| α,ω-dicarboxylic acids(α,ω-DCAs),represented by sebacic acid,are an important intermediate in chemical industry,which have a wide range of applications in food and medicine,agricultural production,especially in the production of polymer materials.The traditional production process of sebacic acid based on chemical oxidation cracking/pyrolysis involves strong acid and alkali,toxic and harmful solvents,and has some defects such as high energy consumption,serious pollution and uncontrollable by-products.The development of biocatalytic technology provides a new opportunity for the green and sustainable production of sebacic acid.However,there are many limiting factors in the multi-enzyme cascade system of sebacic acid production,including:(1)the natural cofactor specificity of Baeyer-Villiger monooxygenase and secondary alcohol dehydrogenase does not match,and the cofactor cannot be self-recycled;(2)the sources of natural Baeyer-Villiger monooxygenases which can be used for biocatalytic synthesis of sebacic acid are rare,and they often have poor stability and low catalytic activity;(3)the optimum reaction conditions of each enzyme in the system are different,and the process parameters of the multi-enzyme system need to be optimized.This study aims to solve the above restrictive problems and develop an efficient and green multi-enzyme cascade system of sebacic acid.Firstly,the cofactor specificity of secondary alcohol dehydrogenase(Ml ADH)from Micrococcus luteus WIUJH20 was switched from NAD~+to NADP~+through directed evolution and semi-rational design based on molecular simulation technology.The yield of sebacic acid was increased by 3.7 times catalyzed by Ml ADH mutant(D37G-A38T-V39K)with NADP~+as a cofactor.Then an in situ NADP~+/NADPH regeneration self-cycling system was successfully constructed between Ml ADH mutant(D37G-A38T-V39K)and Baeyer-Villiger monooxygenase(Pa BVMO)from Pseudomonas aeruginosa and the final yield of sebacic acid in multi-enzyme cascade system was increased to 49.3%.The difficulty of product separation was simplified and the cost of enzymatic transformation was reduced after construction of in situ cofactor regeneration systemSecondly,the oxidation stability of Pa BVMO was improved based on hydrogen peroxide channel prediction and site-specific mutation,and the catalytic conversion rate of single mutant(Y404L)was increased by 37%.The thermal stability of Pa BVMO was improved based on B-factor calculation and directional saturation mutation,and the catalytic conversion of single mutant R354P and R354F was increased by 50%and 41%,respectively.On this basis,the catalytic conversion of double mutant(R354P-Y404L)was increased by 51%by iterative mutation.Finally,the process parameters of multi-enzyme cascade system were optimized.The final yield of sebacic acid reached to 78.34%when the optimal coupling catalytic ratio of Ml ADH mutant(D37G-A38T-V39K)and Pa BVMO mutant(R354P-Y404L)was 1:6.Then combined with single factor optimization and response surface analysis,the final yield of sebacic acid reached to 87.73%catalyzed at the optimal process parameters,including p H 8.8,reaction temperature at 26.5℃and the catalytic ratio of Ml ADH mutant(D37G-A38T-V39K)and Pa BVMO mutant(R354P-Y404L)was 1:6.5.The final yield of sebacic acid wad increased by 2.38 times compared with the multi-enzyme co-catalytic system adding GDH and NOX assisted cofactor regeneration system.In this study,an in situ cofactor regeneration in multi-enzyme cascade system of sebacic acid was constructed and the catalytic stability of key enzyme was improved based on the semi-rational design of enzyme molecules.The optimization of the process parameters of the multi-enzyme system was completed,which effectively improved the catalytic efficiency and yield of sebacic acid.An efficient and green multi-enzyme synthesis system provides a new way for the production of sebacic acid from natural fatty acids and a new technical support for the bio-refining of oil resources. |
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