Antimicrobial activity of histidine-rich peptides is dependent on acidic conditions

Synthetic peptides composed of multiples of the consensus heparin-binding Cardin and Weintraub sequences AKKARA and ARKKAAKA are antimicrobial. Replacement of lysine and arginine by histidine in these peptides completely abrogates their antimicrobial and heparin-binding activities at neutral pH. However, the antibacterial activity against Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) as well as the fungus Candida albicans, was restored at acidic conditions (pH 5.5). Fluorescence microscopy and FACS analysis showed that the binding of the histidine-rich peptides to E. coli and Candida was significantly enhanced at pH 5.5. Likewise, fluorescence studies for assessment of membrane permeation as well as electron microscopy analysis of peptide-treated bacteria, paired with studies of peptide effects on liposomes, demonstrated that the peptides induce membrane lysis only at acidic pH. No discernible hemolysis was noted for the histidine-rich peptides. Similar pH-dependent antimicrobial activities were demonstrated for peptides derived from histidine-rich and heparin-binding regions of human kininogen and histidine-rich glycoprotein. The results demonstrate that the presence of an acidic environment is an important regulator of the activity of histidine-rich antimicrobial peptides.     

Molecular weight and pH aspects of the efficacy of oligochitosan against methicillin-resistant Staphylococcus aureus (MRSA)

Oligochitosan samples varying in molecular weight (M_w) and having narrow polydispersities were prepared by means of depolymerization of chitosan in hydrochloric acid, and their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) was measured at pH values 5.5-8.0. The antibacterial testing of oligochitosans obtained showed that oligochitosans having M_w in the range of 0.73-20.0 kDa could be used both at slightly acidic and neutral pH values, and that the activity against MRSA remained moderate for oligochitosan samples having M_w about 3-5 kDa even at slightly basic pH values. The self-assembling behavior of oligochitosan macromolecules in the dilute solution at various pH values as a function of chain length was investigated. At first it was shown that oligochitosans formed supramolecular aggregates in dilute solutions below the critical pH value 6.5. Despite the aggregation phenomenon, the formation of nano-sized aggregates did not prevent oligochitosan from demonstrating the bactiostatic activity.     

Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli

Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods.     

Structural Basis of pH Dependence of Neoculin,a Sweet Taste-Modifying Protein

Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.     

Design of a bactericidal hydrogel scaffold containing genipin crosslinked HF-18 peptide

Wound healing is a process getting affected by internal and external factors and might be interrupted by infections. To overcome infections during wound healing, novel antibacterial agents such as antimicrobial peptides have gained popularity because of the rising antibiotic resistance. Therefore, in this study, a three-dimensional polymeric scaffold was designed for the controlled release of HF-18 peptide, with the contribution of hyaluronic acid, chondroitin sulfate, and chitosan polymers with the crosslinker genipin. The obtained scaffold structure (OPT) was found to have interconnected pores, was pH-responsive and swelled more in acidic conditions (5446.5% at pH: 5.0). It was observed that HF-18-loaded OPT (P-OPT) was able to release HF-18 peptide both in acidic and neutral conditions in a controlled release manner. This study also demonstrated that both OPT and P-OPT were biocompatible and promoted L929 cell attachment and migration. Antimicrobial activity assessments demonstrated that P-OPT was effectively bactericidal on Staphylococcus aureus and methicillin-resistant S. aureus. Moreover, OPT produced a synergistic effect on the antimicrobial activity of HF-18 peptide, as P-OPT showed activity below the reported MIC value. As a result, OPT is considered a promising scaffold as a carrier for HF-18 for wound healing.     

Antimicrobial activity of histidine-rich peptides is dependent on acidic conditions

Synthetic peptides composed of multiples of the consensus heparin-binding Cardin and Weintraub sequences AKKARA and ARKKAAKA are antimicrobial. Replacement of lysine and arginine by histidine in these peptides completely abrogates their antimicrobial and heparin-binding activities at neutral pH. However, the antibacterial activity against Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) as well as the fungus Candida albicans, was restored at acidic conditions (pH 5.5). Fluorescence microscopy and FACS analysis showed that the binding of the histidine-rich peptides to E. coli and Candida was significantly enhanced at pH 5.5. Likewise, fluorescence studies for assessment of membrane permeation as well as electron microscopy analysis of peptide-treated bacteria, paired with studies of peptide effects on liposomes, demonstrated that the peptides induce membrane lysis only at acidic pH. No discernible hemolysis was noted for the histidine-rich peptides. Similar pH-dependent antimicrobial activities were demonstrated for peptides derived from histidine-rich and heparin-binding regions of human kininogen and histidine-rich glycoprotein. The results demonstrate that the presence of an acidic environment is an important regulator of the activity of histidine-rich antimicrobial peptides.     

Catalytic and structural role of a metal-free histidine residue in bovine Cu-Zn superoxide dismutase

Cu-Zn superoxide dismutase (SOD) contains a conserved, metal-free His residue at an opening of the backbone beta-barrel in addition to six Cu- and/or Zn-bound His residues in the active site. We examined the protonation and hydrogen bonding state of the metal-free His residue (His41) in bovine SOD by UV Raman spectroscopy. Analysis of the His Raman intensity at 1406 cm(-1) in a D2O solution has shown that His41 has a pKa of 9.4, consistent with the NMR and X-ray structures at acidic to neutral pH, in which two imidazole nitrogen atoms of cationic His41 are hydrogen bonded to the main chain C=O groups of Thr37 and His118. Upon deprotonation of His41 at pH 9.4, the Thr37-His41-His118 hydrogen bond bridge breaks on the His118 side and SOD loses 70% of its activity. Concomitantly, hydrogen-deuterium exchange is accelerated for amide groups of beta-strands, indicating an increased conformational fluctuation of the beta-barrel. Thr37 and His41 are in direct contact with Leu36, whose hydrophobic side chain closes off the opening of the beta-barrel, while His118 is indirectly connected to Arg141 that assists the docking of superoxide to Cu. These Raman findings strongly suggest that the His41-mediated hydrogen bond bridge plays a crucial role in keeping the protein structure suitable for highly efficient catalytic reactions. The catalytic and structural role of His41 is consistent with the observation that the mutation of His43 in human SOD (equivalent to His41 in bovine SOD) to Arg largely reduces the dismutase activity and the protein structural stability.     

Activation of lactoperoxidase by heme‐linked protonation and heme‐independent iodide binding

Lactoperoxidase (LPO), a mammalian secretory heme peroxidase, catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, an antibacterial agent. Although LPO is known to be activated at acidic pH and in the presence of iodide, the structural basis of the activation is not well understood. We have examined the effects of pH and iodide concentration on the catalytic activity and the structure of LPO. Electrochemical and colorimetric assays have shown that the catalytic activity is maximized at pH 4.5. The heme Soret absorption band exhibits a small red‐shift at pH 5.0 upon acidification, which is ascribable to a structural transition from a neutral to an acidic form. Resonance Raman spectra suggest that the heme porphyrin core is slightly contracted and the Fe‐His bond is strengthened in the acidic form compared to the neutral form. The structural change of LPO upon activation at acidic pH is similar to that observed for myeloperoxidase, another mammalian heme peroxidase, upon activation at neutral pH. Binding of iodide enhances the catalytic activity of LPO without affecting either the optimum pH of activity or the heme structure, implying that the iodide binding occurs at a protein site away from the heme‐linked protonation site. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 113–120, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Phylogenetic and functional analysis of histidine residues essential for pH-dependent multimerization of von Willebrand factor

von Willebrand factor (VWF) is a multimeric plasma protein that mediates platelet adhesion to sites of vascular injury. The hemostatic function of VWF depends upon the formation of disulfide-linked multimers, which requires the VWF propeptide (D1D2 domains) and adjacent D′D3 domains. VWF multimer assembly occurs in the trans-Golgi at pH ∼6.2 but not at pH 7.4, which suggests that protonation of one or more His residues (pK_a ∼6.0) mediates the pH dependence of multimerization. Alignment of 30 vertebrate VWF sequences identified 13 highly conserved His residues in the D1D2D′D3 domains, and His-to-Ala mutagenesis identified His³⁹⁵ and His⁴⁶⁰ in the D2 domain as critical for VWF multimerization. Replacement of His³⁹⁵ with Lys or Arg prevented multimer assembly, suggesting that reversible protonation of this His residue is essential. In contrast, replacement of His⁴⁶⁰ with Lys or Arg preserved normal multimer assembly, whereas Leu, Met, and Gln did not, indicating that the function of His⁴⁶⁰ depends primarily upon the presence of a positive charge. These results suggest that pH sensing by evolutionarily conserved His residues facilitates the assembly and packaging of VWF multimers upon arrival in the trans-Golgi.     

Quaternization of N-aryl chitosan derivatives: synthesis, characterization, and antibacterial activity

Chemical modification of chitosan by introducing quaternary ammonium moieties into the polymer backbone renders excellent antimicrobial activity to the adducts. In the present study, we have synthesized 17 derivatives of chitosan consisting of a variety of N-aryl substituents bearing either electron-donating or electron-withdrawing groups. Selective N-arylation of chitosan was performed via Schiff bases formed by the reaction between the 2-amino groups of the glucosamine residue of chitosan with aromatic aldehydes under acidic conditions, followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Each of the derivatives was further quaternized using N-(3-chloro-2-hydroxypropyl)trimethylammonium chloride (Quat-188) as the quaternizing agent that reacted with either the primary amino or hydroxyl groups of the glucosamine residue of chitosan. The resulting quaternized materials were water soluble at neutral pH. Minimum inhibitory concentration (MIC) antimicrobial studies of these materials were carried out on Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria in order to explore the impact of the extent of N-substitution (ES) on their biological activities. At ES less than 10%, the presence of the hydrophobic substituent, such as benzyl and thiophenylmethyl, yielded derivatives with lower MIC values than chitosan Quat-188. Derivatives with higher ES exhibited reduced antibacterial activity due to low quaternary ammonium moiety content. At the same degree of quaternization, all quaternized N-aryl chitosan derivatives bearing either electron-donating or electron-withdrawing substituents did not contribute antibacterial activity relative to chitosan Quat-188. Neither the functional group nor its orientation impacted the MIC values significantly.     

Histidine-mediated pH-sensitive regulation of M-ficolin:GlcNAc binding activity in innate immunity examined by molecular dynamics simulations

Background

M-ficolin, a pathogen recognition molecule in the innate immune system, binds sugar residues including N-acetyl-D-glucosamine (GlcNAc), which is displayed on invading microbes and on apoptotic cells. The cis and trans Asp282-Cys283 peptide bond in the M-ficolin, which was found to occur at neutral and acidic pH in crystal structures, has been suggested to represent binding and non-binding activity, respectively. A detailed understanding of the pH-dependent conformational changes in M-ficolin and pH-mediated discrimination mechanism of GlcNAc-binding activity are crucial to both immune-surveillance and clearance of apoptotic cells.

Methodology/Principal Findings

By immunodetection analysis, we found that the pH-sensitive binding of GlcNAc is regulated by a conformational equilibrium between the active and inactive states of M-ficolin. We performed constant pH molecular dynamics (MD) simulation at a series of pH values to explore the pH effect on the cis-trans isomerization of the Asp282-Cys283 peptide bond in the M-ficolin fibrinogen-like domain (FBG). Analysis of the hydrogen bond occupancy of wild type FBG compared with three His mutants (H251A, H284A and H297A) corroborates that His284 is indispensible for pH-dependent binding. H251A formed new but weaker hydrogen bonds with GlcNAc. His297, unlike the other two His mutants, is more dependent on the solution pH and also contributes to cis-trans isomerization of the Asp282-Cys283 peptide bond in weak basic solution.

Conclusions/Significance

Constant pH MD simulation indicated that the cis active isomer of Asp282-Cys283 peptide bond was predominant around neutral pH while the trans bond gradually prevailed towards acidic environment. The protonation of His284 was found to be associated with the trans-to-cis isomerization of Asp282-Cys283 peptide bond which dominantly regulates the GlcNAc binding. Our MD simulation approach provides an insight into the pH-sensitive proteins and hence, ligand binding activity.     

Synthesis of antibacterial pseudopeptides with less hemolytic activity from a cytotoxic peptide and their pH-dependent activity

We synthesized antibacterial pseudopeptides with less hemolytic activity by incorporation of reduced amide bond ψ[CH₂NH] into α helical antibacterial peptide with hemolytic activity. As the pK_a value of reduced amide bond is 7–8, it is protonated depending on the pH. We investigated the secondary structure, the binding affinity and the leakage activity for the vesicles, and the antibacterial activity of the peptide and its pseudopeptides at neutral and basic pH. Unlike the peptide, the pseudopeptides showed a more potent leakage activity when pH increased. The peptide exhibited a lower antibacterial activity at basic pH than at neutral pH, whereas the pseudopeptide showed the same antibacterial activity at basic and neutral pH. Overall results indicated that hydrophobicity of backbone of the pseudopeptide plays an important role in the increase of leakage activity and retention of antibacterial activity at basic pH.     

The coexistence of Ser84 in renin and His13 in angiotensinogen brings a pH profile of two separate peaks to the reaction of human renin and sheep angiotensinogen

The pH dependence and kinetics parameters of renin-angiotensinogen reactions were determined using wild-type and S84G mutant human renins and wild-type and H13Y mutant sheep angiotensinogens. It is explained in this report that (i) renin catalyzes acidic and basic reactions of which the optimum pHs are 5.5 and 7.5-8.2 respectively, both of which produce angiotensin I; (ii) Ser84 specific to human renin accelerates the acidic reaction by 75-110% through elevation of V(max), and shifts the optimum pH of the basic reaction from 7.5 to 8.0-8.2; and (iii) His13 specific to sheep angiotensinogen accelerates the acidic and basic reactions by 25-42% through reduction of K(m). It is concluded from these results that the coexistence of Ser84 in renin and His13 in angiotensinogen brings a pH profile of two separate peaks at pHs 5.5 and 8.2 to the reaction of human renin and sheep angiotensinogen.     

Schistosoma mansoni NAD+ catabolizing enzyme: Identification of key residues in catalysis

Schistosoma mansoni NAD⁺ catabolizing enzyme (SmNACE), a distant homolog of mammalian CD38, shows significant structural and functional analogy to the members of the CD38/ADP-ribosyl cyclase family. The hallmark of SmNACE is the lack of ADP-ribosyl cyclase activity that might be ascribed to subtle changes in its active site. To better characterize the residues of the active site we determined the kinetic parameters of nine mutants encompassing three acidic residues: (i) the putative catalytic residue Glu202 and (ii) two acidic residues within the ‘signature’ region (the conserved Glu124 and the downstream Asp133), (iii) Ser169, a strictly conserved polar residue and (iv) two aromatic residues (His103 and Trp165). We established the very important role of Glu202 and of the hydrophobic domains overwhelmingly in the efficiency of the nicotinamide–ribosyl bond cleavage step. We also demonstrated that in sharp contrast with mammalian CD38, the ‘signature’ Glu124 is as critical as Glu202 for catalysis by the parasite enzyme. The different environments of the two Glu residues in the crystal structure of CD38 and in the homology model of SmNACE could explain such functional discrepancies. Mutagenesis data and 3D structures also indicated the importance of aromatic residues, especially His103, in the stabilization of the reaction intermediate as well as in the selection of its conformation suitable for cyclization to cyclic ADP-ribose. Finally, we showed that inhibition of SmNACE by the natural product cyanidin requires the integrity of Glu202 and Glu124, but not of His103 and Trp165, hence suggesting different recognition modes for substrate and inhibitor.     

Evidence for pH-Dependent Protease Activity in the Adeno-Associated Virus Capsid

Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection.     

Studies of the Physiological and Genetic Basis of Acid Tolerance in Rhizobium leguminosarum biovar trifolii

Acid-tolerant Rhizobium leguminosarum biovar trifolii ANU1173 was able to grow on laboratory media at a pH as low as 4.5. Transposon Tn5 mutagenesis was used to isolate mutants of strain ANU1173, which were unable to grow on media at a pH of less than 4.8. The acid-tolerant strain ANU1173 maintained a near-neutral intracellular pH when the external pH was as low as 4.5. In contrast, the acid-sensitive mutants AS25 and AS28 derived from ANU1173 had an acidic intracellular pH when the external pH was less than 5.5. The acid-sensitive R. leguminosarum biovar trifolii ANU794, which was comparatively more sensitive to low pH than mutants AS25 and AS28, showed a more acidic internal pH than the two mutants when the three strains were exposed to medium buffered at a pH of less than 5.5. The two acid-sensitive mutants had an increased membrane permeability to protons but did not change their proton extrusion activities. However, the acid-sensitive strain ANU794 exhibited both a higher membrane permeability to protons and a lower proton extrusion activity compared with the acid-tolerant strain ANU1173. DNA hybridization analysis showed that mutants AS25 and AS28 carried a single copy of Tn5 located in 13.7-kb (AS25) and 10.0-kb (AS28) EcoRI DNA fragments. The wild-type DNA sequences spanning the mutation sites of mutants AS25 and AS28 were cloned from genomic DNA of strain ANU1173. Transfer of these wild-type DNA sequences into corresponding Tn5-induced acid-sensitive mutants, respectively, restored the mutants to their acid tolerance phenotypes. Mapping studies showed that the AS25 locus was mapped to a 5.6-kb EcoRI-BamHI megaplasmid DNA fragment, whilst the AS28 locus was located in an 8.7-kb BglII chromosomal DNA fragment.     

Green tea polyphenols function as prooxidants to inhibit Pseudomonas aeruginosa and induce the expression of oxidative stress-related genes

Green tea polyphenols (GTP) are widely believed to function as antioxidants and antimicrobial agents. Here we observed that GTP and epigallocatechin gallate, the most abundant catechin in GTP, could also function as prooxidants and produce hydrogen peroxide (H₂O₂) to inhibit the growth of Pseudomonas aeruginosa. pH value of the medium was the key factor that affected prooxidant versus antioxidant property of GTP. Under weakly acidic conditions (pH 5.5–6.5), GTP showed antioxidant activity by eliminating H₂O₂; whereas, under neutral and weakly alkaline conditions (pH 7.0–8.0), GTP showed prooxidant activity and inhibited the growth of P. aeruginosa. Furthermore, we studied the effects of GTP on gene expression profiles of a few oxidative stress-related genes by quantitative real-time PCR analysis. After 10 min to 1 h of exposure under weakly alkaline condition, GTP significantly up-regulated expression levels of katB, sodM, ohr, lexA, and recN gene. These findings highlight that the pH-dependent H₂O₂ production by GTP contributes to the antibacterial activity and can induce oxidative stress-related responses in P. aeruginosa.     

A single histidine residue modulates enzymatic activity in acidic mammalian chitinase

Mammals express two active chitinases, chitotriosidase and AMCase. Only AMCase displays an extremely acidic pH optimum, consistent with its observed presence in the gastro-intestinal tract. A structural model of AMCase reveals the presence of a conserved histidine residue in the active site. Mutational analyses and molecular dynamics simulations show that His187 is responsible for the acidic optimum and suggest pH dependent modulation of the reaction mechanism that is unique to AMCases. Concluding, His187 is a crucial structural component of the active site of AMCase and this unique feature may serve as a lead for the development of specific inhibitors.     

Improving the acidic stability of a β-mannanase from Bacillus subtilis by site-directed mutagenesis

β-Mannanase can randomly hydrolyze the (1→4)-β-d-mannosidic linkages in mannans, galactomannans and glucomannans, yielding manno-oligosaccharides. In this study, the β-mannanase (MAN) from Bacillus subtilis B10-02 was overexpressed successfully in B. subtilis 168 as a hexa-histidine tagged, secreted protein. The recombinant enzyme BsMAN6H was not stable under acidic conditions, which restricts its use in food and feed industry. We aimed to improve the acid stability of BsMAN6H by changing several surface-exposed amino acid residues to acidic or neutral ones. Among the mutations, the His54Asp resulted in a shift in the optimal pH from 6.5 to 5.5. Accordingly, the acid stability was improved by a factor of a negative potential on the structure surface around the mutated site. Furthermore, the H54D variant showed the enzyme activity up to 3207.82 U/mL in bioreactors using the cheap Kojac powder as substrate. As a result, a bacterial β-mannanase was produced efficiently with increased acid stability, improving its applicability in the animal feed industry.     

The Importance of pH in Regulating the Function of the Fasciola hepatica Cathepsin L1 Cysteine Protease

The helminth parasite Fasciola hepatica secretes cathepsin L cysteine proteases to invade its host, migrate through tissues and digest haemoglobin, its main source of amino acids. Here we investigated the importance of pH in regulating the activity and functions of the major cathepsin L protease FheCL1. The slightly acidic pH of the parasite gut facilitates the auto-catalytic activation of FheCL1 from its inactive proFheCL1 zymogen; this process was ∼40-fold faster at pH 4.5 than at pH 7.0. Active mature FheCL1 is very stable at acidic and neutral conditions (the enzyme retained ∼45% activity when incubated at 37°C and pH 4.5 for 10 days) and displayed a broad pH range for activity peptide substrates and the protein ovalbumin, peaking between pH 5.5 and pH 7.0. This pH profile likely reflects the need for FheCL1 to function both in the parasite gut and in the host tissues. FheCL1, however, could not cleave its natural substrate Hb in the pH range pH 5.5 and pH 7.0; digestion occurred only at pH≤4.5, which coincided with pH-induced dissociation of the Hb tetramer. Our studies indicate that the acidic pH of the parasite relaxes the Hb structure, making it susceptible to proteolysis by FheCL1. This process is enhanced by glutathione (GSH), the main reducing agent contained in red blood cells. Using mass spectrometry, we show that FheCL1 can degrade Hb to small peptides, predominantly of 4–14 residues, but cannot release free amino acids. Therefore, we suggest that Hb degradation is not completed in the gut lumen but that the resulting peptides are absorbed by the gut epithelial cells for further processing by intracellular di- and amino-peptidases to free amino acids that are distributed through the parasite tissue for protein anabolism.