协同两类腈水合酶高效催化烟酰胺及丙烯酰胺生物合成

Efficient production of nicotinamide and acrylamide with co-expression of two kinds of nitrile hydratase

腈水合酶(nitrile hydratase,NHase)可以将腈类物质通过水合作用转化生成更有利用价值的酰胺类化合物,在工业上被用于生产烟酰胺、丙烯酰胺等重要化学品。然而,尚无兼具对底物和产物耐受性高以及催化活性高的腈水合酶,导致底物不能被彻底利用、酰胺产物的产量受限。该研究协同表达了高催化活性的低分子质量腈水合酶(L-NHase)和高耐受性的高分子质量腈水合酶(H-NHase),并优化两类酶在质粒上的排列位置。其中BAE-BAG菌株对底物、产物的耐受性与H-NHase菌株相仿;在以烟腈或者丙烯腈为底物进行全细胞催化时,该菌株的催化速率与L-NHase菌株相仿,最终烟酰胺产量可达到516 g/L,是目前报道的最高产量;丙烯酰胺产量比原始BAE或BAG菌株提高19.1%以上。该文成功协同两类腈水合酶,同时提高了合成烟酰胺和丙烯酰胺的催化反应速率及产量,对工业应用具有一定的指导意义。

Nitrile hydratase(NHase)can hydrate nitrile substrates to generate more valuable amides,which are used industrially in the production of important chemicals such as nicotinamide and acrylamide.These amides are very important organic compounds,widely used in medicine,pesticide and chemical related fields.Most nitrile hydrases with high activity have lower thermal stability,and the reaction temperature should be strictly controlled at lower level in industrial application.In addition,nitrile hydrase has low tolerance to nitrile substrates and amide products,and is prone to inactive at the later stage of the reaction,and the amide products can only be attained at a low level.There is no nitrile hydrase with both high tolerance and high catalytic activity.As a result,the substrate could not be completely utilized with limited production of amide products.To the best of our knowledge,nitrile hydrase with higher stability or tolerance is mainly obtained by screening nitrile hydrase from different strains or modifying nitrile hydrase,but there is no report of providing more efficient nitrile hydrase cell catalyst through co-expressing different kinds of nitrile hydrases.In this study,we co-expressed low-molecular-mass nitrile hydrase(L-NHase,encoded by BAE)and high-molecular-mass nitrile hydrase(H-NHase,encoded by BAG),which possess high catalytic activity and high tolerance,respectively.Their positions on plasmids were also optimized to achieve the optimal expression proportion in the cell.Therefore,the respective advantages of L-NHase and H-NHase were combined to improve the reaction rate and the final concentration of amide products and promote the complete conversion of substrate,using whole cell catalyst.The main results are as follows.(1)Analysis of the tolerance of the recombinant strains.When incubated in 0.8 mol/L niconitrile for 30 min,50%of the enzymatic activity of the BAG-BAE and BAE-BAG strains was remained,and the BAE strain almost lost its catalytic activity.When incubated in 1.5 mol/L nicotinamide,approximately 80%of the enzymatic activity of the BAG-BAE and BAE-BAG strains were remained,and the BAE strain almost lost its activity.When incubated in 2 mol/L acrylonitrile,the residual enzymatic activity of the BAG-BAE and BAE-BAG strains were higher than 80%.When incubated in 5 mol/L acrylamide,the residual enzymatic activities of the BAG-BAE and BAE-BAG strains was about 60%,and the BAE strain almost lost its catalytic activity.Therefore,this study synergistically expressed both L-NHase and H-NHase and retained the high tolerance to the substrates and products by the recombinant strains.(2)Whole-cell catalytic synthesis of nicotinamide.By over-expressing L-NHase with H-NHase,the BAE-BAG strain produced 400 g/L nicotinamide at 70 min,and the final nicotinamide titer reached 516 g/L.The BAG-BAE strain produces 300 g/L nicotinamide at 70 min,and the final nicotinamide titer reached 375 g/L.These results demonstrated that the goal of simultaneous increasing catalytic rate and product yield was achieved.(3)Whole-cell catalytic synthesis of acrylamide.The catalytic rate of the BAE strain for acrylamide synthesis from acrylonitrile was significantly higher than the BAG strain.Collaborative expression of L-NHase and H-NHase made the catalytic reaction rates of the BAE+BAG,BAE-BAG and BAG-BAE strains close to that of the BAE strain.The average reaction rate was increased by approximately 92.2%compared to the BAG strain.The reaction time of BAE-BAG and BAG-BAE strains was extended to 150 min.The final acrylamide titer was 1.19-fold higher than the BAE strain and 1.18-fold higher than the BAG strain.These results showed that the synergistic expression of L-NHase and H-NHase significantly improved the catalytic rate and titer of acrylamide.This study successfully co-expressed two kinds of nitrile hydratases,and improved the catalytic reaction rate and titer of nicotinamide and acrylamide,which would be significant for industrial applications.