重组L-门冬酰胺酶的聚乙二醇化学修饰及修饰后酶的稳定性研究

目的单甲氧基聚乙二醇 (mPEG)化学修饰大肠杆菌重组L 门冬酰胺酶 (L ASP) ,考察经过修饰的酶的稳定性。方法N 羟基琥珀酰亚胺 (NHS)活化酯法活化mPEG ,生成的单甲氧基聚乙二醇琥珀酰琥珀酸亚胺酯 (SS mPEG)按不同摩尔比例与L ASP偶联 ,确定适合的反应时间和反应pH值。通过聚乙二醇化学修饰后的酶 (L ASP PEG) ,酶活力和纯度通过奈氏法和丙烯酰胺凝胶电泳 (SDS PAGE)检测 ,高效液相色谱检测L ASP PEG相对分子质量并考察了L ASP PEG体外稳定性等。结果SDS PAGE显示mPEG已经偶联到L ASP分子上 ,以两者摩尔比 1 0∶1为最佳 ,反应pH条件为 8.5 ,获得的L ASP PEG平均比活单位为 6 4 .8IU/mg ,相对分子质量为 30 1 80 0 ,体外稳定性高于L ASP。结论此实验确定了mPEG化学修饰L ASP最佳反应条件为 2 5℃反应 30min ,两者投料摩尔比为 1 0∶1 ,获得的L ASP PEG比L ASP稳定性高

Modification of recombinant L-asparaginase with activated methoxypolyethylene glycol and study on stability of methoxypolyethylene glycol conjugates L-asparaginase

PurposeTo investigate modified enzyme( L ASP PEG) stability through methoxypolyethyethylene glycoly(mPEG) chemical modified recombinant L asparaginase( L ASP).MethodsmPEG was activated by N hydroxysuccinimide(NHS) and conjugated with L ASP in different ratio(mol/mol). Nessler′agent and SDS PAGE were introduced to analyze enzyme activity and purification of L ASP PEG, the relative molecular weight of which was determined by HPLC, respectively. Meanwhile, we also studied stability of L ASP PEG at 4℃ and 37℃ in vitro. ResultsActivated mPEG has been conjugated with L ASP in ratio of 10∶1 and reactive optimal pH was 8.5. The specific activity of L ASP PEG was 64.8 IU/mg on average and relative molecular weight was 301 800. L ASP PEG was more stable than L ASP both in vitro. ConclusionThe optimal condition of mPEG chemical modified L ASP was 25℃,30min and molecular ratio was 10∶1.