γ-Glutamyl transpeptidase from Bacillus pumilus KS 12: decoupling autoprocessing from catalysis and molecular characterization of N-terminal region

Gamma glutamyl transpeptidase from Bacillus pumilus KS12 (GGTBP) was cloned, expressed in pET-28-E. coli expression system as a heterodimeric enzyme with molecular weights of 45 and 20 kDa for large and small subunit, respectively. It was purified by nickel affinity chromatography with hydrolytic and transpeptidase activity of 1.82 U/mg and 4.35 U/mg, respectively. Sequence analysis revealed that GGTBP was most closely related to Bacillus licheniformis GGT and had all the catalytic residues and nucleophiles for autoprocessing recognized from E. coli. It was optimally active at pH 8 and 60°C. It exhibited pH stability from pH 6-9 and high thermostability with t(1/2) of 15 min at 70°C. It had K(m), V(max) of 0.045 mM, 4.35 μmol/mg/min, respectively. Decoupling of autoprocessing by co-expressing large and small subunit in pET-Duet1-E. coli expression system yielded active enzyme with transpeptidase activity of 5.31 U/mg. Though N-terminal truncations of rGGTBP upto 95 aa did not affect autoprocessing of GGT however activity was lost with truncation beyond 63 aa.     

γ-Glutamyl transpeptidase: catalytic,structural and functional aspects

Summary -Glutamyl transpeptidase catalyzes transfer of the -glutamyl moiety of glutathione to amino acids, dipeptides, and to glutathione itself; the enzyme also catalyzes the hydrolysis of glutathione to glutamate and cysteinyl-glycine. This review deals with the tissue distribution and localization of the enzyme in mammals, the catalytic properties of the enzyme (including its inhibition by reversible and irreversible inhibitors), structural studies on the enzyme, and new findings about its physiological function.     

Effect of temperature on some characteristics of the thermostable α-amylase from Bacillus licheniformis

The V _max of an extracellular, thermostable -amylase from Bacillus licheniformis 44MB82 were 5.70×10^-3 and 9.70×10^-3 mM s^-1 at 30 and 90°C, respectively, whereas the K _m values were similar (0.9 mg ml^-1) at both temperatures. Excluding dextrins, the dominant products from soluble starch and amylopectin hydrolysis contained less than six glucose residues. The enzyme hydrolysed amylopectin better than soluble starch. Increasing the temperature from 30 to 90°C was accompanied by an increase in the production of malto-oligosaccharides, especially maltotetrose, and this was related to the secondary hydrolysis of maltopentose and maltohexose.The authors are with the Institute of Microbiology, Bulgarian Academy of Sciences, Sofia 1113. 26 Academician G. Bonchev, Bulgaria     

Operational and storage stability of neutral β-mannanase from Bacillus licheniformis

The stability of neutral -mannanase from Bacillus licheniformis during operation and storage was investigated. The enzyme activity decreased by 70% with a hydrolysate of glucomannan at 20 g l^–1 over 30 min at 25 °C. In an enzymatic membrane reactor operated at 50 °C after 24 h, the loss of enzyme activities were 23% and 9% in the absence/presence of the substrate. The residual activities of the enzyme were 21% and 90%, respectively, when stored in 30% (v/v) glycerol solution and in solid state at 4 °C after one year.     

Hyperproduction of γ-glutamyl transpeptidase from Bacillus licheniformis ER15 in the presence of high salt concentration

Background: Microbial γ-glutamyl transpeptidases (GGTs) have been exploited in biotechnological, pharmaceutical, and food sectors for the synthesis of various γ-glutamyl compounds. But, till date, no bacterial GGTs are commercially available in the market because of lower levels of production from various sources. In the current study, production of GGT from Bacillus licheniformis ER15 was investigated to achieve high GGT titers. Results: Hyperproduction of GGT from B. licheniformis ER15 was achieved with 6.4-fold enhancement (7921.2?±?198.7?U/L) by optimization of culture medium following one-variable-at-a-time strategy and statistical approaches. Medium consisting of Na₂HPO₄: 0.32% (w/v); KH₂PO₄: 0.15% (w/v); starch: 0.1% (w/v); soybean meal: 0.5% (w/v); NaCl: 4.0% (w/v), and MgCl₂: 5?mM was found to be optimal for maximum GGT titers. Maximum GGT titers were obtained, in the optimized medium at 37°C and 200?rpm, after 40?h. It was noteworthy that GGT production was a linear function of sodium chloride concentration, as observed during response surface methodology. While investigating the role of NaCl on GGT production, it was found that NaCl drastically decreased subtilisin concentration and indirectly increasing GGT recovery. Conclusion: B. licheniformis ER15 is proved to be a potential candidate for large-scale production of GGT enzyme and its commercialization.     

Escherichia coli signal peptidase recognizes and cleaves the signal sequence of α-amylase originating from Bacillus licheniformis

The gene encoding the α-amylase from Bacillus licheniformis was cloned, with and without the native signal sequence, and expressed in Escherichia coli, resulting in the production of the recombinant protein in the cytoplasm as insoluble but enzymatically active aggregates. Expression with a low concentration of the inducer at low temperature resulted in the production of the recombinant protein in soluble form in a significantly higher amount. The protein produced with signal sequence was exported to the extracellular medium, whereas there was no export of the protein produced from the gene without the signal sequence. Similarly, the α-amylase activity in the culture medium increased with time after induction in case of the protein produced with signal sequence. Molecular mass determinations by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing of the purified recombinant α-amylase from the extracellular medium revealed that the native signal peptide was cleaved by E. coli signal peptidase between Ala28 and Ala29. It seems possible that the signal peptide of α-amylase from B. licheniformis can be used for the secretion of other recombinant proteins produced using the E. coli expression system.     

Effect of glycerol–water binary mixtures on the structure and dynamics of protein solutions

We have performed 20?ns of fully atomistic molecular dynamics simulations of Hen Egg-White Lysozyme in 0, 10, 20, 30, and 100% by weight of glycerol in water to better understand the microscopic physics behind the bioprotection offered by glycerol to naturally occuring biological systems. The solvent exposure of protein surface residues changes when glycerol is introduced. The dynamic behavior of the protein, as quantified by the incoherent intermediate scattering function, shows a nonmonotonic dependence on glycerol content. The fluctuations of the protein residues with respect to each other were found to be similar in all water-containing solvents, but different from the pure glycerol case. The increase in the number of protein–glycerol hydrogen bonds in glycerol–water binary mixtures explains the slowing down of protein dynamics as the glycerol content increases. We also explored the dynamic behavior of the hydration layer. We show that the short length scale dynamics of this layer are insensitive to glycerol concentration. However, the long length scale behavior shows a significant dependence on glycerol content. We also provide insights into the behavior of bound and mobile water molecules.     

Effect of polyols on the structure and aggregation of recombinant human γ-Synuclein,an intrinsically disordered protein

Polyol osmolytes accumulated in cells under stress are known to promote stability in globular proteins with respect to their increasing hydroxyl groups but their effect on the structure, stability and aggregation of intrinsically disordered proteins (IDPs) is still elusive. The lack of a natively folded structure in intrinsically disordered proteins under physiological conditions results in their aggregation and fibrillation that gives rise to a number of diseases. We have investigated the effect of a series of polyols, ethylene glycol (EG), glycerol, erythritol, xylitol and sorbitol on the fibrillation pathway of recombinant human γ-Synuclein, used as a model, for an IDP known to form fibrils that play a role in neurodegeneration and cancer. With an increase in the number of –OH groups in polyols except EG, we observe a decrease in lag time for fibrillation at equimolar concentrations, suggesting stronger preferential exclusion of polyols that promotes γ-Syn self-association and oligomerization. The polyols act early during nucleation and their diverse effect on the rate of fibrillation suggests the role of favourable solvent-side chain interactions. With increasing –OH group, polyols stabilize the natively unfolded conformation of γ-Syn under non-fibrillating conditions and delay the structural transition to characteristic β-sheet structure by forming an α-helical intermediate during fibrillation. The results, overall suggest that the effect of osmolytes on IDPs is much more complex than their effect on globular protein stability and aggregation and a fine balance between the dominant unfavourable osmolyte-peptide backbone and favourable osmolyte-charged side chain interactions would govern their stability and aggregation properties.     

Significance of Tyr302, His235 and Asp194 in the α-amylase from Bacillus licheniformis

The calcium-binding residues, Tyr302 and His235, and the sodium-binding residue, Asp194, on the activity of Bacillus licheniformis α-amylase were investigated using site-directed mutagenesis. Tyr302 and His235 were replaced by Asn and Asp, respectively, to produce the mutants Y302N and H235D; Asp194 was replaced by Ala to produce D194A. The mutant amylases were purified to homogeneity; each was ~53?kDa. The specific activity of the D194A was 236?U?mg(-1), lower than the specific activity of the wild-type enzyme by 55%. No significant changes of thermostability, optimum temperature, and optimum pH level were observed in D194A. Mutant amylases with H235D and Y302N significantly improved their specific activity by 43% (754?U?mg(-1)) and 7% (563?U?mg(-1)), respectively, compared with the wild-type enzyme. H235D substitution decreased its optimum pH by approx. 0.5-1 pH unit.     

Integration,amplification and expression of the Bacillus licheniformis α-amylase gene in Bacillus subtilis chromosome

We have introduced the α-amylase gene from Bacillus licheniformis (amy gene) in a non-replicative plasmid which can be conveniently integrated and amplified at a specific site of the B. subtilis chromosome. Although we were able to select spontaneous and stable gene amplification of about 20 integrated copies, the amylase secretion remained very low. A DNA fragment presenting a high promoter activity in B. subtilis was therefore inserted upstream from the amy gene coding sequence, leading to a significant increase of amylase production. However, the amplified structures obtained with this construction were found to contain no more than 12 copies of the amy gene and to be rather unstable when cells were grown under non-selective conditions.     

Cloning, Functional Expression and Characterization of an Alkaline Protease from Bacillus licheniformis

A gene (apr 46) encoding a protease was cloned from Bacillus licheniformis RSP-09-37. It had an ORF of 1725 bp, encoding a pre-protein of 575 amino acids (63.2 kDa), which was functionally expressed and processed in E. coli JM 109. The mature protein, Apr 46, consists of 500 amino acids with a calculated molecular mass of 55 kDa. This protease shows 29-50% homology to known serine proteases and conserved domains. N-terminal sequencing suggests that Apr 46 protease is identical to a B. licheniformis RSP-09-37 protease, which is further supported by a similar stability in acetonitrile.     

Effect of NaCl on the conformational stability of the thermophilic γ-glutamyltranspeptidase from Geobacillus thermodenitrificans: Implication for globular protein halotolerance

The transpeptidation activity of γ-glutamyltranspeptidase from Geobacillus thermodenitrificans (GthGT) is negligible and the enzyme is highly thermostable. Here we have examined the effect of concentrated NaCl solutions on structure, stability, dynamics and enzymatic activity of GthGT. The protein exhibited hydrolytic activity over a broad range of NaCl concentrations. Even at 4.0 M NaCl, GthGT retained more than 90% of the initial activity and showed unaltered fluorescence emission, secondary structure and acrylamide quenching on tryptophan fluorescence. Furthermore, at 2.8 M and 4.0 M NaCl the temperature-induced unfolding profiles are dramatically changed with large (> 20 °C) positive shifts in the denaturation temperature. These features make GthGT an ideal system to be used in industrial processes that require high temperatures and high-salt environments. A general explanation of the NaCl effect by means of a statistical thermodynamic model is also provided, together with an analysis of residue distribution between protein surface and interior in 15 non-redundant families of halophilic and non-halophilic proteins. The results are in line with a comparative sequence and structural analysis between halophilic and non-halophilic γ-glutamyltranspeptidases which revealed that a major role in halotolerance should be played by solvent exposed negatively charged residues.     

Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?

We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge −19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT_trs/d[ion] (T_trs; thermal midpoint). We show that dT_trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins.     

Effect of immobilization on stability and properties of N-acetyl-β-d-hexosaminidase from Turbo cornutus

A mixture of glycosidases from the liver of the gastropod Turbo cornutus was co-immobilized with bovine serum albumin and glutaraldehyde, and then cast as membranes. The properties of immobilized N-acetyl-β-d-hexosaminidase were studied. The recovery of N-acetyl-β-d-hexosaminidase after immobilization was unaffected by increasing the concentration of glutaraldehyde, but was decreased by increasing the bovine serum albumin concentration. The immobilized enzyme showed enhanced resistance towards proteolytic and thermal inactivation. While the pH optimum for the soluble enzyme was 4.0, a bimodal pH curve with optima at 3.4 and 5.0 was observed after insolubilization. This bimodality was abolished when the immobilized enzyme was assayed in the presence of M NaCl. The K_m values, for p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside, of the immobilized isoenzymes of N-acetyl-β-d-hexosaminidase were larger than those of their soluble counterparts. No loss of activity could be detected in the membrane after using it for 24 consecutive assays or after storage for at least 50 days at 4°.     

Effect of protein aggregation on the spectroscopic properties and excited state kinetics of the LHCII pigment–protein complex from green plants

Steady-state and time-resolved absorption and fluorescence spectroscopic experiments have been carried out at room and cryogenic temperatures on aggregated and unaggregated monomeric and trimeric LHCII complexes isolated from spinach chloroplasts. Protein aggregation has been hypothesized to be one of the mechanistic factors controlling the dissipation of excess photo-excited state energy of chlorophyll during the process known as nonphotochemical quenching. The data obtained from the present experiments reveal the role of protein aggregation on the spectroscopic properties and dynamics of energy transfer and excited state deactivation of the protein-bound chlorophyll and carotenoid pigments.     

Impact of cysteine variants on the structure,activity, and stability of recombinant human α-galactosidase A

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.     

Effect of zinc salts on the structure–function of actomyosin from pelagic fish

The effect of zinc salts, zinc chloride and zinc sulfate on the structure and ATPase enzyme activity of actomyosin from pelagic fish (Sardinella longiceps) has been investigated. ATPase enzyme activity decreased in the presence of both the zinc salts. The inhibitory effect is present in both pH of 7.0 and 9.0. At concentration of 1 × 10^?3 M of zinc salts, complete inhibition of ATPase enzyme activity is observed. With the increase in temperature from 25 °C to 45 °C the ATPase activity decreased by nearly 80%. The solubility profile of actomyosin in the presence of zinc salts shows a sigmoid pattern as the concentration of both the zinc salts increases. Free SH content of actomyosin decreased with the increase in concentration of zinc salts. Intrinsic fluorescence indicated significant decrease in relative fluorescence intensity of actomyosin. This indicates significant alterations in the structure of actomyosin. Analysis of secondary structure also indicates significant alteration in the α-helical content upon binding of both zinc salts.     

Influence of the SNPs on the structural stability of CBS protein： Insight from molecular dynamics simulations

Cystathionine β-synthase is an essential enzyme of the trans-suifuration pathway that condenses serine with homocysteine to form cystathionine. Missense mutations in CBS are the major cause of inherited homocystinuria, and the detailed effect of disease associated amino acid substitutions on the structure and stability of human CBS is yet unknown. Here, we apply a unique approach in combining in silico tools and molecular dynamics simulation to provide structural and functional insight into the effect of SNP on the stability and activity of mutant CBS. In addition, principal component analysis and free energy landscape were used to predict the collective motions, thermodynamic stabilities and essential subspace relevant to CBS function. The obtained results indicate that C109R, E176K and D376N mutations have the diverse effect on dynamic behavior of CBS protein. We found that highly conserved D376N mutation, which is present in the active pocket, affects the protein folding mechanism. Our strategy may provide a way in near future to understand and study effects of functional nsSNPs and their role in causing homocystinuria.