产大环糊精4-α-糖基转移酶的分离纯化及其性质研究

Purification and Properties of 4-α-Glucanotransferase Producing Large-Ring Cyclodextrin

摘要:

4-α-糖基转移酶能够作用直链淀粉产生大环糊精。作者研究了重组E.coliDH-5α-TA(保藏编号:3093)产生的4-α-糖基转移酶的分离纯化及酶学性质。粗酶液经过65℃处理20min、Ni-NTA亲和层析、生物半透膜脱盐得到目标酶,该酶经SDS-PAGE凝胶电泳呈单一蛋白条带,其相对分子质量为57000。该酶具有较高的转糖基活性且最小作用底物为麦芽糖。酶学性质研究表明:该酶的最适反应温度为75℃,最适pH值为7.5;该酶具有良好的耐热性,在70～85℃酶活维持在80%以上;酶的pH稳定范围为6.0～8.5。

Abstract:

4-α-glucanotransferase has the function of generating large-ring cyclodextrin.The crude enzymes production by the genetic mutant of E.coli DH-5α-TA (CGMCC No.3093)was separated and purified by high-temperature processing (65℃,20 min),Ni-NTA affinity column and dialysis.The purified enzyme was demonstrated by SDS-PAGE to be a homogeneous protein and the molecular weight was estimated as 57KDa.It was proved to be with the high level of transglycosylation activity by HPLC which was always used to measure the oligosaccharide and its smallest substrate was maltose.The enzyme exhibited high stability at the temperature range from 70℃ to 85℃ and the pH range from 6.0 to 8.5.The optimal temperature for the enzyme was 75℃ and optimal pH was 7.5,respectively.