Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6593-2

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Expression, purification, and characterization of a bifunctional 99-kDa peptidoglycan hydrolase from Pediococcus acidilactici ATCC 8042 Israel García-Cano 1 & Manuel Campos-Gómez 1 & Mariana Contreras-Cruz 1 & Carlos Eduardo Serrano-Maldonado 1 & Augusto González-Canto 2 & Carolina Peña-Montes 1 & Romina Rodríguez-Sanoja 3 & Sergio Sánchez 3 & Amelia Farrés 1

Received: 23 January 2015 / Revised: 1 April 2015 / Accepted: 5 April 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract 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 99kDa PGHs of P. acidilactici, a result that suggests synergy

* Amelia Farrés [email protected] 1

Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México D.F., México

2

Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México y Hospital General de México, 06720 México D.F., México

3

Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México D.F., México

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. Keywords Peptidoglycan hydrolase . Pediococcus acidilactici ATCC 8042 . N-acetylmuramoyl-L-alanine amidase . N-acetylglucosaminidase . 4-Nitrophenyl N-acetyl-β-D-glucosamine . Zymogram

Introduction Peptidoglycan hydrolases (PGHs) are enzymes that hydrolyze glycosidic or peptide bonds found in peptidoglycan, the main component of bacterial cell walls, thereby promoting cell lysis. PGHs are found in organisms from all major taxa, including animals, plants, bacteria, and viruses, but their functions vary (Vollmer et al. 2008). Classification of these enzymes is based on the type of peptidoglycan linkage that they hydrolyze, such as Nac ety l glu co s a m i ni d ase , N -acet ylmurami das e, N acetylmuramoyl-L-alanine amidase, and endopeptidases (Vollmer et al. 2008; Layec et al. 2008). In recent years, PGHs have been proposed as an alternative or complementary tool for combating bacterial foodborne diseases, which are rapidly spread and associated with high morbidity, economic loss, and even death (Fischetti 2010). The use of PGHs can assist in the elimination of deleterious bacterial colonization of the gastrointestinal mucosa and the control of pathogenic bacteria in food infections (Hermoso et al. 2007). When added to finished food products, these lytic enzymes extend shelf life, prevent

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deterioration, inhibit the growth of undesirable microorganisms, and improve the organoleptic properties of the final product. A specific example of the use of PGH in the food industry is lysozyme, which is an N-acetylmuramidase, used by plants and animals as a defense mechanism against pathogenic bacteria and generally considered a nonobjectionable food additive (Nakimbugwe et al. 2006; Maidment et al. 2009). Lysostaphin is another example of a useful PGH in industry. This enzyme is produced by Staphylococcus simulans and is highly specific for a five-glycine peptide that joins carbohydrate moieties in the Staphylococcus aureus cell wall, an important pathogen in the food, veterinary, and medical industry (Szweda et al. 2012). It is a Zn2+-dependent metalloprotease with a molecular weight of 27 kDa (Turner et al. 2007). Its main drawback is that it comes from a pathogenic strain; so, in recent years, lysostaphin has been cloned and industrially produced in heterologous systems such as LAB, which are recognized by the Food and Drug Administration (FDA) as generally recognized as safe (GRAS). Pediococcus acidilactici ATCC 8042 is a nonpediocinproducing strain (Mora et al. 2000) that exerts an antibacterial effect due to two lytic proteins with different molecular masses that were partially purified to determine their antibacterial spectra. All tested Gram-positive strains were inhibited, most notably Bacillus cereus, Streptococcus pyogenes, and S. aureus, and some Gram-negative strains, such as Escherichia coli and Salmonella typhimurium (García-Cano et al. 2011). The first enzyme is a putative protein found in the genome of P. acidilactici 7_4 of 110 kDa and of unknown function. This protein shows a conserved region homologous to an ATP-binding cassette transporter and lytic activity. The second protein, with a molecular mass of 99 kDa, was identified as an Nacetylmuramidase and has two putative lytic domains corresponding to those of an N-acetylmuramoyl-L-alanine amidase and N-acetylglucosaminidase, respectively. There are reports of proteins that have double PGH activity, with one example being a Staphylococcus epidermidis enzyme that has a characterized and crystallized N-acetylmuramoyl-Lalanine amidase domain in the intermediate sequence of the protein and an N-acetylglucosaminidase domain at the C-terminus of the protein, which has not been fully explored (Zoll et al. 2010). The aim of this work was to study the 99-kDa bifunctional PGH from P. acidilactici ATCC 8042. This protein is the first PGH from LAB for which a double lytic activity has been proposed. Due to the difficulties involved in the purification process, the cloning, expression, purification, and biochemical characterization were required. The resulting information will be useful for the design of new antibacterial agents to combat foodborne disease and to improve hygienic practices in both the food and clinical sectors.

Materials and methods Bacterial strains and culture conditions The bacterial strains used in this study are listed in Table 1. P. acidilactici ATCC 8042 was cultivated without shaking in MRS broth (De Man Rogosa and Sharp, Oxoid, UK) at 29± 2 °C. E. coli strain BL21 (DE3, Invitrogen, USA) was used as host for expression, and it was grown in Luria-Bertani broth (LB: bacto tryptone 10 g/l, yeast extract 5 g/l, NaCl 10 g/l, pH=7.0) at 37 °C. The LB medium was supplemented with ampicillin (100 μg/ml) when necessary. The other strains were cultivated in BHI broth (Brain Heart Infusion, Difco, USA) without shaking at 37 °C. Construction of pET19-paci99 vector and overexpression The pET19b (+) vector (Novagen, Darmstadt, Germany) was used for cloning and expressing the PCR product. The genomic DNA of P. acidilactici and the pET19b (+) were extracted using kit Genomic DNA Extraction (Fast ID, Fairfield, USA) and Gene JET Plasmid Miniprep Kit (Thermo Scientific, Rockford, USA), respectively, according to the manufacturer’s instructions. PCR primers were designed based on the full protein-coding region of the P. acidilactici 7_4 genomic scaffold supercont 1.3 (accession number GI: 2702800752 ->GenBank GG730085.1) of the 99-kDa Nacetylmuramidase (García-Cano et al. 2011) using OligoCalc Software (Kibbe 2007). To determine the complete sequence, different primers were designed (Table 1). Figure 1 shows gene paci99, the site where the primers were located and the expected size of the amplicon. Expression of the complete gene was achieved using the forward primer Pa99F, which contained a recognition site for XhoI (underlined, Table 1) (Thermo Scientific) and the reverse primer Pa99R, which contained a recognition site for BamHI (underlined, Table 1) (Thermo Scientific). PCR was performed in a Maxygen thermocycler (Axygen, California, USA) using HotStart Taq Polymerase enzyme (Qiagen, Hilden, Germany) under standard conditions: one cycle for denaturation at 95 °C for 15 min, 35 cycles at 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 1 min, followed by a final extension step at 72 °C for 10 min. The 2784-bp PCR product was visualized on a 1 % agarose gel containing 0.01 % of ethidium bromide and was extracted using the kit MinElute BrEt (SigmaAldrich, Missouri, USA). The products were sequenced by Macrogen Inc. (Seoul, Korea). The pET19b (+) plasmid DNA and the PCR product were double digested with XhoI and BamHI. The linearized plasmid and the digested PCR product were used for ligation by T4 DNA ligase (Thermo Scientific). After ligation, E. coli BL21 was transformed with the recombinant pET19-paci99 plasmid selected on LB agar plates containing ampicillin (100 μg/ml).

Appl Microbiol Biotechnol Table 1

Bacterial strains, plasmid, and primers used in this work

Bacterial strains, plasmids, and primers

Description

Source or reference

Strains Pediococcus acidilactici ATCC 8042 Staphylococcus aureus ATCC 6538 Micrococcus lysodeikticus ATCC 4698 Enterococcus faecalis F QB

Target strain, MRS broth Target strain, BHI broth Target strain, BHI broth Target strain, MRS broth

CINVESTAV, IPN, México FESC-UNAM, México Sigma-Aldrich García-Cano et al. (2014)

Target strain, MRS broth

García-Cano et al. (2014)

Enterococcus faecium G QB Lactobacillus paracasei CFQ-B-90

Target strain, MRS broth

García-Cano et al. (2011)

Bacillus subtilis ATCC 6633

Target strain, BHI broth

García-Cano et al. (2011)

Bacillus cereus CFQ-B-230

Target strain, BHI broth

García-Cano et al. (2011)

Streptococcus pyogenes CFQ-B-218

Target strain, BHI broth

García-Cano et al. (2011)

Listeria monocytogenes CFQ-B-103

Target strain, BHI broth

García-Cano et al. (2011)

Pseudomonas aeruginosa ATCC 27853

Target strain, BHI broth

García-Cano et al. (2011)

S. typhimurium ATCC 14028

Target strain, BHI broth

García-Cano et al. (2011)

E. coli DH5α

Target strain, BHI broth

Invitrogen

E. coli BL21(DE3)

F− mpT hsdSB (rB- mB-) gal dcm, LB broth

Novagen

Expression vector, T7 lac promotor, ampR, production of recombinant proteins with an N-terminal 10-His tag

Dr. B. González-Pedrajo IFC-UNAM

5′-CTACACCTCGAGTTTAAGTCAGGGAAG-3′ 5′-ATCTCAGGATCCACCAACGAAATTGC-3′ 5′-GGGCAACTGGTCAAAATATTTATAGCGG-3′ 5′-CCGCTATAAATATTTTGACCAGTTGCC-3′ 5′-CGTTAACTTACTCCGTGATTCCGC-3′ 5′-CGGAATCACGGAGTAAGTTAACGT-3′

This study This study This study This study This study This study

Plasmid pET19b (+)

Primers Pa99F Pa99R PaF3 PaR2 PaF2 PaR1 ampR ampicillin resistance

Ten colonies were assayed by colony PCR with primers complementary to the T7 promoter and T7 terminator and the specific primers Pa99F and Pa99R. To determine the complete sequence, different primers were designed (Table 1). Figure 1 shows gene paci99, the site where the primers were located and the expected size of the amplicon. After selecting the recombinant clones, the plasmid DNA was extracted from the overnight culture, and it was sequenced at Macrogen Inc., Korea. Expression of the pET19-paci99 gene product was achieved by growing E. coli BL21 in LB broth supplemented with ampicillin (100 μg/ml) at 37 °C with orbital shaking at 200 rpm. At midexponential phase of growth Fig. 1 Scheme of the amplified internal regions of the gene paci99

(O.D.600nm of approx. 0.5), protein expression was induced by the addition of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After 2 h of incubation, the supernatant was collected by centrifugation at 8500 rpm for 20 min and stored at 4 °C for further characterization (the activity was determined in the supernatant and cells, the highest activity was found in the cell-free extracts). PGH activity 4-Nitrophenyl N-acetyl-β- D -glucosamine (NP-GlcNAc, Sigma-Aldrich) was used as the substrate in the enzyme

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activity assays. Upon N-acetylglucosaminidase catalysis, pnitrophenol is released, which forms the p-nitrophenolate ion under alkaline conditions. This ion can be measured spectrophotometrically at 405 nm. Ten microliters of a 1-mg/ml NPGlcNAc solution was placed in a 96-well plate along with 10 μl of the assayed enzyme and 80 μl of sodium citrate buffer (100 mM, pH 4.8). Additionally, 10 μg/ml of the β-Nacetylglucosaminidase from Canavalia ensiformis (SigmaAldrich) was used as a positive control. The reaction mixture was incubated at 37 °C for 10 min, and 100 μl of sodium carbonate (140 mM) was used to stop the reaction and to increase the color. Absorbance at 405 nm was determined in an Epoch microplate spectrophotometer (BioTek, Vermon, USA). Unit definition: one unit will hydrolyze 1.0 μmole of 4-nitrophenyl N-acetyl-β-D-glucosamine to p-nitrophenol and N-acetyl-β-D-glucosamine per minute at pH 4.7 at 37 °C. In order to evaluate the N-acetylmuramoyl-L-alanine amidase activity, L-alanine-p-nitroanilide hydrochloride (SigmaAldrich) was used as a substrate (Yoke-Ming et al. 2015). Ten microliters of a 1-mg/ml solution was placed in a 96-well plate along with 10 μl of the assayed enzyme and 80 of μl Tris-HCl buffer (100 mM, pH 7.6). The reaction mixture was incubated at 37 °C for 10 min. Absorbance at 405 nm was determined. One unit was defined as the amount of enzyme that will hydrolyze 1.0 μmole of L-alanine-p-nitroanilide to p-nitroaniline per minute at pH 7.6 at 37 °C. Additionally, 10 μg/ml of β-Nacetylglucosaminidase and 1 mg/ml of lysostaphin and lysozyme were used as negative controls. A recombinant protein generated at our laboratory that contains only the Nacetylmuramoyl-L-alanine amidase dominion was used as a positive control. Purification of recombinant protein The cell-free extract was concentrated by ultrafiltration and stirring in an Amicon Ultrafiltration Cell using an Amicon YM-10 membrane (NMWCO 10 kDa, M i ll i p o re, U S A ). T h e mo l ecu l ar si ev e colum n Superdex® 200 (Pharmacia Biotech) (2.6×60 cm) was used to fractionate 20-mg samples of the previously concentrated extract using Biologic LP (Bio-Rad) with a 1-ml/min flux in a buffer containing 100 mM of TrisHCl, pH 8.0. Then, 5-ml fractions were collected, and absorbance at 280 nm (Abs280nm) was assayed with a BioMate 3 UV–Visible spectrophotometer (Thermo Scientific, Madison, USA) to locate protein. Enzyme activity was assayed using the substrate 4-nitrophenyl N-acetyl-β-D-glucosamine. The fractions that showed activity were dried by lyophilization (Labconco Freezone 4.5, Kansas City, USA) and resuspended in 500 μl of H2O. These fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms.

Protein profile and zymograms The protein profile was obtained as described previously (García-Cano et al. 2011). Ten percent of polyacrylamide SDS gels were prepared according to Laemmli (1970). The lytic activity against Micrococcus lysodeikticus was determined as described by Leclerc and Asselin (1989). The molecular masses of the bands that showed lytic activity were determined using high range molecular weight markers (Bio-Rad). The analysis of the gels was performed, and the intensity of each band was calculated using a Gel Doc™ XR+ System (Bio-Rad).

Protein assay Proteins were quantified according to Bradford (1976) using a commercial kit (Bio-Rad) and bovine serum albumin as protein standard.

Effects of pH on enzyme activity and stability Optimal pH was determined using the following buffers: 20 mM citric acid (for buffers in the 2.1–4.1 pH range), 20 mM acetic acid (for buffers in the 3.8–5.8 pH range), 20 mM MES (for buffers in the 5.5–6.7 pH range), 20 mM HEPES (for buffers in the 6.8–8.2 pH range), and 20 mM CHES (for the buffers in the pH 8.6–10.0 range). The pH was adjusted to values 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. Enzyme activity was assayed using 10 μl of substrate (1 mg/ml) and 80 μl of buffer. The reaction was initiated by the addition of 10 μl of pure enzyme with no preincubation. Stability studies were performed using 10 μl of enzyme solution and 90 μl of each buffer. The reactions were allowed to incubate for 60 min, and then, 10 μl of this reaction mixture was added to 80 μl of buffer at pH 7.0, and the reaction was initiated by substrate addition. The enzyme with no added buffers was used as a control.

Optimal temperature of activity and stability Ten microliters of purified protein, 10 μl of substrate (1 mg/ml), and 80 μl of buffer solution at pH 7.0 were mixed together. The mixture was incubated for 10 min at different temperatures (37, 45, 50, 60, 70, 80, and 90 °C). Twentymicroliter samples were taken and subjected to different temperatures: 37, 45, 50, 60, 70, 80, and 90 °C, where they were incubated for 60 min. The activity was then assayed using 10-μl samples. Enzyme that was not subjected to thermal treatment was used as a control.

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Effect of metallic ions, protease inhibitor, and chelating agents on activity Mg2+, Zn2+, Ca2+, Na+, EDTA, ethylene glycol tetraacetic acid (EGTA), and phenylmethylsulfonyl fluoride (PMSF) at 1 and 10 mM final concentrations were used to evaluate the effect on enzyme activity (Morner and Braun 1984). Ten microliters of each solution was added to 10 μl of the enzyme and incubated at room temperature for 60 min. The enzyme with no added effectors was used as a control. Antibacterial spectrum of the 99-kDa recombinant clone The antibacterial activity was assayed by agar diffusion, as described by García-Cano et al. (2014). All bacteria, growing conditions, and culture media used in this study are described in Table 1. Gene sequences submitted to GenBank The complete paci99 gene cloned in this work was submitted to GenBank, and its accession number is KP728239.

Results Cloning, expression, and purification of the recombinant protein DNA from P. acidilactici ATCC 8042 was extracted and amplified by PCR with designed primers (Table 1). An amplicon of 2784 bp, corresponding to the complete paci99 gene (GenBank accession number KP728239), was obtained along with other amplicons of sizes 940, 890, and 1000 bp, corresponding to intermediate gene sequences. The resulting sequences were subject to a multiple alignment against the genomes of different P. acidilactici strains, using the Multaline software (Corpet 1988). The amino acid sequence was obtained after a virtual translation, performed using the software provided by the Translate Tool in the ExPASy Proteomics Server (Swiss Institute of Bioinformatics). The deduced amino acid sequence was compared to that found in the National Center for Biotechnology Information database. Figure 2a shows the two different amino acids (black shadow) found in P. acidilactici ATCC 8042 compared to those found in the database and multiple amino acid sequence alignment of 99PGH from different strains of P. acidilactici. Figure 2b shows the total amino acid sequence of the 99-kDa protein. The Nacetylmuramoyl-L-alanine amidase domain is highlighted in green, along with the amino acids involved in catalysis, substrate binding, and Zn2+ binding. In the same figure, the Nacetylglucosaminidase domain is shown in magenta color. Figure 2c shows a schematic drawing the 99-kDa protein.

The putative signal peptide (SP) is represented by a yellow box, and it was found in two strains of P. acidilactici. The Nacetylmuramoyl-L-alanine amidase domain (green box) is reported as a peptidoglycan recognition protein (PGRP). The gray box corresponds to an asparagine-rich sequence (N-rich). This amino acid represents 13.5 % of the total sequence and 22.5 % in the 403 amino acid N-rich region. The magenta box indicates the N-acetylglucosaminidase domain, which is included within the lysozyme-like superfamily. Cell-free extract containing 99-kDa protein was concentrated by ultrafiltration and analyzed by SDS-PAGE and zymograms. In Fig. 3, lane 2 shows the cell-free extract, and lane 3 shows the expressed product after ultrafiltration. In the same way, lanes 4 and 5 show the lytic activity against M. lysodeikticus, respectively, exhibiting bands of approximately 99 kDa. The concentrated sample was applied to a molecular exclusion column, and all the fractions were monitored for N-acetylglucosaminidase activity. As can been seen in the chromatogram in Fig. 4a, fractions 26–36 show activity, and the highest activity was found in fraction 29. This fraction was subject to electrophoresis. The protein profile shows three bands: one with the expected molecular mass of 99 kDa corresponding to the full recombinant protein and to 23 % of the protein present in the preparation. The second and third bands correspond to 66 and 55 kDa molecular weight, with 7 and 67 % of the protein present, respectively. Based on the apparent molecular weight, this protein is most likely a degradation product of the highmolecular weight band. The remaining 3 % corresponds to bands that cannot be visualized (Fig. 4b, lane 2). It must be noted that only the 99-kDa band shows lytic activity against M. lysodeikticus (Fig. 4b, lane 3), suggesting that the lower molecular weight band is a degradation product and does not contain the active site. This result was confirmed by Western blotting (Anti-His (C-term) Mouse Monoclonal Antibody, Novex®, NY, USA), where a faint band was observed at 99 kDa and an intense but inactive band was observed at approximately 55 kDa (data not shown). The culture medium did not show any detectable proteolytic activity (data not shown), and there are reports that indicate that SDS-PAGE and zymograms from other peptidoglycan hydrolases show several bands, which may represent proteolytically processed forms of the same enzyme (McLaughlan and Foster 1997). The purity of each band was calculated using the Image Lab software (Bio-Rad), based on optical density readings. Table 2 shows that with this rapid and easy procedure, a highly enriched fraction of the 99-kDa protein was obtained, with 6 % of activity recovery and 50.6-fold enriched PGH activity. Biochemical characterization of PGH The cell-free extract used to purify the recombinant enzyme was used to assay the lytic activities whose domains are

Appl Microbiol Biotechnol Fig. 2 Amino acid sequence of the 99-kDa protein. a Multiple amino acid sequence alignment of PGH from P. acidilactici ATCC 8042 compared to PGHs proteins from different P. acidilactici. In black shadow the different amino acid in P. acidilactici ATCC 8042. In gray the amino acid that are different between species of P. acidilactici. b Sequence of P. acidilactici ATCC 8042. In green, the N-acetylmuramoyl-Lalanine amidase domain is indicated. The boxes show the amino acid residues involved in catalysis, the bold letters exhibit the substrate binding site and the amino acid residues with a dot show the Zn2+ binding site. Region 777 to 915 (in magenta) indicates the putative Nacetylglucosaminidase domain. c Schematic representation of the domains structure of the 99-kDa PGH. In yellow, signal peptide (SP); in green, Nacetylmuramoyl-L-alanine amidase domain; in gray, N-rich sequence; and in magenta, the Nacetylglucosaminidase domain

inferred from the reported genomic sequence. The specific activity (U/mg protein) of N-acetylglucosaminidase was of 4.9± 0.3; however, the N-acetylmuramoyl-L-alanine amidase activity was 0.23±0.09. Therefore, the N-acetylglucosaminidase activity was further characterized, assessing initially the optimal pH and temperature of activity using 4-nitrophenyl N-acetyl-βD-glucosamine as a substrate.

at pH 3.0, 9.0, and 10.0 after an incubation time of 60 min (Fig. 5a, inset). Optimal temperature of the recombinant enzyme was 50 °C. Thirty percent of activity was lost at 37 °C, while 70 % of the activity is lost after 70 °C, and interestingly, at 90 °C, the activity remained (Fig. 5b). The enzyme remained stable in the 37–60 °C range. At 70 °C, the activity was completely lost after incubation for 60 min (Fig. 5b, inset).

Effects of pH and temperature Effect of ions, chelating agents, and protease inhibitor Optimal pH was 6.0. The enzyme showed only 50 % residual activity at acidic pH (3.0–4.0), while alkaline pH resulted in a 20 % loss of activity at pH 9.0 and 80 % loss of activity at pH 10.0 (Fig. 5a). The protein was stable at pH 6.0 and even showed a 60 % increase in activity after incubation, while at pH 5.0, activity levels increased 30 %, and at pH 7.0, activity levels equal to those of the enzyme without incubation were observed. Thirty percent of the enzyme activity was retained

The addition of divalent ions causes a decrease in activity from 10 to 40 % (Fig. 5c). The only cation that did not cause any effect on activity at 1 and 10 mM was Na+. PGH activity is not affected by incubation with 1 or 10 mM of PMSF and EDTA for 60 min, but 1 mM EGTA resulted in the loss of 20 % PGH activity, and when 10 mM was used, loss increases to 80 %.

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Fig. 3 Protein profiles and zymograms. Lane 1 molecular weight high range; lanes 2 and 3 show the SDS-PAGE (10 %) of the cell-free extract and the same concentrated by ultrafiltration, respectively; lanes 4 and 5 zymograms against M. lysodeikticus

Antibacterial spectrum of the 99-kDa recombinant enzyme The antibacterial effect of the 99-kDa recombinant enzyme was evaluated using pathogenic bacteria or culture collection strains, as illustrated in Table 3. Antibacterial activity against seven species was found, notably against S. pyogenes, Listeria monocytogenes, and S. aureus.

Discussion The use of PGH to provide consumer safety in the food sector has been explored recently (Callewaert et al. 2011; Garcia et al. 2010). PGHs from LAB are particularly interesting as Fig. 4 Purification of the 99-kDa recombinant protein. a Chromatogram obtained after passing the sample through the Superdex® 200 column. Squares represent the Abs280nm and the triangles represent the specific Nacetylglucosaminidase activity. b Protein profiles. Lane 1 molecular weight high range, lane 2 fraction number 29 subjected to SDSPAGE (10 %), lane 3 zymogram against M. lysodeikticus

this bacterial group is categorized as GRAS (Zhang 2012). We previously reported that a 99-kDa protein from P. acidilactici ATCC 8042 exerted lytic activity against several pathogenic and undesirable bacteria in the food industry. In this work, the gene that encodes this protein was analyzed. It showed 99.8 % identity with the reported sequence in the genome of P. acidilactici 7_4 (NCBI: WP_002829921.1). In silico translation showed two different amino acid residues when compared to three sequences reported in database from different genomes of P. acidilactici. These amino acids are not found in the N-acetylmuramoyl-L-alanine amidase or Nacetylglucosaminidase domains; so, the differences are not expected to affect the lytic activity of the recombinant enzyme. In addition, the physicochemical properties of the found amino acids are similar: in the Val-144 position, Ala was found, while in the Ala-208, a Val is present. Both amino acids confer hydrophobic properties; therefore, the amino acid substitutions do not result in significant changes in enzyme activity. Multiple amino acid sequence alignment shows that some other amino acids are different among P. acidilactici strains; for example, position 84 is a Pro in P. acidilactici 7_4 and P. acidilactici DSM20284, while it is Ser in P. acidilactici ATCC 8042 and P. acidilactici MA18/5 M. However, as both amino acids are uncharged polar, it is not likely that the protein folding and the lytic activity of the 99kDa recombinant protein reported in this study will be affected. Not all the reported putative sequences from P. acidilactici possess a signal peptide, and in the case of the PGH from P. acidilactici ATCC 8042, we do not know if there is one as the work was performed only with the final protein and the genome is not known. The N-acetylmuramoyl-L-alanine amidase domain has been reported as a member of the PGRP family, which is divided into three classes: short PGRPs (PGRP-S), which are small (20 kDa) extracellular proteins; intermediate PGRPs (PGRP-I) that are 40–45 kDa and are predicted to be transmembrane proteins; and long PGRPs

Appl Microbiol Biotechnol Table 2

Purification of 99-kDa PGH from P. acidilactici ATCC 8042

Purification step

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Times of purification

Yield (%)

Culture supernatant Ultrafiltration 10 kDa Superdex® 200

42±2 6±1.5 0.05±0.002

199±4 129±2.1 12.1±0.5

4.9±0.3 22.3±1.3 248±0.5

1 4.5 50.6

100 65 6

Data represent the mean and standard deviation of at least three independent experiments

(PGRP-L), up to 90 kDa, which may be either intracellular or transmembranal. This family includes Zn-dependent Nacetylmuramoyl-L-alanine amidase (Dziarski and Gupta 2006). The 99-kDa PGH in this study probably lies within the third class because of its molecular weight, which is above 90-kDa and it is membrane-bound, as was previously described (García-Cano et al. 2011). There are few reports which indicate the function of proteins with repeated regions of asparagines. The parasite Plasmodium falciparum has a proteome rich in asparagine repeats containing proteins. One possible explanation is that these repeats provide a selective advantage, such as acting as tRNA sponges or having a role in immune evasion and antigenic variation or protein-protein interactions (Muralidharan and Goldberg 2013). The role of this N-rich region in the 99kDa sequence from P. acidilactici ATCC 8042 is not known. The N-acetylglucosaminidase domain is reported in the database as a member of the lysozyme-like superfamily. This contains several members including soluble lytic transglycosylases (SLT), goose egg-white lysozymes Fig. 5 Biochemical characterization of 99-kDa PGH. a, b Effect of pH and temperature over lytic activity, insets show the pH and thermal stability, respectively. c Effect of different ions, chelating agents, and protease inhibitor; the control C indicates that the sample was treated without agents. Error bars represent standard deviation of three independent experiments

(GEWL), hen egg-white lysozymes (HEWL), chitinases, bacteriophage lambda lysozymes, endolysins, autolysins, and chitosanases. All the family members are involved in the hydrolysis of β-1,4-linked polysaccharides (Koraimann 2003). Most reported N-acetylglucosaminidase domains are putative. Among those characterized are the autolysin or PGH Auto (Lmo1076) from L. monocytogenes, which was crystallized and characterized; finding that residues Glu-122 and Glu-156 are catalytically essential (Bublitz et al. 2009). We performed an alignment of Lmo1076 and the 99-kDa Nacetylglucosaminidase domain, and they had low similarity, but they share the location of the abovementioned glutamic acid residues (data not shown). The purified protein was biochemically characterized using the substrate involved in the N-acetylglucosaminidase reaction because it is the highest one when assayed spectrophotometrically. The optimal pH for activity was 6.0, which indicates that an amino acid which ionizes at neutral pH might be present in the catalytic site (Ho-Shing and Ming-Ju 2004; Basu et al. 2008). The behavior of the enzyme at acidic or

Appl Microbiol Biotechnol Table 3

Inhibition of bacterial strains by recombinant PGH

Bacterial strains Gram-positive Streptococcus pyogenes CFQ-B-218 Enterococcus faecium QB Lactobacillus paracasei CFQ-B-90 Listeria monocytogenes CFQ-B-103 Pediococcus acidilactici ATCC 8042 Enterococcus faecalis QB Staphylococcus aureus ATCC 6538 Bacillus cereus CFQ-B-230 Bacillus subtilis ATCC 6633 Gram-negative Salmonella typhimurium ATCC 14028 Escherichia coli DH5α Pseudomonas aeruginosa ATCC 27853

mm/mg prot.

40.8±2.1 25.8±1.2 24.7±1.3 23.6±2.0 22.1±1.2 19.9±1.3 11.4±1.1 0.0 0.0 0.0 0.0 0.0

Data represent the mean and standard deviation of at least three independent experiments

alkaline pH values corresponds to the reports on several similar enzymes that require neutral or slightly acidic reaction conditions to achieve maximal activity, which is drastically reduced at pH over 7.5–8.0 (Cheng and Fischetti 2007; Fukushima et al. 2007; Sugahara et al. 2007). This property allows the use of the enzymes in the food industry to prevent the growth of undesirable microorganisms as most food products have a pH close to 6.0–7.0 (Mendes de Souza et al. 2010). The 99-kDa recombinant protein showed values in optimal and stability temperatures similar to those shown by PGHs that exhibit biotechnological potential (Kyomuhendo et al. 2007). The 99-kDa PGH was still lost active if the incubation time at high temperatures was increased, suggesting an irreversible denaturation. This is probably because no disulfide bonds can be found in the 99-kDa protein according to the predictions made with the Dianna web server (Ferré and Clote 2005) (http://Clavius.bc.edu/~clotelab/DiANNA). Thermostable PGHs, like that produced by Pseudomonas aeruginosa bacteriophage φKMV, have four disulfide bonds and show 26 % residual activity after incubation for 2 h at 100 °C and resist autoclaving for 20 min at 120 °C (Lavigne et al. 2004). Some lytic enzymes are known to require the binding of an ion in the catalytic domain to display activity. Endolysin LysB4 from the B4 bacteriophage requires Zn2+ or Mn2+ (Son et al. 2012). Some authors have reported that treatment with EDTA reduces lytic activity because this chelating agent sequesters the metallic ion required in the catalytic site of the protein, commonly Zn 2 + (Morner and Braun 1984; Schmelcher et al. 2012). The 99-kDa protein was exposed to different chelating agents at multiple concentrations and was not inhibited by EDTA despite containing a putative Zn2+

binding site, possibly because the enzyme has different affinities toward the different metallic ions in the coordination site of the chelating agent (McCafferty et al. 1997). This behavior differs from other PGHs that possess a Zn2+ binding site, where this ion has been reported as essential for activity (Schmelcher et al. 2012; Sabala et al. 2012). The role of Zn2+ and chelating agents in the N-acetylmuramoyl-L-alanine amidase activity remains to be explored. PMSF (inhibitor of serine proteinases) did not affect the lytic activity. This result was expected as in the studied enzyme, the amino acids involved in the catalytic Nacetylmuramoyl-L-alanine amidase site are not serine residues. In the N-acetylglucosaminidase, the amino acids involved in catalysis are unknown, and the alignment with other N-acetylglucosaminidases does not show which residues are involved either in carbohydrate binding or in catalysis. EGTA, a Ca2+ chelating agent, inhibited activity by 20 % when used at 1 mM and 80 % when used at 10 mM, suggesting that the 99-kDa enzyme requires Ca2+ to perform substrate hydrolysis (Morner and Braun 1984; Yoshimura et al. 2006). However, no divalent ion exerted a significant effect on activity, unlike other PGHs. The endopeptidase CHAPK from S. aureus is completely inhibited when these divalent ions are present at 100 mM (Fenton et al. 2011), while Nacetylmuramoyl-L-alanine amidase from L. monocytogenes can recover 100 % activity by the addition of 10 mM Mg2+ and Ca2+ (Schmelcher et al. 2012), a behavior that might be explained because Ca 2+ increases substrate affinity by interacting with two aspartate residues found in the enzyme and in the substrate lateral acid chain (Claperon et al. 2008). The antibacterial spectrum of the recombinant enzyme is more restrictive than that shown by the partially purified native PGH. It does not inhibit Gram-negative bacteria, a result that suggests that this lytic activity is performed by the 110kDa protein (García-Cano et al. 2011). Therefore, the role of the 110-kDa lytic protein and its uncharacterized lytic domain must be explored, as well as the roles of the 99-kDa protein double lytic domain in the lysis of specific bacteria. It must be stressed that PGHs with two lytic domains have been reported, but they are mainly produced by Bacillus subtilis and by pathogenic bacteria, such as S. aureus, Streptococcus pneumoniae, and Bacillus anthracis (Xu et al. 2014; Mellroth et al. 2014; DeWitt and Grossman 2014). The fact that the recombinant lytic enzyme comes from a GRAS organism represents an advantage in future applications.

Acknowledgments This work was partially funded by PAPIITDGAPA-UNAM IN210606. I.G.-C. received a scholarship from CONACyT 208631. We appreciate English review by American Journal Experts. Conflict of interest The authors declare that they have no conflict of interest.

Appl Microbiol Biotechnol

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