Synthesis Of Sodium Pyruvate From L-lactate By Whole Cell Transformation Of Recombinant Pichia Pastoris

Pyruvic acid is an important organic small molecules in biological system,oxidation function is cells play a key role in the process of the intermediate product,in the three major nutrients metabolism contact plays an important role in hub,is widely used in medicine and chemical industry,foodstuff industry,agricultural chemicals and the daily industrial products and other industries,the growing demand for its business.At present,the industrial production of pyruvate is mainly by chemical synthesis and fermentation methods,but the industrial production of pyruvate is limited due to the problems of low conversion rate,many by-products and difficulty in separation and extraction in chemical synthesis and fermentation methods.Therefore,it is of great economic and social significance to develop a new whole-cell conversion process for the production of sodium pyruvate and to achieve industrial production.This topic with high yield of glycolic acid oxidase recombinant pichia GS115-GO for starting strain,fermentation in 5 l fermenter process by moving kind of quantity,p H,culture temperature,methanol,methanol concentration,methanol induction time start induction time such as process optimization,improve the recombinant pichia GS115-GO fermented liquid of thalli concentration and ethanol acid oxidase activity.Under the conditions of methanol induction with a concentration of 0.3-1.5g/L at a transfer rate of15%,p H control at 4.5-5.0,fermentation temperature of 30?,and culture at 25 h,the wet weight of the recombinant pichia pichia gs115-go reached 241.47g/L for 60 h on a 5L fermentation tank,increasing by 33.8%,and the activity of glycolate oxidase reached8.9U/g,increasing by 59.3%.In order to further study the industrial application value of the strains,we of recombinant pichia GS115-GO seed pot culture medium optimization,seed expanding training and multistage fermentation process validation,and then,in accordance with the principle of dissolved oxygen consistency for 5 t fermentation tank recombinant pichia GS115-GO pilot magnification 60 h fermentation,recombinant pichia GS115-the biggest bacteria GO wet weight of 273.83 g/L,13.4% higher than that of 5 L fermentation level,glycolic acid oxidase activity of 9.16 U/g,2.92% higher than that of 5 L fermentation level.In the process of preparing sodium pyruvate by transforming l-lactic acid from gs115-go by recombinant pichia pichia,the cell permeability treatment process of gs115-go by recombinant pichia pichia was optimized to improve the cell transformation efficiency.The optimal permeability treatment process was 0.06% benzalkonium chloride for 60 min.On this basis,the substrate l-lactic acid concentration,cell concentration,p H,conversion temperature and other conversion conditions of the conversion system were optimized to improve the yield of sodium pyruvate and the conversion rate of l-lactic acid.Under the conditions of substrate l-lactic acid concentration of 90g/L,recombinant pichia pichia cell concentration of 80g/L,p H value of 7.5-8.0,and conversion temperature of 15?,the concentration of sodium pyruvate reached 187.9g/L,the l-lactic acid conversion rate reached 91.15%,and the reaction rate reached 2.93g/L/h.In view of the whole cell biological catalysis by-products of less,easy purification and pyruvic acid ion unstable characteristics,to establish the extraction and purification technology of sodium pyruvate and decoloring technique in the process of extraction,enrichment cooling crystallization process and alcohol crystallization process is optimized,the preparation of sodium pyruvate crystals,content of 99.8%,the purity was 99.7%,and the yield was 72%.This project USES whole cell biocatalysis to prepare sodium pyruvate,the by-product is few,the conversion rate is high,the pollution to the environment is small,conforms to the idea of green development.The extraction and purification of sodium pyruvate by alcohol crystallization is simple and can save production cost.At the same time,it lays a foundation for the industrial application of sodium pyruvate and further meets the domestic and foreign market demand for sodium pyruvate products.