纤维素酶液中β-葡萄糖苷酶的分离纯化

Isolation and purification of β-glucosidase from cellulase solution

通过(NH4)2SO4分级沉淀、HiPrep 26/10 Desalting脱盐柱、Source15Q阴离子交换柱、Source 15 S阳离子交换柱、HiTrap 16/60 Sephacryl S-200 HR凝胶过滤等技术,分离纯化3种来源里氏木霉、黑曲霉、里氏木霉与黑曲霉混合纤维素酶液中的β-葡萄糖苷酶。结果表明,经SDS-PAGE电泳鉴定均为电泳纯,测得黑氏木霉、黑曲霉单独培养β-葡萄糖苷酶相对分子质量分别为68、129 ku,而混合菌培养分离得到两种β-葡萄糖苷酶分子质量大小分别为66.2、134 ku。与单独培养的β-葡萄糖苷酶相似。3种来源的β-葡萄糖苷酶经多步分离纯化后的纯化倍数分别为37.25、40.21、30.12,酶活回收率分别为20.1 2%、23.21%、28.56%。

The β-glucosidase were separated and purified from cellulase solutions produced by Trichoderma reesei,Aspergillus niger,and mixed enzymes of T.reesei and A.niger through（NH4）2SO4 fractional precipitation,HiPrep 26/10 gel filtration,Source 15 Q and source 15 S ion-exchange columns,and HiTrep 16/60 Sephacryl S-200 HR gel column.Both β-1,4-glucosidase showed apparent homogeneity in SDS-PAGE.The estimated molecular masses of β-1,4-glucosidase were 68 and 129 ku from T.reesei and A.niger,respectively.Two kinds of β-glucosidases were purified from mixed culture of T.reesei and A.niger,two single subunit molecular masses were about 134 ku and 66.2 ku which are similar to the single culture.Three sources of β-GA through multi-step purification the purification multiple were 37.25,40.21,30.12 respectively.The recovery of enzyme activity were 20.12 %,23.21 %,28.56 %.