Expression Of Recombinant AiiA Protein And Analysis Of Its Enzymatic Properties

The gram-negative bacteria are the mainly pathogens of plant, pathogenic bacteria regulate the expression of their virulence factors through quorum sensing (QS). And N-acyl homoserine lactone (AHL) is the key signaling molecule of QS system. In a relatively closed environment, when the concentration of AHL continues to accumulate and reaches a certain threshold, the promoter of pathogenic agents in the QS system will be activited, and to promote the expression of pathogenic factors, to cause the pathogenicity of plants. Reducing the concentration of AHL is the key to prevention and treatment of such diseases. The AiiA protein expressed by gene aiiA from Bacillus thuringiensis can hydrolyze the AHL on the lactone ring, thereby using AiiA to reduce the concentration of AHL, blocking or even destroying the quorum sensing system of Gram-negative bacteria is expected to become a new way of biological prevention and control of plant diseases caused by gram-negative bacteria.A composite experimental design was used to optimize the fermentation conditions for maximizing the productivity of AiiA protein from engineering bacterial E.coli BL21 (DE3)-pET29a-aiiA. It was found that the optimal fermentation media for the soluble expression of AiiA is beef extract peptone, Higher yields were obtained after optimizing media components and conditions of fermentation. Maximum soluble AiiA production was obtained at pH7.0,37.0℃,20mmol/L trace elements of Na2HPO4,250mL flask with 70mL media, and 20h induction fermentation by 0.6 mmol/L IPTG added into the media when the strains grown to OD6oo0.5. Under the best conditions, the relative amount of soluble AiiA protein was 4.54 mg/mL, and the soluble AiiA constituted up to 60.3%of the target protein, compare to 2.23 mg/mL soluble AiiA constituted up to 31.4%of the target protein before optimizing.AiiA protein was purified after the culture and induction of recombinant bacteria E.coli BL21 (DE3)-pET29a-aiiA. The enzymatic characteristics of AiiA protein was investigated. It was found that the optimum temperature of AiiA catalyzed reaction is 28℃, the optimum reaction pH is 6.5; AiiA protein is sensitive to temperature, the enzyme activity remained about 81.8%after incubated at 28℃for 30min, but the enzyme activity remained about 19.1%after incubated at 37℃for 30min. The enzyme activity was almost completely lost after incubated at 45℃for 30min. The remaining relatively enzyme activity of AiiA protein maintained at 85%after stored 24h at 4℃. After 96h, the remaining activity is only 20%and the activity is almost completely lost activity when after 120h;Under the measure system of pH6.5,28℃, keep the AHL substrate (S) concentration on 0.4mmol/L,0.6 mmol/L,1.0 mmol/L, 2.0 mmol/L, the initial velocity of enzymatic reaction (V0) was determined.1/[S] do the Lineweaver-Burk diagram on 1/Vo. It was found that the Km and Vmax of AiiA protein were respectively 1.25mmol/L and 25umol/L/min. The reseach of enzymatic properties of AiiA protein was not only good for further study of enzyme structure and function, providing useful data to comparative enzymology of biological differences between different sources, but also good for exploring the future role of protein in the mechanism AiiA the theoretical foundation, for Transformation of the structural characteristics of AiiA protein provides a theoretical basis.The amino acids located in 57th,62th,65th,195th and 206th of AiiA protein were site-directed mutated. The 57th amino acids was transformed from the Ala into Pro; the 62th amino acids from Gly to Ala; the 65th amino acids from Asn to Lys; the195th amino acids from Thr to Asp; the 206th Amino acids from Ala to Glu. It was found that the activity of AiiA-65-12, AiiA-195-12 and AiiA-206-6 had improved in some extent. Compared to the wild type, the optimal temperature of AiiA-206-6 was 37℃, increased 9℃. The improvement of optimal temperature of AiiA-206-6 maybe due to the strong negative charged amino acids Glu which imcrease the binding force between AiiA and Zn2+that improved the stability of the enzyme activity center. After incubated at 37℃for 30min, the enzyme activity of AiiA-65-12 still maintained 90%, compared to wild-type 19.1%. The enzyme activity of AiiA-195-12 was no dramatic decline in the process of storage at 4℃, indicated that the stability of the enzyme was increased. Compare to the wild-type AiiA, either the temperature stability or the 4℃storage stability of AiiA-65-12 and AiiA-195-12 mutant have been obvious increased.The recombinant vector of GS115-pPIC9K-aiiA was constructed by the eukaryotic expression vector pPIC9K, and preliminary identification the expression of AiiA protein.