Expression And Purification Of Oxalate Decarboxylase As Well As In Vitro Oxalate Degradation By Bacterial Ghost Encapsulated Oxalate Decarboxylase

Kidney stone is one of the most common urological disease.The per capital prevalence is about 6%-10%according to statistics.With the development of extracorporeal shock wave lithotripsy and endoscopic surgery,urinary calculi treatment has been greatly improved.However,postoperative recurrence risk did not significantly reduce.Data showed that the recurrence rate of five years was 50%,and the recurrence rate of ten years was as high as 80%to 90%.Calcium oxalate stone is the most common of the stone composition in urolithiasis,accounting for about 70%?80%of all urinary calculi and as high as 90%in some areas.Hyperoxaluria is a major pathogenic factors of urinary calcium oxalate stone,there is no effective treatments at presentand studies have indicated that high oxalate urinary played an important role in the forming of calcium oxalate stone.Oxalic acid is a substance that can not be degradation in the body and food-borne oxalic acid is an important source of urinary oxalate,which is widely existed in daily fruits and vegetables Enzyme therapy is a new method for diseases in medicine.There are two kinds of oxalate degrading enzyme in nature:oxalate decarboxylase(OxdC)and oxalate oxidase(OxO).Due to their specific degradation of oxalic acid and has the advantages of stable structure,high temperature resistance,acid resistance,etc,and indicated that they had a good potential in hyperoxaluria treatment.Studies have shown that oxalate decarboxylase can keep 80%enzyme activity in the enviroment of pH3.0 and pH6.5,indicated it could play the role of oxalate degradation in the stomach and intestines pH environment respectively,thus solving the barrier of enzyme reaction in the stomach and intestines.But oxalate decarboxylase as a heterologous protein,intravenous injection not only easily lead to the body's immune response,organ damage,but also bring great pain to the patient.Relatively speaking,oral administration is simple and high patient compliance,but the enzyme is easily be digested by tract protease that can not effectively play the biological degradation role,therefore its application is very limited.Bacterial ghost(BG)is a kind of vaccine carrier system with outstanding natural assistant agent.Is a gram negative bacterium lacking the cytoplasm,and structural features remain the same as a cell membrane sac.Simultaneously,BG has intact surface membrane composition,so it could be designed as a carrier of active substances and exogenous antigen(protein or DNA),so using BG envelope protein enzyme capsule is an ideal method.of oral administration.In this study,oxalate decarboxylase was expressed by prokaryotic expression system and purified to homogeneity.,and wrapped by using the BG as drug carrier that build a sustained-release drug delivery carrier which can protect enzyme from gastointestinal protease degradation.And then the stability and effective in vitro were verified.This study can provide a new way for the treatment of hyperoxaluria.The current results obtained are as follow:1.According to the reported genome information of Bacillus subtilis str.168 strain in NCBI database.Then primers were designed to clone the fragment of the gene,cloned genes and expression plasmid pGEX-6p-1 were connected to form a recombinant plasmid,and then transformed into E.coli Transta cells,the protein was expressed and purified.The enzyme was purified by GST affinity chromatography,and it can be purified 20 mg pure enzyme from 1 L bacteria cultures.2.The enzyme kinetics studies have been carried on the purification of oxalate decarboxylase It if found that the enzyme that the optimum reaction temperature is 37 ?,the optimum pH value is 3.0 and stability is the best in pH 8.0 After incubated in pH 3.0 and pH 8.0 buffer respectively for 15h,it also have 80%of the original activity.At the same time,the enzyme has good thermal stability,enzyme incubated in 4 ? as a control,the enzyme had still contain more than 60%of the activity when deal with in 37? for 20 h or incubated in 60 ? for 1 h,and could sustain more than 50%of the activity when handle in 80 ? for 20 minutes.And the ions of Mn2+,Cu2+,Mg2+,Zn2+,K+ had no great impacts on the enzymatic reaction.3.The E protein coding sequence has been obtained by polymerase chain reaction(PCR)according to the sequence of phiX174 bacterial virus E protein gene in NCBI database and phage genome as templates.Then the cds was subcloned into the and tranfered into expression E.c.oli BL21(DE3)to prepare BG.The cracking rate of BG was 99.0%by preliminary statistics.And the capacity of loading oxalate decarboxylase protein have been tested.The enzyme catalyzed reaction was carried out with the BG that wrapped oxalate decarboxylase.and the reactionof purified enzyme as a control.The results showed that the BG wrapped oxalate decarboxylase could degrade oxalate.So the oxalate decarboxylase BG have some feasibility in oral administration on oxalate degradation.