Construction Of Hybrid Multiple-Microcompartment Containing Coacervate And The Regulation Of Enzymatic Reaction

Compartmentalization provides a stable chemical environment for various chemical reactions in cells.In recent years,a variety of microcompartments have been constructed to mimic cell structures and functions with the development of synthetic biology.Among them,coacervate,as a kind of membrane-free compartment,has been extensively studied by researchers because of their ability to spontaneous formation and sequestration.However,real cells are not single compartments,but contain many internal regions and compartments with different compositions and physical properties,which are more complex in structure and play important roles in many cellular functions,making it more difficult to study.Therefore,it is necessary to construct a multiple compartmentalization model to mimic this structural feature for further understanding of cell function.In this paper,by designing hybrid multiplemicrocompartments containing with coacervate core step by step,the spatial position of active molecules,the formation of subcompartments,and the morphology of the internal subcompartment structure were studied to regulate the enzymatic reaction,in order to understand the role of cellular hierarchical structure in regulating cell functions such as biochemical reactions.The specific contents are as follows:A hierarchical structure of protein-membraned coacervate was designed and prepared by the self-assembly of pegylated proteins on the surface of coacervate,which was formed by the electrostatic interaction between positively charged quaternized amylose(Q-Am)and negatively charged succinylated amylose(Su-Am).The protein membrane on the surface of coacervate was formed by self-assembly of PEGylated bovine serum albumin(PEGy BSA),which could be in-situ monitored.It was found that PEGy BSA membrane could effectively improve the stability of the coacervate.By adding the molecules of different molecular weights,it was verified that the protein membrane had selective permeability to molecules and that the coacervate coated with PEGy BSA still remained the ability to sequester molecules.By introducing pegylated enzymes(PEGy GOx,PEGy HRP,PEGy Amylase),it was found that by regulating the spatial position of the enzymes on the membrane or inside the coacervate,the rate regulation of enzymatic cascade reaction and the "switch" control of enzymatic reaction were successfully achieved.The Pickering emulsion and phase transfer methods were used to realize the construction of a core-shell structure that proteinosome encapsulating coacervate to further increase the complexity of the artificial compartment.Spontaneous formation of coacervate core was realized by encapsulating oppositely charged polydiene dimethyl ammonium chloride(PDDA)and succinylated dextran(Su-Dex)within proteinosome during the process of phase transfer.It was found that the coacervate size was proportional to the concentration of the encapsulated components.With the concentration of the components increasing from 5 mg/m L to 50 mg/m L,the size of the coacervate increased from 5.47 μm to 10.01 μm.Based on the ionic strength response of the coacervate,dynamic behavior of the coacervate could be realized by regulating ionic strength,further realized the spatial dispersion and reunion of horseradish peroxidase.Utilizing the enrichment of coacervate on molecules,the increased local concentration of enzymes and the accelerated rate of enzymatic reaction were realized.Compared with hollow proteinosomes,the core-shell structure achieved a 15-fold enhancement in the ability to detect low-concentration toxin uric acid molecules.Based on the above-mentioned core-shell structure,a method for constructing a multiphase coexisting artificial compartment model was proposed by introducing a two-phase aqueous system to further enrich the internal compartments.Dextran phase around the coacervate core was induced by the addition of polyethylene glycol(PEG)into the proteinosomes co-encapsulating with quaternized dextran(Q-Dex),succinylated dextran(Su-Dex),and neutral dextran.It had been proved that coacervate phase and dextran phase had different fluidity and partition for DNA,HRP and PEGy GOx.Dextran had a protective effect on the DNA molecules within the coacervate,reducing the rate of enzymatic hydrolysis by half.Based on the regulation of the molecular weight of PEG on the morphology of multi-compartment,it was revealed that the interfacial tension was the main mechanism.The procedural adjustment of the internal multi-compartment was realized by adjusting the ionic strength,temperature and PEG concentration.In this process,the procedural adjustment of the enzymatic reaction rate was further realized by adjusting the spatial position of the enzyme and the substrate molecule.