Extraction And Spectrum Analysis Of Adenosine Phosphorylase Of Mycoplasma Mycoides Subsp Capricolum

At present,drug-resistance of mycoplasma become more and more serious.Mycoplasma pneumonia does not invade into tissue and blood,its pathopoiesis processes are as follows:firstly,it adheres to the surface of the host cell by summit configuration,and exserts microbule to insert into the cell to imbibe nutrition,thereby damages the cell membrane,accordingly emits neuropoison,phosphatase,nuclease,antigen,hydrogen dioxide and so on to cause cell to lysis,epithelial cell to engorgement and necrosis,moreover,antibody induced by it could also participate in the above pathopoiesis processes.In conclusion,enzyme produced by mycoplasma during metabolism is important to the pathopoiesis processes of mycoplasma and need be further investigated.At present, reports of research on metabolic enzyme of mycoplasma are seldom abroad while there is no formal reports in China.Contagious Caprine Pleuropneumonia,(CCPP),one of the most serious and dramatic diseases of goats,is caused by Mycoplasma mycoides subsp.capri(PG3).CCPP is widespread in central Asia,north Africa,and the Middle East and so on where keep a lot of sheeps,thereby,CCPP is a serious hazard to sheep husbandry.This essay is aim to provide theoretic gist for revealing pathogenetic theory and diagnosis and treatment of Mycoplasma.1.Determine the optimization time to extract adenosine phosphorylaseTo determine the optimization time to extract most activity adenosine phosphorylase,0.1 ml of the culture fluid of type strain(PG3)and isolation one of mycoplasma capri which were cultivanted for 48h,were inoculated to 10ml of Hartley's medium,each inoculated to 48 cuvettes,cultivanted at 37℃,every 6h OD value and pH value of 3 cuvettes(2ml/cuvette) were measured.The result indicated that the lag phase of mycoplasma Capri was at 0～18h, log phase was at 18～60h,stationary phase was at 60～72h,then reached decline phase after 72h,within 60h the pH changed from7.5 to 6.671,from 60h to 96h,pH value kept at 6.671～6.510.Therefore,mycoplasma cultivanted for 48h reach the midanaphase of log phase,Mycoplasma was most vigorous at this phase which was determined as the optimal time to extract adenosine phosphorylase2.Extract and purify adenosine phosphorylaseTo prepare to study the enzymatic properties and to detect the feasibility of purification method of adenosine phosphorylase,adenosine phosphorylase was purificated by a series of centrifugalization,supersonic spallation,salting out,dislysis,ultrafiltration,ion exchange layer,gel filtration chromatography(GFC)and affinity chromatography.Purification level was measured by SDS—PAGE,enzyme activity,protein contents purification multiples.The result of the purification multiple was up to 117.The result of SDS—PAGE of the eribble adenosine phosphorylase showed a single strap,the molecular mass was 29.1 kDa.Then we could conclude that adenosine phosphorylase was purificated and the above purification method was feasible.3.Study the enzymatic properties of adenosine phosphorylaseTo study the enzymatic properties of adenosine phosphorylase,K₃PO4 buffer which pH values were 6.0,6.5,6.8,7.0,7.3,7.5 and 8.0 was prepared.Enzyme activities at different pHvalues and at the temperature of 30,34,37,40,45,50,55,60,65 and 70℃were measured.Catalytic activity of adenosine phosphorylase to adenosine,carnine,uridine(1.5 mmol/l)was measured.The result showed adenosine phosphorylase was heat stability,enzyme activity changed little at 37℃,optimal reaction temperature was 50℃, over55℃,descended quickly.The optimal pH value of adenosine phosphorylase was 7.0, enzyme activity of adenosine phosphorylase descended little in alky condition,but dropped quickly in acor condition.At pH6.0,The enzyme activity was only a half when at the optimal pH value.Adenosine phosphorylase displayed maximal catalytic activity to adenosine, while equation to inosine which was just 5.6%of adenosine,scarcely to uridine.In conclusion,the pH value of the culture medium after cultivanted for 48h was 7.0,while the optimal pH value of the enzyme activity was also 7.0,which demonstrated that the optimization time to extract adenosine phosphorylase at 48h was correct.4.Spectral analyze adenosine phosphorylaseTo detect the construction subunits and isoenzyme of adenosine phosphorylase,the coarse liquid of adenosine phosphorylase was analyzed by S D S—PAGE(0.1%adenosine was added in separation gel).After electrophoresis,adenosine phosphorylase was renaturated by 25% dimethyl carbinol,kept at 40℃overnight,colored by 1%Congo red,bleached by 1mol / L NaC1.As a result,at the localization corresponding enzyme action showed pellucid zone。In conclusion,this bacterium generated only one adenosine phosphorylase of which the molecular mass was 29.1kDa.Forthmore,this adenosine phosphorylase was a single subunit protein and there was no existence of isoenzyme.