绿豆（Vigna radiata L.）淀粉分支酶 I 的分子特征。

Molecular characterization of mungbean (Vigna radiata L.) starch branching enzyme I.

机构信息

Department of Food Science, Biotechnology Division, School of Life Sciences, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan, ROC.

出版信息

J Agric Food Chem. 2010 Oct 13;58(19):10437-44. doi: 10.1021/jf102129f.

DOI:10.1021/jf102129f

PMID:20822148

Abstract

Mungbean (Vigna radiata L. cv. Tainan no. 5) starch branching enzyme I (SBE, EC 2.4.1.18) cDNA, VrsbeI, was cloned, and its expression was characterized. Conserved regions of the family B SBE were used to amplify a full length cDNA of 2208 bp. Phylogeny was analyzed, and the partial 3D structure and functional features were predicted. Catalytic residues were identified in the (α/β)(8)-fold, and a unique loop from F365 to F376 between β3/α3 was located. Gene expression of VrsbeI in seeds during growth showed that the transcript appeared from week 1 and increased substantially at week 3-4. It was cloned into the pET30 vector and expressed in E. coli BL21(DE3) pLysS cells as a soluble recombinant protein. The affinity-purified recombinant VrSBEI exhibited a specific activity of 314.6 U/mg as an active enzyme with 114-fold activity enrichment from the crude extract.

摘要

绿豆（Vigna radiata L. cv. Tainan no. 5）淀粉分支酶 I（SBE，EC 2.4.1.18）cDNA，VrsbeI，被克隆，并对其表达特性进行了研究。使用家族 B SBE 的保守区扩增了一个全长为 2208bp 的 cDNA。对其进行了系统进化分析，并预测了部分 3D 结构和功能特征。在（α/β）（8）折叠中鉴定出了催化残基，在β3/α3 之间定位了一个独特的 F365 到 F376 环。在种子生长过程中 VrsbeI 的基因表达表明，该转录本从第 1 周开始出现，并在第 3-4 周显著增加。将其克隆到 pET30 载体中，并在 E. coli BL21(DE3) pLysS 细胞中作为可溶性重组蛋白表达。亲和纯化的重组 VrSBEI 表现出 314.6 U/mg 的比活性，比粗提物的活性富集了 114 倍。