Expression and characterization of a fusion protein-containing cyclodextrin glycosyltransferase from Paenibacillus sp. A11.
A recombinant cyclodextrin glycosyltransferase (CGTase) gene fused with thioredoxin (Trx), hexa-histidine (His(6)) and S-protein (S) at the N terminus and a proline-rich peptide (PRP) at the C terminus, was constructed using the wild-type gene from Paenibacillus sp. A11, the pET-32a vector and Escherichia coli BL21(DE3) as the host cell. The expression levels and enzyme characteristics of the Trx-His(6)-CGTase-PRP fusion protein, the recombinant CGTase without fusion peptides, and the wild-type CGTase were compared. The maximum specific activity for the Trx-His(6)-CGTase-PRP fusion enzyme was 2.7 fold higher than that of the non-fusion form at the optimal IPTG concentration. The Trx-His(6)-CGTase-PRP fusion protein was purified to homogeneity by starch adsorption and Ni-NTA affinity chromatography, with a specific activity of 2,268 units/mg protein at a 61% yield. The ease of purification and the higher enzyme yield were obtained with the fusion form when compared to the non-fusion and wild-type enzymes. The fusion enzyme was superior than its wild-type counterpart in terms of stability against high temperature and organic solvents. Moreover, the fusion enzyme could catalyze the synthesis of cyclodextrins in 20% (v/v) dimethylformamide with a higher product yield of CD(7) and CD(8) compared to that of the wild-type enzyme in the same buffer-solvent system.
Kaulpiboon
Jarunee
J
Department of Pre-Clinical Science Biochemistry, Faculty of Medicine, Thammasat University, Patumthani, Thailand. jkaulpiboon@yahoo.com
Prasong
Wanida
W
Rimphanitchayakit
Vichien
V
Murakami
Shuichiro
S
Aoki
Kenji
K
Pongsawasdi
Piamsook
P
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012997:Solvents; D013696:Temperature
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D012997:Solvents; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase