| Microfluidic biofilm catalytic reaction technology can be used to transform substrates to form target products,which is expected to be used in the directional transformation of natural products to prepare rare pharmacological molecules.However,due to the lack of whole-cell catalysts with fluorescence labeling,good catalytic performance and stability,its application is greatly limited.In this paper,the?-L-rhamnosidase obtained by heterologous expression of GH78 family gene rha B1 screened from elephant fecal metagenome in E.coli?Escherichia coli,E.coli?BL21?DE3?was used as the research object.The recombinant strain BL21-p ET-28-rha B1-?N1-EGFP was constructed for the first time by truncated mutation strategy to realize the fusion expression of Rha B1-?N1 and EGFP.The enzyme-producing bacterial biofilm can be controlled by self-designed segmented microfluidic technology and can be used for continuous biocatalytic hydrolysis of rutin to isoquercitrin.The main contents and results are as follows:?1?Under the guidance of structural analysis of?-L-rhamnosidase Rha B1,truncated mutants with good enzyme activity and stability were screened.The results showed that the molecular simulation Rha B1 has five domains,the C-terminal contains two conserved domains,and N1 is a redundant peptide.The predicted catalytic sites of Rha B1 are Asp655and Glu918,respectively.Eight truncated mutants were constructed around the catalytic domain of Rha B1,and four of them had enzyme activity?Rha B1-?N1,Rha B1-?N1-d E1,Rha B1-?N1-d E2,Rha B1-?N1-d E3?.Among them,Rha B1-?N1 had the highest enzyme activity,its molecular weight was 77.05 k Da,and the enzyme activity was 190.55 U/ml,which was 12.12%higher than that of Rha B1.The optimum temperature of Rha B1-?N1was 35°C,which was 5°C lower than that of Rha B1,and the residual relative enzyme activity was more than 90.12%after 60min treated at 30°C and 35°C.The optimum p H of Rha B1-?N1 was 6.5,and the remaining relative enzyme activity between p H6.0 and 6.5 was more than 92.32%.The catalytic performance of Rha B1-?N1 was tested with p NPR as substrate,and the Km of Rha B1-?N1 was 1.12mg/m L,which was 47.41%lower than that of Rha B1,and the catalytic efficiency Kcat/Km was 7.2 times higher.Therefore,the mutant Rha B1-?N1 of Rha B1 truncated by N1 has higher enzyme activity and stronger stability.?2?A simple and visual whole-cell catalyst for catalytic hydrolysis of rutin to isoquercitrin was constructed,and the affinity of Rha B1 and Rha B1-?N1-EGFP to rutin was analyzed by surface plasmon resonance?SPR?.The results showed that the optimum temperature of recombinant Rha B1-?N1-EGFP was 35°C,and the remaining relative enzyme activity was 89.24%after 60min treated at 35°C.the optimum p H was 6.5 and incubated at 35°C for 60 minutes,and the residual relative enzyme activity was above 94.15%at p H 6.0-6.5.the kinetic parameter of catalytic hydrolysis reaction with p NPR as substrate was 1.31mg/m L,which was 38.49%lower than that of Rha B1,indicating that its affinity to the substrate was higher.In the reaction of biocatalytic hydrolysis of rutin to isoquercitrin,the crude enzyme solution of Rha B1-?N1-EGFP was used as the catalyst,the optimum temperature was 35°C,and the optimum p H was 6.5.Under these conditions,the maximum yield was 93.69±0.39%after 8 h reaction.Using Rha B1-?N1-EGFP whole cell as catalyst,the optimum temperature is 40°C and the optimum p H is 6.0.Under these conditions,the maximum yield is 90.78±6.4%after 1.5h reaction,which is 81.25%shorter than that of crude enzyme solution.The equilibrium dissociation constants of Rha B1-?N1-EGFP and Rha B1with rutin were 7.05×10-6mol L-1 and 2.14×10-6mol L-1,respectively,indicating that both enzymes could specifically bind to the substrate.Therefore,Rha B1-?N1-EGFP whole-cell catalyst not only has tracer performance,but also has better catalytic performance and stability.?3?A microfluidic biofilm system with recombinant E.coli BL21 p ET-28-rha B1-?N1-EGFP was constructed and applied to catalyze the hydrolysis of rutin to isoquercitrin.The results showed that the best growth conditions of recombinant biofilm in microfluidic chip were as follows:the strain was modified by silane,the culture mode was"water-air"sectional flow,and the flow rate of water phase and air phase was 8?L/min,culture medium p H 7.0;The biofilm in the microchannel was characterized by fluorescence inversion microscope?Fluorescence inverted microscope,FIM?,Fourier transform infrared spectroscopy?Fourier transform infrared spectrometer,FTIR?and confocal laser scanning microscope?Confocal lasers canning microscope,LSCM?.It was found that the fluorescence intensity of the biofilm modified by silanization on the surface of the microchannel increased by 74%,and showed compact and flat film characteristics after being cultured under the condition of sectional flow for 48 hours.The recombinant strain was planted on the inner surface of microfluidic chip channel,and the biofilm was prepared by segmented microfluidic technology.When the concentration of rutin was 0.6g/L,the temperature was 35°C and p H was 6.5,the yield of isoquercitrin was 0.79?g/Ltube/d,which was increased by 2.25 times compared to the mutant Rha B1-?N1-d E1.Therefore,the microfluidic recombinant BL21-p ET-28-rha B1-?N1-EGFP biofilm can achieve controllable and stable growth,and can effectively continuously transform the substrate to form the target product.In summary,a new enzyme Rha B1-?N1 with high catalytic performance and stability with N1 domain truncated was obtained,and the whole cell catalyst E.coli BL21-rha B1-?N1-EGFP was successfully constructed,and the microfluidic biofilm catalytic system could be controlled to continuously transform rutin to isoquercitrin,which provided a new technology for glycoside orientation transformation of mulberry flavonoid glycosides. |
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