The gene encoding α-cyclodextrin glycosyltransferase (α-CGTase) was amplified by degenerate primer from Paenibacillus macerans YLW and was inserted into expression vector pET28a (+),and a recombinant strain α-CGTase-pET28a/BL21 (DE3) was constructed.After induction for 15h at 16℃ with 1 mM IPTG,α-CGTase was expressed in solution and the activity of α-CGTase in the periplasm was reached to 10046 U/mL,which was approximately 3.25-fold with that from the parent strain.The recombinant α-CGTase was purified by one-step nickel affinity chromatography,and purification fold and yield was 6.05 and 28.82％,respectively.The enzyme catalytic tests demonstrated that the yield of total CD reached 40.7％ after 15h incubation with 5％ potato starch,and α-CD:β-CD:γ-CD ratio was 43.6:41.8:14.6.Therefore,the recombinant α-CGTase showed better special enzymatic characterization and have potential utilizaton in industrial production of α-CD with further op timized transformation condition in the future.%通过设计简并引物,从Paenibacillus macerans YLW菌株中克隆到其α-环糊精葡萄糖基转移酶(α-CGTase)基因,构建重组质粒α-CGTase-pET28a(+),转化大肠杆菌BL21 (DE3),得到重组菌株α-CGTase-pET28a/BL21 (DE3).在16℃,1mM IPTG条件下诱导15 h,实现了α-CGTase的可溶性表达,胞内酶活达到10046U/mL,是野生菌株胞外酶活的3.25倍.经镍柱一步法亲和纯化α-CGTase后,酶蛋白纯化了6.05倍,酶收率28.82％,通过SDS-PAGE检测获得表观电泳纯酶蛋白.酶催化转化实验表明:重组α-CGTase酶转化质量分数为5％的马铃薯淀粉15 h后,环糊精的总转化率可达40.7％,转化生成α-CD,ββ-CD,γ-CD比例分别为:43.6％、41.8％和14.6％.该重组酶对α-CD具有较好的转化专一性,通过转化条件的进一步优化将具有非常好的产业化开发前景.
展开▼
