Screening And Preliminary Research Of Calcineurin Inhibitor From Microorganism

Calcineurin as a member of protein phosphatase family is the only known Ser/Thr protein phosphatase which is regulated by cellar second messenger Ca²⁺and Calmodulin?CaM?.Calcineurin play a important role in physical activity like cell signal transduction,immune regulation,learning and memory.CsA?Cyclosporin A?and Fk506?Tacrolimus?as Calcineurin inhibitor is the most widely used clinical immunotherapy drug.This study has screened more than 6000 strains from soil.Five strains?CZW-15?CZW-17?ZL-130?W-4?W-53?with high calcineurin inhibitory activity were screened by a yeast report gene?CDRE::LacZ?based high throughput screening model.The fermentation supernatant of CZW-15 with the highest activity,it inhibited yeast calcineurin-dependent gene?CDRE::LacZ?expression with IC₅₀=7.4?L/mL,as to Fk506?65 ng/mL?,with the inhibition rate of 33%in a Human Calcineurin Phosphatase Assay Kit.CZW-15 were indentified by morphological,physiological and biochemical characteristics and 16S rDNA sequence analysis.The evolutionary relationship of CZW-15 is similar to Streptomyces galbus DSM40480 and Streptomyces sp.SAP193.2.And it belongs to the Streptomyces spp.And the fermentation conditions were optimized by single factor and orthogonal experiment.The optimum fermentation conditions of CZW-15 were as follows:2%corn flour,0.1%yeast extracts,0.04%K₂HPO₄,0.04%MgSO₄,0.05%NaCl,0.001%FeSO₄,at initial pH of7.5,180 r/min,28?,liquid volume 50 mL in 250 mL flask,inoculation size of 4%for 4 d.At the optimum conditions,the calcineurin inhibitory activity of 1.5?L fermentation supernatant of CZW-15 reached 117%,increased 4.5 times.Physic-chemical property analysis of active material showed that active material has characteristic absorption peak in 210 nm and the molecular weight of active material may belongs to 3500 Da-8000 Da.Active material can be stored in 4?for 30d with 31%decline and is stable under mild pH?6-8?and tempreture?20?-40??;Analysis of active material with protease K,Biuret reagents and fehling reagents showed active material is not a kind of protein or peptides and doesn't contain reducing sugars;Active material can be extracted from fermentation supernatant by N-butyl alcohol,the rate of extraction is83%Tentative study of active material's isolation and purification showed active material can be adsorbed by macroporous adsorbent resin AB-8.And then active material can be eluted by N-butyl alcohol,the yeild of elution is 67%;Active material can combie to ion exchange resins D115?H-Type?with iro-exchange,but with bad elution result.The highest rate of static elution is 2.8%?eluent:0.02mol/L NH₃.H₂O?.This study has screened 5 strains with high calcineurin inhibitory activity.The CZW-15 is belong to Streptomyces spp with the highest inhibitory activity.The active substance produced by CZW-15 has inhibitory activity both on calcineurin of yeast and calcineurin of human.Follow-up experiments can continue to study for CZW-15strains identification,the separation and purification of the active substance.