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Expression and characterization of a fusion protein‐containing cyclodextrin glycosyltransferase from Paenibacillus sp. A11
Author(s) -
Kaulpiboon Jarunee,
Prasong Wanida,
Rimphanitchayakit Vichien,
Murakami Shuichiro,
Aoki Kenji,
Pongsawasdi Piamsook
Publication year - 2010
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.200900039
Subject(s) - fusion protein , biochemistry , chemistry , affinity chromatography , enzyme , escherichia coli , recombinant dna , thioredoxin , microbiology and biotechnology , biology , gene
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. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)