The Expression And Purification Of Microcystinase' Family And Character Study Of Enzymology

Frequent outbreaks of blue-green algae blooms have a serious impact on the environment and people's drinking water safety,on the one hand,algae and other aquatic life thrive,led to the decrease of the dissolved oxygen in water which is responsible for death of aquatic organisms.on the other hand,cyanobacteria in growth or death will release a large amount of MCs,these toxins can seriously affect the lives of humans and animals, long-term consumption of drinking water containing MCs can cause a person's heart, kidney, liver, reproductive disease, eventually to promote the occurrence of cancer. these toxins molecule has a good stability, they can still keep a long time even under the condition of more than 300 degrees, and the traditional way of treatment is difficult to be clean I,classical physics can be a good method to degrade MCs, but just move MCs from one place to another place, and costly, cumbersome process; Classic chemical method well treatment MCs, but easy to cause secondary pollution.so we urgently need find an effective and safe method to degrade of MCs. Because of the characteristics of the enzyme degradation has its high specificity and safe way of degradation by Environmentally friendly, which has been widespread attention by the people, the paper mainly by enzymatic degradation of MCs has done the following work:(1) the construction of expression vector of Microcystinase' family, First, we find MCs degrading enzyme gene (mlrA, mlrB, mlrC, mlrD) at NCBI,in order to induce the impaction of the rare codons to the process of E. coli expression, we optimized genes of encoding enzymes and get the optimized gene sequence (opt-mlrA, opt-mlrB, opt-mlrC, opt-mlrD),we respectively clone these sequences to pCold expression vector. not only mlrA protein cannot be expressed on pCold vector, and fusion expression is required, but also mlrA is the primary enzyme of family,we clone mlrA gene to pMAL-C2X expression vector, and finally we get the pMAL C2X-mlrA,pCold-opt-mlrA, pCold-opt-mlrB, pCold-opt-mlrC, pCold-opt-mlrD these five kinds of expression vector.(2)expression and purification of Microcystinase' family:we established the expression and purification system of Microcystinase' family, First the induction conditions of mlrA protein is:When the OD600 of bacteria reached 0.6-0.8, we added IPTG to a final concentration of 0.5mM to induce, cultured at 22? for 8h, we will disrupt bacteria by ultrasonic, and affinity chromatography, in the end,1L LB medium can purify 8mg the purity of 98% mlrA; Secondly the induction conditions of mlrB and mlrC protein is:When the OD600 of bacteria reached 0.5-0.6, we added tetracycline to a final concentration of 50?g/mL, cultured at 37? for Half an hours, we added IPTG to a final concentration of 0.5mM to induce, cultured at 15? for 20h, we will disrupt bacteria by ultrasonic, and affinity chromatography, in the end,1L LB medium can purify 11.6mg the purity of 98% mlrB;and 4.48mg the purity of 99% mlrC, Unfortunately mlrD protein is not expressed in the pCold.(3) the analysis of the enzymology properties of Microcystinase' family:the enzyme activity measurement system of MC - RR is:Sample volume is 10 ?L, the mobile phase is 60% methanol and 40% of secondary water, column temperature is 25 ?, the flow rate of 0.8 mL/min, detection wavelength of 236 nm; the enzyme activity measurement system of MC - RR is:Sample volume is 10 ?L, mobile phase is 70%aqueous solution which contain the volume fraction of 0.1% formic acid and 30% acetonitrile, column temperature is 30 ?, the flow rate of 0.8 mL/min, detection wavelength of 236 nm. On the basis of the system we measured the mlrA' enzyme activity for MC-RR is 68.7 U and the mlrA' enzyme activity for MC-LR is 53.9 U (1 mg protein degrade 1 ?g substrate per minute to 1 U). the best conditions for the removal of MC-RR/MC-LR by mlrA were PH 7.0 and 50?, the optimum test temperature 30 ?, In addition, the MBP tag has no effect on the activity of mlrA protein; The degradation activity of mlrB protein on the A-RR/A-LR is lower, so there is no way to research the enzymology properties; we measured the mlrA' enzyme activity for A-RR is 46.4U and the mlrA'enzyme activity for A-LR is 55.6 U(1 mg protein degrade 1?g substrate per minute to 1 U), the best conditions for the removal of A-RR/A-LR by mlrC were 50?, the optimum test temperature 30?.(4)the analysis of degradation mechanism:By HPLC and high-resolution mass spectrometry in conjunction, we detected the main function of the mlrA enzyme is disconnect a peptide into chain? mlrB/mlrC cannot degradat the MC-RR/MC-LR directly, but they can continue disconnect A-RR/A-LR to a four peptide?...