Enzymatic characterization of Bacillus licheniformis γ-glutamyltranspeptidase fused with N-terminally truncated forms of Bacillus sp. TS-23 α-amylase.
Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was fused at its C-terminal end with N-terminally truncated forms of Bacillus sp. TS-23 α-amylase. BlGGT and six fusion enzymes, BlGGT/SBD, BlGGT/AMYΔN476, BlGGT/AMYΔN443, BlGGT/AMYΔN376, BlGGT/AMYΔN195, and BlGGT/AMYΔN34, were over-expressed in Escherichia coli M15 cells and purified to apparent homogeneity by metal-affinity chromatography. The fusion constructions had no significant effect on the autocatalytic processing of BlGGT. Progressive decrease in the GGT activity of fusion proteins was associated with an increasing level of truncation, and only BlGGT/AMYΔN34 reserved the amylolytic activity. The protein fusions did not alter the optimal temperature and pH of BlGGT. However, as compared with the parental BlGGT, a significant change in circular dichorism and fluorescence spectra was observed in the fusion enzymes. Thermal unfolding of BlGGT, BlGGT/AMYΔN476, BlGGT/AMYΔN443, and BlGGT/AMYΔN376 followed the two-state unfolding process with a transition point (T(m)) of 61.3-63.1 °C, whereas BlGGT/AMYΔN195 and BlGGT/AMYΔN34 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 50mM Tris-HCl (pH 9.0) containing 2% soluble starch.
Department of Food Science and Technology, Hungkuang University, Shalu, Taichung City, Taiwan.
Hui-Yu
HY
Hu
Jia-Ci
JC
Yang
Jiau-Hua
JH
Chen
Meng-Chun
MC
Chi
Long-Liu
LL
Lin
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013213:Starch; D005723:gamma-Glutamyltransferase; D000516:alpha-Amylases