Heterologous expression and characterization of glycogen branching enzyme from Synechocystis sp. PCC6803.
A gene (sll0158) putatively encoding a glycogen branching enzyme (GBE, E.C. 2.4.1.18) was cloned from Synechocystis sp. PCC6803, and the recombinant protein expressed and characterized. The PCR-amplified putative GBE gene was ligated into a pET-21a plasmid vector harboring a T7 promoter, and the recombinant DNA transformed into a host cell, E. coli BL21(DE3). The IPTG-induced enzymes were then extracted and purified using Ni-NTA affinity chromatography. The putative GBE gene was found to be composed of 2,310 nucleotides and encoded 770 amino acids, corresponding to approx. 90.7 kDa, as confirmed by SDS-PAGE and MALDI-TOF-MS analyses. The optimal conditions for GBE activity were investigated by measuring the absorbance change in iodine affinity, and shown to be pH 8.0 and 30 degrees in a 50 mM glycine-NaOH buffer. The action pattern of the GBE on amylose, an alpha-(1,4)-linked linear glucan, was analyzed using high-performance anion-exchange chromatography (HPAEC) after isoamylolysis. As a result, the GBE displayed alpha-glucosyl transferring activity by cleaving the alpha-(1,4)-linkages and transferring the cleaved maltoglycosyl moiety to form new alpha-(1,6)- branch linkages. A time-course study of the GBE reaction was carried out with biosynthetic amylose (BSAM; Mp approximately = 8,000), and the changes in the branch-chain length distribution were evaluated. When increasing the reaction time up to 48 h, the weight- and number-average DP (DPw and DPn) decreased from 19.6 to 8.7 and from 17.6 to 7.8, respectively. The molecular size (Mp, peak Mw approximately = 2.45-2.75 x 10(5)) of the GBE-reacted product from BSAM reached the size of amylose (AM) in botanical starch, yet the product was highly soluble and stable in water, unlike AM molecules. Thus, GBE-generated products can provide new food and non-food applications, owing to their unique physical properties.
Lee
Byung-Hoo
BH
Department of Food Science and Technology and Carbohydrate Bioproduct Research Center, Sejong University, Seoul 143-747, Korea.
Yoo
Young-Hee
YH
Ryu
Je-Hoon
JH
Kim
Tae-Jip
TJ
Yoo
Sang-Ho
SH