Study On The Construction And Optimization Of Glucuronic Acid Biotransformation System

D-glucuronic acid,also referred to as glucuronlactone,is a very promising compound.Due to its good detoxification properties,it is currently widely used in medicine,health food and cosmetics.At present,the main preparation method of glucuronic acid is the starch-oxidation method with concentrated nitric acid,which has problems such as relatively severe reaction conditions,poor substrate selectivity,many by-products,serious environmental pollution,high energy consumption,and low yield.In recent years,with the requirements of environmental protection,biological methods have become a trend to replace chemical synthesis of this product.This thesis aims to construct a glucuronic acid bioconversion system and optimize it to establish an environmentally friendly,cost-effective bioconversion system under mild reaction conditions.The main contents in the present thesis are as follows:1.Construction of an glucuronic acid producing engineered bacteria and its expression study.Construct the recombinant plasmid p ETDuet-miox and transfer the recombinant plasmid into BL21 to construct the recombinant strain BL21/p ETDuet-miox.Through enzyme digestion experiments,it was verified that the recombinant vector was successfully constructed and the recombinant strain was induced to express the target protein.The results of electrophoresis showed that the MIOX protein was expressed.The optimal conditions for inducing expression of inositol oxidase were optimized,and the optimal induction conditions were obtained at an OD₆₀₀of 0.5;and induced at 26°C for 8 hours with 0.2 m M IPTG as inducer.2.The construction of a biocatalytic conversion system for the production of glucuronic acid from inositol.The inositol oxidase vector plasmid p ETDuet-miox was transformed into different expression hosts,and the best expression host was E.coli Rosetta(DE3)according to the bioconversion yield.Due to the complex composition of the fermentation broth in the laboratory shake flask fermentation,the concentration of the target product is low and the liquid phase determination of the fermentation broth has many impurity peaks which affect the accurate determination of the product.Therefore,a biocatalytic system with inositol oxidase as a catalyst is used,and the best catalytic form of inositol oxidase to catalyze the production of glucuronic acid by inositol oxidase is freeze-broken bacteria,and the catalytic reaction system is optimized.The best catalyzing conditions were as follows:in the MOPS buffer system with p H 7.5,the catalyzing temperature was 30?,the amount of bacteria added was 100 g/L wet bacteria,and the substrate concentration was 2 g/L.Under these conditions,the conversion rate of inositol was 80.3%,and the output of glucuronic acid reached 1.606g/L.3.Exploring the effect of Vitreoscilla hemoglobin on the glucuronic acid conversion system and the optimization of output.Construct the recombinant strain Rosetta/p ETDuet-miox-vgb.Exploring the effect of Vitreoscilla hemoglobin on the growth of E.coli,the strain Rosette/p ETDuet-miox-vgb compared with the strain Rosette/p ETDuet-miox,regardless of the high dissolved oxygen or low dissolved oxygen,the growth of the bacteria has been improved to a certain extent.Under high dissolved oxygen speed(250 r/min),the cell density increased by 5.08%,and under low dissolved oxygen speed(150 r/min),the cell density increased by 12.37%.It is proved that VHb can obviously promote the growth of bacteria under the condition of low dissolved oxygen.Through the determination of crude enzyme activity of the two strains,the crude enzyme activity of Rosette/p ETDuet-miox-vgb group reached 65.13 k U/L,which was increased by 62.61%compared with Rosette/p ETDuet-miox,indicating that VHb can significantly increase the activity of inositol oxidase..After the introduction of VHb,the catalytic conversion system was optimized.The optimal catalytic system substrate concentration was 4 g/L,the glucuronic acid output reached3.672 g/L,and the conversion rate reached 90.68%.Micro-addition of toluene can slightly increase the conversion rate of inositol,and the yield of glucuronic acid of 17.87 g/L can be obtained in the subsequent experiment of intermittent addition of bacteria.This project has successfully constructed a glucuronic acid bioconversion system,which not only provides a new method for glucuronic acid biosynthesis,but also lays a theoretical foundation for the low-cost industrial production of glucuronic acid.At same time,the present study also provide usable experience for other high value-added chemical product preparations with synthetic biology methods.