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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 (t 1/2 ) 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 Mg 2+ 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 MgCl 2 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 γ-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 Mg 2+ ions to the reaction mixture improves L-theanine production., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A highly conserved 458 PLSSMXP 464 sequence in the small subunit (S-subunit) of an industrially important Bacillus licheniformis γ-glutamyltranspeptidase ( Bl GGT) was identified by sequence alignment. Molecular structures of the precursor mimic and the mature form of Bl GGT 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 Bl GGT were created through a series of deletion and alanine-scanning mutagenesis. SDS-PAGE and densitometric analyses showed that the intrinsic ability of Bl GGT 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 Bl GGT, S460A and S461S showed greatly enhanced k cat / K m 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 Bl GGT. 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 Bl GGT can be altered upon mutations.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the present study, the stabilizing effect of four different biological osmolytes on Bacillus licheniformis γ-glutamyl transpeptidase ( Bl GGT) was investigated. Bl GGT 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 Bl GGT, and sucrose was found to be the most effective among these. The use of circular dichroism spectroscopy for studying the secondary structure of Bl GGT 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 Bl GGT. Taken together, we observed evidently that some well-known biological osmolytes, especially sucrose, make a dominant contribution to the structural stabilization of Bl GTT.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Several γ-glutamyl compounds are known to have favorable features for a variety of biotechnological and pharmaceutical applications. However, so far only a limited number of γ-glutamyl compounds have been synthesized through γ-glutamyltranspeptidase (GGT). With the help of thin layer chromatography and computer-assisted image analysis techniques, we performed an enzymatic method involving Bacillus licheniformis GGT (BlGGT) for the synthesis of γ-glutamyl-L-leucine (γ-Glu- Leu). The optimum conditions for a biocatalytic synthesis of γ- Glu-Leu were determined to be 200 mM glutamine, 200 mM leucine, 50 mM Tris-HCl buffer (pH 9.0), and BlGGT at a working concentration of 2.0 U/mL. Upon a 5-h incubation of the reaction mixture at 50°C, the product yield could reach approximately 52%. The preparative synthesis of γ-Glu-Leu by BlGGT was done under optimal conditions at millimol scale level. The reaction products were subjected to mass spectrometry/nuclear magnetic resonance analyses to ensure the successful synthesis of γ-Glu-Leu. Taken together, the present study demonstrates the high potentiality of BlGGT-catalyzed reaction in the synthesis of γ-glutamyl compounds. [ABSTRACT FROM AUTHOR]

Several γ-glutamyl compounds are known to have attractive features for food industry applications. In this study, we report a straightforward procedure for the biocatalytic synthesis of γ-glutamyl-phenylalanine (γ-Glu-Phe) using recombinant Bacillus licheniformis γ-glutamyltranspeptidase ( Bl GGT). The qualitative analysis of reaction products was carried out by thin-layer chromatography technique and computer-assisted image analysis. The optimized conditions for the biocatalytic process were pH 9.0, 50 °C, l -glutamine and l -phenylalanine in a 1:1 ratio, and Bl GGT at a working concentration of 2.0 U/ml. Preparative synthesis of γ-Glu-Phe was also performed at mmol scale. The desired product from the reaction mixture was then verified by mass spectrometry/nuclear magnetic resonance analyses. Strikingly, the experimental results suggest the high potentiality of Bl GGT-catalyzed reaction in the enzymatic synthesis of γ-glutamyl peptides. [ABSTRACT FROM AUTHOR]

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 Bacillus licheniformis TreA ( Bl BlTreA) and its R201Q mutant complexed with p p-nitrophenyl-α- d d-glucopyranoside (R201Q- p pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure of Bl BlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site of Bl BlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions of Bacillus cereus Bacillus cereus oligo-1,6-glucosidase ( Bc BcOgl) and Erwinia rhapontici Erwinia rhapontici isomaltulose synthase (NX-5), the surface potential of the Bl 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 Bl BlTreA. Strikingly, the281 281HHLK284 284 motif and Lys292 play critical roles in substrate discrimination by Bl BlTreA. [ABSTRACT FROM AUTHOR]

Here, the crystallization and preliminary X-ray diffraction studies of Bacillus licheniformisγ-glutamyl transpeptidase ( BlGT) are reported. The serendipitous finding of heterogeneous nucleants in the initial experiments provided the first crystallization conditions for the protein. Crystals were grown by hanging-drop vapour diffusion using a precipitant solution consisting of 20%( w/ v) PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M Tris-HCl pH 8.2. The protein crystallized in the orthorhombic space group P212121, with one heterodimer per asymmetric unit and unit-cell parameters a = 60.90, b = 61.97, c = 148.24 Å. The BlGT crystals diffracted to 2.95 Å resolution. [ABSTRACT FROM AUTHOR]

Abstract: 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 () 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 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. [Copyright &y& Elsevier]

γ-Glutamyl transpeptidase (GGT) catalyzes the transfer of glutathione's γ-glutamyl group and related γ-glutamyl amides to water, amino acids or peptides, and utilizes a conserved Thr residue to process its own polypeptide chain into a large and a small subunit that then assemble to produce a catalytically competent enzyme. In this study, the magnetic cross-linked enzyme aggregates (mCLEAs) of a transpeptidase-specialized variant (N450D) of Bacillus licheniformis GGT were successfully prepared with optimized process parameters viz.1.25:1 (v/v) of isopropanol to N450D (0.3 mg/mL) ratio/0.02:1 (w/w) of enzyme to 3-aminopropyl triethoxysilane (APTES)-coated magnetic nanoparticle ratio/20 mM of glutaraldehyde. The prepared magnetic nanoparticles and immobilized enzyme (N450D-mCLEAs) were characterized by X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy, field-emission scanning electron microscope integrated with energy dispersive X-ray spectroscopy (FESEM/EDS), and superparamagnetic analysis. As compared with free enzyme, N450D-mCLEAs displayed significantly higher heat resistance at temperatures of 55 and 60 °C, and had a greater stability over a storage period of one month. The immobilized enzyme could also be reused for 10 consecutive biocatalytic cycles with no significant reduction in the percent yield of l-theanine. Conclusively, this immobilization strategy surely provides a meaningful glance of developing N450D-mediated biocatalysis for the production of physiologically important γ-glutamyl compounds. [ABSTRACT FROM AUTHOR]

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 ( Bl HtpG) were 45 °C and pH 7.0 in the presence of 0.5 mM Mg 2+ ions. The molecular architecture of this protein was stable at higher temperatures with a transition point ( T m ) of 45 °C at neutral pH, whereas the T m value was reduced to 40.8 °C at pH 10.5. Acrylamide quenching experiment further indicated that the dynamic quenching constant ( K sv ) of Bl HtpG became larger at higher pH values. Bl HtpG 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 Bl HtpG and identify the alteration of protein structure to forfeit the ATPase activity at alkaline conditions. [ABSTRACT FROM AUTHOR]

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 ( Bl GT), obtained by mutating catalytically important threonine to alanine (T399A- Bl GT), 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. [ABSTRACT FROM AUTHOR]

In the practical application of Bacillus licheniformis γ-glutamyltranspeptidase ( Bl GGT), 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 Bl GGT 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 Bl GGT. [ABSTRACT FROM AUTHOR]

A tryptophan-free Bacillus licheniformis nucleotide exchange factor ( Bl GrpE) 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 ( Bl DnaK) from the same bacterium by 3.1–14.7-fold, which are significantly lower than the synergistic stimulation (18.9-fold) of Bl GrpE. Protein–protein interaction analysis revealed that Trp mutants relevant to amino acid positions 103 and 149 lost the ability to bind Bl DnaK. Circular dichroism measurements indicate that F70W displayed a comparable level of secondary structure to that of Bl GrpE, 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 Bl GrpE was calculated to be 1.25 M corresponding to Δ G 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 Δ G 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 Bl GrpE has been proven to be detrimental to structural integrity and proper function of the protein. [ABSTRACT FROM AUTHOR]

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 ( Bl GT), 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 Bl GT 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. [ABSTRACT FROM AUTHOR]

Abstract: Site-directed mutagenesis together with biochemical and biophysical techniques were used to probe effects of single-tryptophan-incorporated mutations on a bacterial molecular chaperone, Bacillus licheniformis DnaK (BlDnaK). Specifically, five phenylalanine residues (Phe120, Phe174, Phe186, Phe378 and Phe396) of BlDnaK were individually replaced by single tryptophans, thus creating site-specific probes for the fluorescence analysis of the protein. The steady-state ATPase activity for BlDnaK, F120W, F174W, F186W, F378W, and F396W was determined to be 76.01, 52.82, 25.32, 53.31, 58.84, and 47.53nmolPi/min/mg, respectively. Complementation test revealed that the single mutation at codons 120, 186, and 378 of the dnaK gene still allowed an Escherichia coli dnaK756-Ts strain to grow at a stringent temperature of 44°C. Simultaneous addition of co-chaperones and NR-peptide did not synergistically stimulate the ATPase activity of F174W and F396W, and these two proteins were unable to assist the refolding of GdnHCl-denatured luciferase. The heat-induced denaturation of all variants could be fitted adequately to a three-state model, in agreement with the observation for the wild-type protein. By CD spectral analysis, GdnHCl-induced unfolding transition for BlDnaK was 1.51M corresponding to ΔG N–U of 1.69kcal/mol; however, the transitions for mutant proteins were 1.07–1.55M equivalent to ΔG N–U of 0.94–2.93kcal/mol. The emission maximum of single-tryptophan-incorporated variants was in the range of 333.2–335.8nm. Acrylamide quenching analysis showed that the mutant proteins had a dynamic quenching constant of 3.0–4.2M−1. Taken together, these results suggest that the molecular properties of BlDnaK have been significantly changed upon the individual replacement of selected phenylalanine residues by tryptophan. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: An unidentified Bacillus licheniformis trehalose-6-phosphate hydrolase (BlTreA) gene was cloned and heterologously expressed in Escherichia coli M15 cells. The over-expressed BlTreA was purified to apparent homogeneity by metal-affinity chromatography and its molecular mass was determined to be approximately 65.9kDa. The temperature and pH optima for BlTreA were 30°C and 8.0, respectively. The enzyme hydrolyzed p-nitrophenyl-α-d-glucopyranoside (pNPG) and trehalose-6-phosphate efficiently, but it was inactive toward five other p-nitrophenyl derivatives. Steady-state kinetics with pNPG showed that BlTreA had a K M value of 5.2mM and a k cat value of 30.2s−1. Circular dichroism analysis revealed that the secondary structures of BlTreA did not altered by 5–10% acetone and 10–20% ethanol, whereas 5–10% SDS had a detrimental effect on the folding of the enzyme. Thermal unfolding of this enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ∼0.14M guanidine hydrochloride (GdnHCl) and reached the unfolded intermediates, [GdnHCl]0.5,N–I and [GdnHCl]0.5,I–U, at 1.02 and 2.24M, respectively. BlTreA was unfolded completely by 8M urea with [urea]0.5,N–U of 4.98M, corresponding to a free energy change of 4.29kcal/mol for the N→U process. Moreover, the enzyme was unfolded by GdnHCl through a reversible pathway and the refolding reaction exhibited an intermediate state. Taken together, the characterization data provide a foundation for the future structure–function studies of BlTreA, a typical member of glycoside hydrolase family 13. [Copyright &y& Elsevier]

Abstract: A DNA fragment encoding Bacillus licheniformis GrpE (BlGrpE) with double mutations at codons 52 and 134 was obtained during PCR cloning. Leu52 and Leu134 in BlGrpE were individually replaced with Pro and His to generate BlGrpE-L52P and BlGrpE-L134H. BlGrpE and BlGrpE-L52P synergistically stimulated the ATPase activity of B. licheniformis DnaK (BlDnaK); however, BlGrpE-L134H and the double-mutated protein (BlGrpE-L52P/L134H) had no co-chaperone function. BlGrpE, BlGrpE-L52P, and BlGrpE-L134H mainly interacted with the monomer of BlDnaK but non-specific interaction was observed for BlGrpE-L52P/L134H. Measurement of intrinsic fluorescence revealed a significant alteration of the microenvironment of aromatic acid residues in the mutant proteins. As compared with BlGrpE, quenching of 208-nm and 222-nm signals were observed in the mutant BlGrpEs and the single-mutated proteins were more sensitive to thermal denaturation. [Copyright &y& Elsevier]