Design,Synthesis,Purification And Immobilization Of Recombinase

Enzymes,a natural catalyst,have the characteristics of high substrate and product specificity and high catalytic efficiency in the process of catalysis.However,the complicated separation and purification process,unstable and unrecyclable shortcomings hinder the industrial application of enzymes.In order to overcome these weaknesses and make the most advantages of enzyme's excellent performance,an integrated strategy was proposed for production,purification and catalytic application of enzyme.In this study,two?-glucosidases?GLEGB,GLEH?containing fusion tags were constructed and expressed.On the one hand,the one-step rapid separation and purification of target enzyme molecule was achieved by using the temperature-responsive behavior of ELP-tag.On the other hand,the immobilization of enzyme on support materials was enhanced by GB and His-tags.The details are as follows:?1?A binary ELP&GB-tag recombinant?-glucosidase vector pET-GLEGB,ELP&His-tagged recombinase vector pET-GLEH.The construction of recombinant plasmid was verified by double restriction enzyme digestion and PCR,and the correct gene sequence was obtained by gene sequencing..?2?The?-glucosidase vector was transfected into the receptor strain BL21 and induced to express at 25°C.The crude extract containing the target enzyme was purified by ELP under the conditions of 25°C and different concentrations of?NH₄?₂SO₄.The optimum concentration of?NH₄?₂SO₄ was 0.5 M according to the recovery rate of enzyme activity and purification fold.The activity recovery of the target enzyme was 97.2%by one-round ITC method,which was better than 80.6%of the commercial affinity Ni-NTA resin.?3?The strong hydrophobicity of the GB-tag was utilized to generate a strong adsorption force with the hydrophobic carbon material,thereby realizing the stable immobilization of GLEGB onto the surface of support materials by simple adsorption method.The loading amount of immobilized GLEGB using GO and C₃N₄ can reach The loading amount of GLEGB immobilized on GO and C₃N₄ reached 698.2 and 527.3 mg g^-1,respectively.At the same time,the ELP-tag also improved the thermal stability of?-glucosidase.After 30 min in water bath at 60°C,the remaining relative activities of free GLE and GLEGB were 45.6%and56.3%,respectively,while that of free Glu was only 4.0%.The storage stability and reusability of free enzymes and immobilized enzymes containing ELP and GB tags have been significantly improved.?4?In order to further enhance the operational stability and recycling performance of the immobilized enzyme,the biomineralization technology was applied to the immobilization of recombinase into microscale hybrid organic-inorganic nanoflowers self-assembled by nanoscale petals.The effects of copper ions concentrations,enzyme concentrations,reaction temperature and time on the morphology and catalytic properties of composite nanoflowers GLEH-NF were analyzed,and its formation mechanism was proposed.The biomineralization of the GLEH-NF was achieved by ultrasonic assisted method within 15 min with a high encapsulation yield?81.2%?and activity recovery?90.3%?.Compared to free GLEH,GLEH-NF had a relative enzyme activity of 128.7%.The immobilized enzyme prepared by the proposed method showed high stability and retained nearly 70%of its original activity after repeated use for 16 times..