交联脲酶聚集体的制备和初步应用

The Preparation and Application of Cross-linked Urease Aggregates

为提高游离脲酶的稳定性 ,将游离脲酶用硫酸铵沉淀下来后 ,以戊二醛作为交联剂对其进行化学交联 ,制备新型的固定化脲酶 交联脲酶聚集体 ,并对其酶学性质进行研究。交联脲酶聚集体的最适pH、最适温度和Km 值分别为 :pH 8 0、70℃和 0 0 2 1mol L。在对交联脲酶聚集体的热稳定性 ,储存稳定性 ,和对抗蛋白水解酶的能力的研究中 ,交联脲酶聚集体均显示了比游离脲酶更高的稳定性。为考察其使用效果和稳定性 ,将其与包醛氧淀粉联合 ,用于慢性肾衰动物模型的口服治疗。以腺嘌呤灌胃法 (每天 30 0mg kg ,共 30d)制备慢性肾衰动物模型 ,将 2 3只大鼠随机分为模型对照组 (每天以 10mL kg蒸馏水灌胃 )、单纯包醛氧淀粉组 (给予含包醛氧淀粉饲料 ,10mL kg蒸馏水灌胃 )和包醛氧淀粉 +交联脲酶聚集体组 (给予含包醛氧淀粉饲料 ,交联脲酶聚集体悬浮液 10mL kg灌胃 ) ,经 2周治疗后 ,模型对照组、治疗对照组和治疗组实验前后的肌酐含量均有小幅下降 ,但差异不显著 (P值分别为0 92 2、0 972和 0 2 2 5 >0 0 5 )。模型对照组的尿素氮含量变化不明显 (P =0 2 11>0 0 5 )。治疗对照组和治疗组实验前后的尿素氮含量均明显下降 (P值分别为 0 0 0 4和小于 0 0 0 1,均小于 0 0 1)。

Urease was immobilized in a simple and effective way by physical aggregation using a precipitant ammonium sulfate, followed by chemical cross linking using a bifunctional reagent glutaraldehyde to form insoluble Cross linked urease aggregates (CLUAs). The optimum pH, optimum temperature and K m of CLUAs were 8 0, 70℃ 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 CLUAs for the treatment of rats with chronic renal failure was also studies. The rats with chronic renal failure caused by adenine were divided into 3 groups randomly:the control group (fed with 10mL water /kg per day), Coated Aldehyde Oxystarch (CAO) group (fed with 20g CAO /kg and 10mL water /kg per day) and CLUAs+CAO group (fed with 20g CAO /kg and 10mL CLUAs /kg per 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+CAD system was more efficient than the CAO system for the removal of urea in serum.