Preparation And Application Of Cross-linked Urease Aggregates

Enzymes can be immobilized in different ways, among them, crosslinking is a very attractive technique because no carrier is necessary. A major breakthrough in this field was the development of cross-linked enzyme crystals (CLECs), but the preparation of CLECs was by no means trial and involves high costs because the CLECs requires the crystallization of the enzyme prior to crosslinking. The cross-linked enzyme aggregates (CLEAs) technologies offers an easy,cheap and broadly applicable method for the enzyme immobilization. In this paper, Urease was immobilized in a simple and effective way by physical aggregation using precipitant (ammonium sulfate), followed by chemical crosslinking using a bifunctional reagent(glutaraldehyde) to form insoluble Cross-linked urease aggregates (CLUAs). The optimum pH, optimum temperature and Km of the CLUAs were 8.0, 70°C and 0.021 mol/L, respectively. Compared with that of free urease, the thermal stability, storage stability and resistance of cross-linked urease aggregates to the exogenous proteolysis were enhanced. The efficacy of the CLUAs for the treatment of rats with chronic renal failure was also studied. The rats with chronic renal failure caused by adenine were divided into 3 groups randomly: the control group (fed with10mL water /kg every day), Coated Aldehyde Oxystarch (CAO) group (fed with20g CAO /kg and 10mL water /kg every day) and CLUAs+CAO group (fed with 20g CAO /kg and 10mL CLUAs /kg every day) in which CAO was used to absorb the ammonia produced from urea. The contents of BUN and Scr in serum before and after 2 weeks treatment were determined. In three groups, the level of Scr decreased slightly (p=0.922, 0.972 and 0.225>0.05 respectively) after treatment. The level of BUN was not changed (p=0.211>0.05) In the control group, but decreased greatly BUN in both CAO group and CLUAs+CAO group (p=0.004<0.05 and p<0.001, respectively). Furthermore, the decrease of the BUN level after treatment in the CLUAs+CAO group was more remarkable than that in the CAO group (p=0.016<0.05), which showed that the CLUAs+CAO system was more efficient than the CAO system for the removal of urea in serum. In one word, removal of urea by oral delivery of the product suggests that the final obstacle to a possible oral treatment forkidney failure should have been overcome, by using a combination of CAO and CLUAs systems. With the final obstacle of urea removal solved, there is now the real potential of an oral pharmaceutical approach for kidney failure to replace the expensive and cumbersome treatment using dialysis.