(合肥工业大学 食品与生物工程学院/安徽省农产品精深加工省级实验室, 安徽 合肥 230009)
(School of Food and Biological Engineering/Anhui Key Laboratory of Intensive Processing of Agricultural Products, Hefei University of Technology, Hefei 230009, China)
D-阿洛酮糖-3-差向异构酶能够催化D-果糖转化为D-阿洛酮糖。为实现D-阿洛酮糖-3-差向异构酶的异源表达,设计引物,克隆并分离其序列,通过生物信息学软件分析D-阿洛酮糖-3-差向异构酶DNA和蛋白质的结构特点。结果表明,该基因开放阅读框870bp,编码289个氨基酸；蛋白质二级结构中α-螺旋占38.41%,β-折叠占47.06%,无规则卷曲占14.53%；该蛋白为亲水性蛋白,不含信号肽,无跨膜区,定位于细胞膜；构建原核表达载体并导入E. coli BL21宿主中,表达的D-阿洛酮糖-3-差向异构酶分子质量约为33kDa,1mmol/L IPTG诱导E. coli BL21重组菌28h后,目的蛋白表达量和酶活分别为0.32g/L和3.8U/mL。根癌农杆菌D-阿洛酮糖-3-差向异构酶基因能够在大肠杆菌中实现表达。
D-Psicose can be converted from D-fructose catalyzed by D-psicose-3-epimerase. To realize the heterologous expression of D-psicose-3-epimerase (DPEase), the primers were designed to clone and isolate the gene. The DNA sequences and structural characteristics of DPEase were analyzed by bioinformatics. The results showed the gene possessed 870bp open reading frame and encoded 289 amino acids. Alpha helix, beta folding, and random coil respectively accounted for 38.41%, 47.06%,and 14.53% in the secondary structure of the protein. The protein located at the cell membrane belonged to a hydrophilic protein without the signal peptide and transmembrane region. The prokaryotic expression vector was constructed and transferred into the E. coli BL21. The molecular weight of DPEase was appropriately 33kDa. After an induction of 1mmol/L IPTG for 28h, the protein amount and enzymatic activity of the recombinant E. coli BL21 were 0.32g/L and 3.8U/mL. The DPEase gene of A. tumefaciens could be expressed in the engineered E. coli.