Effects of Sodium Dodecyl Sulfate on the Enzyme Catalysis and Conformation of a Recombinant γ-Glutamyltranspeptidase from Bacillus licheniformis.

The study of interactions between proteins and surfactants is of relevance in a diverse range of applications including food, enzymatic detergent formulation, and drug delivery. In spite of sodium dodecyl sulfate (SDS)-induced unfolding has been studied in detail at the protein level, deciphering the conformation-activity relationship of a recombinant γ-glutamyltranspeptidase (BlrGGT) from Bacillus licheniformis remains important to understand how the transpeptidase activity is related to its conformation. In this study, we examined the enzyme catalysis and conformational transition of BlrGGT in the presence of SDS. Enzymatic assays showed that the transpeptidase activity of BlrGGT was greatly affected by SDS in a concentration-dependent manner with approximately 90% inactivation at 6 mM. Native polyacrylamide gel electrophoresis of SDS-treated samples clearly revealed that the heterodimeric enzyme was apparently dissociated into two different subunits at concentrations above 2 mM. The study of enzyme kinetics showed that SDS can act as a mixed-type inhibitor to reduce the catalytic efficiency of BlrGGT. Moreover, the t1/2 value of the enzyme at 55 °C was greatly reduced from 495.1 min to 7.4 min in the presence of 1 mM SDS. The I3/I1 ratio of pyrene excimer fluorescence emission changed around 3.7 mM SDS in the absence of BlrGGT and the inflection point of enzyme samples was reduced to less than 2.7 mM. The Far-UV CD spectrum of the native enzyme had two negative peaks at 208 and 222 nm, respectively; however, both negative peaks increased in magnitude with increasing SDS concentration and reached maximal values at above 4.0 mM. The intrinsic fluorescence spectra of tryptophan further demonstrated that the SDS-induced enzyme conformational transition occurred at approximately 5.1 mM. Tween 20 significantly suppressed the interaction of BlrGGT with SDS by forming mixed micelles at a molar ratio of 1.0. Taken together, this study definitely promotes our better understanding of the relationship between the conformation and catalysis of BlrGGT.

Activation and thermal stabilization of a recombinant γ-glutamyltranspeptidase from Bacillus licheniformis ATCC 27811 by monovalent cations.

The regulation of enzyme activity through complexation with certain metal ions plays an important role in many biological processes. In addition to divalent metals, monovalent cations (MVCs) frequently function as promoters for efficient biocatalysis. Here, we examined the effect of MVCs on the enzymatic catalysis of a recombinant γ-glutamyltranspeptidase (BlrGGT) from Bacillus licheniformis ATCC 27,811 and the application of a metal-activated enzyme to L-theanine synthesis. The transpeptidase activity of BlrGGT was enhanced by Cs+ and Na+ over a broad range of concentrations with a maximum of 200 mM. The activation was essentially independent of the ionic radius, but K+ contributed the least to enhancing the catalytic efficiency. The secondary structure of BlrGGT remained mostly unchanged in the presence of different concentrations of MVCs, but there was a significant change in its tertiary structure under the same conditions. Compared with the control, the half-life (t1/2) of the Cs+-enriched enzyme at 60 and 65 °C was shown to increase from 16.3 and 4.0 min to 74.5 and 14.3 min, respectively. The simultaneous addition of Cs+ and Mg2+ ions exerted a synergistic effect on the activation of BlrGGT. This was adequately reflected by an improvement in the conversion of substrates to L-theanine by 3.3-15.1% upon the addition of 200 mM MgCl2 into a reaction mixture comprising the freshly desalted enzyme (25 µg/mL), 250 mM L-glutamine, 600 mM ethylamine, 200 mM each of the MVCs, and 50 mM borate buffer (pH 10.5). Taken together, our results provide interesting insights into the complexation of MVCs with BlrGGT and can therefore be potentially useful to the biocatalytic production of naturally occurring γ-glutamyl compounds. KEY POINTS: • The transpeptidase activity of B. licheniformis Î³-glutamyltranspeptidase can be activated by monovalent cations. • The thermal stability of the enzyme was profoundly increased in the presence of 200 mM Cs+. • The simultaneous addition of Cs+and Mg2+ions to the reaction mixture improves L-theanine production.

Mutational Analysis of a Highly Conserved PLSSMXP Sequence in the Small Subunit of Bacillus licheniformis γ-Glutamyltranspeptidase.

A highly conserved 458PLSSMXP464 sequence in the small subunit (S-subunit) of an industrially important Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was identified by sequence alignment. Molecular structures of the precursor mimic and the mature form of BlGGT clearly reveal that this peptide sequence is in close spatial proximity to the self-processing and catalytic sites of the enzyme. To probe the role of this conserved sequence, ten mutant enzymes of BlGGT were created through a series of deletion and alanine-scanning mutagenesis. SDS-PAGE and densitometric analyses showed that the intrinsic ability of BlGGT to undergo autocatalytic processing was detrimentally affected by the deletion-associated mutations. However, loss of self-activating capacity was not obviously observed in most of the Ala-replacement mutants. The Ala-replacement mutants had a specific activity comparable to or greater than that of the wild-type enzyme; conversely, all deletion mutants completely lost their enzymatic activity. As compared with BlGGT, S460A and S461S showed greatly enhanced kcat/Km values by 2.73- and 2.67-fold, respectively. The intrinsic tryptophan fluorescence and circular dichroism spectral profiles of Ala-replacement and deletion mutants were typically similar to those of BlGGT. However, heat and guanidine hydrochloride-induced unfolding transitions of the deletion-associated mutant proteins were severely reduced as compared with the wild-type enzyme. The predictive mutant models suggest that the microenvironments required for both self-activation and catalytic reaction of BlGGT can be altered upon mutations.

Affinity Immobilization of a Bacterial Prolidase onto Metal-Ion-Chelated Magnetic Nanoparticles for the Hydrolysis of Organophosphorus Compounds.

In this study, silica-coated magnetic nanoparticles (SiMNPs) with isocyanatopropyltriethoxysilane as a metal-chelating ligand were prepared for the immobilization of His6-tagged Escherichia coli prolidase (His6-EcPepQ). Under one-hour coupling, the enzyme-loading capacity for the Ni2+-functionalized SiMNPs (NiNTASiMNPs) was 1.5 mg/mg support, corresponding to about 58.6% recovery of the initial activity. Native and enzyme-bound NiNTASiMNPs were subsequently characterized by transmission electron microscopy (TEM), superparamagnetic analysis, X-ray diffraction, and Fourier transform infrared (FTIR) spectroscopy. As compared to free enzyme, His6-EcPepQ@NiNTASiMNPs had significantly higher activity at 70 °C and pH ranges of 5.5 to 10, and exhibited a greater stability during a storage period of 60 days and could be recycled 20 times with approximately 80% retention of the initial activity. The immobilized enzyme was further applied in the hydrolysis of two different organophosphorus compounds, dimethyl p-nitrophenyl phosphate (methyl paraoxon) and diethyl p-nitrophenyl phosphate (ethyl paraoxon). The experimental results showed that methyl paraoxon was a preferred substrate for His6-EcPepQ and the kinetic behavior of free and immobilized enzymes towards this substance was obviously different. Taken together, the immobilization strategy surely provides an efficient means to deposit active enzymes onto NiNTASiMNPs for His6-EcPepQ-mediated biocatalysis.

High-level expression and molecular characterization of a recombinant prolidase from Escherichia coli NovaBlue.

Long-term use of organophosphorus (OP) compounds has become an increasing global problem and a major threat to sustainability and human health. Prolidase is a proline-specific metallopeptidase that can offer an efficient option for the degradation of OP compounds. In this study, a full-length gene from Escherichia coli NovaBlue encoding a prolidase (EcPepQ) was amplified and cloned into the commercially-available vector pQE-30 to yield pQE-EcPepQ. The overexpressed enzyme was purified from the cell-free extract of isopropyl thio-ß-D-galactoside IPTG-induced E. coli M15 (pQE-EcPepQ) cells by nickel-chelate chromatography. The molecular mass of EcPepQ was determined to be about 57 kDa by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the result of size-exclusion chromatography demonstrated that the enzyme was mainly present in 25 mM Tris-HCl buffer (pH 8.0) as a dimeric form. The optimal conditions for EcPepQ activity were 60 °C, pH 8.0, and 0.1 mM Mn2+ ion. Kinetic analysis with Ala-Pro as the substrate showed that the K m and k cat values of EcPepQ were 8.8 mM and 926.5 ± 2.0 s-1, respectively. The thermal unfolding of EcPepQ followed a two-state process with one well-defined unfolding transition of 64.2 °C. Analysis of guanidine hydrochloride (GdnHCl)-induced denaturation by tryptophan emission fluorescence spectroscopy revealed that the enzyme had a [GdnHCl]0.5,N-U value of 1.98 M. The purified enzyme also exhibited some degree of tolerance to various water/organic co-solvents. Isopropanol and tetrahydrofuran were very detrimental to the enzymatic activity of EcPepQ; however, other more hydrophilic co-solvents, such as formamide, methanol, and ethylene glycol, were better tolerated. Eventually, the non-negative influence of some co-solvents on both catalytic activity and structural stability of EcPepQ allows to adjust the reaction conditions more suitable for EcPepQ-catalyzed bioprocess.

Protective Effect of Biological Osmolytes against Heat- and Chaotropic Agent-Induced Denaturation of Bacillus licheniformis γ-Glutamyl Transpeptidase.

In the present study, the stabilizing effect of four different biological osmolytes on Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT) was investigated. BlGGT appeared to be stable under temperatures below 40°C, but the enzyme retained less than 10% of its activity at 60°C. The tested osmolytes exhibited different degrees of effectiveness against temperature inactivation of BlGGT, and sucrose was found to be the most effective among these. The use of circular dichroism spectroscopy for studying the secondary structure of BlGGT revealed that the temperature-induced conformational change of the protein molecule could be prevented by the osmolytes. Consistently, the molecular structure of the enzyme was essentially conserved by the osmolytes at elevated temperatures as monitored by fluorescence spectroscopy. Sucrose was further observed to counteract guanidine hydrochloride (GdnHCl)- and urea-induced denaturation of BlGGT. Taken together, we observed evidently that some well-known biological osmolytes, especially sucrose, make a dominant contribution to the structural stabilization of BlGTT.

Functional role of the conserved glycine residues, Gly481 and Gly482, of the γ-glutamyltranspeptidase from Bacillus licheniformis.

Six mutants bearing single amino acid substitutions in the small subunit of Bacillus licheniformis γ-glutamyltranspeptudase (BlGGT) have been constructed by site-directed mutagenesis. The resultant enzymes were overexpressed in Escherichia coli and purified by affinity chromatography for biochemical and biophysical characterizations. Replacing Gly481 by either Ala or Glu did affect both autocatalytic processing and catalytic activity of the enzyme, but the substitution of this residue to arginine resulted in an unprocessed enzyme with insignificant catalytic activity. The replacement of another conserved glycine residue, Gly482, by either Ala or Glu caused a significant change in the functional integrity of the enzyme. Moreover, the mutation of Gly482 to arginine led to a marked reduction in the autocatalytic processing. Structural analyses revealed that the fluorescence and circular dichroism properties of mutant proteins were basically consistent with those of BlGGT. However, guanidine hydrochloride (GdnHCl)-induced transitions of most mutants were profoundly reduced in comparison with that of wild-type enzyme. Molecular modeling suggests that the conserved Gly481 and Gly482 residues of BlGGT are located at critical positions to create an environment suitable for both autoprocessing and catalytic reactions.

Facile immobilization of Bacillus licheniformis γ-glutamyltranspeptidase onto graphene oxide nanosheets and its application to the biocatalytic synthesis of γ-l-glutamyl peptides.

For the practical application of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT), we illustrated a simple and efficient approach to fabricate a biocatalytic system by immobilizing the enzyme onto graphene oxide (GO) nanosheets via both non-covalent (GO-BlGGT) and covalent (GO/GA-BlGGT) bonds. The enzyme-loading capacity for the prepared GO/GA nanomaterial was 3.47 mg/mg support, corresponding to 68.7% recovery of the initial activity. Native and enzyme-bound layered GOs were characterized by X-ray diffraction, followed by Raman and Fouier transform infrared spectroscopy, elemental analysis and thermogram analysis. As compared to the free form of BlGGT, the immobilized enzymes exhibited significantly higher activity, possibly due to the beneficial effect of the layered GO carrier. The kinetic behaviors of GO-BlGGT and GO/GA-BlGGT were mostly consistent with those of free enzyme. The covalently immobilized enzyme had a comparable stability respective to free enzyme during a storage period of 30 days and could be recycled nine times with 45.3% retention of the initial activity. Besides, the biocatalytic synthesis of γ-l-glutamyl-phenylalanine and γ-l-glutamyl-leucine by immobilized enzymes resulted in the product yield of more than 31%. Taken together, these results suggest that the facile strategy is an economical means of depositing bioactive enzymes upon GO nanosheets for BlGGT-mediated biocatalysis.

Site-directed mutagenesis of a conserved Asn450 residue of Bacillus licheniformis γ-glutamyltranspeptidase.

Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) belongs to N-terminal nucleophile hydrolase superfamily in which all inclusive members are synthetized as single-chain precursors, and then self-processed to form mature enzymes. Here we investigated the role of a conserved Asn450 residue in BlGGT through site-directed mutagenesis and molecular characterization of four relevant variants. Substitution of Asn450 by arginine resulted in a significant reduction in the catalytic activity of BlGGT. Conversely, N450A and N450D displayed an enhanced activity. The catalytic efficiency of BlGGT was calculated to be 16.04mM(-1)s(-1), but this value was either decreased to 8.93mM(-1)s(-1) in N450K or increased to more than 123.65mM(-1)s(-1) in N450A and N450D. In addition, the ratio of transpeptidation to hydrolysis was increased from 3.5 to more than 7.6 by the mutations. Structural analyses showed that fluorescence, circular dichroism spectra and thermal denaturation profiles of mutant proteins were essentially consistent with those of BlGGT. However, guanidine hydrochloride (GdnHCl)-induced transition was significantly reduced in comparison with the wild-type enzyme. Molecular modeling suggests that residue Asn450 of BlGGT is important to create suitable environments for both autoprocessing and catalytic reactions.

Bacillus licheniformis trehalose-6-phosphate hydrolase structures suggest keys to substrate specificity.

Trehalose-6-phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to yield glucose and glucose 6-phosphate. The products of this reaction can be further metabolized by the energy-generating glycolytic pathway. Here, crystal structures of Bacillus licheniformis TreA (BlTreA) and its R201Q mutant complexed with p-nitrophenyl-α-D-glucopyranoside (R201Q-pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure of BlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site of BlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions of Bacillus cereus oligo-1,6-glucosidase (BcOgl) and Erwinia rhapontici isomaltulose synthase (NX-5), the surface potential of the BlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity of BlTreA. Strikingly, the (281)HHLK(284) motif and Lys292 play critical roles in substrate discrimination by BlTreA.

Gene expression and molecular characterization of a chaperone protein HtpG from Bacillus licheniformis.

Heat shock protein 90 (Hsp90/HtpG) is a highly abundant and ubiquitous ATP-dependent molecular chaperone consisting of three flexibly linked regions, an N-terminal nucleotide-binding domain, middle domain, and a C-terminal domain. Here the putative htpG gene of Bacillus licheniformis was cloned and heterologously expressed in Escherichia coli M15 cells. Native-gel electrophoresis, size exclusion chromatography, and cross-linking analysis revealed that the recombinant protein probably exists as a mixture of monomer, dimer and other oligomers in solution. The optimal conditions for the ATPase activity of B. licheniformis HtpG (BlHtpG) were 45°C and pH 7.0 in the presence of 0.5mM Mg(2+) ions. The molecular architecture of this protein was stable at higher temperatures with a transition point (Tm) of 45°C at neutral pH, whereas the Tm value was reduced to 40.8°C at pH 10.5. Acrylamide quenching experiment further indicated that the dynamic quenching constant (Ksv) of BlHtpG became larger at higher pH values. BlHtpG also experienced a significant change in the protein conformation upon the addition of ATP and organic solvents. Collectively, our experiment data may provide insights into the molecular properties of BlHtpG and identify the alteration of protein structure to forfeit the ATPase activity at alkaline conditions.

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.

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.

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.

Residues Phe103 and Phe149 are critical for the co-chaperone activity of Bacillus licheniformis GrpE.

A tryptophan-free Bacillus licheniformis nucleotide exchange factor (BlGrpE) and its Trp mutants (F70W, F103W, F149W, F70/103W, F70/149W, F103/149W and F70/103/149W) were over-expressed and purified to near homogeneity. Simultaneous addition of B. licheniformis DnaJ, NR-peptide and individual variants synergistically stimulated the ATPase activity of a recombinant DnaK (BlDnaK) from the same bacterium by 3.1-14.7-fold, which are significantly lower than the synergistic stimulation (18.9-fold) of BlGrpE. Protein-protein interaction analysis revealed that Trp mutants relevant to amino acid positions 103 and 149 lost the ability to bind BlDnaK. Circular dichroism measurements indicate that F70W displayed a comparable level of secondary structure to that of BlGrpE, and the wild-type protein and the Trp mutants as well all experienced a reversible behavior of thermal denaturation. Guanidine hydrochloride (GdnHCl)-induced unfolding transition for BlGrpE was calculated to be 1.25 M corresponding to ΔG(N-U) of 4.29 kcal/mol, whereas the unfolding transitions of mutant proteins were in the range of 0.77-1.31 M equivalent to ΔG(N-U) of 2.41-4.14 kcal/mol. Taken together, the introduction of tryptophan residue, especially at positions 103 and 149, into the primary structure of BlGrpE has been proven to be detrimental to structural integrity and proper function of the protein.

Immunomodulatory Potential of the Polysaccharide-Rich Extract from Edible Cyanobacterium Nostoc commune.

A dry sample of Nostoc commune from an organic farm in Pingtung city (Taiwan) was used to prepare polysaccharide-rich (NCPS) extract. The conditioned medium (CM) from NCPS-treated human peripheral blood (PB)-mononuclear cells (MNC) effectively inhibited the growth of human leukemic U937 cells and triggered differentiation of U937 monoblast cells into monocytic/macrophagic lines. Cytokine levels in MNC-CMs showed upregulation of granulocyte/macrophage-colony stimulatory factor and IL-1ß and downregulation of IL-6 and IL-17 upon treatment with NCPS. Moreover, murine macrophage RAW264.7 cells treated with NCPS exhibited the stimulatory effects of nitric oxide and superoxide secretion, indicating that NCPS might activate the immunity of macrophages. Collectively, the present study demonstrates that NCPS from N. commune could be potentially used for macrophage activation and consequently inhibited the leukemic cell growth and induced monocytic/macrophagic differentiation.

γ-Glutamyl transpeptidase architecture: Effect of extra sequence deletion on autoprocessing, structure and stability of the protein from Bacillus licheniformis.

γ-Glutamyl transpeptidases (γ-GTs, EC 2.3.2.2) are a class of ubiquitous enzymes which initiate the cleavage of extracellular glutathione (γ-Glu-Cys-Gly, GSH) into its constituent glutamate, cysteine, and glycine and catalyze the transfer of its γ-glutamyl group to water (hydrolysis), amino acids or small peptides (transpeptidation). These proteins utilize a conserved Thr residue to process their chains into a large and a small subunit that then form the catalytically competent enzyme. Multiple sequence alignments have shown that some bacterial γ-GTs, including that from Bacillus licheniformis (BlGT), possess an extra sequence at the C-terminal tail of the large subunit, whose role is unknown. Here, autoprocessing, structure, catalytic activity and stability against both temperature and the chemical denaturant guanidinium hydrochloride of six BlGT extra-sequence deletion mutants have been characterized by SDS-PAGE, circular dichroism, intrinsic fluorescence and homology modeling. Data suggest that the extra sequence has a crucial role in enzyme activation and structural stability. Our results assist in the development of a structure-based interpretation of the autoprocessing reaction of γ-GTs and are helpful to unveil the molecular bases of their structural stability.

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 3Šresolution 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.

Biophysical characterization of a recombinant aminopeptidase II from the thermophilic bacterium Bacillus stearothermophilus.

In the present study, the biophysical properties of His6-tagged Bacillus stearothermophilus aminopeptidase II (His6-tagged BsAmpII) are characterized in detail by gel-filtration, analytical ultracentrifugation, and various spectroscopic techniques. Using size-exclusion chromatography and analytical ultracentrifugation, we demonstrate that His6-tagged BsAmpII exists predominantly as a dimer in solution. The enzyme is active and stable at pHs ranging from 6.5 to 8.5. Far-UV circular dichroism analysis reveals that the secondary structures of His6-tagged BsAmpII are significantly altered in the presence of SDS, whereas the presence of 5-10% acetone and ethanol was harmless to the folding of the enzyme. Thermal unfolding of His6-tagged BsAmpII was found to be irreversible and led to the formation of aggregates. The native enzyme started to unfold beyond 0.6 M guanidine hydrochloride and had a midpoint of denaturation at 1.34 M. This protein remained active at concentrations of urea below 2.7 M but experienced an irreversible unfolding by >5 M denaturant. Taken together, this work lays a foundation for potential biotechnological applications of His6-tagged BsAmpII.

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.