Carrier-free Immobilization And Chemical Modification Of Oxalate Decarboxylase

Oxalic acid (HOOC-COOH) is the most acidic organic dibasic acid in nature. It takes the form of oxalate to exist in a wide range of plants.microbes and animals including human. People eat the foods which have high contents of oxalate will easy to cause oxalic acid accumulation in body, and further causing a variety of pathological conditions. Many primary hyperuricemia patients require intensive dialysis and organ transplantation; it greatly increases the patient’s pain and the economic pressure. So we need to look for a safe method of removing oxalate from the body.Experiment optimized the conditions induced E.coliBL21(DE3)/pET32a/Yvr Kto espress oxalate decarboxylase. The crude enzyme was extracted from the cells with the enzyme activity was36.8U/mL, protein content was1.12mg/mL, specific activity was32.86U/mg.In order to obtain enzyme preparation for relieving the oxalate urinary system stones disease, cross-linked oxalate decarboxylase aggregates was prepared by using carrier-free immobilization method cross-linked enzyme aggregates(CLEAs).66.2%impurity protein was removed by using30%ethanol precipitation, and the recovery of enzyme activity was91.2%. The cross-linked oxalate decarboxylase aggregates was prepared under the condition of adding0.06%glutaraldehyde, pH5, adding bovine serum albumin (BSA) to0.5g/L,4℃and36r/min oscillating for2hours, the recovery of enzyme activity was95.4%. Compared with free oxalate decarboxylase, cross-linked aggregates of oxalate decarboxylase’s resistance to acid, heat and trypsin degradation was increased. This paper created a foundation for the enzyme’s application development.In order to reduce the immunogenicity of oxalate decarboxylase and to improve its stability.Using fast protein liquid chromatography system (AKTA FPLC) to purify oxalate decarboxylase.Optimized the conditions of the dextran-modified oxalate decarboxylase.characterized the Changes in molecular weight and structure with SDS-PAGE and IR.studyed the thermal stability, pH stability and resistance to trypsin after the modification of oxalate decarboxylase.results:the mass ratio of odium periodate and dextran is1:1,the mass ratio of dextran and oxalate decarboxylase is200:1.Modifing6h obtained enzymes.SDS-PAGE and IR show that the molecular weight and protein structure significantly change after modified.the enzyme stability significantly improved.It has potential for development of a new drug to treat urinary tract stones.