Unfolding analysis of the mature and unprocessed forms of Bacillus licheniformis γ-glutamyltranspeptidase.

Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) undergoes an autocatalytic process to generate 44.9 and 21.7 kDa subunits; however, a mutant protein (T399A) loses completely the processing ability and mainly exists as a precursor.

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.

Biophysical characterization of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases: A comparative analysis.

The oligomeric states of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases (BlGGT and EcGGT) in solution have been investigated by analytical ultracentrifugation. The results showed that BlGGT has a sedimentation coefficient of 5.12S, which can be transformed into an experimental molecular mass of approximately 62,680Da.

Preparation of carboxylated magnetic particles for the efficient immobilization of C-terminally lysine-tagged Bacillus stearothermophilus aminopeptidase II.

This article reports the synthesis and use of surface-modified iron oxide particles for the simultaneous purification and immobilization of Bacillus stearothermophilus aminopeptidase II (BsAPII) tagged C-terminally with either tri- or nona-lysines (BsAPII-Lys(3/9)). The carboxylated magnetic particles were prepared by the simple co-precipitation of Fe(3+)/Fe(2+) in aqueous medium and then subsequently modified with adipic acid. Transmission electron microscopy (TEM) micrographs showed that the carboxylated magnetic particles remained discrete and had no significant change in size after binding BsAPIIs.

Significance of the conserved Tyr352 and Asp380 residues in the catalytic activity of Bacillus stearothermophilus aminopeptidase II as evaluated by site-directed mutagenesis.

The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.