多黏芽孢杆菌产ɑ-环糊精葡萄糖基转移酶在大肠杆菌中的可溶性表达及其转化产物特异性研究  

Study of soluble expression in Escherichia coli of α-cyclodextrin glycosyl transferase produced by Paenibacillus macerans and properties of its derivatives

作　　者：王琰[1] 李皎[1] 杨国武[1] 王军[1] 邓媛[1] 万一[1] 

机构地区：[1]陕西省微生物研究所,西安710043

出　　处：《中国食品添加剂》2017年第2期81-86,共6页China Food Additives

基　　金：陕西省科学院应用基础研究项目(2013K-09);陕西省科学院科学技术平台项目(2015k-33)

摘　　要：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-p ET28a(+),转化大肠杆菌BL21(DE3),得到重组菌株α-CGTase-p ET28a/BL21(DE3)。在16℃,1m M 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/m L,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,β-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。The gene encoding a-cyclodextrin glycosyltransferase （a-CGTase） was amplified by degenerate primer fromPaenibacillus macerans YLW and was inserted into expression vector pET28a （＋）, and a recombinant strain a-CGTase-pET28a/BL21 （DE3） was constructed. After induction for 15h at 16℃ with 1 mM IPTG, a-CGTase was expressed insolution and the activity of a-CGTase in the periplasm was reached to 10046 U/mL, which was approximately 3.25-foldwith that from the parent swain. The recombinant a-CGTase was purified by one-step nickel affinity chromatography, andpurification fold and yield was 6.05 and 28.82%, respectively. The enzyme catalytic tests demonstrated that the yield of totalCD reached 40.7% after 15h incubation with 5% potato starch, and a-CD ： fl-CD .. TCD ratio was 43.6 ： 41.8 ： 14.6.Therefore, the recombinant a-CGTase showed better special enzymatic characterization and have potential utilization inindustrial production of a-CD with further optimized transformation condition in the future.

关 键 词：Α-环糊精葡萄糖基转移酶 可溶性表达 纯化 转化性质 

分 类 号：Q555[生物学—生物化学]