Photo-controlled Reversible Self-assembly Of Nitrilase

Enzyme assembly is one of the hotspots areas in biocatalysis,bio-nanomaterials and synthetic biology.There are many methods of enzyme assembly,but one of the common drawbacks of these enzyme assembly methods is that they can not be precisely and reversibly controlled in the assembly process.Optical biology has become one of the new popular research fields because of its ability to achieve precise and reversible control over the time and space of biological processes.In this study,we combined enzyme assembly with optical biology,took use of the photoswitch protein AsLOV2 to achieve the enzyme assembly that can be precisely and reversibly controlled.The nitrilase gene BCNIT was fused with the photoswitch AsLOV2-SsrA into recombinant protein BNAS.In this work,a method for the photo-controlled reversible assembly and depolymerization of dodecamer nitrilase was developed by using the photoswitch-improved light-inducible dimer(iLID)system,which could induce protein-protein dimerization by blue light.The morphology of the assembly was characterized by scanning electron microscopy(SEM).The change of the particle size of the assembly of the extracellular light was analyzed by the dynamic light scattering experiment.The experimental results show that BNAS can polymerize into larger aggregates in a few seconds under 450 nm light conditions,but under dark conditions,the aggregates can be completely dissociated within 5 minutes.In addition,assembly under the light and dissociation in the dark maintain a cycle of more than five times.When BNAS and SspB protein were co-expressed,the formed assembly was labeled by a bimolecular fluorescent fragment.A stronger fluorescence signal was found to indicate that 450 nm blue light could induce the formation of intracellular assembly.The analysis of the activity of intracellular and extracellular light-controlled assemblies showed that the assembly kept good enzyme activity,and the assembly had better recovery and recycling that still maintain more than 90%of the enzyme activity after four cycles.This study describes the method of light controlled enzyme assembly,not only for homologous enzyme assembly,but also can be applied to the assembly of heterologous enzymes and cascade enzyme reactions that need to precisely control the biological process in the body.