Study On Immobilization Of Creatinine Degrading Enzymes Using Saccharomyce Cerevisiae Spores And Its Application

Creatinine is widely used as the glomerular filtration rate indicator.Creatininase(CA),creatinase(CI)and sarcosine oxidase(SOX)can convert creatinine to the H2O2 which can be detected by chromogenic reaction.Based on this strategy,these enzymes are employed in clinic creatinine assay kits to detect the patients' creatinine concerctration.But the preparation of pure enzymes makes cost high.In Saccharomyce cerevisiae spore microencapsulated enzyme skill,enzyme gene is transformed into yeast.After sporulation,spore encapsulated enzyme can be harvested.This skill does not need the preparation of pure enzyme,and is easy to operate.In this study,CA,CI and SOX were encapsulated into spore wall to develop low cost and high efficiency creatinine assay kit.Our study can be summarized as follow:Firstly,the CA from Pseudomonas sp.PS-7,CI from Pseudomonas putida KT2440 or SOX from Bacillus sp.NS-129 was immobilized in spores separately.The Western blotting and enzyme activity assay showed that those enzymes were immobilized successfully in the spore wall separately,which gained more resistant to harsh environments,higher temperature and wider range of reaction pH,and the enzymatic properties were greatly improved than the free enzyme.They could be stored for a long time at room temperature and showed good reusability.Though glycosylation increased the molecular weight of CI and SOX,it did not affect the immobilization and enzymatic properties of the enzymes.Then,in the screening of the best mutant for immobilization,wt,osw2?,dit1? and chs3? were employed and their enzymatic properties were compared.The results indicated that,the osw2? could trip massive amount of enzymes and showed higher activity than wt,because the holes of its dityrosine layer were bigger than wt.It also showed a better resistance,reusability and room temperature storage compared to dit1?,which lost its dityrosine layer,though had a higher Km.Considering the enzymatic properties together,osw2? was picked as the best candidate.Finally,the best candidate,osw2? mutant was used in the co-immobilization research,which consisted of “one spore two enzymes” and “one spore three enzymes”.The former one co-immobilized CA and CI in one spore,which showed 2 times urea yield as the optimized separately immbolized one using creatinine as the substrate.The latter one co-immobilized CA,CI and SOX showed a higher enzymatic efficiency than the former one.In creatinine assay,the detection threshold of “one spore three enzymes” was 0.1 mmol·L-1,50 times higher than separately immobilized one,and the linearity range of creatinine concentration was 0.1~50 mmol·L-1.It also showd a long storage time at room temperature.Finally,the high throughput samples assay were researched.