Whole-Cell Catalytic Production Of N-Acetylneuraminic Acid And Its Isolation And Purification

N-acetylneuraminic acid（Neu5Ac）has a variety of biological functions and is widely used in the fields of medicine and food.The production of Neu5Ac by sustainable biotechnological methods has attracted more and more attention.The enzymatic method has greatly increased its production cost due to some disadvantages（e.g,low stability of enzymes and addition of cofactors）.Whole-cell catalysis has the advantages of simple operation process and easy product separation,but there are still problems of low conversion rate and high substrate cost.In this study,the crude N-acetylglucosamine（GlcNAc）fermentation broth was used to replace high-purity substrate GlcNAc for whole-cell catalytic production of Neu5Ac,and the catalytic conditions were further optimized to improve the conversion rate of the substrate.Neu5Ac in the transformation solution was separated and purified,and the optimal process conditions were determined.This study provided a certain reference for large-scale production and purification of Neu5Ac.The main findings of this study are as follows:（1）Two key enzymes,N-acetylglucosamine-2-epimerase（AGE）and N-acetylneuraminic acid aldolase（Nan A）,which can catalyze the synthesis of Neu5Ac from GlcNAc,were expressed in recombinant E.coli△NTE/p NSAS.When high-purity GlcNAc as the substrate was catalyzed by E.coli△NTE/p NSAS whole cells,the yield of Neu5Ac reached 319.5mmol·L^-1at 36 h,and the conversion rate was 53.2%.（2）For the recombinant E.coli ATLWX strain with high production of GlcNAc constructed in our previous work,its ability to produce GlcNAc was firstly investigated in shake flask.The results showed that the yield of GlcNAc reached 5.28 mmol·L^-1(1.17 g·L^-1)at 36 h.When GlcNAc was produced using a fed-batch in a 5 L fermentor,the optimal fermentation conditions were obtained as follows:the inoculum amount was 16%,the fermentation period was 45 h.The cell density OD₆₀₀ reached 135,and the yield of GlcNAc could reach 275.76mmol·L^-1(61.00 g·L^-1).（3）The GlcNAc fermentation broth obtained above was preliminarily separated and used as a substrate,and was whole-cell catalyzed to produce Neu5Ac.The cell density,the concentration of substrate sodium pyruvate,the initial pH of the transformation solution,the concentration of the surfactant Triton X-100,and temperature were optimized.The cell concentration,sodium pyruvate concentration,and the amount of surfactant added were further optimized by orthogonal experiments.The optimal transformation conditions were obtained as follows:the cell density OD₆₀₀ was 30,the sodium pyruvate concentration was 1.38 mol·L^-1,and the surfactant addition amount was 0.4%.Using the optimized conditions,the whole-cell catalytic amplification reaction was carried out in a 5 L fermenter.When the catalytic reaction was carried out for 70 h,the maximum yield of Neu5Ac reached 180 mmol·L^-1(55.67 g·L^-1),which was increased by 3 times compared with that before optimization,and its conversion rate was 65.45%.（4）The effect of different pHs on the stability of Neu5Ac in the transformation solution was studied,and the optimum pH was 4.0.The effect of the addition of activated carbon on adsorption and decolorization was investigated,and the optimum addition amount was 0.5%,and the decolorization time was 30 min.The separation and purification conditions of the conversion solution were systematically optimized,and the optimal conditions were obtained as follows:HZ-201 resin,the optimal static adsorption time of 90 min,and formic acid as the eluent.When the above optimal conditions were used to carry out dynamic adsorption,the flow rate of 1.5 m L·min^-1 and the linear elution of 0～0.65 mol·L^-1 formic acid were used to collect the eluent.Neu5Ac was concentrated and crystallized.The purified Neu5Ac was pure,purity of which reached 98.3%.Its crystallization recovery rate reached 68.3%.