高浓度变性-还原溶菌酶的流加复性动力学特性

DYNAMIC FED-BATCH REFOLDING BEHAVIOR OF DENATURED-REDUCED LYSOZYME AT HIGH CONCENTRATIONS

研究了高浓度变性 还原溶菌酶的流加复性过程动力学特性 ,重点考察了影响复性速率和复性收率的主要因素 ,包括盐酸胍浓度、酶浓度和氧化还原剂浓度 .结果表明 ,与直接稀释复性相比 ,流加操作可有效降低肽链分子间聚集 ,提高蛋白质的复性收率 ;在流加复性条件下 ,随着酶浓度的提高 ,复性速率和复性收率均有所下降 ,但适当提高复性液中盐酸胍浓度仍可获得高浓度蛋白质的高复性收率 .另外 ,随着变性酶浓度的提高 ,需要适当提高复性液中氧化型谷胱甘肽浓度 ,以加快溶菌酶分子内二硫键的形成 。

Protein refolding process by fed-batch dilution is a course of protein dilution and refolding at the same time. In this work, the dynamic fed-batch refolding behavior of denatured-reduced lysozyme at concentrations up to 7mg·ml -1 was investigated to find the influence of some important factors, including the concentrations of denaturant (guanidine hydrochloride), lysozyme and redox reagents. The fed-batch refolding was operated by 60min fed-batch dilution and 24h incubation at 37℃. In the fed-batch process, the denatured-reduced protein could rapidly reach homogeneous distribution and be kept at low concentration in the refolding buffer, so the formation of protein aggregates was greatly suppressed. Thus, significantly higher refolding yield was obtained by the fed-batch dilution than direct dilution. With the increase of final lysozyme concentration, the fed-batch refolding yield decreased somewhat. However, this decrease could be alleviated by properly increasing guanidine hydrochloride concentration in the refolding buffer. In addition, at lysozyme concentrations higher than 3mg·ml -1, increased oxidizer concentration was needed to enhance the rate of intramolecular disulfide bond formation and thus the rate of protein refolding.