Construction And Expression Of Fluorinase Engineering Strain

It is common believed that fluorinated nucleosides have significant advantages in anticancer and antiviral therapy.Fluorinated modification of nucleaside analogues has become an important research fields of fluorinated chemistry.However,due to the complicated steps and harsh conditions of chemical fluorine modification,enzymatic conversion becomes potential choice because of environment friendly and simple process.While,only six strains have been reported to catalyze natural fluoride metabolites with low catalytic efficiency.This study attempts to screen the strains from natural environment.At the same time,the engineering bacteria with synthetic fluorinase gene were constructed to transform S-adenosyl-L-methionine(SAM).Also the microbial community was analyzed and the catalytic parameters were optimized.Site directed mutagenesis was used to improve the activity of fluorinase and the product 5?-FDA was primary purified and identified.The main results are as follows:(1)Strains isolation and microbial community analysis.Soil and water samples were collected from fluorite mine and fluorinated chemical products factory,the microbial community from obtained environmental samples were analyzed.Fluorinase primers were designed to detect the possibility of the fluorinase colonies in the environmental samples.Different dominant bacteria and actinomycetes were existed under the contaminated environment.No strains with known fluorinase was detected even in this fluorine contaminated environment.(2)The gene sequence of fluorinase was synthesized according to whole genome information of Streptomyces catleya and other known fluorinase genes.The engineering bacteria BL21(DE3)-p ET28a(fl A)and BL21(DE3)-p HY300 p LK(fl A)were constructed.The bioconversion catalyzed by engineering fluorinase was tested using SAM and flurione ion as substrats.Enzyme activity was measured by HPLC and LC-MS.(3)The optimal fluorinase induction and reaction parameters of the engineering strain BL21(DE3)-p ET28a(fl A)were studied.The optimal induction conditions were as follows: adding 0.01mm/l IPTG at the middle logarithmic phase,inducing overnight at 16 ?.The highest activity of fluorinase was obtained at 56 ? and p H8.0 in Tris-HCL buffer.The existence of metal ions,EDTA and SDS could reduce the enzyme activityobviously.(4)Single primer mutation specific PCR was used to construct mutants aimed to site L17,T80 and S158 of fluorinase.14.8 % of fluorinase mutant's still behaved SAM conversion activity and were 20% of the original.Activity of the mutant L17 C was 1.03 times higher than wild enzyme.The enzyme activity of the other mutant either single site or double sites,was obviously lower than wild enzyme.It is speculated that different expression parameters of mutant and wild enzyme were existed.(5)The crude fluorinase enzyme was prepared for fluorination reaction and the product was verified.The engineering bacteria was broken and centrifuged,20 % ammonium sulfate was added to the supernatant and placed at 4 ? overnight.The salted out protein was dialyzed and desalted to obtain crude enzyme solution containing fluorinase.65% SAM was conversed within one hour under optimal condition.The reaction solution was concentrated and the product was purified by column chromatography.The results showed that the product contained carbon fluorine(C-F)bond,and 5?-FDA was identified by LC-MS.