Trehalose analogues: latest insights in properties and biocatalytic production

Trehalose (alpha-d-glucopyranosyl alpha-d-glucopyranoside) is a non-reducing sugar with unique stabilizing properties due to its symmetrical, low energy structure consisting of two 1,1-anomerically bound glucose moieties. Many applications of this beneficial sugar have been reported in the novel food (nutricals), medical, pharmaceutical and cosmetic industries. Trehalose analogues, like lactotrehalose (alpha-d-glucopyranosyl alpha-d-galactopyranoside) or galactotrehalose (alpha-d-galactopyranosyl alpha-d-galactopyranoside), offer similar benefits as trehalose, but show additional features such as prebiotic or low-calorie sweetener due to their resistance against hydrolysis during digestion. Unfortunately, large-scale chemical production processes for trehalose analogues are not readily available at the moment due to the lack of efficient synthesis methods. Most of the procedures reported in literature suffer from low yields, elevated costs and are far from environmentally friendly. "Greener" alternatives found in the biocatalysis field, including galactosidases, trehalose phosphorylases and TreT-type trehalose synthases are suggested as primary candidates for trehalose analogue production instead. Significant progress has been made in the last decade to turn these into highly efficient biocatalysts and to broaden the variety of useful donor and acceptor sugars. In this review, we aim to provide an overview of the latest insights and future perspectives in trehalose analogue chemistry, applications and production pathways with emphasis on biocatalysis

Characterization of modular bacteriophage endolysins from giant phiKZ related myoviridae phages OBP, 201phi2-1 and PVP-SEl

Peptidoglycan lytic enzymes (endolysins) of bacteriophages have a major role in bacterial lysis at the end of the phage replication cycle. These endolysins turned out to be potential antibacterial compounds to combat a broad range of Gram-positive pathogens, yet Gram-negative bacteria remain unharmed due to their impermeable outer membrane. With this background, we recently characterized three new endolysins from Gramnegative origin: OBPgp279 (Pseudomonas fluorescens phage OBP), PVP-SElgpl46 (Salmonella Enteritidis phage PVP-SEl) and 20lphi2-lgp229 (Pseudomonas chlororaphis phage 201phi2-1). These endolysins share a modular structure with anNterminal cell wall binding domain and a C-terrninal catalytic domain, a unique property of endolysins belonging to giant phiKZ related phages and some other giant, non-related myoviruses. All three endolysins showed strong muralytic activity on the peptidoglycan of a broad range of Gram-negative bacteria, a feature linked with their modular composition. In case of OBPgp279, the presence of the cell wall binding domain is responsible for 38 % of the total muralytic activity. Moreover, the binding domain of PVP-SE1gp146 has a binding affinity for Salmonella peptidoglycan that falls within the range of typical cell adhesion molecules. Remarkably, PVP-SElgp146 shows thermoresistant properties up to temperatures of 90°C, making it a potential candidate as antibacterial in hurdle technology for food preservation. OBPgp279, on the other hand. is able to pass the outer membrane of P. aeruginosa PAO 1 using an unknown mechanism, thereby gaining access to its peptidoglycan and reduce the bacterium with !logarithmic unit. Addition of the outer membrane permeabilizer EDTA significantly increased the antibacterial activity of the three endolysins up to 2-3 logarithmic units. This research offers perspectives towards elucidation of the structural differences explaining the unique biochemical and antibacterial properties ofOBPgp279, PVP-SE1gp146 and 201phi2- lgp229. Furthermore, these endolysins extensively enlarge the pool of potential antibacterial compounds used for treatment of Gram-negative bacterial infections

Antibacterial activity on opportunistic Pseudomonas aeruginosa pathogen by a novel Salmonella phage endolysin

The Gram-negative pathogen Pseudomonas aeruginosa can cause severe infections of burn wound or cystic fibrosis on patients. Bacteriophage endolysin based strategy can offer a new alternative antimicrobial therapy. Endolysins are lytic enzymes that break down the peptidoglycan of bacterial cell wall at the late phage lytic cycle, however they are inactive on their own against Gram-negative bacteria when applied exogenously as recombinant proteins due to the peptidoglycan (endolysin substrate) protective outer membrane.We propose an innovative strategy to target Gram-negative Ps. aeruginosa based on the combination of endolysin enzymes and an outer membrane permeabilizing agent - ethylenediamine tetraacetic acid (EDTA).To validate this approach, we have isolated a novel Salmonella phage endolysin (68gpLys). Cloning this gene into E. coli expression system and subsequent large scale protein expression led to a high soluble yield of 14,3 mg/L of expression culture. In order to characterized it, muralytic assays on chloroform/Tris-HCl pretreated Ps. aeruginosa strain PAO1k (to remove the outer membrane) were made to check activity levels on substrate (398.05 Units/mM). The pH range was also determined with pH 7 being the optimum for the endolysin activity. For antimicrobial test, in vitro assays showed that incubation of 106 Ps. aeruginosa cells/mL with 0.5 mM EDTA and 5000 nM of 68gpLys, led to a strain inactivation of 3.42 ± 0.02 logarithmic reduction units in a time-frame of 30 min.Here we prove that the synergistic effect of endolysin 68gpLys with EDTA can significantly reduce Ps. aeruginosa contamination. These results suggests, the great potential of this strategy for prevention and/or control of other Gram-negative pathogens.Current work has been also development to engineer new endolysins with incorporated cell penetrating peptides (CPP), employing sited- and random-mutagenesis molecular techniques, to further enhance outer membrane permeabilization

Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa

Artilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophage-encoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and kill Pseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (similar to 5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect against P. aeruginosa persisters (up to > 4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections

A thermostable salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.This work was supported by the projects FCOMP-01-0124-FEDER-019446, FCOMP-01-0124-FEDER-027462 and PEst-OE/EQB/LA0023/2013 from "Fundacao para a Ciencia e Tecnologia" (FCT), Portugal. The authors thank the Project "BioHealth - Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte), QREN, FEDER. Hugo Oliveira acknowledges the FCT grant SFRH/BD/63734/2009. Maarten Walmagh held a PhD scholarship of the IWT Vlaanderen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Characterization of Modular Bacteriophage Endolysins from Myoviridae Phages OBP, 201ϕ2-1 and PVP-SE1

Peptidoglycan lytic enzymes (endolysins) induce bacterial host cell lysis in the late phase of the lytic bacteriophage replication cycle. Endolysins OBPgp279 (from Pseudomonas fluorescens phage OBP), PVP-SE1gp146 (Salmonella enterica serovar Enteritidis phage PVP-SE1) and 201ϕ2-1gp229 (Pseudomonas chlororaphis phage 201ϕ2-1) all possess a modular structure with an N-terminal cell wall binding domain and a C-terminal catalytic domain, a unique property for endolysins with a Gram-negative background. All three modular endolysins showed strong muralytic activity on the peptidoglycan of a broad range of Gram-negative bacteria, partly due to the presence of the cell wall binding domain. In the case of PVP-SE1gp146, this domain shows a binding affinity for Salmonella peptidoglycan that falls within the range of typical cell adhesion molecules (Kaff = 1.26×106 M−1). Remarkably, PVP-SE1gp146 turns out to be thermoresistant up to temperatures of 90°C, making it a potential candidate as antibacterial component in hurdle technology for food preservation. OBPgp279, on the other hand, is suggested to intrinsically destabilize the outer membrane of Pseudomonas species, thereby gaining access to their peptidoglycan and exerts an antibacterial activity of 1 logarithmic unit reduction. Addition of 0.5 mM EDTA significantly increases the antibacterial activity of the three modular endolysins up to 2–3 logarithmic units reduction. This research work offers perspectives towards elucidation of the structural differences explaining the unique biochemical and antibacterial properties of OBPgp279, PVP-SE1gp146 and 201ϕ2-1gp229. Furthermore, these endolysins extensively enlarge the pool of potential antibacterial compounds used against multi-drug resistant Gram-negative bacterial infections

Development and evaluation of engineered bacteriophage endolysins to inactivate Gram-negative bacteria.

Bacteriophages, viruses infecting bacteria, disrupt the bacterial cell wall at the end of their replication cycle to release newly produced virions. The major constituent of the bacterial cell wall is the peptidoglycan. To degrade this rigid layer, bacteriophages encode peptidoglycan hydrolases, called endolysins, that hydrolyze specific bonds in the peptidoglycan. This dissertation specifically focuses on endolysins, isolated from phages infecting Gram-negative bacterial species, including Pseudomonas aeruginosa, Salmonella Typhimurium, Escherichia coli, Klebsiella pneumoniae and Citrobacter rodentii. Most of these bacteria are opportunistic pathogens that are of increasing concern in hospitals due to their high intrinsic and acquired antibiotic resistance. In a first part of this study, we extend the pool of available endolysins from Gram-negative origin and analyze their potential and applicability as alternative antibacterial agents for antibiotics to combat these Gram-negative pathogens. The Gram-negative outer membrane prevents exogenously applied endolysins from reaching the peptidoglycan layer and protects bacteria against their lytic activity. We therefore evaluate in the second part of this dissertation an approach that allows the endolysin to efficiently destabilize the outer membrane and subsequently reach the peptidoglycan. This approach consists of the fusion of a set of outer membrane-permeabilizing antimicrobial peptides to the endolysin to allow for an autonomous interaction with the outer membrane. To sketch the background, this dissertation starts with an overview of the literature concerning bacteriophage endolysins (history and structural diversity), antimicrobial peptides (types and mode of action) and outer membrane diversity present among Gram-negative bacteria.From an in silico analysis of fully sequenced phage genomes, a selection of fifteen interesting candidate endolysins is made (Chapter 4). Six single-domain (Chapter 5) and three modular (Chapter 6) endolysins with the highest maximal muralytic activity under physiological conditions, are selected for extensive characterization on biochemical (pH-dependency, enzymatic activity, activity upon heating) and antibacterial level. In this way, we aim to prove their lytic role and to reveal enzyme-specific characteristics interesting from an application perspective. In silico, the single-domain endolysins consist of a catalytic domain, whereas the modular ones feature an N-terminal peptidoglycan binding domain and a C-terminal catalytic domain, hitherto a unique property present in a few endolysins from Gram-negative origin. In addition, the predicted peptidoglycan binding domains are experimentally verified.The modular endolysins in this study are shown to be enzymatically more active than the single-domain endolysins, an observation that was translated into their in vitro antibacterial activity. Of all tested endolysins, the modular endolysin from Pseudomonas fluorescens phage OBP, OBPgp279, shows the highest muralytic and antibacterial activity, followed by PVP-SE1gp146, the endolysin from Salmonella Enteritidis phage PVP-SE1. The peptidoglycan binding domain present in modular endolysins accounts for their strong lytic action since the contribution of this domain (38 to 56 %) to the total enzymatic activity is considerable. In addition, the enzymatic activity is consistent for the different Gram-negative bacterial species due to their conserved peptidoglycan (A1gamma chemotype). This characterization also revealed various interesting biochemical properties. OBPgp279 shows intrinsic antibacterial activity on P. aeruginosa PAO1 (± 1 log unit), probably by destabilizing the Pseudomonas outer membrane. PVP-SE1gp146 remains active up to temperatures of 90°C with 60 % residual enzymatic activity after 40 minutes. This last property makes the enzyme a potential candidate as antibacterial component in hurdle technology for food preservation. At the start of the second part, OBPgp279 and PVP-SE1gp146, the two most promising endolysins, are selected to evaluate the proposed fusion approach for passage of the outer membrane (Chapter 7). The N-terminal fusion of a polycationic PK peptide (KRKKRKKRK) composed of lysine and arginine residues, turns out to be the most effective fusion to improve the antibacterial activity of both endolysins. The highest activity is reached for P. aeruginosa with maximal 2.61 log units. Addition of minor EDTA concentrations enhances activity and extends the activity range with E. coli (maximal 1.70 log units) and S. Typhimurium (maximal 0.91 log units). This fused PK peptide is believed to compete with the Achilles heel of the outer membrane: the stabilizing divalent cations. A double N-terminal fusion of this promising PK peptide with other antimicrobial peptides only increases the antibacterial activity of OBPgp279 against E. coli to maximal 2.22 log units (for PP-PK double fusion), but is detrimental for the activity against other Gram-negative species. Analysis for the impact of the N-terminal PK fusion on endolysin characteristics reveals a protein-dependent inhibition of the enzymatic activity (with 52 to 94 %), a reduced heat resistance and a switch in pH-dependency to slightly more alkaline values (Chapter 8). Due to a more hydrophobic outer membrane, the antibacterial efficacy of the PK fusion is limited for Enterobacteriaceae. Additionally, the PK fusion also confers biofilm-degrading activity to PVP-SE1gp146. Extension of the linker length between the PK peptide and endolysin partly reconstitutes the reduced muralytic activity due to the PK peptide fusion, leading to an improved antibacterial activity against Pseudomonads and Enterobacteriaceae. Switching the PK peptide to the C-terminal end does not improve activity. These data nicely illustrate that endolysins can be turned into effective anti-Gram-negative compounds by an N-terminalfusion approach and subsequent optimization of the linker length.In the last part, we evaluate the PK-fused endolysin approach on an in vitro human keratinocyte monolayer and an in vivo Caenorhabditis elegans model. PK-PVP-SE1gp146 is able to protect the keratinocyte monolayer from a P. aeruginosa PA14 infection (Chapter 9). In addition, PK-PVP-SE1gp146 improves the survival of PA14-infected C. elegans with 60 % after five days of treatment (Chapter 10). These results prove the in vitro and in vivo applicability of the PK-fused endolysin approach against P. aeruginosa, offering promising perspectives towards prophylactic and therapeutic applications in human health and veterinary and towards microbial decontamination purposes in the food industry.Table of contents Dankwoord I Summary III Samenvatting V List of abbreviations VIII List of publications IX Chapter 1 Introduction and background 1 1.1 General introduction 1 1.2 Bacteriophage-encoded peptidoglycan hydrolases 2 1.2.1 History on bacteriophage-encoded peptidoglycan hydrolases 3 1.2.2 Natural role of peptidoglycan hydrolases in bacterial lysis mechanisms 5 1.2.3 Structural composition of phage endolysins 9 1.3 Gram-negative outer membrane as a natural barrier for external agents 17 1.3.1 Structure and diversity of lipopolysaccharide 17 1.3.2 Outer membrane-based resistance mechanisms 24 1.3.3 Possible strategies to overcome the outer membrane barrier 27 1.4 Antimicrobial peptides 32 1.4.1 Structure of antimicrobial peptides 32 1.4.2 Mode of action of antimicrobial peptides 33 1.4.3 Optimizing activity of antimicrobial peptides 40 Chapter 2 Aims and study objectives 41 Chapter 3 Materials and methods 43 3.1 Bacterial strains and Caenorhabditis elegans SS104 43 3.2 Cloning, recombinant large scale expression and protein purification 44 3.2.1 Cloning methodology 50 3.2.2 Recombinant large scale expression and purification protocols 52 3.3 Biochemical and biophysical assays 54 3.3.1 Peptidoglycan degrading or muralytic assay 54 3.3.2 Fluorescence binding assay 55 3.3.3 Surface Plasmon Resonance 56 3.4 In vitro assays 56 3.4.1 In vitro antibacterial assay 56 3.4.2 In vitro biofilm assay 57 3.4.3 In vitro human keratinocyte assays 58 3.5 In vivo Caenorhabditis elegans assays 59 3.5.1 Cytotoxic effects of compounds on C. elegans 59 3.5.2 C. elegans infection and survival assays 59 Chapter 4 Preface: selecting most promising Gram-negative phage endolysins 61 Chapter 5 Characterization of six novel, single-domain endolysins from Gram-negative infecting bacteriophages 65 5.1 In silico structural analysis of six novel, single-domain endolysins 66 5.2 Biochemical properties of six novel, single-domain endolysins 68 5.2.1 pH-dependency 68 5.2.2 Quantification of muralytic activity 69 5.2.3 Stability at 4°C and 50°C 71 5.3 In vitro antibacterial activity 72 5.4 Discussion 74 Chapter 6 Characterization of modular bacteriophage endolysins from Myoviridae phages OBP, 201ϕ2-1 and PVP-SE1 77 6.1 Modularity feature in OBPgp279, 201ϕ2-1gp229 and PVP-SE1gp146 78 6.1.1 In silico domain prediction in OBPgp279, 201ϕ2-1gp229 and PVP-SE1gp146 78 6.1.2 The predicted peptidoglycan binding domains in OBPgp279, 201ϕ2-1gp229 and PVP-SE1gp146 80 6.2 Biochemical properties of OBPgp279, 201ϕ2-1gp229 and PVP-SE1gp146 83 6.2.1 Substrate specificity 83 6.2.2 Quantification of muralytic activity 84 6.2.3 Activity after heat treatment 87 6.3 In vitro antibacterial activity 88 6.4 Discussion 90 Chapter 7 N-terminal single and double fusion modifications of OBPgp279 and PVP-SE1gp146 97 7.1 Introduction 97 7.1.1 Hydrophobic peptides: pentapeptide and ArtiMW1 peptides 97 7.1.2 Polycationic peptide: PK peptide 99 7.1.3 Combining the best of both: amphipathic peptides 99 7.2 Cloning, expression and purification of N-terminally fused OBPgp279 and PVP-SE1gp146 variants 100 7.3 In vitro antibacterial activity of N-terminal OBPgp279 and PVP-SE1gp146 fusion variants 102 7.4 Cloning, expression and purification of N-terminal double fusion variants of OBPgp279 and PVP-SE1gp146 105 7.5 In vitro antibacterial activity of N-terminal double fusion variants of OBPgp279 and PVP-SE1gp146 107 7.6 Discussion 109 Chapter 8 Evaluation and optimization of PK peptide fusion approach 113 8.1 Introduction 113 8.2 Influence of N-terminal PK peptide fusion on endolysin characteristics 113 8.2.1 Influence on expression yield 113 8.2.2 Influence on muralytic activity 115 8.2.3 Influence on pH-dependency 116 8.2.4 Influence on activity of OBPgp279 and PVP-SE1gp146 after heat treatment 117 8.2.5 Influence on in vitro antibacterial activity 118 8.3 Biofilm-degrading potential of PK peptide fusion approach 121 8.4 Strategies to optimize the PK peptide fusion approach 123 8.4.1 By switching the PK peptide to the C-terminal end of OBPgp279 and PVP-SE1gp146 123 8.4.2 By extension of linker length between N- or C-terminal PK peptide and endolysin 128 8.5 Discussion 133 Chapter 9 Antibacterial potential and cytotoxicity of PK-fused endolysin on an in vitro human keratinocyte infection model 137 9.1 A keratinocyte monolayer as alternative in vitro model 137 9.2 Cytotoxicity assessment on growing human keratinocytes 137 9.3 Antibacterial efficacy on a keratinocyte infection model 140 9.4 Discussion 142 Chapter 10 Antibacterial potential and cytotoxicity of PK fusion endolysins on an in vivo Caenorhabditis elegans nematode model 147 10.1 C. elegans as model organism for bacterial pathogenesis 147 10.2 Cytotoxicity assessment on C. elegans 148 10.3 In vivo antibacterial efficacy on C. elegans 150 10.3.1 Selection of appropriate P. aeruginosa strain for infection of C. elegans SS104 150 10.3.2 Survival of P. aeruginosa PA14-infected C. elegans upon endolysin treatment 151 10.4 Discussion 153 Chapter 11 General conclusions and future perspectives 157 11.1 From fundamental conclusions and perspectives 157 11.1.1 Modular versus single-domain endolysins of Gram-negative infecting phages 157 11.1.2 Extending the base of the endolysin pyramid 158 11.1.3 Structural analysis of characterized endolysins and their interactions 159 11.2 …to application-oriented conclusions and perspectives 159 11.2.1 PK peptide fusion as ideal starting point to tackle the outer membrane in vitro 159 11.2.2 Biofilm-degrading potential of PK peptide fusion approach 161 11.2.3 Safety and antibacterial efficacy in keratinocyte and C. elegans infection models 162 11.2.4 Taking the PK peptide fusion approach to a next level 164 11.2.5 Issues in future development of PK fusion approach 164 11.3 Widening the possibilities: from phage therapy to natural predators 167 References 171nrpages: 206status: publishe

Use of bacteriophage endolysin EL188 and outer membrane permeabilizers against Pseudomonas aeruginosa

Aims:  To select and evaluate an appropriate outer membrane (OM) permeabilizer to use in combination with the highly muralytic bacteriophage endolysin EL188 to inactivate (multi-resistant) Pseudomonas aeruginosa. Methods and Results:  We tested the combination of endolysin EL188 and several OM permeabilizing compounds on three selected Ps. aeruginosa strains with varying antibiotic resistance. We analysed OM permeabilization using the hydrophobic probe N-phenylnaphtylamine and a recombinant fusion protein of a peptidoglycan binding domain and green fluorescent protein on the one hand and cell lysis assays on the other hand. Antibacterial assays showed that incubation of 10(6) Ps. aeruginosa cells ml(-1) in presence of 10 mmol l(-1) ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA) and 50 μg ml(-1) endolysin EL188 led to a strain-dependent inactivation between 3·01 ± 0·17 and 4·27 ± 0·11 log units in 30 min. Increasing the EL188 concentration to 250 μg ml(-1) further increased the inactivation of the most antibiotic resistant strain Br667 (4·07 ± 0·09 log units). Conclusions:  Ethylene diamine tetraacetic acid disodium salt dihydrate was selected as the most suitable component to combine with EL188 in order to reduce Ps. aeruginosa with up to 4 log units in a time interval of 30 min. Significance and Impact of the Study:  This in vitro study demonstrates that the application range of bacteriophage encoded endolysins as 'enzybiotics' must not be limited to gram-positive pathogens.status: publishe

Food applications of bacterial cell wall hydrolases

Bacterial cell wall hydrolases (BCWHs) display a remarkable structural and functional diversity that offers perspectives for novel food applications, reaching beyond those of the archetype BCWH and established biopreservative hen egg white lysozyme. Insights in BCWHs from bacteriophages to animals have provided concepts for tailoring BCWHs to target specific pathogens or spoilage bacteria, or, conversely, to expand their working range to Gram-negative bacteria. Genetically modified foods expressing BCWHs in situ showed successful, but face regulatory and ethical concerns. An interesting spin-off development is the use of cell wall binding domains of bacteriophage BCWHs for detection and removal of foodborne pathogens. Besides for improving food safety or stability, BCWHs may also find use as functional food ingredients with specific health effects.status: publishe

Novel endolysin (for gram negative pathogens)

World Intellectual Property Organization (WIPO); Patent Cooperation Treaty (PCT)Patent nº WO 2012/059545 A1The present invention relates to a polypeptide with endolysin activity comprising an amino acid sequence according to SEQ ID No. 1 and fragments or derivatives thereof, or fusion proteins derived thereof. Moreover, the present invention relates to nucleic acid molecules encoding said polypeptide or fusion protein, vectors comprising said nucleic acid molecules and host cells comprising either said nucleic acid molecules or said vectors. In addition, the present invention relates to said polypeptide, fragment, derivative or fusion protein for use as a medicament, in particular for the treatment or prevention of Gram-negative bacterial infections, as diagnostic means, as cosmetic substance or as sanitizing agent. The present invention also relates to the use of said polypeptide, fragment, derivative or fusion protein for the treatment or prevention of Gram-negative bacterial contamination of foodstuff, of food processing equipment, of food processing plants, of surfaces coming into contact with foodstuff, of medical devices, of surfaces in hospitals and surgeries. Furthermore, the present invention relates to a pharmaceutical composition comprising said polypeptide, fragment, derivative or fusion protein