Enzymatic characterization of Bacillus licheniformis γ-glutamyltranspeptidase fused with N-terminally truncated forms of Bacillus sp. TS-23 α-amylase

摘要

Bacillus licheniformis 7-glutamyltranspeptidase (B/GGT) was fused at its C-terminal end with N-terminally truncated forms of Bacillus sp. TS-23 a-amylase. B/GGT and six fusion enzymes, B/GGT/SBD, B/GGT/AMYAN476, B/GGT/AMYAN443, B/GGT/AMYAN376, B/GGT/AMYAN195, and B/GGT/AMYAN34, were over-expressed in Escherichia coll Ml 5 cells and purified to apparent homogeneity by metal-affinity chromatography. The fusion constructions had no significant effect on the autocatalytic processing of B/GGT. Progressive decrease in the GGT activity of fusion proteins was associated with an increasing level of truncation, and only B/GGT/AIV1YAN34 reserved the amylolytic activity. The protein fusions did not alter the optimal temperature and pH of B/GGT. However, as compared with the parental B/GGT, a significant change in circular dichorism and fluorescence spectra was observed in the fusion enzymes. Thermal unfolding of B/GGT, B/GGT/AMYAN476, B/GGT/AMYAN443, and B/GGT/AMYAN376 followed the two-state unfolding process with a transition point (T_m) of 61.3-63.1 C, whereas B/GGT/AMYΔN195 and B/GGT/AMYAN34 displayed two temperature transitions at 40.6 and 46.7 C as well as at 62.8 and 62.9 C, respectively. All of the fusion enzymes exhibited the raw-starch-binding ability, and the adsorbed proteins could be eluted from the adsorbent by 50 mM Tris-HCl (pH 9.0) containing 2% soluble starch.