Site-mutation Of Malic Enzyme Gene From Escherichia Coli K8 And Malic Acid Fermentation By Lactobacillus Delbrueckii

Malic acid is an important intermediate metabolite in the process of metabolic reactions of plants,animals and microorganisms.Because of its easy metabolism through citric acid cycle as well as special flavor and good anti-bactericidal activity,malic acid is widely used in food,medicine and household chemicals as additives and preservatives.Because of the extensive demand for malic acid,relative research has gained great progress.At present,the production of malic acid has developed from simple extraction from fruits to chemical synthesis,biological fermentation,enzyme catalysis and else.Chemical synthesis of malic acid has the advantage of briefness,controllable conditions.However,there are also obvious shortcomings,such as the great consumption of energy,complexity of separation and purification.Biological fermentation is another method that produce malic acid directly using the intrinsic metabolic pathway of malic acid in yeast,Aspergillus and other microorganisms.Through induced mutation,researchers screen high-yield strains,and fermented sugar and other substances to produce malic acid.At present,part of the methods have been applied in industrial production.Research and optimization of malic acid-biological production get more and more popular.Lactobacillus is a kind of bacteria producing an amount of lactic acid from pyruvic acid.Through changing the metabolic pathway of lactic acid to malic acid production pathway,Lactobacillus may be a good candidate in malic acid production.And the metabolites of Lactobacillus generally consist of kinds of acidic substances,which can react with carbonates to produce carbon dioxide to fulfill the production of malic acid.At the same time,it can also effectively alleviate the accumulation of acid in the fermentation broth to guarantee the growth of bacteria.This article is based on the work of malic acid fermentation metabolic pathway and mechanism analysis,using genetic engineering means to control the construction of malic enzyme mutated gene.Escherichia coli K-12 as the starting strain,using experimental methods to raise its genome,then amplifying the target fragment by polymerase chain reaction,combined with overlapping PCR to make it mutagenesis,lead to the No.104 Tyrosine mutate to Glutamine,and the malic enzyme mutations will be named SM-NAD-Y104Q.By cutting with restriction enzymes,put the SM-NAD-Y104Q post pET24b expression plasmid into Escherichia coli BL21(DE3),the strain which was used for highly expressing cloning the gene in an expression vector containing the phage T7promoter.It can be screened out the engineered strains which expressing SM-NAD-Y104Q by Kanamycin.By IPTG induced,Escherichia coli BL21(DE3)successfully expressed SM-NAD-Y104Q,after separation and purification of this protein,designed a redox reaction system by measuring NADH+H~+decreasing value at the reducing reaction in340nm absorbance to calculate ME-NAD-Y104Q reactivity,the results showed that the reverse reactivity of mutated malic enzyme had been successful promoted,means that SM-NAD-Y104Q can effectively combine pyruvate and carbon dioxide,converted them to L-malic acid.Making use of the shuttle plasmid pMG36e connect to SM-NAD-Y104Q to express inLactobacillus delbrueckii,and measured growth curve and malic acid fermentation characteristics of Lactobacillus delbrueckii for the future use of Lactobacillus delbrueckii as genetically engineered microorganism to produce L-malic acid,which provides empirical and experimental basis.