| Methionine hydroxy analogue is the ananlogue of methionine,in which the a-amino(-NH2)of methionine was substituted by the hydroxy(-OH).Methionine hydroxy analogue was first developed as the alternative of methionine by Monsanto Co.It can be absorbed by animals and transformed to the active L-methionine with the catalysis of various enzymes,and it plays the same biological role as methionine.It is of great significance to develop the production of methionine hydroxy analogue.Compared to the chemical methods,biocatalysis provides some advantages such as mild reaction condition,less by-products,and environment-friendly.In this paper,the efficient hydrolysis of 2-hydroxy-4-(methylthio)butyronitrile to methionine hydroxy analogue by nitrilase was developed.In this work,the recombinant strain E.coli BL21(D3)/pET-28b-NIT was screened to prepare methionine hydroxy analogue.The product was determined as the methionine hydroxy analogue by high performance liquid chromatography,mass spectra(MS)and nuclear magnetic resonance(NMR).The catalytic conditions for hydrolysis of 2-hydroxy-4-(methylthio)butyronitrile to methionine hydroxy analogue were investigated.It was showed the optimal temperature was 65℃,and the apparent activation energy Ea was 20.14 kJ-mol-1.The half-lives of nitrilase activity at 50℃,55℃,60℃,65℃ and 70℃ were 44.9h,18.8h,15.2h,5.7 hand 2.2 h,respectively.The optimal pH of the reaction was 7.0.The batch experiments showed that only 36.7%of the initial activity was remained after five batches.The maxium reaction rate of 1346 μmol·min-1·g-1DCW was obtained when the substrate concentration was 150 mM and the reaction rate would decreased at concentration above 150 mM.Furthermore,the substrate specificity analysis indicated that the nitrilase also exhibited high activity towards aromatic nitriles.The process of the nitrilase immobilization and the catalytic property of immobilized enzyme were investigated.Separate solution of sodium metasilicate and amines were mixed to obtain a 30 mM silicic acid with a molar ratio of[Si]:[N]=1:1 and 1.0 mg·mL-1 free enzyme concentration.The immobilization process was performed in 80 mM PBS buffer at pH 7.0(or water).The immobilized enzyme showed the highest activity at 70℃ and pH 6.5(100 mM Citric acid/Sodium citrate buffer).It showed the stabilities of immobilized enzyme including thermostability,pH stability,storage stability and operational stability were obviously improved.The maxium substrate concentration could reach 200 mM with immobilized enzyme. |
