Synthesis, Expression And Purification Of Microcystin-degrading Enzyme Mir A Gene Optimized By Lactococcus Lactis Preference Codon

Microcystin-LR (MC-LR) is a naturally occurring toxin released after the algalcells break down, which can do great damage to liver, very stable and in someextent is very resistant to high temperature and pH change, so it brings thewater purification into a very embarrassing situation.The use of the biodegradation of the microcapsules has become one of themain ways to remove the toxins from the algae. In nature, there exist suchmicroorganisms, but the degradation process is very slow. The degradation ofthe enzyme gene by the degradation of the enzyme gene was cloned and theconstruction of the gene engineering bacteria was the effective way to solvethe problems of physical and chemical treatment and the slow degradation ofnatural microorganism.This paper synthesized a of microcystin degrading enzyme gene which optimizated byLactococcus lactis bias codon, constructed a Mlr gene engineering bacteriaLactococcus lactis L6to expression. In order to solve the increasingly severepollution of the lake reservoir water body and provide a solution for the safety ofdrinking water.The MLR A gene from Sphingomonas sp. ACM-3962was composed of336aminoacid residues. The Lactococcus lactis encoding codons preference GC content lower,according to the2504code region and696252codons from Lactococcus lactis SK11,choose the abundant and preferred codons to optimize. Total246codons was replaced.After codon optimization, the MLR A expression in Lactococcus lactis increased37.14%.Sphingomonas ACM-3962MC-LR degradation gene cluster, between the mlrC andMLR a gene, there is a positive promoter and a reverse promoter, respectively, tocontrol the expression of a MLR and mlrC. The fragment was cloned and the greenfluorescent protein and the red fluorescent protein were added to the E.coli to detectand verify its function. The results show that the promoter of promoter MLR can startthe promoter of A MLR gene expression, and it can be expressed in the downstreamproteins of LR microcystin..Insert the promoter-GFP MLR gene and the A MLR gene into the pMG36e vector,and construct the recombinant vector pMG-mlr. After amplification and verification, the recombinant vector pMG-mlr was transformed into E.coli BL21and L.lactisML23through electric shock method, and the gene engineering bacteria L.lactis L6was constructed to express MlrA.The culture medium was placed in the darkroom of UV light irradiation, to observethe color of bacteria liquid, visible bacteria liquid issued a green fluorescence, showthat in the presence of microcystin LR and MLR promoter can start GFP-mlr a fusionexpression, the bacterium liquid in L.lactis ML23emits green fluorescence under UVlight. The results showed that the positive promoter of promoter GFP-mlr and theexpression ofAMLR fusion protein could also be initiated in Lactobacillus..Adding the freeze-dried MLR A powder to the MC-LR solution, MC-LR can behydrolyzed to linear molecule in3h by more than97%, and the toxicity of MC-LR isgreatly reduced.Adding the lyophilized Lactococcus lactis powder to the MC-LR solution, MC-LRcan be hydrolyzed to linear molecule in3h by more than99%. Acoording to theunlyophilized Lactococcus lactis, the enzyme activity of MLR A was lossed30%,The death number of the cell is about48%, so the directly use of freeze-driedLactococcus lactis has good application prospects.Using the freeze-dried Lactococcus lactis to degradate MC-LR in natural water,compared with laboratory buffer conditions, the enzyme activity was greatly reduced.This is because of the lacking of nutrients in natural water and pH is demanding. Italso shows that, MC-LR degradation in natural water by Lactococcus lactislyophilized, also need to be studied.