Development Of Crude Enzyme Immobilization-based Cell-free System For Efficient N-acetylneuraminic Acid Biosynthesis

N-acetylneuraminic acid（Neu Ac）is a negatively charged functional monosaccharide widely found in a variety of plants,animals and microorganisms.Neu Ac has potential pharmaceutical applications owing to its anti-inflammatory,anti-cancer,antiviral,and anti-adhesion activities and the potential to modify drug carriers.Neu Ac is also an important additive in dairy products,which can enhance the immunity of infants and promote the development of brain and bone.The low yield and production efficiency of Neu Ac synthesized by traditional methods and environmental pollution hinder its industrial application.At the same time,the current research is to use the purified enzyme to synthesize Neu Ac by enzymatic method,which increases the production cost and is not conducive to industrial production.In this study,we modified the thermal stability of N-acetylneuraminic acid aldolase（Nan A）and developed a crude enzyme immobilization-based cell-free system（CEICFS）for the biosynthesis of Neu Ac.The main research contents of this paper are as follows:The main results of this study are as follows:（1）Heterologous expression of recombinant enzyme in Escherichia coli.Firstly,the gene bage encoding N-acetylglucosamine 2-epimerase（AGE）from Anabaena sp.CH1 and the gene nan A encoding Neu Ac aldolase（Nan A）from Staphylococcus hominis EFS20452.1 were cloned into the expression vector p ET28a and heterologously expressed in Escherichia coli.After that,the expression conditions of the recombinant enzyme were optimized.The crude enzyme obtained by heterologous expression was used for enzymatic synthesis of Neu Ac.Using 0.4mol·L^-1 Glc NAc and 1.0 mol·L^-1 pyruvic acid as substrates for 10 h,43.6 g·L^-1 Neu Ac was obtained,and the molar conversion rate of Glc NAc was 35.3%.（2）In order to improve the expression level of AGE and Nan A,the ability of solubilization tags,chaperones and N-terminal coding sequence（NCS）were used to improve protein expression.The results showed that although the solubilization tag could increase the expression of the protein,the activity of the recombinant enzyme was decreased.Molecular chaperones have little effect on increasing the expression of enzymes.Finally,NCS with a length of 30 nt was screened from the NCS library with green fluorescent protein（GFP）as the reporter protein,which could increase the expression of recombinant enzyme.The expression of AGE enzyme was increased by 1.5-fold,reaching 14.82 U·m L^-1,and the expression of Nan A enzyme was also increased by 1.2-fold,reaching 12.65 U·m L^-1.（3）In order to improve the thermal stability of AGE and Nan A,the semi-rational modification of enzyme was carried out to improve its thermal stability based on its protein sequence and structure.Site-directed mutagenesis of the screening sites of the two methods showed that the thermal stability of AGE was not significantly improved.Compared with Nan A,the activity retention of the mutant Nan A_S188C-A197C at 50°C was increased by 3.9-fold,and the half-life at 65°C was increased from 48.5 min to 95.3 min,the specific activity was increased by 1.25-fold.The kinetic parameters K_m,k_cat and V_max were 93.88 mg·m L^-1,16.52min^-1,and 5.20μmol·min^-1·mg^-1,respectively.The catalytic efficiency improved by 142%.Structural analysis and molecular dynamics simulation showed that the improvement of thermal stability of the mutant was mainly attributed to the increase of enzyme structural rigidity.（4）Study on immobilization of cell-free system.The carrier was screened according to the immobilization method,and the best immobilized carrier was determined to be amino resin LXTE-703.After the optimization of immobilization conditions,the immobilization conditions were determined as follows:25 m L AGE（Nan A）crude enzyme was added with 5 g LXTE-703resin and immobilized in a shaker at 25（30）°C,170 rpm for 12 h.The enzymatic properties of the CEICFS showed that the optimum reaction temperature and p H were consistent with the free enzyme,the temperature stability was higher than that of the free enzyme,and the p H adaptability was wider than that of the free enzyme.The K_m of the immobilized enzyme increased,indicating that the affinity of the enzyme to the substrate decreased.The yield of Neu Ac synthesized by CEICFS reached 68 g·L^-1,and about 78%of the enzyme activity could be retained after repeated use for 5 times.