末端环化疏棉状嗜热丝孢菌脂肪酶的稳定性

Study on the stability of lipase of terminally cyclized Thermomyces lanuginosus

目的:获取活性高、稳定性好的工业脂肪酶。方法:利用哺乳动物细胞和大肠杆菌分别表达Butelase 1和梳棉状嗜热丝孢菌脂肪酶(Thermomyces lanuginosus lipase,TLL),通过Flag亲和层析和His亲和层析柱进行纯化;通过纯化的Butelase 1连接酶对重组TLL(rTLL)进行末端连接,以形成首尾相连的环状TLL(cTLL),通过测定rTLL和cTLL活性、热稳定性、蛋白酶稳定性、抗热聚沉能力等,研究环化处理对脂肪酶的影响。结果:纯化后cTLL与rTLL的比活基本相当;70℃下处理180 min,cTLL大部分可保持可溶状态,相同条件下线性蛋白TLL几乎完全沉淀或降低;环化后的脂肪酶cTLL粒径均一、分子量分布均匀,具有更稳定的蛋白特性。结论:Butelase 1可有效地环化梳棉状嗜热丝孢菌脂肪酶,环化后的蛋白酶酶活未改变,热稳定性大大提高,蛋白特性更好。

Objective:To obtain industrial standard lipase with high activity and stability,Butelase 1 ligase was used to connect the N-and C-termini of Thermomyces lanuginosus lipase(TLL),and then the protein characteristics,including heat stability,protease resistance,and enzymatic kinetics were analyzed.Methods:His6 affinity column was used to purify recombinant Butelase 1(rButelase 1)and TLL(rTLL)from mammalian cells and E.coli;rButelase 1 to was utilized connect the N-and C-termini of rTLL to generate circular rTLL(cTLL);the enzymatic activity,the heat stability,the protease resistance,the resistance to heat-induced precipitation,and the enzymatic kinetics were analyzed to determine the difference of rTLL and cTLL.Results:Recombinant rButelase 1 and rTLL expressed in mammalian cells and E.coli were purified;cTLL was obtained,and the comparable enzymatic activity was found in both cTLL and rTLL.After heat treatment at 70℃for 180 min,most of cTLL remained soluble and 95%of its activity,while rTLL was almost completely precipitated and lost most of its activity.cTLL has characteristics of stable proteins,such as uniform particle size and molecular weight distribution.Conclusion:Butelase 1 can effectively circularize lipase from T.lanuginosus.cTLL maintained its enzymatic activity.Meanwhile,its protein characteristics,including thermal stability and protease resistance,has also been improved.