不同宿主来源的α-环糊精葡萄糖基转移酶分离纯化及化学修饰提高其热稳定性

摘要

研究了不同宿主来源的α-环糊精葡萄糖基转移酶(α-CGT)经化学修饰后的热稳定变化,首先通过阴离子交换和疏水色谱分离纯化了来源于Paenibacillus macerans的天然α-CGT酶以及来源于Escherichia coli和Bacillus subtilis的重组α-CGT酶,再将α-CGT酶置于50℃水浴30min测残酶活,发现α-CGT酶残酶活分别为28％,30％和25％.首次采用戊二醛交联的方法研究了化学修饰对提高重组α-CGT酶稳定性的影响,E.coli重组表达的α-CGT酶经戊二醛交联成大分子聚合物后,与对照相比50℃水浴30 min后酶的热稳定性明显提高,水浴后仍有78％的酶活,酶活保有率相对未交联的酶提高了2.9倍.%The -cyclodextrin glycosyltransferase (α -CGTase) from Paenibacillus macerans, Escherichia coli,Bacillus subtilis were purified respectively through a combination of ion-exchange and hydrophobic interaction chromatography. Specific activities of the CGTase from these three sources were 201 U/mg,200 U/mg and 199 U/mg,respectively. Chemical modification was further performed to improve the thermal stability of α-CGTase. The α-CGTase expressed in E. coli was cross-linked into a stable macromoleculs by glutaraldehyde. After water bathed at 50 ℃ for 30 min the stability of α-CGTase was improved as the cross-linked α-CGTase exhibit 78% left enzyme activity,which was 3.9 folds higher than that of dissociated α-CGTase.