A 1, 4-benzoquinone derivative isolated from Ardisia crispa (Thunb.) A. DC. root suppresses angiogenesis via its angiogenic signaling cascades.

The root hexane extract of Ardisia crispa (ACRH), which belongs to the Primulaceae family, has been reported to possess anti-inflammatory, chemopreventive, anti-arthritic, and antiangiogenic activities. In this study, we isolated a p-benzoquinone derivative, 2-methoxy-6-undecyl-1,4-benzoquinone (AC2), from ACRH and investigated its potential antiangiogenic activity in human umbilical vein endothelial cells (HUVECs) and zebrafish embryo models. Prior to this study, AC2 was characterized using 1H NMR spectroscopy and MS. AC2 significantly suppressed HUVEC proliferation in a time-independent manner, with an IC50 value of 1.35 ± 0.05, 1.15 ± 0.02, and 1.00 ± 0.01 µg/mL at 24, 48, and 72 h, respectively. AC2 also induced apoptosis in HUVECs and significantly suppressed their migration, invasion, and tube formation in a concentration-dependent manner. Additionally, AC2 significantly attenuated most of the analyzed protein markers, including pro-MMP-2, VEGF-C, VEGF-D, angiopoietin-2, endothelin-1, fibroblast growth factor (FGF)-1, FGF-2, follistatin, heparin-binding epidermal growth factor-like growth factor (HB-EGF), and hepatocyte growth factor (HGF) at all tested concentrations. Furthermore, AC2 significantly inhibited zebrafish embryo intersegmental vessels (ISVs), confirming its antiangiogenic role. In conclusion, AC2 exhibits a potential anti-angiogenic effect by suppressing several proangiogenic and growth factors. Further studies are needed to investigate their effects on other excessive angiogenic diseases.

Bacteriolytic activity in saliva of the hematophagous Triatoma infestans (Reduviidae) and novel characterization and expression site of a third lysozyme.

Saliva of hematophagous insects contains many different compounds, mainly acting as anticoagulants. Investigating the bacteriolytic compounds of the saliva of the bloodsucking Triatoma infestans photometrically between pH 3 and pH 10 using unfed fifth instars and nymphs up to 15 days after feeding, we found bacteriolytic activity against lyophilized Micrococcus luteus was stronger at pH 4 and pH 6. After feeding, the activity level at pH 4 was unchanged, but at pH 6 more than doubled between 3 and 7 days after feeding. In zymographs of the saliva and after incubation at pH 4, bacteriolytic activity against Micrococcus luteus was present at eight lysis zones between 14.1 and 38.5 kDa, showing the strongest activity at 24.5 kDa. After incubation at pH 6, lysis zones only appeared at 15.3, 17, and 31.4 kDa. Comparing zymographs of the saliva of unfed and fed nymphs, bacteriolytic activity at 17 kDa increased after feeding. In total nine lysis bands appeared, also at >30 kDa, so far unreported in the saliva of triatomines. Reverse transcription polymerase chain reaction using oligonucleotides based on the previously described lysozyme gene of T. infestans, TiLys1, verified expression of genes encoding TiLys1 and TiLys2 in the salivary glands, but also of an undescribed third lysozyme, TiLys3, of which the cloned cDNA shares characteristics with other c-type lysozymes of insects. While TiLys1 was expressed in the tissue of all three salivary glands, transcripts of TiLys2 and of TiLys3 seem to be present only in the gland G1 and G3, respectively.

In-vitro anti-inflammatory activity of serine protease inhibitor from Cassia siamea and Dolichos biflorus: A comparative study.

Cassia siamea is a nonedible legume belonging to Fabaceae. The seed of C. siamea contains ~16% of protein. The study reports the biochemical characterization of purified novel serine protease inhibitor from seeds of C. siamea, aimed with assessing the anti-inflammatory activity. The seed extract was subjected to ammonium sulfate precipitation followed by fast protein liquid chromatography (FPLC)-anion exchange chromatography and affinity-chromatography to obtain a relative pure protease inhibitor. Thirty-fivefold purification with the specific activity of 250 U/mg of trypsin inhibitory unit was obtained. The characterization of protease inhibitor for optimum temperature, pH, and metal ions were measured using N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) assay and casein zymogram. The C. siamea trypsin inhibitor (CsTI) has a relative molecular mass of 25.540 kDa. Purified CsTI and Dolichos biflorus were tested for anti-inflammatory efficacy against A549 and RAW264.7 cell lines. The inhibitory activity of both purified inhibitors are comparable and are potent toward anti-inflammatory activity. The purified inhibitor shows to be a promising candidate as anti-inflammatory agent by targeting the serine proteases.

Comparative study on electrocardiograms and serological examinations of acute pulmonary embolism and acute non-ST elevation myocardial infarction.

BACKGROUND: The aim of this study was to investigate the value of electrocardiograms (ECGs) and serological examinations in the differential diagnosis of acute pulmonary embolism (APE) and acute non-ST elevation myocardial infarction (NSTEMI) in order to reduce the rate of clinical misdiagnosis. METHODS: The clinical data of 37 patients with APE and 103 patients with NSTEMI admitted to our hospital were retrospectively analyzed. The differences in the clinical manifestations, ECGs, myocardial zymograms, D-dimers, and troponin (cTn) of the two groups were compared. RESULTS: In the patients with APE, the main symptom-found in 25 cases (67.56%)-was dyspnea, while in the patients with NSTEMI, the main symptom-found in 52 cases (50.49%)-was chest tightness. The incidences of sinus tachycardia and SI QIII TIII in the group of patients with APE were higher than in the group of patients with NSTEMI, and the difference was statistically significant (p < .05). There was no statistical significance in the difference of aspartate aminotransferase and lactate dehydrogenase (LDH) in the two groups (p > .05), although there was a statistically significant difference of creatine kinase (CK) and the creatine kinase isoenzyme-MB (CK-MB) in the two groups (p < .05). The levels of D-dimers and cTn were increased in both groups, but the level of D-dimers in the group of patients with APE was higher than that in the group of patients with NSTEMI. CONCLUSION: With the occurrence of clinical manifestations like dyspnea, chest tightness, chest pain, and palpitation of unknown causes, the possibility of APE and NSTEMI should be considered.

Proteomic analysis revealed the biofilm-degradation abilities of the bacteriophage UPMK_1 and UPMK_2 against Methicillin-resistant Staphylococcus aureus.

OBJECTIVE: The degradation activity of two bacteriophages UPMK_1 and UPMK_2 against methicillin-resistant Staphylococcus aureus phages were examined using gel zymography. METHODS: The analysis was done using BLASTP to detect peptides catalytic domains. Many peptides that are related to several phage proteins were revealed. RESULTS: UPMK_1 and UPMK_2 custom sequence database were used for peptide identification. The biofilm-degrading proteins in the bacteriophage UPMK_2 revealed the same lytic activity towards polysaccharide intercellular adhesin-dependent and independent of Methicillin-resistant Staphylococcus aureus (MRSA) biofilm producers in comparison to UPMK_1, which had lytic activity restricted solely to its host. CONCLUSION: Both bacteriophage enzymes were involved in MRSA biofilm degradation during phage infection and they have promising enzybiotics properties against MRSA biofilm formation.

New alkali tolerant ß-galactosidase from Paracoccus marcusii KGP - A promising biocatalyst for the synthesis of oligosaccharides derived from lactulose (OsLu), the new generation prebiotics.

The enzyme ß-galactosidase can synthesise novel prebiotics such as oligosaccharides derived from lactulose (OsLu) which can be added as a supplement in infant food formula. In this study, the intracellular ß-galactosidase produced by the alkaliphilic bacterium Paracoccus marcusii was extracted and purified to homogeneity using hydrophobic and metal affinity chromatography. The purification resulted in 18 U/mg specific activity, with a yield of 8.86% and an 18-fold increase in purity. The purified enzyme was a monomer with an 86 kDa molecular weight as determined by SDS PAGE and Q-TOF-LC/MS. ß-Galactosidase was highly active at 50 °C and pH 6-8. The enzyme displayed an alkali tolerant nature by maintaining more than 90% of its initial activity over a pH range of 5-9 after 3 h of incubation. Furthermore, the enzyme activity was enhanced by 37% in the presence of 5 M NaCl and 3 M KCl, indicating its halophilic nature. The effects of metal ions, solvents, and other chemicals on enzyme activity were also studied. The kinetic parameters KM and Vmax of ß-galactosidase were 1 mM and 8.56 µmoles/ml/min and 72.72 mM and 11.81 µmoles/ml/min on using oNPG and lactose as substrates. P. marcusii ß-galactosidase efficiently catalysed the transgalactosylation reaction and synthesised 57 g/L OsLu from 300 g/L lactulose at 40 °C. Thus, in this study we identified a new ß-galactosidase from P. marcusii that can be used for the industrial production of prebiotic oligosaccharides.

A Novel Lipase from Lasiodiplodia theobromae Efficiently Hydrolyses C8-C10 Methyl Esters for the Preparation of Medium-Chain Triglycerides' Precursors.

Medium-chain triglycerides (MCTs) are an emerging choice to treat neurodegenerative disorders such as Alzheimer's disease. They are triesters of glycerol and three medium-chain fatty acids, such as capric (C8) and caprylic (C10) acids. The availability of C8-C10 methyl esters (C8-C10 ME) from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs. However, there are few reports on enzymes that can efficiently hydrolyse C8-C10 ME to industrial specifications. Here, we report the discovery and identification of a novel lipase from Lasiodiplodia theobromae fungus (LTL1), which hydrolyses C8-C10 ME efficiently. LTL1 can perform hydrolysis over pH ranges from 3.0 to 9.0 and maintain thermotolerance up to 70 °C. It has high selectivity for monoesters over triesters and displays higher activity over commercially available lipases for C8-C10 ME to achieve 96.17% hydrolysis within 31 h. Structural analysis by protein X-ray crystallography revealed LTL1's well-conserved lipase core domain, together with a partially resolved N-terminal subdomain and an inserted loop, which may suggest its hydrolytic preference for monoesters. In conclusion, our results suggest that LTL1 provides a tractable route towards to production of C8-C10 fatty acids from methyl esters for the synthesis of MCTs.

Zymography for Picogram Detection of Lipase and Esterase Activities.

Lipases and esterases are important catalysts with wide varieties of industrial applications. Although many methods have been established for detecting their activities, a simple and sensitive approach for picogram detection of lipolytic enzyme quantity is still highly desirable. Here we report a lipase detection assay which is 1000-fold more sensitive than previously reported methods. Our assay enables the detection of as low as 5 pg and 180 pg of lipolytic activity by direct spotting and zymography, respectively. Furthermore, we demonstrated that the detection sensitivity was adjustable by varying the buffering capacity, which allows for screening of both high and low abundance lipolytic enzymes. Coupled with liquid chromatography-mass spectrometry, our method provides a useful tool for sensitive detection and identification of lipolytic enzymes.

Probucol recovers pathological damage in viral Myocarditis through improvement of myocardium-related proteins.

This study explored the effects of probucol on myocardial injury, oxidative stress, and Cav-3 and Smad3 expression in myocardial tissues by establishing VMC rat models, in order to provide a basis for exploring the mechanism of probucol in treatment of VMC. Sixty rats were randomly divided into control group, model group, probucollowdose group, andprobucol highdose group, with 15 in each group. Except for the control group, rats in each group were intraperitoneally injected coxsackievirus B3 diluent (0.2 ml) to replicate VMC models every 4 days. The results showed that Caspase-3 and Caspase-9, myocardial enzymes, cTn I, and MDA levels in the model group significantly increased (P < 0.05), while the SOD level significantly decreased (P < 0.05); and after probucol treatment, Caspase-3 and Caspase-9, myocardial enzymes, cTn I and MDA levels significantly decreased (P < 0.05), and the SOD level significantly increased (P < 0.05). Compared with the control group, there was an increase in myocardial fibers with significant lesions in the model group, and the pathological scores and the mRNA and protein expression levels of Cav-3 and Smad3 in myocardial cells significantly increased (P < 0.05). Compared with the control group, the myocardial tissue lesions were improved in the probucol low dose group and highdose group, and the pathological scores and the mRNA and protein expression levels of Cav-3 and Smad3 in myocardial cells were significantly reduced (P < 0.05). In conclusion, probucol can significantly improve the pathological damage of myocardial tissue in VMC rats, and its mechanism may be related to improving the expression of myocardium-related proteins Caspase-3 and Caspase-9, inhibiting oxidative stress response, and down-regulating Cav-3 and Smad3 gene expression in myocardial tissue of VMC rats.

Crystal violet stains proteins in SDS-PAGE gels and zymograms.

Coomassie brilliant blue R250, an anionic dye is the most popular stain to detect proteins resolved in SDS-PAGE gels. Crystal violet, a cationic dye was found to be versatile and stained proteins in SDS-PAGE gels and in zymograms. Stained proteins can be transferred to nitrocellulose and the stained proteins on the western detected with enzyme coupled antibodies. Staining can be reversed. Staining takes 3 h at RT or 30 min at 60 °C. Crystal violet stained some E. coli high and low molecular weight proteins not stained by Coomassie blue R250. Crystal violet stained down to 16 ng of protein, some five-fold lower than Coomassie blue, though the two stains had a similar linear dynamic range. The staining sensitivity could be increased to 2 ng when crystal violet and Coomassie blue were combined in a double staining/counterion dye formulation. The low concentrations of the dye without a destaining step reduces the costs of the technique and results in a more environmentally friendly stain compared to traditional staining methods.

False positives in using the zymogram assay for identification of peptidoglycan hydrolases.

Zymogram assays have been used extensively to identify novel peptidoglycan hydrolases. In this study it is reported that the zymogram is susceptible to false positive results when highly positively charged proteins are assayed. As an example, we report on the case of the ChiZ membrane protein from the Mycobacterium tuberculosis divisome, which previously was described as a peptidoglycan hydrolase. Even though the full length ChiZ protein was able to produce positive assay results, other direct methods for measuring peptidoglycan hydrolysis do not provide convincing evidence that ChiZ has peptidoglycan hydrolysis activity. We show that the false positive result is produced by the highly positively charged N-terminal region of ChiZ. Thus, we developed a zymogram control that can be used to identify false positives results. This control assay lacks the refolding step in the normal zymogram assay. For lysozyme the control assay shows no activity, while the N-terminal region of ChiZ shows a false positive result. Given the limitations of the zymogram assay to reliably identify peptidoglycan hydrolases, we recommend using the zymogram control assay together with other methods to evaluate possible peptidoglycan hydrolysis activity.

Lignolytic mushroom Lenzites elegans WDP2: Laccase production, characterization, and bioremediation of synthetic dyes.

A mycoremedial study was undertaken for decolourization of synthetic dyes using wood rot fungal culture Lenzites elegans WDP2. The culture was isolated from decaying wood as fruiting body, and identified on the basis of 5.8S ITS rRNA gene sequence analysis. Qualitative plate screening of culture showed extracellular laccase and lignin peroxidase production, while only laccase enzyme was produced in higher amount (156.793 Uml-1) in minimal salt broth medium containing glucose and veratryl alcohol. Laccase activity was increased up to 189.25 Uml-1 after optimization of laccase production by optimization of one variable at a time approach. Molecular characterization of laccase enzyme was done using SDS PAGE and Native PAGE based isozyme analyses. The culture was able to decolorize three synthetic dying compounds (congo red, Malachite green and brilliant green) in broth media, while showed very less decolourization in plate assay. The fungal culture varied in their dye decolourizing potential in broth culture, showing 92.77%, 21.27% and 98.8% maximum decolourization of brilliant green, malachite green and congo red respectively. The congo red dye was completely bio-absorbed by fungal culture within one month. The fungal decolourized broth also revealed the extracellular laccase activity; varied from 10 Uml-1 to 68.5 Uml-1 in all the three cases, supports the involvement of laccase enzyme in decolorization. Phase contrast microscopy clearly revealed bio-sorption of the dyes by fungal culture into the mycelium/spores in the photomicrographs.

Isolation and characterization of a cold-active, alkaline, detergent stable α-amylase from a novel bacterium Bacillus subtilis N8.

A cold-active alkaline amylase producer Bacillus subtilis N8 was isolated from soil samples. Amylase synthesis optimally occurred at 15°C and pH 10.0 on agar plates containing starch. The molecular weight of the enzyme was found to be 205 kDa by performing SDS-PAGE. While the enzyme exhibited the highest activity at 25°C and pH 8.0, it was highly stable in alkaline media (pH 8.0-12.0) and retained 96% of its original activity at low temperatures (10-40°C) for 24 hr. While the amylase activity increased in the presence of ß-mercaptoethanol (103%); Ba2+, Ca2+, Na+, Zn2+, Mn2+, H2O2, and Triton X-100 slightly inhibited the activity. The enzyme showed resistance to some denaturants: such as SDS, EDTA, and urea (52, 65, and 42%, respectively). N8 α-amylase displayed the maximum remaining activity of 56% with 3% NaCl. The major final products of starch were glucose, maltose, and maltose-derived oligosaccharides. This novel cold-active α-amylase has the potential to be used in the industries of detergent and food, bioremediation process and production of prebiotics.

One-step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme.

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production.

Quantification of Bacillus thuringiensis Vip3Aa16 Entomopathogenic Toxin Using Its Hemolytic Activity.

Vegetative insecticidal proteins produced by some Bacillus thuringiensis strains are specifically toxic to different agricultural pests such as the polyphagous Spodoptera and several other Lepidopteran insects, but one of the major problems found in the use of these biopesticides was the lack of an easy and credible method of quantification of such secreted toxins. Heterologous expression of B. thuringiensis Vip3Aa16 toxin was performed in Escherichia coli then the protein was purified by chromatography. Using blood agar as well as blood agar overlay (zymogram assay), we reported, for the first time, the capacity of Vip3Aa16 to induce hemolysis. The hemolytic activity of this protein was shown to be relatively stable after treatment at 40 °C and at a range of pH between 6.5 and 9. Moreover, a linear relationship was shown between hemolysis levels and Vip3Aa16 concentrations. The model established in the present study could quantify Vip3A toxin as a function of hemolytic activity and the assay proposed showed to be a simple and low-cost method to readily assess Vip3A toxins in liquid cultures and facilitate the use of this kind of bioinsecticides in pest management programs.

Purification and properties of beta-cyclomaltodextrin glucanotransferase from Bacillus flexus SV 1.

Cyclomaltodextrin glucanotransferase is a unique enzyme that degrades starch into cyclic oligosaccharides called cyclodextrins, which have numerous applications in various industries such as pharmaceutical, textile, agricultural, cosmetics etc. Due to its wide applications, microorganism producing one type of cyclodextrin is of interest as it simplifies the down streaming process of separating mixture of cyclodextrins. In the present study, ß-CGTase was isolated from Bacillus flexus SV 1 and biochemically characterized. Enzyme was purified by starch adsorption followed by DEAE cellulose column chromatography which resulted in a fold purification of 6.1, with a yield of 44.07%. Molecular weight of the purified enzyme was found to be 96.68 kDa, enzyme was monomeric in nature with a Km and Vmax of 0.08976 µmol mL-1 and 585.1 µmol/ml/min, respectively. Optimum pH and temperature of the purified enzyme was found to be 8.0 and 60 °C. Ca2+ showed significant increase in enzyme activity. The inhibition of enzyme by EDTA indicates that CGTase is a metalloenzyme. CGTase produced majorly ß-CD and was alkalotolarent and active at high temperatures which is a promising candidate for various industries such as textile, food, agriculture, and pharmaceuticals.

Multi-Approach Analysis for the Identification of Proteases within Birch Pollen.

Birch pollen allergy is highly prevalent, with up to 100 million reported cases worldwide. Proteases in such allergen sources have been suggested to contribute to primary sensitisation and exacerbation of allergic disorders. Until now the protease content of Betula verrucosa, a birch species endemic to the northern hemisphere has not been studied in detail. Hence, we aim to identify and characterise pollen and bacteria-derived proteases found within birch pollen. The pollen transcriptome was constructed via de novo transcriptome sequencing and analysis of the proteome was achieved via mass spectrometry; a cross-comparison of the two databases was then performed. A total of 42 individual proteases were identified at the proteomic level. Further clustering of proteases into their distinct catalytic classes revealed serine, cysteine, aspartic, threonine, and metallo-proteases. Further to this, protease activity of the pollen was quantified using a fluorescently-labelled casein substrate protease assay, as 0.61 ng/mg of pollen. A large number of bacterial strains were isolated from freshly collected birch pollen and zymographic gels with gelatinase and casein, enabled visualisation of proteolytic activity of the pollen and the collected bacterial strains. We report the successful discovery of pollen and bacteria-derived proteases of Betula verrucosa.

Enzymatic activity of fungi isolated from crops.

AIM: To detect and assess the activity of extracellular hydrolytic enzymes and to find differences in enzymograms between fungi isolated from wheat and rye samples and grown on Czapek-Dox Broth and Sabouraud Dextrose Broth enriched with cereal (wheat or rye). Isolated strains were also classified in the scale of biosafety levels (BSL). MATERIAL AND METHODS: The study used 23 strains of fungi cultured from samples of wheat and rye (grain, grain dust obtained during threshing and soil) collected in the Lublin region (eastern Poland). API ZYM test (bioMérieux) was carried out according to the manufacturer's instructions. Classification of BSL (Biosafety levels) was based on the current literature. RESULTS: High enzymatic activity was found in strains cultured in media containing 1% of wheat grain (Bipolaris holmi, Penicillium decumbens) and with an addition of 1% of rye grain (Cladosporium herbarum, Aspergillus versicolor, Alternaria alternata). The total number of enzymes varied depending on the type of media, and in most cases it was higher in the culture where an addition of cereal grains was used. CONCLUSIONS: Isolated strains of fungi reveal differences in the profiles of the enzyme assay. It can be assumed that the substrate enriched in grains stimulate the higher activity of mold enzymes.

Reliable simultaneous zymographic method of characterization of cellulolytic enzymes from fungal cellulase complex.

A method for zymographic detection of specific cellulases in a complex (endocellulase, exocellulase, and cellobiase) from crude fermentation extracts, after a single electrophoretic separation, is described in this paper. Cellulases were printed onto a membrane and, subsequently, substrate gel. Cellobiase isoforms were detected on the membrane using esculine as substrate, endocellulase isoforms on substrate gel with copolymerized carboxymethyl cellulose (CMC), while exocellulase isoforms were detected in electrophoresis gel with 4-methylumbelliferyl-ß-d-cellobioside (MUC). This can be a useful additional tool for monitoring and control of fungal cellulase production in industrial processes and fundamental research, screening for particular cellulase producers, or testing of new lignocellulose substrates.

Expression, purification, and characterization of a bifunctional 99-kDa peptidoglycan hydrolase from Pediococcus acidilactici ATCC 8042.

Pediococcus acidilactici ATCC 8042 is a lactic acid bacteria that inhibits pathogenic microorganisms such as Staphylococcus aureus through the production of two proteins with lytic activity, one of 110 kDa and the other of 99 kDa. The 99-kDa one has high homology to a putative peptidoglycan hydrolase (PGH) enzyme reported in the genome of P. acidilactici 7_4, where two different lytic domains have been identified but not characterized. The aim of this work was the biochemical characterization of the recombinant enzyme of 99 kDa. The enzyme was cloned and expressed successfully and retains its activity against Micrococcus lysodeikticus. It has a higher N-acetylglucosaminidase activity, but the N-acetylmuramoyl-L-alanine amidase can also be detected spectrophotometrically. The protein was then purified using gel filtration chromatography. Antibacterial activity showed an optimal pH of 6.0 and was stable between 5.0 and 7.0. The optimal temperature for activity was 60 °C, and all activity was lost after 1 h of incubation at 70 °C. The number of strains susceptible to the recombinant 99-kDa enzyme was lower than that susceptible to the mixture of the 110- and 99-kDa PGHs of P. acidilactici, a result that suggests synergy between these two enzymes. This is the first PGH from LAB that has been shown to possess two lytic sites. The results of this study will aid in the design of new antibacterial agents from natural origin that can combat foodborne disease and improve hygienic practices in the industrial sector.