amidase from Bacillus cereus ZJB-07112 were investigated in this paper.NHase from Nocardia sp.108 was purified to 10.8 fold with a yield of 24.3%through ultrasonication,precipitation,IEX and HIC.The enzyme is composed of two kinds of subunits:αsubunit(27.0-kDa)andβsubunit(32.1-kDa).Their N-terminal amino acid sequences were determined to be SEHVNKYTEY and MDGI,respectively.Its optimum pH and temperature for acrylonitrile hydration were pH 7.5 and 32℃respectively.It was sensitive to heat with a half-life of 1.29h at 40℃. Ca²⁺and Co²⁺can improve the enzyme activity,whereas most metal ions and EDTA caused obvious inhibition.The K_m and V_maxvalues of NHase for acrylonitrile were 172.42 mmol/L and 344.83μmol/(min·ml), respectively.Amidase from Bacillus cereus ZJB-07112 was purified to 33.3 fold with a yield of 21.8%through ultrasonication,IEX and HIC.It has a molecular mass of 60.6-kDa,estimated by SDS-PAGE.Its N-terminal sequence was ATIRPDDKAI.Its optimum pH and temperature for acrylamide hysrolysis were pH 7.5 and 35℃,respectively.The enzyme was unstable at temperatures above 50℃and its half-life reduced from 7.33h to 2.15h with temperature increased from 20℃to 40℃.Most of metal ions and EDTA had no significant effect on the enzyme activity, whereas Hg⁺,Ag⁺ and urea caused obvious inhibition.The K_m and V_max values of amidase for acrylamide were 2.64 mmol/L and 0.6μmol/(min·ml),respectively.