Solubilization of Phospholipid by Surfactin Leading to Lipid Nanodisc and Fibrous Architecture Formation.

Nanodiscs belong to a category of water-soluble lipid bilayer nanoparticles. In vivo nanodisc platforms are useful for studying isolated membrane proteins in their native lipid environment. Thus, the development of a practical method for nanodisc reconstruction has garnered consider-able research interest. This paper reports the self-assembly of a mixture of bio-derived cyclic peptide, surfactin (SF), and l-α-dimyristoylphosphatidylcholine (DMPC). We found that SF induced the solubilization of DMPC multilamellar vesicles to form their nanodiscs, which was confirmed by size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy analyses. Owing to its amphiphilic nature, the self-assembled structure prevents the exposure of the hydrophobic lipid core to aqueous media, thus embedding ubiquinol (CoQ10) as a hydrophobic model compound within the inner region of the nanodiscs. These results highlight the feasibility of preparing nanodiscs without the need for laborious procedures, thereby showcasing their potential to serve as promising carriers for membrane proteins and various organic compounds. Additionally, the regulated self-assembly of the DMPC/SF mixture led to the formation of fibrous architectures. These results show the potential of this mixture to function as a nanoscale membrane surface for investigating molecular recognition events.

Hydrogenolysis of Furfuryl Alcohol to 1,2-Pentanediol Over Supported Ruthenium Catalysts.

Hydrogenolysis of the furan rings of furfural and furfuryl alcohol, which can be obtained from biomass, has attracted attention as a method for obtaining valuable chemicals such as 1,2-pentanediol. In this study, we examined the hydrogenolysis of furfuryl alcohol to 1,2-pentanediol over Pd/C, Pt/C, Rh/C, and various supported Ru catalysts in several solvents. In particular, we investigated the effects of combinations of solvents and supports on the reaction outcome. Of all the tested combinations, Ru/MgO in water gave the best selectivity for 1,2-pentanediol: with this catalyst, 42 % selectivity for 1,2-pentanediol was achieved upon hydrogenolysis of furfuryl alcohol for 1 h at 463 K. In contrast, reaction in water in the presence of Ru/Al2 O3 afforded cyclopentanone and cyclopentanol by means of hydrogenation and rearrangement reactions.

Enrichment and Isolation of Surfactin-degrading Bacteria.

A total of 100 environmental samples were investigated for their ability to degrade 1 g/L surfactin as a substrate. Among them, two enrichment cultures, which exhibited microbial growth as well as surfactin degradation, were selected and further investigated. After several successive cultivations, nanopore sequencing of full-length 16S rRNA genes with MinIONTM was used to analyze the bacterial species in the enrichment cultures. Variovorax spp., Caulobacter spp., Sphingopyxis spp., and Pseudomonas spp. were found to be dominant in these surfactin-degrading mixed cultures. Finally, one strain of Pseudomonas putida was isolated as a surfactin-degrading bacterium. This strain degraded 1 g/L surfactin below a detectable level within 14 days, and C13 surfactin was degraded faster than C15 surfactin.

Direct entry of cell-penetrating peptide can be controlled by maneuvering the membrane curvature.

A biomembrane's role is to be a barrier for interior cytosol from an exterior environment to execute the cell's normal biological functions. However, a water-soluble peptide called cell-penetrating peptide (CPP) has been known for its ability to directly penetrate through the biomembranes into cells (cytolysis) without perturbating cell viability and expected to be a promising drug delivery vector. Examples of CPP include peptides with multiple arginine units with strong cationic properties, which is the key to cytolysis. Here we show the conclusive evidence to support the mechanism of CPP's cytolysis and way to control it. The mechanism we proposed is attributed to biomembrane's physicochemical nature as lamellar liquid crystal (Lα). Cytolysis occurs as the temporal and local dynamic phase transitions from Lα to an undulated lamellar with pores called Mesh1. We have shown this phase transfer of Lα composed of dioleoyl-phosphatidylcholine (DOPC) with water by adding oligo-arginine (Rx) as CPP at the equilibrium. Using giant unilamellar vesicle composed of DOPC as a single cell model, we could control the level of cytolysis of CPP (FITC-R8) by changing the curvature of the membrane through osmotic pressure modulation. The cytolysis of CPP utilizes biomembrane's inherent topological and functional flexibility corresponding to the stimuli.

Au(i)-, Ag(i)-, and Pd(ii)-coordination-driven diverse self-assembly of an N-heterocyclic carbene-based amphiphile.

Au(i)-, Ag(i)-, and Pd(ii)-coordination-driven diverse self-assembly of an N-heterocyclic carbene (NHC)-based amphiphile was demonstrated herein. The transition metals had significant effects over the whole system, setting the self-assembly direction of the NHC-based amphiphile. More specifically, Au(i)- and Ag(i)-coordination to the NHC-based amphiphile promoted the formation of spherical and hexagonal structures, while Pd(ii)-coordination promoted the formation of cylindrical and lamellar structures.

Synthesis of a Bolaamphiphilic Alkenyl Phosphonic Acid by Ru-catalyzed Olefin Cross Metathesis Reaction.

We report the synthesis of bolaamphiphilic alkenyl phosphonic acid (BPC12) through the olefin crossmetathesis reaction of vinylphosphonic acid with 1,11-dodecadiene in the presence of a Ru-carbene catalyst. BPC12 possesses two trans-P-C=C moieties and is thus readily soluble in water up to 3.4 g L-1, as confirmed by 1H nuclear magnetic resonance (NMR) measurements. Surface tension measurements revealed that BPC12 reduced the surface tension of water from 72.0 to 47.0 mN m‒1. The occupied area per molecule at the air/water interface (A) of BPC12 (216 Å2) was ten times larger than that of dodecenyl phosphonic acid PC12 (23 Å2). Moreover, dynamic light scattering measurement of an aqueous BPC12 solution (5 mM) revealed the formation of large aggregates with an average diameter of 81.8±27.0 nm.

Synthesis of an N-Heterocyclic Carbene-based Au(I) Coordinate Surfactant: Application for Alkyne Hydration Based on Au Nanoparticle Formation.

In this study, an N-heterocyclic carbene (NHC)-based metal coordinate surfactant (MCS), NHC-Au-MCS, in which the NHC framework afforded the bonding of the Au(I) at the linkage of the hydrophilic and hydrophobic moieties, was synthesized. The structure of NHC-Au-MCS was confirmed by 1H and 13C NMR spectroscopic measurements together with elemental analysis. Matrix-assisted laser desorption/ionization (MALDI), laser desorption/ionization (LDI), and electrospray ionization mass spectrometry (ESI-MS) indicated the distinct reactivity of NHC-Au-MCS, such as the exchange of Br to Cl and the formation of a cationic Au complex, where the two NHC ligands were coordinated to an Au(I) center upon laser activation. The surface tension and dynamic light scattering (DLS) measurements revealed that the coordination of Au(I) to NHC reduced the critical micelle concentration (CMC) of NHC-Au-MCS (1.3×10-5 M), which resulted in the formation of micelles at concentrations higher than the CMC in water. We also confirmed that the surface-active Au(I) complex of NHC-Au-MCS catalyzed the hydration of 1-dodecyne to 2-dodecanone in water in the absence of an organic solvent. On the basis of the detailed mechanistic investigations regarding the reactivity of NHC-Au-MCS, we revealed that NHC-Au-MCS partially translated into Au nanoparticles (AuNPs), which facilitated alkyne hydration. These mechanistic studies were supported by UV-vis measurements, transmission electron microscopy (TEM), and LDI-MS.

Spontaneous Vesicle Formation of Monododecenyl Phosphonic Acid in Water.

We report the synthesis of amphiphilic dodecenyl phosphonic acid PC12 from vinylphosphonic acid, a reactive phosphonic acid intermediate. The trans-P-C=C moiety enabled PC12 to disperse well in water. Surface tension and dynamic light scattering measurements revealed that PC12 exhibited high surface activity and reduced the surface tension of water from 72.0 to 23.6 mN/m, thereby resulting in the spontaneous formation of aggregates even in a dilute aqueous solution (critical aggregation concentration (CAC) = 4.8 × 10-4 M). In contrast to modern lipids with double-tailed structures, the PC12 of simple singletailed structure spontaneously formed bilayered vesicles, without an external energy supply. Compared with the strength of hydrogen bonds formed by the long, saturated alkyl chain of dodecyl phosphonic acid (DPA), the strength of PC12 intermolecular hydrogen bonds was weaker. The melting point of PC12 was approximately 20°C lower than that of DPA. These results indicate that the trans-P-C=C moiety was considerably important for spontaneous vesicle formation in water. Preliminary modeling of the morphological transitions of the closed bilayer structures in the vesicles was then conducted, by varying the pH and adding an α-helical peptide scaffold.

Evaluation of Yield and Surface Tension-lowering Activity of Iturin A Produced by Bacillus subtilis RB14.

Bacillus subtilis RB14 produces the lipopeptide antibiotic iturin A by submerged and biofilm fermentation. In this study, we optimized the conditions for iturin A production in a jar fermentor. The maximum yield of iturin A was 932 mg L-1 after 120 h. The surface tension of water decreased from 72.0 to 39.0 mN m-1 as the concentrations of C14 iturin A increased, indicating that C14 iturin A behaves as a surfactant in water. The critical micellar concentration obtained from the intersection of two fitted lines was 1.2 × 10-4 M. Moreover, the surface tension of water decreased as the length of the alkyl chain of iturin A increased.

High Internal Phase Emulsion Gels Stabilized by Natural Casein peptides.

The surface and interfacial properties of casein-hydrolyzed peptides were evaluated using measurement of surface and interfacial tensions, surface viscosity, dynamic light scattering (DLS), and freeze-fracture transmission electron microscopy (FF-TEM). In this study, high internal oil phase emulsion (HIPE) gels were successfully prepared, using the surface and interfacial properties of casein peptides. The casein peptides exhibited surface and interfacial activities. The estimated critical micelle concentration (CMC) and γCMC values were 3.0 mg/mL and 47.8 mN/m, and the average size of casein peptide micelles was 13.2 ± 1.7 nm. The surface shear viscosity of an aqueous casein peptide solution at 10 mg/mL was 1603 µPa ms, which is fifteen times larger than that of sodium dodecyl sulfate (SDS, 106 µPa ms). The larger surface viscosity of casein peptide adsorbed layer could stabilize emulsions and prevent flocculation and coalescence. High internal oil phase gel emulsions were then prepared by slowly adding oil and polyisobutene into an aqueous casein peptide solution/glycerol mixture with different compositions. Based on the pseudo ternary 15 wt% aqueous casein peptide solution/polyisobutene/glycerol phase diagram, the HIPE containing the maximum 88.1 wt% (91.5 vol%) of oil is obtained by the addition of 0.36 wt% of casein peptides. The use of only a small amount of protein-hydrolyzed peptides instead of the commonly used synthetic surfactants for HIPE preparation has great advantages for the widespread application of HIPE technology.

Surface Activity and Ca2+-Dependent Aggregation Property of Lichenysin Produced by Bacillus licheniformis NBRC 104464.

Bacillus licheniformis NBRC 104464 produces a cyclic lipopeptide different from surfactin. After we performed liquid chromatography fractionation and purification, we used structural analyses to identify the cyclic lipopeptide as lichenysin. Surface tension measurements of lichenysin sodium salt in water yielded a critical micelle concentration (CMC) of 1.0×10-5 M. The surface tension at the CMC was 28.9 mN/m. Comparative analysis of Ca2+-influenced micellar aggregation of lichenysin and surfactin revealed that the formation rate of the lichenysin-Ca2+ complex aggregate remained low up to a [Ca2+]/[lichenysin] molar ratio of 80, whereas the surfactin-Ca2+ complex formed micellar aggregates at the same molar ratio. Further excessive addition of Ca2+ to the micellar solution of lichenysin induced higher turbidity than surfactin.

Self-assembling Properties of an N-Heterocyclic Carbene-based Metallosurfactant: Pd-Coordination Induced Formation of Reactive Interfaces in Water.

In this study, an N-heterocyclic carbene (NHC)-based metallosurfactant (MS), NHC-PdMS, was synthesized, where Pd(II) was bound to the NHC framework via a robust Pd-carbene bond with NEt3 as a co-ligand. Surface tension measurements revealed that the critical micelle concentration (CMC) of NHC-PdMS (1.8×10-4 M) was one order of magnitude lower than that of its MS precursor (imidazolium bromide). Coordination of the MS precursor and NEt3 to Pd(II) also influenced micelle size; the hydrodynamic diameters of NHC-PdMS and the MS precursor were observed to be 25.8±5.6 nm and 2.5±0.3 nm, respectively. Furthermore, small angle X-ray scattering measurements indicated that NHC-PdMS exhibited liquid crystalline behavior above 26 wt%, with a spacing ratio of 1:2:3 for the first, second, and third Bragg peaks. To understand the role of the reactive interface, NHC-PdMS was also applied to aqueous catalytic reactions. Owing to its low CMC value, a catalytic amount of NHC-PdMS (3 mol%) provided the reactive interface, which facilitated the aqueous Mizoroki-Heck reaction of various aryl iodides and styrene in good yields (72-95%). These results suggest that MS formation results in a drastic change in selfassembling properties, which are important for the development of highly reactive chemical interfaces in water.

Lipid Nanodisc Formation using Pxt-5 Peptide Isolated from Amphibian (Xenopus tropicalis) Skin, and its Altered Form, Modify-Pxt-5.

Nanodiscs are self-assembled discoidal nanoparticles composed of amphiphilic α-helical scaffold proteins or peptides that accumulate around the circumference of a lipid bilayer. In this study, Pxt-5, which is an antimicrobial peptide isolated from the skin of Xenopus tropicalis, and its modified peptide (Modify-Pxt-5) were synthesized by solid-phase peptide synthesis (SPPS).Their surface properties, which are an important factor in inducing nanodisc formation, were investigated by circular dichroism (CD) spectroscopy, surface tension measurement, phospholipid vesicle clearance assay, and negative-staining transmission electron microscopy (NS-TEM). The α-helicity of Pxt-5 (8.4%) improved drastically to 45.6% by four amino-acid substitutions (Modify-Pxt-5). Both the peptides, having hydrophobic and hydrophilic faces, behaved like general surfactants, and the surface activity of Modify-Pxt-5 (CAC: 9.5×10-5 M, γCAC: 30.3 mN·m-1) was much higher than that of Pxt-5 (CAC: 7.9×10-5 M, γCAC: 38.1 mN·m-1). A turbid L-α-dimyristoylphosphatidylcholine (DMPC) vesicle solution (T = 0.3%) quickly turned transparent upon addition of Pxt-5 or Modify-Pxt-5. After twelve hours, the transmittance of vesicle solution with Modify-Pxt-5 (T = 96.2%) was found to be higher than that of vesicle solution with Pxt-5 (T = 83.5%), and then the micro-solubilized solutions were observed by NS-TEM. Interestingly, nanodisc structures were found in the vicinity of DMPC vesicles in both the images, and the average diameter of the nanodiscs was 11.2 ± 6.0 nm for those containing Pxt-5 and 10.8 ± 5.8 nm for those containing Modify-Pxt-5. It was also found that Modify-Pxt-5 effectively self-assembles into nanodiscs compared to Pxt-5 without any substitutions.

Efficient Production of Acid-Form Sophorolipids from Waste Glycerol and Fatty Acid Methyl Esters by Candida floricola.

We discovered that Candida floricola ZM1502 is capable of selectively producing the promising hydrophilic biosurfactants, acid-form sophorolipids (SLs), from glycerol. However, productivity was very low (approximately 3.5 g L-1) under the initial culture conditions. Here, we describe the design of culture medium for abundant production of acid-form SLs by C. floricola ZM1502 using waste glycerol and hydrophobic substrates in order to develop a method for SL production and disposal of waste glycerol produced by oleo-chemical industries. Urea provided the best nitrogen source for acid-form SL production from glycerol among four nitrogen sources tested [urea, NaNO3, NH4NO3, and (NH4)2SO4]. Among carbon sources we compared, hydrophobic substrates (soybean oil and oleic acid) led to productivities of approximately 20 g L-1, indicating that hydrophobic substrates provided fatty acid moieties for SL production. Addition of olive oil and oleic acid to waste glycerol enhanced acid-form SL production to 42.1 ± 0.9 and 37.5 ± 3.4 g L-1, respectively. To develop a potential industrial process, we explored other suitable hydrophobic substrates for SL production, which were obtained on site from oleo-chemical industries. Alkyl C18 esters (Pastell M-182), along with waste glycerol, increased acid-form SL production to 48.0 ± 3.4 g L-1 over a 7-d period. Furthermore, we demonstrated abundant production of acidic SLs at the mini-jar fermenter scale, obtaining 169 g L-1 over 180 h using a fed-batch cultivation technique. Efficient acid-form SL production by C. floricola could have a great impact on the development of bio-industrial processes using waste glycerol as a substrate.

Selective Production of Acid-form Sophorolipids from Glycerol by Candida floricola.

Biosurfactants (BSs) are produced in abundance from various feedstocks by diverse microorganisms, and are used in various applications. In this paper, we describe a new yeast isolate that produces glycolipid-BSs from glycerol, with the aim of enhancing the utilization of the surplus glycerol produced by the oleo-chemical industry. As a result of the screening, strain ZM1502 was obtained as a potential producer of BS from glycerol. Based on TLC analysis, the strain produced glycolipid BSs. According to structural analyses (NMR, MALDI-TOF MS, and GC-MS), the main component of the glycolipids was 6',6"-di-O-acetylated acid-form sophorolipid (SL). Interestingly, the strain produced only acid-form SL, without lactone-form SLs, although the conventional SL-producing yeast, Starmerella bombicola, produces lactone-form SLs with small amounts of the acid-form. Based on taxonomy, the strain was identified as Candida floricola. It produced 3.5 g L-1 of acid-form SLs in 20% (w/v) glycerol. In addition, C. floricola CBS7290 and NBRC10700T also produced only acid-form SLs from glycerol. These results suggest that C. floricola would enhance the utilization of waste glycerol as a fermentation feedstock and facilitate a broad range of applications for SLs.

Screening of a Bacillus subtilis Strain Producing Multiple Types of Cyclic Lipopeptides and Evaluation of Their Surface-tension-lowering Activities.

Sixty Bacillus subtilis strains were investigated for their ability to produce cyclic lipopeptides (CLPs). Among them, B. subtilis NBRC 109107 produced at least three types of CLPs by high-performance liquid chromatography (HPLC) analysis, and these CLPs were thought to be surfactin, iturin A, and fengycin by polymerase chain reaction amplification of respective CLP synthetase-encoding genes. However, after HPLC fractionation and purification, structural analysis of the CLPs revealed that these were surfactin homologues, iturin A, and unknown CLPs, whose surface-tension-lowering activities were 29.4, 56.7, and 48.6 mN/m, respectively. By contrast, fengycin was not detected.

Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata.

BACKGROUND: Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner. In this study, this peptide profiling approach is successfully applied to explore the venom peptides of a Japanese solitary carpenter bee, Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). Although interesting biological effects of the crude venom of carpenter bees have been reported, the structure and biological function of the venom peptides have not been elucidated yet. METHODS: The venom peptide profiling of the crude venom of X. appendiculata was performed by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. The venom was purified by a reverse-phase HPLC. The purified peptides were subjected to the Edman degradation, MS/MS analysis, and/or molecular cloning methods for peptide sequencing. Biological and functional characterization was performed by circular dichroism analysis, liposome leakage assay, and antimicrobial, histamine releasing and hemolytic activity tests. RESULTS: Three novel peptides with m/z 16508, 1939.3, and 1900.3 were isolated from the venom of X. appendiculata. The peptide with m/z 16508 was characterized as a secretory phospholipase A2 (PLA2) homolog in which the characteristic cysteine residues as well as the active site residues found in bee PLA2s are highly conserved. Two novel peptides with m/z 1939.3 and m/z 1900.3 were named as Xac-1 and Xac-2, respectively. These peptides are found to be amphiphilic and displayed antimicrobial and hemolytic activities. The potency was almost the same as that of mastoparan isolated from the wasp venom. CONCLUSION: We found three novel biologically active peptides in the venom of X. appendiculata and analyzed their molecular functions, and compared their sequential homology to discuss their molecular diversity. Highly sensitive mass analysis plays an important role in this study.

pH-induced conformational change of natural cyclic lipopeptide surfactin and the effect on protease activity.

The cyclic lipopeptide surfactin (SF) is one of the promising environmental friendly biosurfactants abundantly produced by microorganisms such as Bacillus subtilis. SF shows excellent surface properties at various pH, together with lower toxicity and higher biodegradability than commonly used petroleum-based surfactants. However, the effect of the dissociation degree of SF on self-assembly is still incompletely understood, even though two acidic amino acid residues (Asp and Glu) are known to influence eventual surface and biological functions. Here, we report changes in the secondary structure of SF induced by increased pH, and the effect on protease activity. We found that the ß-sheet and ß-turn formation of SF are significantly enhanced through increased dissociation of Asp and Glu as revealed by a titration experiment of SF solution to estimate apparent pK1 and pK2 values together with circular dichroism spectroscopy. We also studied the activity of the common detergent enzyme subtilisin in SF solution at above its pK2 (pH 7.6) to understand the role of the dissociation degree in the interaction with the protein. The mixing of SF having a unique cyclic topological feature with subtilisin suppressed the decrease in protease activity observed in the presence of synthetic surfactants such as sodium dodecyl sulfate and polyoxyethylene alkyl ether. Thus, SF has great potential for use in laundry detergent formulations, to improve the stability and reliability of detergent enzymes.

Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata

Background Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner. In this study, this peptide profiling approach is successfully applied to explore the venom peptides of a Japanese solitary carpenter bee, Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). Although interesting biological effects of the crude venom of carpenter bees have been reported, the structure and biological function of the venom peptides have not been elucidated yet. Methods The venom peptide profiling of the crude venom of X. appendiculata was performed by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. The venom was purified by a reverse-phase HPLC. The purified peptides were subjected to the Edman degradation, MS/MS analysis, and/or molecular cloning methods for peptide sequencing. Biological and functional characterization was performed by circular dichroism analysis, liposome leakage assay, and antimicrobial, histamine releasing and hemolytic activity tests. Results Three novel peptides with m/z 16508, 1939.3, and 1900.3 were isolated from the venom of X. appendiculata. The peptide with m/z 16508 was characterized as a secretory phospholipase A2 (PLA2) homolog in which the characteristic cysteine residues as well as the active site residues found in bee PLA2s are highly conserved. Two novel peptides with m/z 1939.3 and m/z 1900.3 were named as Xac-1 and Xac-2, respectively. These peptides are found to be amphiphilic and displayed antimicrobial and hemolytic activities. The potency was almost the same as that of mastoparan isolated from the wasp venom. Conclusion We found three novel biologically active peptides in the venom of X. appendiculata and analyzed their molecular functions, and compared their sequential homology to discuss their molecular diversity. Highly sensitive mass analysis plays an important role in this study.(AU)

Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata

Abstract Background Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner. In this study, this peptide profiling approach is successfully applied to explore the venom peptides of a Japanese solitary carpenter bee, Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). Although interesting biological effects of the crude venom of carpenter bees have been reported, the structure and biological function of the venom peptides have not been elucidated yet. Methods The venom peptide profiling of the crude venom of X. appendiculata was performed by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. The venom was purified by a reverse-phase HPLC. The purified peptides were subjected to the Edman degradation, MS/MS analysis, and/or molecular cloning methods for peptide sequencing. Biological and functional characterization was performed by circular dichroism analysis, liposome leakage assay, and antimicrobial, histamine releasing and hemolytic activity tests. Results Three novel peptides with m/z 16508, 1939.3, and 1900.3 were isolated from the venom of X. appendiculata. The peptide with m/z 16508 was characterized as a secretory phospholipase A2 (PLA2) homolog in which the characteristic cysteine residues as well as the active site residues found in bee PLA2s are highly conserved. Two novel peptides with m/z 1939.3 and m/z 1900.3 were named as Xac-1 and Xac-2, respectively. These peptides are found to be amphiphilic and displayed antimicrobial and hemolytic activities. The potency was almost the same as that of mastoparan isolated from the wasp venom. Conclusion We found three novel biologically active peptides in the venom of X. appendiculata and analyzed their molecular functions, and compared their sequential homology to discuss their molecular diversity. Highly sensitive mass analysis plays an important role in this study.