Molecular characterization of mungbean (Vigna radiata L.) starch branching enzyme I.
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.
Chang
Jia-Wei
JW
Department of Food Science, Biotechnology Division, School of Life Sciences, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan, ROC.
Li
Sing-Chung
SC
Shih
Yun-Chi
YC
Wang
Reuben
R
Chung
Pei-Shan
PS
Ko
Yuan-Tih
YT
D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics; D012639:Seeds / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment
D018076:DNA, Complementary; D018744:DNA, Plant; D011994:Recombinant Proteins; D015061:1,4-alpha-Glucan Branching Enzyme