Meng-Chun Chi

Engineering of a truncated α-amylase of Bacillus sp. strain TS-23 for the simultaneous improvement of thermal and oxidative stabilities

BACDeltaNC/Delta RS is a thermostable variant derived from the truncated alpha-amylase (BAC Delta NC) of alkaliphilic Bacillus sp. strain TS-23. With the aim of enhancing its resistance towards chemical oxidation, Met231 of BAC Delta NC/Delta RS was replaced by leucine to create BAC Delta NC/Delta RS/M231L. The functional significance of the 31 C-terminal residues of BAC Delta NC/Delta RS/M231L was also explored by site-directed mutagenesis of the 483 th codon in the gene to stop codon (TAA), thereon the engineered enzyme was named BAC Delta NC/Delta RS/M231L/Delta C31. BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 were very similar to BAC Delta NC in terms of specific activity, kinetic parameters, pH-activity profile, and the hydrolysis of raw starch; however, the engineered enzymes showed an increased half-life at 70 degrees C. The intrinsic fluorescence and circular dichroism spectra were nearly identical for wild-type and engineered enzymes, but they exhibited a different sensitivity towards GdnHCl-induced denaturation. This implicates that the rigidity of the enzyme has been changed as the consequence of mutations. Performance of the engineered enzymes was evaluated in the presence of commonly used detergent compounds and some detergents from the local markets. A high compatibility and performance of both BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 may be desirable for their practical uses in the detergent industry.

Covalent Immobilization of Bacillus licheniformis γ-Glutamyl Transpeptidase on Aldehyde-Functionalized Magnetic Nanoparticles

This work presents the synthesis and use of surface-modified iron oxide nanoparticles for the covalent immobilization of Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT). Magnetic nanoparticles were prepared by an alkaline solution of divalent and trivalent iron ions, and they were subsequently treated with 3-aminopropyltriethoxysilane (APES) to obtain the aminosilane-coated nanoparticles. The functional group on the particle surface and the amino group of BlGGT was then cross-linked using glutaraldehyde as the coupling reagent. The loading capacity of the prepared nanoparticles for BlGGT was 34.2 mg/g support, corresponding to 52.4% recovery of the initial activity. Monographs of transmission electron microscopy revealed that the synthesized nanoparticles had a mean diameter of 15.1 ± 3.7 nm, and the covalent cross-linking of the enzyme did not significantly change their particle size. Fourier transform infrared spectroscopy confirmed the immobilization of BlGGT on the magnetic nanoparticles. The chemical and kinetic behaviors of immobilized BlGGT are mostly consistent with those of the free enzyme. The immobilized enzyme could be recycled ten times with 36.2% retention of the initial activity and had a comparable stability respective to free enzyme during the storage period of 30 days. Collectively, the straightforward synthesis of aldehyde-functionalized nanoparticles and the efficiency of enzyme immobilization offer wide perspectives for the practical use of surface-bound BlGGT.

Effects of C-terminal truncation on autocatalytic processing of Bacillus licheniformis γ-glutamyl transpeptidase

The role of the C-terminal region of Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT) was investigated by deletion analysis. Seven C-terminally truncated BlGGTs lacking 581–585, 577–585, 576–585, 566–585, 558–585, 523–585, and 479–585 amino acids, respectively, were generated by site-directed mutagenesis. Deletion of the last nine amino acids had no appreciable effect on the autocatalytic processing of the enzyme, and the engineered protein was active towards the synthetic substrate L-γ-glutamyl-p-nitroanilide. However, a further deletion to Val576 impaired the autocatalytic processing. In vitro maturation experiments showed that the truncated BlGGT precursors, pro-Δ(576–585), pro-Δ(566–585), and pro-Δ(558–585), could partially precede a time-dependent autocatalytic process to generate the L- and S-subunits, and these proteins showed a dramatic decrease in catalytic activity with respect to the wild-type enzyme. The parental enzyme (BlGGT-4aa) and BlGGT were unfolded biphasically by guanidine hydrochloride (GdnCl), but Δ(577–585), Δ(576–585), Δ(566–585), Δ(558–585), Δ(523–585), and Δ(479–585) followed a monophasic unfolding process and showed a sequential reduction in the GdnCl concentration corresponding to half effect and ΔG0 for the unfolding. BlGGT-4aa and BlGGT sedimented at ∼4.85 S and had a heterodimeric structure of approximately 65.23 kDa in solution, and this structure was conserved in all of the truncated proteins. The frictional ratio (f/fo) of BlGGT-4aa, BlGGT, Δ(581–585), and Δ(577–585) was 1.58, 1.57, 1.46, and 1.39, respectively, whereas the remaining enzymes existed exclusively as precursor form with a ratio of less than 1.18. Taken together, these results provide direct evidence for the functional role of the C-terminal region in the autocatalytic processing of BlGGT.

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. The monomeric conformation is conserved in EcGGT. SDS-PAGE analysis and cross-linking studies further proved that the autocatalytically processed BlGGT and EcGGT form a heterodimeric association. Unfolding analyses using circular dichroism and tryptophan emission fluorescence revealed that these two proteins had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlGGT and EcGGT had a T(m) value of 59.5 and 49.2°C, respectively, and thermal unfolding of both proteins was found to be highly irreversible. Chemical unfolding of BlGGT was independent to the pH value ranging from 5 to 10, whereas the pH environment was found to significantly influence the GdnHCl-induced denaturation of EcGGT. Both enzymes did not reactivate from the completely unfolded states, accessible at 6M GdnHCl. BlGGT was active in the presence of 4M NaCl, whereas the activity of EcGGT was significantly decreased at the high-salt condition. Taken together, these findings suggest that the biophysical properties of the homologous GGTs from two mesophilic sources are quite different.

Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: Opened active site cleft and a cluster of acid residues potentially involved in the recognition of a metal ion

γ-Glutamyltranspeptidases (γ-GTs) cleave the γ-glutamyl amide bond of glutathione and transfer the released γ-glutamyl group to water (hydrolysis) or acceptor amino acids (transpeptidation). These ubiquitous enzymes play a key role in the biosynthesis and degradation of glutathione, and in xenobiotic detoxification. Here we report the 3Å resolution crystal structure of Bacillus licheniformis γ-GT (BlGT) and that of its complex with l-Glu. X-ray structures confirm that BlGT belongs to the N-terminal nucleophilic hydrolase superfamily and reveal that the protein possesses an opened active site cleft similar to that reported for the homologous enzyme from Bacillus subtilis, but different from those observed for human γ-GT and for γ-GTs from other microorganisms. Data suggest that the binding of l-Glu induces a reordering of the C-terminal tail of BlGT large subunit and allow the identification of a cluster of acid residues that are potentially involved in the recognition of a metal ion. The role of these residues on the conformational stability of BlGT has been studied by characterizing the autoprocessing, enzymatic activity, chemical and thermal denaturation of four new Ala single mutants. The results show that replacement of Asp568 with an Ala affects both the autoprocessing and structural stability of the protein.

Enzymatic synthesis of γ-l-glutamyl-S-allyl-l-cysteine, a naturally occurring organosulfur compound from garlic, by Bacillus licheniformis γ-glutamyltranspeptidase

In the practical application of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT), we describe a straightforward enzymatic synthesis of γ-L-glutamyl-S-allyl-L-cysteine (GSAC), a naturally occurring organosulfur compound found in garlic, based on a transpeptidation reaction involving glutamine as the γ-glutamyl donor and S-allyl-L-cysteine as the acceptor. With the help of thin layer chromatography technique and computer-assisted image analysis, we performed the quantitative determination of GSAC. The optimum conditions for a biocatalyzed synthesis of GSAC were 200 mM glutamine, 200 mM S-allyl-L-cysteine, 50 mM Tris-HCl buffer (pH 9.0), and BlGGT at a final concentration of 1.0 U/mL. After a 15-h incubation of the reaction mixture at 60 °C, the GSAC yield for the free and immobilized enzymes was 19.3% and 18.3%, respectively. The enzymatic synthesis of GSAC was repeated under optimal conditions at 1-mmol preparative level. The reaction products together with the commercially available GSAC were further subjected to an ESI-MS/MS analysis. A significant signal with m/z of 291.1 and the protonated fragments at m/z of 73.0, 130.1, 145.0, and 162.1 were observed in the positive ESI-MS/MS spectrum, which is consistent with those of the standard compound. These results confirm the successful synthesis of GSAC from glutamine and S-allyl-L-cysteine by BlGGT.

Identification of Amino Acid Residues Essential for the Catalytic Reaction of Bacillus kaustophilus Leucine Aminopeptidase

The functional significance of amino acid residues Lys-265, Asp-270, Lys-277, Asp-288, Asp-347, Glu-349, and Arg-351 of Bacillus kaustophilus leucine aminopeptidase was explored by site-directed mutagenesis. Variants with an apparent molecular mass of approximately 54 kDa were overexpressed in Escherichia coli and purified to homogeneity by nickel-chelate chromatography. The purified mutant enzymes had no LAP activity, implying that these residues are important for the catalytic reaction of the enzyme.

The maturation mechanism of γ-glutamyl transpeptidases: Insights from the crystal structure of a precursor mimic of the enzyme from Bacillus licheniformis and from site-directed mutagenesis studies

γ-Glutamyl transpeptidases (γ-GTs) are members of N-terminal nucleophile hydrolase superfamily. They are synthetized as single-chain precursors, which are then cleaved to form mature enzymes. Basic aspects of autocatalytic processing of these pro-enzymes are still unknown. Here we describe the X-ray structure of the precursor mimic of Bacillus licheniformis γ-GT (BlGT), obtained by mutating catalytically important threonine to alanine (T399A-BlGT), and report results of autoprocessing of mutants of His401, Thr415, Thr417, Glu419 and Arg571. Data suggest that Thr417 is in a competent position to activate the catalytic threonine (Thr399) for nucleophilic attack of the scissile peptide bond and that Thr415 plays a major role in assisting the process. On the basis of these new structural results, a possible mechanism of autoprocessing is proposed. This mechanism, which guesses the existence of a six-membered transition state involving one carbonyl and two hydroxyl groups, is in agreement with all the available experimental data collected on γ-GTs from different species and with our new Ala-scanning mutagenesis data.

Beneficial Effect of Sugar Osmolytes on the Refolding of Guanidine Hydrochloride-Denatured Trehalose-6-phosphate Hydrolase from Bacillus licheniformis

The influence of three sugar osmolytes on the refolding of guanidine hydrochloride- (GdnHCl-) denatured trehalose-6-phosphate hydrolase of Bacillus licheniformis (BlTreA) was studied by circular dichroism (CD) spectra, fluorescence emission spectra, and the recovery of enzymatic activity. These experimental results clearly indicated that sorbitol, sucrose, and trehalose at a concentration of 0.75 M improved the refolding yields of GdnHCl-denatured  BlTreA, probably due to the fact that these sugars favored the formation of tertiary architectures. Far-UV CD measurements demonstrated the ability of sugar osmolytes to shift the secondary structure of GdnHCl-denatured enzyme towards near-native conformations. ANS fluorescence intensity measurements revealed a reduction of exposed hydrophobic surfaces upon the treatment of denatured enzyme with sugar osmolytes. These observations suggest that sugar osmolytes possibly play a chaperone role in the refolding of chemically denatured BlTreA.

Experimental evidence for the involvement of amino acid residue Glu398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase

The role of glutamate 398 in the autocatalytic processing of Bacillus licheniformis γ‐glutamyltranspeptidase (BlGGT) was explored by site‐directed mutagenesis. This glutamate was substituted by either alanine, aspartate, arginine or glutamine and the expressed mutant enzymes were purified to apparent homogeneity with metal‐affinity chromatography. SDS–PAGE analysis showed that E398A, E398D and E398K were unable to process themselves into a large and a small subunit. However, E398Q was not only able to process itself, but also had a catalytic activity comparable to that of BlGGT. As compared with the wild‐type enzyme, no significant change in circular dichroism spectra was observed for the mutant proteins. Thermal unfolding of BlGGT, E398A, E398D, E398K and E398Q followed the two‐state unfolding process with a transition point (T m) of 47.7–69.4 °C. Tryptophan fluorescence spectra of the mutant enzymes were different from the wild‐type protein in terms of fluorescence intensity. Native BlGGT started to unfold beyond ∼1.92 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]0.5, N–U, at 3.07 M equivalent to free energy change ( Δ ⁢ G N − U H 2 ⁢ O ) of 14.53 kcal/mol for the N → U process, whereas the denaturation midpoints for the mutant enzymes were 1.31–2.99 M equivalent to Δ ⁢ G N − U H 2 ⁢ O of 3.29–12.05 kcal/mol. Taken together, these results strongly suggest that the explored glutamate residue is indeed important for the autocatalytic processing of BlGGT.