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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Identification, characterization, immobilization of a novel type hydrolase (LmH) from Listeria monocytogenes
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Hansol Ju, Bum Han Ryu, T. Doohun Kim ∗ Department of Applied Chemistry and Biological Engineering, College of Engineering, Ajou University, Suwon 443-749, South Korea
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Article history: Received 7 May 2014 Accepted 28 July 2014 Available online xxx
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Keywords: LmH CLEA Hydrogel
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1. Introduction
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A novel type of hydrolase (LmH) from Listeria monocytogenes was identified, characterized, and immobilized for biotechnological applications. Primary sequence analysis indicated that LmH had a catalytic triad (Ser91 -Asp192 -His222 ) with a molecular weight of 27.8 kDa. Homologs of this enzyme are produced by many Gram-positive bacteria including Bacillus, Staphylococcus, and Enterococcus. Biochemical properties of LmH were investigated by performing mass spectrometry, dynamic light scattering (DLS), enzyme assays, enantioselective analysis, circular dichroism (CD) spectroscopy, fluorescence analysis, and macroscopic hydrogel formations. Interestingly, cross-linked enzyme aggregates (CLEAs) of LmH exhibited enhanced stability and good recycling abilities compared to free LmH. These molecular characteristics of LmH highlight its great potential for the pharmaceutical, biotechnological, and chemical industries. © 2014 Elsevier B.V. All rights reserved.
Lipolytic enzymes (esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3)), which can catalyze the formation and cleavage of ester bonds in a wide range of substrates, are one of the most useful biocatalysts for industrial applications [1,2]. Due to their importances in numerous reactions such as hydrolysis, alcoholysis, esterification, and aminolysis, these enzymes are already of widespread use in the preparation of fine chemicals, cosmetics, pharmaceuticals, and other chemical compounds [3–6]. In particular, enzymes from microorganisms are highly attractive due to their stability against extreme conditions as well as their high specificity [7,8]. To date, lipolytic enzymes of bacterial origin have been classified into eight families (family I–VIII) on the basis of sequence identity and functional highlights [9]. Family I is principally involved in the catalysis of substrates with long acylglycerols, while other families (II–VIII) catalyze the hydrolysis short chain aliphatic or aromatic esters. Although primary sequence similarities are low, all these enzymes share characteristic ␣/ hydrolase fold (-strands surrounded by ␣-helices) with similar catalytic mechanisms. Three amino acid residues (Ser-Asp/Glu-His) constitute catalytic active sites with invariant serine in G-X-S-X-G motif [10,11]. In addition, extensive research has been undertaken to identify novel lipolytic
∗ Corresponding author. E-mail address: [email protected] (T. Doohun Kim).
enzymes, which can lead to biotechnological progress and economical advantages. Furthermore, novel enzymes will expand the diversity of lipolytic enzymes, and provide more suitable biocatalysts for industrial applications. Recently, a number of enzymes that could not be classified into any of the conventional eight families were identified. These include EstOF4 from Bacillus pseudofirmus [12], BL28 from Bacillus licheniformis [13], EstA3 from Thermoanaerobacter tengcongensis [14], PDF1 from Anoxybacillus sp. [15], CEGk from Geobacillus kaustophilus [16], EstD from Thermotoga maritima [17], LipG and LipEH166 from metagenomic libraries [18,19], and Est30 from Geobacillus stearothermophilus [20]. Here we report the identification, characterization, and immobilization of a novel-type hydrolase, designated LmH, from Listeria monocytogenes. L. monocytogenes is a pathogenic Gram-positive bacterium that causes severe devastating diseases to humans [21,22]. This bacterium can tolerate drastic conditions as extreme temperatures, high salt concentrations, extreme pHs, and drying. The extraordinary capacity of L. monocytogenes to adapt to environmental changes is highly related to distinct virulence factors including several hydrolases [23]. However, there have been a limited number of reports regarding these proteins of L. monocytogenes. Sequence analysis clearly indicated that LmH could not be grouped into any previously identified families (I–VIII). After expression of the LmH gene, catalytic properties and hydrogel formations of LmH were extensively determined. Furthermore, carrier-free immobilization of LmH was also investigated for a number of biotechnological applications.
http://dx.doi.org/10.1016/j.ijbiomac.2014.07.058 0141-8130/© 2014 Elsevier B.V. All rights reserved.
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2. Materials and methods
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2.1. Bacterial strains and biological reagents
containing 10 M of 4-methyl-umbelliferyl (MUF)-acetate or 4methyl-umbelliferyl (MUF)-phosphate [25]. 2.5. Enzyme assays
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Two E. coli strains DH5␣ and BL21(DE3) were used for cloning and protein expression (Stratagene, La Jolla, CA; Novagen; Madison, WI). PD-10 and Ni-NTA columns were purchased from GE Healthcare (Seoul, Korea). The expression vector pET-21a from Novagen (Madison, WI, USA) was used for recombinant proteins. Molecular biology enzymes were obtained from New England Biolabs (NEB, Ipswich, MA, USA). Linalyl acetate, linalool, Isopropylthio-d-galactoside (IPTG), ampicillin, enzyme substrates including p-nitrophenyl acetate (pNA, C2), and 4-methyl-umbelliferylacetate were all purchased from Sigma Aldrich Korea.
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2.2. Cloning and expression of LmH
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Genomic DNA from L. monocytogenes (Korean Collection for Type Cultures (WDCM 597)) was isolated by heating bacterial colonies at 100 ◦ C for 5 min. Cellular debris was discarded by centrifugation at 10,000 rpm and the supernatant containing genomic DNA was used for PCR reactions. The LmH gene was amplified from the chromosomal DNA of L. monocytogenes. Restriction enzyme sites were added to both ends (5 -XhoI and 3 -BamHI) to allow subcloning in expression vector pET-21a. The PCR conditions were as follows: a hot start at 94 ◦ C for 10 min followed by 30 cycles of 60 s at 94 ◦ C, 45 s at 58 ◦ C and 1 min at 72 ◦ C, and finally 5 min at 72 ◦ C. After PCR, the products were verified by electrophoresis on a 1% agarose gel. The PCR-amplified fragments were purified using the Qiagen PCR purification kit, cloned into pET-21a, and transformed into E. coli DH 5␣. The integrity of plasmid was confirmed by DNA sequencing using T7 universal primers. The recombinant plasmid (pET-LmH) was then transformed to obtain LmH protein in E. coli BL21 (DE3) cells. 2.3. Expression and purification of LmH
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A single colony of E. coli BL21(DE3) was inoculated into LB medium containing ampicillin (100 g/mL) and incubated on a rotary shaker at 37 ◦ C until the optical density at 600 nm (OD600 ) reached about 0.5. Then, isopropyl -d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the bacterial culture was further grown for 4 h. The resulting cells were harvested and sonicated in a buffer solution containing 20 mM Tris–HCl (pH 7.5), 150 mM sodium chloride, and 10 mM imidazole. Cellular debris was removed by centrifugation at 6000 rpm for 20 min at 4 ◦ C. The supernatants were loaded onto a Ni-NTA column followed by extensive washing with 20 mM imidazole. LmH proteins were then eluted with a buffer containing 200 mM imidazole, and desalted using a PD-10 column. The purity of LmH was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The final protein was stored at −20 ◦ C without further modifications. The molecular weight of LmH was confirmed by mass spectrometry (on a VoyagerTM DE STR (Applied Biosystems; National Collaborative Inter-University Research Facilities, Seoul, Korea) [24].
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2.4. Electrophoresis and zymogram analysis
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SDS-PAGE was performed on vertical slab gels and protein bands were stained with Coomassie Brilliant Blue R250. Non-denaturing SDS-PAGE was performed under non-reducing conditions (without SDS, 2-mercaptoethanol and heat). The lipolytic activity of LmH was visualized under UV illumination box (Intron Biotechnologies; Seoul, Korea) after a 10 min incubation in a solution
Hydrolytic specificity was investigated with chromogenic p-nitrophenyl esters with different alkyl chains including pnitrophenyl phosphate (p-NP), p-nitrophenyl acetate (C2 , p-NA), p-nitrophenyl butyrate (C4 , p-NB), p-nitrophenyl octanoate (C8 , p-NO), p-nitrophenyl decanoate (C10 , p-NDec), and p-nitrophenyl dodecanoate (C12 , p-NDo). A typical reaction solution contained 1 mL of 0.3 mM substrate solution in 50 mM sodium phosphate buffer, pH 7.5, and 0.1 g of LmH protein. The reaction mixture was incubated for 10 min at room temperature and the absorbance was determined with a UV/VIS Spectrometer Biochrom 3330 (Biochrom; Cambridge, UK). The release of pnitrophenol was monitored at 405 nm. Furthermore, substrate specificities of LmH were also tested with 1-naphthyl phosphate (1-NP), 1-naphthyl acetate (1-NA), 2-naphthyl acetate (2-NA), and 1-naphthyl butyrate (1-NB). The blue fluorescence of 4methylumbelliferone (4-MU), which is detected under ultraviolet illumination, was used to estimate the hydrolytic activity of LmH. Hydrolysis of 4-MU acetate was observed in a UV-incubation box. The reaction solution, which was incubated for 5 min at 37 ◦ C, consisted of 0.5 mL of 50 mM sodium phosphate buffer (pH 7.5) containing 10 M 4-MU acetate or phosphate, and the purified enzyme. For pH experiments, p-NB was used instead of p-NA to minimize spontaneous hydrolysis at higher pH and the following buffers were used: glycine–HCl (pH 2.5–3.5), citrate (pH 3.0–6.0), phosphate (pH 6.0–8.0), Tris–HCl (pH 7.5–9.0) and glycine–NaOH (pH 9.0–11.0). To determine thermostability of LmH, 0.1 g of enzyme was incubated for 1 h at different temperatures (40–70 ◦ C). The residual activities of LmH withdrawn at different time intervals were measured. Effect of chemical compounds, detergents, and organic solvents on enzyme activity of LmH was determined after 1 h incubation at room temperature. In a standard assay, activity was measured using 10 mM p-NA as a substrate in 20 mM Tris–HCl (pH 8.5). For enantioselectivity analysis experiments, LmH was added to (R)- or (S)-solutions. The (R)- and (S)-solutions contained 300 mM (R)- or (S)-methyl-3-hydroxy-2-methylpropionate and phenol red (2 g/L). The absorbances of (R)- and (S)-solutions were recorded at 350–600 nm [26]. 2.6. Hydrogel formation of LmH LmH (1 mg/mL) was incubated with Cu2+ ions (0, 0.5, 1.0, and 2.0 mM) for 10 min at 60 ◦ C and subsequently cooled at room temperature. For color staining, phenol red (1 mg/mL) was included before heat treatment. The effects of several chemical compounds (10% EtOH, 1 M NaCl, 30% TFE, 5 mM dithiothreitol (DTT), 1% SDS, 2 M urea, and 2 M GdnHCl) on the hydrogel formation of LmH were also investigated. 2.7. Circular dichroism (CD) and fluorescence analysis Far-UV CD spectra were recorded in a J-715 spectropolarimeter (Jasco, Japan) with protein concentration of 0.20 mg/mL in 50 mM sodium phosphate pH 7.5 with a path length of 0.1 cm. The data were converted into mean residue ellipticity per residue (MRE, []) taking into account path length and concentration. Three scans were accumulated with a bandwidth of 2.0 nm from 190 to 250 nm. The spectra were averaged scans and baseline corrected by subtracting a buffer spectrum. Fluorescence spectra were measured using FP-6200 spectrofluorometer (Jasco, Japan) with a protein concentration of
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Fig. 1. Multiple sequence alignments of LmH and related enzymes (1R1D; a carboxylesterase from Bacillus stearothermophilus; 1TQH: Est30 from G. stearothermophilus; 3DKR: esterase D from Lactobacillus rhamnosus).
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0.05 mg/mL. For denaturation experiments, fluorescences were recorded with final GuHCl concentration of 0–5 M using a 1.0 cm quartz cell. LmH protein was excited at 295 nm, and emission spectra were recorded in the range of 300–400 nm. In addition, LmH was incubated with urea (from 0 to 8 M) or guanidine hydrochloride (from 1 to 4 M) for 1 h and incubated samples were assayed for residual activity.
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2.8. Aggregates formation of LmH
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ammonium sulfate (AMS)) in 20 mM Tris–HCl (pH 8.5) at room temperature. The fluorescence of thioflavin T (ThT, 9 M) was used to investigate the aggregates of LmH. The aggregates of LmH were investigated with fluorescence microscope (Motic; UK) and images were processed using its associated software (ext 475 nm/emi 530 nm). 2.9. Cross-linked enzyme aggregates (CLEAs)
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For aggregate formation, LmH samples (1 mg/mL) were incubated with various chemical compounds (10% (v/v) 1-butyl3-methylimidazolium trifluoromethanesulfonate, 1% (w/v) SDS, 1 M NaCl, 2 M GdnHCl, 30% (v/v) trifluoroethanol (TFE), and 80%
LmH (500 g) was precipitated with ammonium sulfate (80%) in 50 mM phosphate buffer (pH 7.5). Then, 50 mM glutaraldehyde was included, and the reaction mixtures were further incubated for 16 h at 4 ◦ C. The resulting suspension was centrifuged at 12,500 × g at 4 ◦ C for 15 min. The resulting CLEA-LmH was washed
Fig. 2. Biochemical characterization of LmH. (A) SDS-PAGE analysis of LmH. Lane M, molecular marker, lane 1 & 2; E. coli crude extracts before and after induction; lane 3, supernatants; lane 4, pellets; lane 5, LmH after dialysis. (B) Mass spectrum of LmH. Two [M+H]+ and [M+2H]2+ peaks were observed at m/z values of 29.56 and 14.73 kDa. (C) Dynamic light scattering (DLS) results of LmH. (D) Non-denaturing PAGE analysis of LmH. Left: Coomassie staining of non-denaturing gel. Right: non-denaturing gel was soaked and fluorescence was observed under UV light.
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Fig. 3. Enzymatic activities of LmH. (A) Substrate specificity of LmH toward different chain lengths of p-NP esters. (B) Hydrolysis of 4-methylumbelliferyl-phosphate (upper) and -acetate (lower) by LmH was investigated under UV light. (C) Thermostability of LmH. LmH was incubated at 20 ◦ C (), 40 ◦ C (), 50 ◦ C (), and 60 ◦ C (䊉) for 1 h, and the residual activities were measured. (D) pH stability of LmH. 208 209 210 211
until no significant catalytic activity was observed in the supernatant. The surface structure of CLEA-LmH was examined with scanning electron microscope (SEM) (SUPRA 55VP, Carl Zeiss, Germany).
To investigate reusability, CLEA-LmH was recovered by simple centrifugation and washed repeatedly (usually 5 times). Then, fresh substrate was then reintroduced for another cycle and activity was measured. Effects of chemical compounds, detergents, and organic
Fig. 4. Chemical stabilities of LmH. (A) Enzymatic activities of LmH with various concentrations of urea. (B) Chemical stabilities of LmH. The residual activities of LmH after 1 h incubation are expressed relative to the initial activity (100%). (C) Far-UV CD spectrum of LmH. (D) Fluorescence spectra were recorded with increasing concentrations of urea.
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Fig. 5. Regio- and stereo-selectivity of LmH. (A) Regioselectivity of LmH was determined using naphthyl derivatives. (B) pH shift assay for enantioselectivity analysis was conducted with (R)- and (S)-methyl-3-hydroxy-2-methylpropionate. 1: (R) substrate, 2: LmH with (R) substrate, 3: (S) substrate, 4: LmH with (S) substrate. Absorbance spectra for each solution were measured. (C) Hydrogel formation of LmH with Cu2+ ions. LmH was incubated with Cu2+ ions (0, 0.5, 1.0, and 2.0 mM) for 10 min at 60 ◦ C. (D) Scanning electron microscope (SEM) images of Cu2+ -induced hydrogel. Representative images at 50 kX (main) and 100 kX (upper) are shown.
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solvents (ethanol, SDS, Triton X-100, Tween 20, phenylmethylsulfonyl fluoride (PMSF), and urea) on the activities of CLEA-LmH and free LmH were also compared by measuring the residual activity after 1 h of incubation. The initial activity without any chemical compounds was defined as 100%. 2.10. Sequence analysis and molecular modeling To identify related lipolytic hydrolases of bacterial origins, primary sequence of LmH was searched for similarities using PSIBLAST [27]. Sequences homologous to LmH were retrieved in FASTA format, and multiple sequence alignments were generated by the CLUSTALW program and then rendered with ESPript [28]. The conserved domains present in the LmH sequence were analyzed
using the NCBI Conserved Domain Database (CDD) [29]. A molecular model of LmH was constructed by homology modeling using the SWISS-MODEL software using G. stearothermophilus (PDB ID: 1TQH) as template [30]. The 3D structures of LmH were analyzed using PYMOL program (PyMOL Molecular Graphics System). A phylogenetic tree was built with 5000 iterations for bootstrap confidence levels [31].
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3. Results and discussion
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3.1. Bioinformatic in silico analysis of LmH
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In the genome sequence of L. monocytogenes, an open reading frame (ORF; locus tag: NP 465973) of 741 bp was identified through
Fig. 6. Aggregate formation of LmH with 10% (v/v) 1-butyl-3-methylimidazolium trifluoromethanesulfonate (A), 1% (w/v) SDS (B), 30% (v/v) trifluoroethanol (TFE) (C), and 80% ammonium sulfate (AMS) (D). Representative images of light (left) and fluorescence (right) microscopy are shown for comparison.
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Fig. 7. CLEA-LmH. Scanning electron microscope (SEM) images of aggregates and crosslinked aggregates of LmH. (A and B) SEM images of cross-linked enzyme aggregates of LmH (CLEA-LmH), 50 kX and 100 kX, respectively. Glutaraldehyde was used for effective crosslinking. (C) Reusability of CLEA-LmH was investigated for 18 cycles. (D) Comparisons of chemical stabilities of CLEA-LmH and free soluble LmH.
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bioinformatic analysis. The putative hydrolase gene (LmH) encoded a protein of 248 amino acids with 27.8 kDa. A conserved domain search detected an ␣/ hydrolase fold [COG1647], which is found in a large variety of enzyme families including lipases, esterases, peroxidases, and dehalogenases [7,29,32]. Homologs of this enzyme are produced by many strains of Gram-positive bacteria including Bacillus or Staphylococcus. In phylogenic analysis, LmH and its most closely related proteins were grouped in a different branch separate from other bacterial lipolytic families (family I–VIII) (Fig. S1). For example, LmH shared only 3–4% sequence identity with EstII from Pseudomonas fluorescens (AAC60403, family IV) and Est2 from Acetobacter pasteurianus (BAA25795, family V). A multiple sequence alignment was performed for LmH using Est30 from G. stearothermophilus (54% sequence identity, PDB ID: 1TQH), a carboxylesterase from Bacillus stearothermophilus (52% sequence identity, PDB ID: 1R1D), and esterase D from Lactobacillus rhamnosus (31% sequence identity, PDB ID: 3DKR). From the sequence alignment we found that LmH has a conserved hexapeptide GLSLGG in these proteins and the putative catalytic triad (Ser91 -Asp192 -His222 ) (Fig. 1). The crystal structure of Est30 from G. stearothermophilus (PDB ID: 1TQH) was used as a template to construct a structural model of LmH. The model of LmH showed an ␣/-hydrolase fold with a seven-stranded -sheet surrounded by ␣-helices, which had a small cap region covering the active site (Fig. S2). The hydrogen bond distances in the catalytic triad were 3.0 A˚ for Ser91 O␥ to His222 N2 and 2.8 A˚ for His333 N␦1 to Asp303 O␦2. In addition, the oxyanion hole of LmH was mediated by Phe24 and Leu94 . Supplementary figures related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.ijbiomac.2014.07.058.
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To investigate the catalytic properties of LmH, recombinant LmH was purified using Ni-NTA affinity chromatography. The purified LmH was homogeneous as indicated by a single protein band on the SDS-PAGE gel (Fig. 2A). Mass spectrometric analysis was performed to confirm molecular weight of LmH, which showed a major peak of 28,813 Da (Fig. 2B). In dynamic light scattering
(DLS) analysis, the hydrodynamic radius (RH ) of LmH was observed to be 3.25 nm (Fig. 2C). Furthermore, the enzymatic activity of LmH was determined by activity staining of native PAGE using 4methylumbelliferyl (4-MU) acetate (Fig. 2D). High fluorescence due to the formation of 4-methylumbelliferone was clearly seen, which confirmed the hydrolytic activity of LmH. The hydrolyzing activities of LmH toward p-NP esters of different acyl chain lengths were analyzed in Tris–HCl buffer (25 mM, pH 7.5). Substrates included 1 mM p-NP ester compounds of acetate (C2 ), butyrate (C4 ), octanoate (C8 ), decanoate (C10 ), and dodecanoate (C12 ). LmH showed a strong preference to p-nitrophenyl acetate (p-NA) or butyrate (p-NB), although it could not hydrolyze effectively p-nitrophenyl phosphate (p-NP) (Fig. 3A) However, only approximately 3.5% of the relative activity was detected toward pNDec, and almost no activity was observed for p-NDo. Furthermore, hydrolysis of 4-MU acetate by LmH was observed by measuring the fluorescence of 4-methylumbelliferone. However, no activity against 4-MU phosphate was detected (Fig. 3B). The thermostability of LmH was investigated by pre-incubating LmH at different temperatures for 1 h. LmH was completely stable at 20 ◦ C, and retained more than 96% activity at 40 ◦ C after 1 h incubation. In addition, LmH retained ∼45% of its maximum activity at 50 ◦ C, but it was extremely thermolabile at temperatures above 60 ◦ C, with its activity almost completely abolished within less than 15 min at 60 ◦ C (Fig. 3C). This temperature profile was similar to other novel type of hydrolases including Est30 from EstA3 from Thermoanaerobacter tengcogensis [14], CEGk Geobacillus kaustophilus [16], and Geobacillus stearothermophilus [20]. The pH dependencies of LmH activity were investigated by measuring the enzymatic activity after incubation over the pH range of 3.0–10.0 (Fig. 3D). LmH displayed its maximal activity at pH 8.0 and ∼75% of its activity at pH 8.0 was observed at pH 7.0. However, only 20% of the maximal activity was retained at pH 6.0, and no significant activities were observed at a very low or high pH. 3.3. Chemical stabilities of LmH The effect of urea on the activity of LmH was examined by measuring catalytic activity after 1 h incubation (Fig. 4A). LmH was
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largely resistant to 3.0 M urea and ∼70% of activity was retained at this concentration. However, at 4.0 M urea, the enzymatic activity was decreased to ∼30% of the initial value. At 5.0 M urea, almost no activity of LmH was observed. In addition, chemical stability of LmH was measured in reaction mixtures containing different concentrations of detergents, organic solvents, or chemicals. As shown in Fig. 4B, LmH showed ∼70% of its initial activity with 10% (v/v) ethanol, but it retained only approximately 10% activity with 30% (v/v) ethanol. The residual activity of LmH was ∼40% of its initial activity with 1.0% (v/v) Tween 20, whereas it showed only ∼15% of its original activity with 1.0% (v/v) Triton X-100. However, LmH lost most of its activity with 0.1% (w/v) SDS. LmH was almost inactive with the retention of ∼8% activity in 30% isopropanol (i-PrOH). Similar inhibition was also shown in 10 mM PMSF. To analyze the secondary structure of LmH, CD analysis was done at room temperature (Fig. 4C). The far-UV CD spectra of LmH showed strong negative bands at 210–220 nm (-sheet contents), and a positive band below 200 nm (␣-helical contents) [33]. The spectrum showed a minimum at 225 nm (−7000 deg cm2 dmol−1 ), and a maximum at 193 nm (20,500 deg cm2 dmol−1 ). The chemical stability of LmH was also investigated by monitoring the intrinsic fluorescence with urea addition [34]. In native state of LmH, the four tryptophan residues are well-buried with a maximum wavelength (max ) at 338 nm (Fig. 4D). However, there were noticeable decreases in the intensity with a red shift of max . A shift of max from 338 to 347 nm was observed with 4.0 M urea, implying that at this condition all tryptophan residues became largely exposed to the solvents.
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Using naphthyl derivatives as substrates, LmH exhibited activities for 1-NA, 1-NB, and 2-NA, respectively (Fig. 5A). The highest activities were observed when using 2-NA, followed by 1-NB and 1-NA. However, no activity against 1-NP was detected. For enantioselectivity analysis, a pH shift assay was employed with (R)- and (S)-methyl--hydroxyisobutyrate. Lipolytic activity was detected based on the color change in the phenol red indicator due to acid release [35]. After incubation with LmH, the color of mixture turned dark yellow in both (R)- and (S)-enantiomer containing solutions, which was also confirmed by spectral readings (Fig. 5B). An interesting feature of LmH is its ability to form hydrogels with Cu2+ ions (Fig. 5C). Although a large variety of compounds (10% EtOH, 1 M NaCl, 30% TFE, 5 mM DTT, 1% SDS, 2 M Urea, 2 M GdnHCl, and 1 mM Cu2+ ) were investigated, only Cu2+ ions resulted in the formation of a three-dimensional network of hydrogels. In SEM images, the surface morphology of hydrogels showed threedimensional networks of globular beads (Fig. 5D). 3.5. Aggregates formation and CLEA of LmH We investigated the propensities of aggregate formation of LmH using several chemical compounds. To investigate aggregates formation, LmH was incubated with several chemical compounds (10% 1-butyl-3-methylimidazolium trifluoromethanesulfonate, 1% SDS, 30% TFE, and 80% AMS), and images of light and fluorescence microscope were recorded (Fig. 6A–D). Thioflavin T, which specifically binds to intermolecular -sheets, was used for the characterization of aggregates [13,24,36]. With 10% 1-butyl-3-methylimidazolium trifluoromethanesulfonate, 1% SDS, or 30% TFE, small aggregates with low ThT fluorescence were observed (Fig. 6A-C). Similar behaviors were also observed for 1 M NaCl and 2 M GdnHCl (data not shown). However, in the presence of 80% AMS, aggregates with high ThT fluorescence intensities were observed (Fig. 6D). To investigate the efficiency of LmH immobilization, CLEAs of LmH were (CLEA-LmH) prepared by precipitating the LmH with
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80% AMS followed by chemical crosslinking with glutaraldehyde [37,38]. The scanning electron microscopic images of CLEA-LmH showed large fibrous structures of high surface density (Fig. 7A and B). The immobilized LmH showed negligible leaching from the crosslinked enzyme aggregates. In the recycling process, CLEALmH was reused for 18 successive cycles, retaining ∼80% of the initial activity (Fig. 7C). Furthermore, the effects of several chemical compounds on the activities of CLEA-LmH and free LmH were compared. As shown in Fig. 7D, CLEA-LmH showed more resistance against these chemical compounds (EtOH, SDS, Triton X-100, Tween 20, PMSF, and urea) compared to free LmH. Specifically, in the presence of 1.0% (v/v) Tween 20, the free LmH retained only ∼25% of its initial activity, as compared to ∼70% for CLEA-LmH. Moreover, although 10 mM PMSF almost completely inactivated free LmH, the residual activity of CLEA-LmH was as high as ∼85% of its initial one. Therefore, crosslinking of LmH (CLEA-LmH) could enable LmH to be used efficiently in industrial conditions. 4. Conclusions This study reports the molecular characteristics of LmH in pathogenic L. monocytogenes. LmH is a novel type of hydrolase produced by several Gram-positive bacterial species. Although several hydrolases are known to be involved in the pathogenesis by this enteric bacteria, molecular characterization of these proteins are largely unknown. Here biochemical characterization and enzymatic properties of LmH were extensively characterized. To the best of our knowledge, this is the first report of the functional and biochemical characterization of a novel type of hydrolase from L. monocytogenes. Acknowledgments This work was supported by National Research Foundation of Korea Grants funded by the Korean Government Q2 (NRF-2012S1A2A1A01028907, NRF-2013K2A1A2053659, NRF2012R1A1A2000910) to T.D.K. References [1] L. Casas-Godoy, S. Duquesne, F. Bordes, G. Sandoval, A. Marty, Methods Mol. Biol. 861 (2012) 3. [2] F. Hasan, A.A. Shah, A. Hameed, Biotechnol. Adv. 27 (2009) 782. [3] T. Tan, J. Lu, K. Nie, L. Deng, F. Wang, Biotechnol. Adv. 28 (2010) 628. [4] A. Robles-Medina, P.A. González-Moreno, L. Esteban-Cerdán, E. Molina-Grima, Biotechnol. Adv. 27 (2009) 398. [5] T. Kobayashi, Biotechnol. Lett. 33 (2011) 1911. [6] T. Hudlicky, J.W. Reed, Chem. Soc. Rev. 38 (2009) 3117. [7] U.T. Bornscheuer, FEMS Microbiol. Rev. 26 (2002) 73. [8] F. Niehaus, C. Bertoldo, M. Kahler, G. Antranikian, Appl. Microbiol. Biotechnol. 51 (1999) 711. [9] J.L. Arpigny, K.E. Jaeger, Biochem. J. 343 (1999) 177. [10] P.D. Carr, D.L. Ollis, Protein Pept. Lett. 16 (2009) 1137. [11] Z. Qian, C.J. Fields, Y. Yu, S. Lutz, Biotechnol. J. 2 (2007) 192. [12] L. Rao, Y. Xue, Y. Zheng, J.R. Lu, Y. Ma, PLOS ONE 8 (2013) e60645. [13] H. Ju, E. Jang, B.H. Ryu, T.D. Kim, Bioresour. Technol. 128 (2013) 81. [14] L. Rao, Y. Xue, C. Zhou, J. Tao, G. Li, J.R. Lu, Y. Ma, Biochim. Biophys. Acta 1814 (2011) 1695. [15] F. Ay, H. Karaoglu, K. Inan, S. Canakci, A.O. Belduz, Protein Expr. Purif. 80 (2011) 74. [16] S. Montoro-García, I. Martínez-Martínez, J. Navarro-Fernández, H. Takami, F. García-Carmona, A. Sánchez-Ferrer, J. Bacteriol. 191 (2009) 3076. [17] M. Levisson, J. van der Oost, S.W. Kengen, FEBS J. 274 (2007) 2832. [18] M.H. Lee, C.H. Lee, T.K. Oh, J.K. Song, J.H. Yoon, Appl. Environ. Microbiol. 72 (2006) 7406. [19] E.Y. Kim, K.H. Oh, M.H. Lee, C.H. Kang, T.K. Oh, J.H. Yoon, Appl. Environ. Microbiol. 75 (2009) 257. [20] H.E. Ewis, A.T. Abdelal, C.D. Lu, Gene 329 (2004) 187. [21] S.C. Corr, L.A. O’Neill, Cell. Microbiol. 11 (2009) 703. [22] M.J. Gray, N.E. Freitag, K.J. Boor, Infect. Immun. 74 (2006) 2505. [23] M. Hamon, H. Bierne, P. Cossart, Nat. Rev. Microbiol. 4 (2006) 423. [24] E. Jang, B.H. Ryu, H. Ju, T.D. Kim, Bioresour. Technol. 143 (2013) 691. [25] S.Y. Bae, B.H. Ryu, E. Jang, S. Kim, T.D. Kim, Appl. Microbiol. Biotechnol. 97 (2013) 1637.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Identification, characterization, immobilization of a novel type hydrolase (LmH) from Listeria monocytogenes
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Hansol Ju, Bum Han Ryu, T. Doohun Kim ∗ Department of Applied Chemistry and Biological Engineering, College of Engineering, Ajou University, Suwon 443-749, South Korea
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Article history: Received 7 May 2014 Accepted 28 July 2014 Available online xxx
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Keywords: LmH CLEA Hydrogel
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1. Introduction
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A novel type of hydrolase (LmH) from Listeria monocytogenes was identified, characterized, and immobilized for biotechnological applications. Primary sequence analysis indicated that LmH had a catalytic triad (Ser91 -Asp192 -His222 ) with a molecular weight of 27.8 kDa. Homologs of this enzyme are produced by many Gram-positive bacteria including Bacillus, Staphylococcus, and Enterococcus. Biochemical properties of LmH were investigated by performing mass spectrometry, dynamic light scattering (DLS), enzyme assays, enantioselective analysis, circular dichroism (CD) spectroscopy, fluorescence analysis, and macroscopic hydrogel formations. Interestingly, cross-linked enzyme aggregates (CLEAs) of LmH exhibited enhanced stability and good recycling abilities compared to free LmH. These molecular characteristics of LmH highlight its great potential for the pharmaceutical, biotechnological, and chemical industries. © 2014 Elsevier B.V. All rights reserved.
Lipolytic enzymes (esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3)), which can catalyze the formation and cleavage of ester bonds in a wide range of substrates, are one of the most useful biocatalysts for industrial applications [1,2]. Due to their importances in numerous reactions such as hydrolysis, alcoholysis, esterification, and aminolysis, these enzymes are already of widespread use in the preparation of fine chemicals, cosmetics, pharmaceuticals, and other chemical compounds [3–6]. In particular, enzymes from microorganisms are highly attractive due to their stability against extreme conditions as well as their high specificity [7,8]. To date, lipolytic enzymes of bacterial origin have been classified into eight families (family I–VIII) on the basis of sequence identity and functional highlights [9]. Family I is principally involved in the catalysis of substrates with long acylglycerols, while other families (II–VIII) catalyze the hydrolysis short chain aliphatic or aromatic esters. Although primary sequence similarities are low, all these enzymes share characteristic ␣/ hydrolase fold (-strands surrounded by ␣-helices) with similar catalytic mechanisms. Three amino acid residues (Ser-Asp/Glu-His) constitute catalytic active sites with invariant serine in G-X-S-X-G motif [10,11]. In addition, extensive research has been undertaken to identify novel lipolytic
∗ Corresponding author. E-mail address: [email protected] (T. Doohun Kim).
enzymes, which can lead to biotechnological progress and economical advantages. Furthermore, novel enzymes will expand the diversity of lipolytic enzymes, and provide more suitable biocatalysts for industrial applications. Recently, a number of enzymes that could not be classified into any of the conventional eight families were identified. These include EstOF4 from Bacillus pseudofirmus [12], BL28 from Bacillus licheniformis [13], EstA3 from Thermoanaerobacter tengcongensis [14], PDF1 from Anoxybacillus sp. [15], CEGk from Geobacillus kaustophilus [16], EstD from Thermotoga maritima [17], LipG and LipEH166 from metagenomic libraries [18,19], and Est30 from Geobacillus stearothermophilus [20]. Here we report the identification, characterization, and immobilization of a novel-type hydrolase, designated LmH, from Listeria monocytogenes. L. monocytogenes is a pathogenic Gram-positive bacterium that causes severe devastating diseases to humans [21,22]. This bacterium can tolerate drastic conditions as extreme temperatures, high salt concentrations, extreme pHs, and drying. The extraordinary capacity of L. monocytogenes to adapt to environmental changes is highly related to distinct virulence factors including several hydrolases [23]. However, there have been a limited number of reports regarding these proteins of L. monocytogenes. Sequence analysis clearly indicated that LmH could not be grouped into any previously identified families (I–VIII). After expression of the LmH gene, catalytic properties and hydrogel formations of LmH were extensively determined. Furthermore, carrier-free immobilization of LmH was also investigated for a number of biotechnological applications.
http://dx.doi.org/10.1016/j.ijbiomac.2014.07.058 0141-8130/© 2014 Elsevier B.V. All rights reserved.
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2. Materials and methods
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2.1. Bacterial strains and biological reagents
containing 10 M of 4-methyl-umbelliferyl (MUF)-acetate or 4methyl-umbelliferyl (MUF)-phosphate [25]. 2.5. Enzyme assays
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Two E. coli strains DH5␣ and BL21(DE3) were used for cloning and protein expression (Stratagene, La Jolla, CA; Novagen; Madison, WI). PD-10 and Ni-NTA columns were purchased from GE Healthcare (Seoul, Korea). The expression vector pET-21a from Novagen (Madison, WI, USA) was used for recombinant proteins. Molecular biology enzymes were obtained from New England Biolabs (NEB, Ipswich, MA, USA). Linalyl acetate, linalool, Isopropylthio-d-galactoside (IPTG), ampicillin, enzyme substrates including p-nitrophenyl acetate (pNA, C2), and 4-methyl-umbelliferylacetate were all purchased from Sigma Aldrich Korea.
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2.2. Cloning and expression of LmH
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Genomic DNA from L. monocytogenes (Korean Collection for Type Cultures (WDCM 597)) was isolated by heating bacterial colonies at 100 ◦ C for 5 min. Cellular debris was discarded by centrifugation at 10,000 rpm and the supernatant containing genomic DNA was used for PCR reactions. The LmH gene was amplified from the chromosomal DNA of L. monocytogenes. Restriction enzyme sites were added to both ends (5 -XhoI and 3 -BamHI) to allow subcloning in expression vector pET-21a. The PCR conditions were as follows: a hot start at 94 ◦ C for 10 min followed by 30 cycles of 60 s at 94 ◦ C, 45 s at 58 ◦ C and 1 min at 72 ◦ C, and finally 5 min at 72 ◦ C. After PCR, the products were verified by electrophoresis on a 1% agarose gel. The PCR-amplified fragments were purified using the Qiagen PCR purification kit, cloned into pET-21a, and transformed into E. coli DH 5␣. The integrity of plasmid was confirmed by DNA sequencing using T7 universal primers. The recombinant plasmid (pET-LmH) was then transformed to obtain LmH protein in E. coli BL21 (DE3) cells. 2.3. Expression and purification of LmH
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A single colony of E. coli BL21(DE3) was inoculated into LB medium containing ampicillin (100 g/mL) and incubated on a rotary shaker at 37 ◦ C until the optical density at 600 nm (OD600 ) reached about 0.5. Then, isopropyl -d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the bacterial culture was further grown for 4 h. The resulting cells were harvested and sonicated in a buffer solution containing 20 mM Tris–HCl (pH 7.5), 150 mM sodium chloride, and 10 mM imidazole. Cellular debris was removed by centrifugation at 6000 rpm for 20 min at 4 ◦ C. The supernatants were loaded onto a Ni-NTA column followed by extensive washing with 20 mM imidazole. LmH proteins were then eluted with a buffer containing 200 mM imidazole, and desalted using a PD-10 column. The purity of LmH was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The final protein was stored at −20 ◦ C without further modifications. The molecular weight of LmH was confirmed by mass spectrometry (on a VoyagerTM DE STR (Applied Biosystems; National Collaborative Inter-University Research Facilities, Seoul, Korea) [24].
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2.4. Electrophoresis and zymogram analysis
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SDS-PAGE was performed on vertical slab gels and protein bands were stained with Coomassie Brilliant Blue R250. Non-denaturing SDS-PAGE was performed under non-reducing conditions (without SDS, 2-mercaptoethanol and heat). The lipolytic activity of LmH was visualized under UV illumination box (Intron Biotechnologies; Seoul, Korea) after a 10 min incubation in a solution
Hydrolytic specificity was investigated with chromogenic p-nitrophenyl esters with different alkyl chains including pnitrophenyl phosphate (p-NP), p-nitrophenyl acetate (C2 , p-NA), p-nitrophenyl butyrate (C4 , p-NB), p-nitrophenyl octanoate (C8 , p-NO), p-nitrophenyl decanoate (C10 , p-NDec), and p-nitrophenyl dodecanoate (C12 , p-NDo). A typical reaction solution contained 1 mL of 0.3 mM substrate solution in 50 mM sodium phosphate buffer, pH 7.5, and 0.1 g of LmH protein. The reaction mixture was incubated for 10 min at room temperature and the absorbance was determined with a UV/VIS Spectrometer Biochrom 3330 (Biochrom; Cambridge, UK). The release of pnitrophenol was monitored at 405 nm. Furthermore, substrate specificities of LmH were also tested with 1-naphthyl phosphate (1-NP), 1-naphthyl acetate (1-NA), 2-naphthyl acetate (2-NA), and 1-naphthyl butyrate (1-NB). The blue fluorescence of 4methylumbelliferone (4-MU), which is detected under ultraviolet illumination, was used to estimate the hydrolytic activity of LmH. Hydrolysis of 4-MU acetate was observed in a UV-incubation box. The reaction solution, which was incubated for 5 min at 37 ◦ C, consisted of 0.5 mL of 50 mM sodium phosphate buffer (pH 7.5) containing 10 M 4-MU acetate or phosphate, and the purified enzyme. For pH experiments, p-NB was used instead of p-NA to minimize spontaneous hydrolysis at higher pH and the following buffers were used: glycine–HCl (pH 2.5–3.5), citrate (pH 3.0–6.0), phosphate (pH 6.0–8.0), Tris–HCl (pH 7.5–9.0) and glycine–NaOH (pH 9.0–11.0). To determine thermostability of LmH, 0.1 g of enzyme was incubated for 1 h at different temperatures (40–70 ◦ C). The residual activities of LmH withdrawn at different time intervals were measured. Effect of chemical compounds, detergents, and organic solvents on enzyme activity of LmH was determined after 1 h incubation at room temperature. In a standard assay, activity was measured using 10 mM p-NA as a substrate in 20 mM Tris–HCl (pH 8.5). For enantioselectivity analysis experiments, LmH was added to (R)- or (S)-solutions. The (R)- and (S)-solutions contained 300 mM (R)- or (S)-methyl-3-hydroxy-2-methylpropionate and phenol red (2 g/L). The absorbances of (R)- and (S)-solutions were recorded at 350–600 nm [26]. 2.6. Hydrogel formation of LmH LmH (1 mg/mL) was incubated with Cu2+ ions (0, 0.5, 1.0, and 2.0 mM) for 10 min at 60 ◦ C and subsequently cooled at room temperature. For color staining, phenol red (1 mg/mL) was included before heat treatment. The effects of several chemical compounds (10% EtOH, 1 M NaCl, 30% TFE, 5 mM dithiothreitol (DTT), 1% SDS, 2 M urea, and 2 M GdnHCl) on the hydrogel formation of LmH were also investigated. 2.7. Circular dichroism (CD) and fluorescence analysis Far-UV CD spectra were recorded in a J-715 spectropolarimeter (Jasco, Japan) with protein concentration of 0.20 mg/mL in 50 mM sodium phosphate pH 7.5 with a path length of 0.1 cm. The data were converted into mean residue ellipticity per residue (MRE, []) taking into account path length and concentration. Three scans were accumulated with a bandwidth of 2.0 nm from 190 to 250 nm. The spectra were averaged scans and baseline corrected by subtracting a buffer spectrum. Fluorescence spectra were measured using FP-6200 spectrofluorometer (Jasco, Japan) with a protein concentration of
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Fig. 1. Multiple sequence alignments of LmH and related enzymes (1R1D; a carboxylesterase from Bacillus stearothermophilus; 1TQH: Est30 from G. stearothermophilus; 3DKR: esterase D from Lactobacillus rhamnosus).
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0.05 mg/mL. For denaturation experiments, fluorescences were recorded with final GuHCl concentration of 0–5 M using a 1.0 cm quartz cell. LmH protein was excited at 295 nm, and emission spectra were recorded in the range of 300–400 nm. In addition, LmH was incubated with urea (from 0 to 8 M) or guanidine hydrochloride (from 1 to 4 M) for 1 h and incubated samples were assayed for residual activity.
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ammonium sulfate (AMS)) in 20 mM Tris–HCl (pH 8.5) at room temperature. The fluorescence of thioflavin T (ThT, 9 M) was used to investigate the aggregates of LmH. The aggregates of LmH were investigated with fluorescence microscope (Motic; UK) and images were processed using its associated software (ext 475 nm/emi 530 nm). 2.9. Cross-linked enzyme aggregates (CLEAs)
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For aggregate formation, LmH samples (1 mg/mL) were incubated with various chemical compounds (10% (v/v) 1-butyl3-methylimidazolium trifluoromethanesulfonate, 1% (w/v) SDS, 1 M NaCl, 2 M GdnHCl, 30% (v/v) trifluoroethanol (TFE), and 80%
LmH (500 g) was precipitated with ammonium sulfate (80%) in 50 mM phosphate buffer (pH 7.5). Then, 50 mM glutaraldehyde was included, and the reaction mixtures were further incubated for 16 h at 4 ◦ C. The resulting suspension was centrifuged at 12,500 × g at 4 ◦ C for 15 min. The resulting CLEA-LmH was washed
Fig. 2. Biochemical characterization of LmH. (A) SDS-PAGE analysis of LmH. Lane M, molecular marker, lane 1 & 2; E. coli crude extracts before and after induction; lane 3, supernatants; lane 4, pellets; lane 5, LmH after dialysis. (B) Mass spectrum of LmH. Two [M+H]+ and [M+2H]2+ peaks were observed at m/z values of 29.56 and 14.73 kDa. (C) Dynamic light scattering (DLS) results of LmH. (D) Non-denaturing PAGE analysis of LmH. Left: Coomassie staining of non-denaturing gel. Right: non-denaturing gel was soaked and fluorescence was observed under UV light.
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Fig. 3. Enzymatic activities of LmH. (A) Substrate specificity of LmH toward different chain lengths of p-NP esters. (B) Hydrolysis of 4-methylumbelliferyl-phosphate (upper) and -acetate (lower) by LmH was investigated under UV light. (C) Thermostability of LmH. LmH was incubated at 20 ◦ C (), 40 ◦ C (), 50 ◦ C (), and 60 ◦ C (䊉) for 1 h, and the residual activities were measured. (D) pH stability of LmH. 208 209 210 211
until no significant catalytic activity was observed in the supernatant. The surface structure of CLEA-LmH was examined with scanning electron microscope (SEM) (SUPRA 55VP, Carl Zeiss, Germany).
To investigate reusability, CLEA-LmH was recovered by simple centrifugation and washed repeatedly (usually 5 times). Then, fresh substrate was then reintroduced for another cycle and activity was measured. Effects of chemical compounds, detergents, and organic
Fig. 4. Chemical stabilities of LmH. (A) Enzymatic activities of LmH with various concentrations of urea. (B) Chemical stabilities of LmH. The residual activities of LmH after 1 h incubation are expressed relative to the initial activity (100%). (C) Far-UV CD spectrum of LmH. (D) Fluorescence spectra were recorded with increasing concentrations of urea.
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Fig. 5. Regio- and stereo-selectivity of LmH. (A) Regioselectivity of LmH was determined using naphthyl derivatives. (B) pH shift assay for enantioselectivity analysis was conducted with (R)- and (S)-methyl-3-hydroxy-2-methylpropionate. 1: (R) substrate, 2: LmH with (R) substrate, 3: (S) substrate, 4: LmH with (S) substrate. Absorbance spectra for each solution were measured. (C) Hydrogel formation of LmH with Cu2+ ions. LmH was incubated with Cu2+ ions (0, 0.5, 1.0, and 2.0 mM) for 10 min at 60 ◦ C. (D) Scanning electron microscope (SEM) images of Cu2+ -induced hydrogel. Representative images at 50 kX (main) and 100 kX (upper) are shown.
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solvents (ethanol, SDS, Triton X-100, Tween 20, phenylmethylsulfonyl fluoride (PMSF), and urea) on the activities of CLEA-LmH and free LmH were also compared by measuring the residual activity after 1 h of incubation. The initial activity without any chemical compounds was defined as 100%. 2.10. Sequence analysis and molecular modeling To identify related lipolytic hydrolases of bacterial origins, primary sequence of LmH was searched for similarities using PSIBLAST [27]. Sequences homologous to LmH were retrieved in FASTA format, and multiple sequence alignments were generated by the CLUSTALW program and then rendered with ESPript [28]. The conserved domains present in the LmH sequence were analyzed
using the NCBI Conserved Domain Database (CDD) [29]. A molecular model of LmH was constructed by homology modeling using the SWISS-MODEL software using G. stearothermophilus (PDB ID: 1TQH) as template [30]. The 3D structures of LmH were analyzed using PYMOL program (PyMOL Molecular Graphics System). A phylogenetic tree was built with 5000 iterations for bootstrap confidence levels [31].
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3. Results and discussion
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3.1. Bioinformatic in silico analysis of LmH
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In the genome sequence of L. monocytogenes, an open reading frame (ORF; locus tag: NP 465973) of 741 bp was identified through
Fig. 6. Aggregate formation of LmH with 10% (v/v) 1-butyl-3-methylimidazolium trifluoromethanesulfonate (A), 1% (w/v) SDS (B), 30% (v/v) trifluoroethanol (TFE) (C), and 80% ammonium sulfate (AMS) (D). Representative images of light (left) and fluorescence (right) microscopy are shown for comparison.
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Fig. 7. CLEA-LmH. Scanning electron microscope (SEM) images of aggregates and crosslinked aggregates of LmH. (A and B) SEM images of cross-linked enzyme aggregates of LmH (CLEA-LmH), 50 kX and 100 kX, respectively. Glutaraldehyde was used for effective crosslinking. (C) Reusability of CLEA-LmH was investigated for 18 cycles. (D) Comparisons of chemical stabilities of CLEA-LmH and free soluble LmH.
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bioinformatic analysis. The putative hydrolase gene (LmH) encoded a protein of 248 amino acids with 27.8 kDa. A conserved domain search detected an ␣/ hydrolase fold [COG1647], which is found in a large variety of enzyme families including lipases, esterases, peroxidases, and dehalogenases [7,29,32]. Homologs of this enzyme are produced by many strains of Gram-positive bacteria including Bacillus or Staphylococcus. In phylogenic analysis, LmH and its most closely related proteins were grouped in a different branch separate from other bacterial lipolytic families (family I–VIII) (Fig. S1). For example, LmH shared only 3–4% sequence identity with EstII from Pseudomonas fluorescens (AAC60403, family IV) and Est2 from Acetobacter pasteurianus (BAA25795, family V). A multiple sequence alignment was performed for LmH using Est30 from G. stearothermophilus (54% sequence identity, PDB ID: 1TQH), a carboxylesterase from Bacillus stearothermophilus (52% sequence identity, PDB ID: 1R1D), and esterase D from Lactobacillus rhamnosus (31% sequence identity, PDB ID: 3DKR). From the sequence alignment we found that LmH has a conserved hexapeptide GLSLGG in these proteins and the putative catalytic triad (Ser91 -Asp192 -His222 ) (Fig. 1). The crystal structure of Est30 from G. stearothermophilus (PDB ID: 1TQH) was used as a template to construct a structural model of LmH. The model of LmH showed an ␣/-hydrolase fold with a seven-stranded -sheet surrounded by ␣-helices, which had a small cap region covering the active site (Fig. S2). The hydrogen bond distances in the catalytic triad were 3.0 A˚ for Ser91 O␥ to His222 N2 and 2.8 A˚ for His333 N␦1 to Asp303 O␦2. In addition, the oxyanion hole of LmH was mediated by Phe24 and Leu94 . Supplementary figures related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.ijbiomac.2014.07.058.
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3.2. Biochemical characterization of LmH
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To investigate the catalytic properties of LmH, recombinant LmH was purified using Ni-NTA affinity chromatography. The purified LmH was homogeneous as indicated by a single protein band on the SDS-PAGE gel (Fig. 2A). Mass spectrometric analysis was performed to confirm molecular weight of LmH, which showed a major peak of 28,813 Da (Fig. 2B). In dynamic light scattering
(DLS) analysis, the hydrodynamic radius (RH ) of LmH was observed to be 3.25 nm (Fig. 2C). Furthermore, the enzymatic activity of LmH was determined by activity staining of native PAGE using 4methylumbelliferyl (4-MU) acetate (Fig. 2D). High fluorescence due to the formation of 4-methylumbelliferone was clearly seen, which confirmed the hydrolytic activity of LmH. The hydrolyzing activities of LmH toward p-NP esters of different acyl chain lengths were analyzed in Tris–HCl buffer (25 mM, pH 7.5). Substrates included 1 mM p-NP ester compounds of acetate (C2 ), butyrate (C4 ), octanoate (C8 ), decanoate (C10 ), and dodecanoate (C12 ). LmH showed a strong preference to p-nitrophenyl acetate (p-NA) or butyrate (p-NB), although it could not hydrolyze effectively p-nitrophenyl phosphate (p-NP) (Fig. 3A) However, only approximately 3.5% of the relative activity was detected toward pNDec, and almost no activity was observed for p-NDo. Furthermore, hydrolysis of 4-MU acetate by LmH was observed by measuring the fluorescence of 4-methylumbelliferone. However, no activity against 4-MU phosphate was detected (Fig. 3B). The thermostability of LmH was investigated by pre-incubating LmH at different temperatures for 1 h. LmH was completely stable at 20 ◦ C, and retained more than 96% activity at 40 ◦ C after 1 h incubation. In addition, LmH retained ∼45% of its maximum activity at 50 ◦ C, but it was extremely thermolabile at temperatures above 60 ◦ C, with its activity almost completely abolished within less than 15 min at 60 ◦ C (Fig. 3C). This temperature profile was similar to other novel type of hydrolases including Est30 from EstA3 from Thermoanaerobacter tengcogensis [14], CEGk Geobacillus kaustophilus [16], and Geobacillus stearothermophilus [20]. The pH dependencies of LmH activity were investigated by measuring the enzymatic activity after incubation over the pH range of 3.0–10.0 (Fig. 3D). LmH displayed its maximal activity at pH 8.0 and ∼75% of its activity at pH 8.0 was observed at pH 7.0. However, only 20% of the maximal activity was retained at pH 6.0, and no significant activities were observed at a very low or high pH. 3.3. Chemical stabilities of LmH The effect of urea on the activity of LmH was examined by measuring catalytic activity after 1 h incubation (Fig. 4A). LmH was
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largely resistant to 3.0 M urea and ∼70% of activity was retained at this concentration. However, at 4.0 M urea, the enzymatic activity was decreased to ∼30% of the initial value. At 5.0 M urea, almost no activity of LmH was observed. In addition, chemical stability of LmH was measured in reaction mixtures containing different concentrations of detergents, organic solvents, or chemicals. As shown in Fig. 4B, LmH showed ∼70% of its initial activity with 10% (v/v) ethanol, but it retained only approximately 10% activity with 30% (v/v) ethanol. The residual activity of LmH was ∼40% of its initial activity with 1.0% (v/v) Tween 20, whereas it showed only ∼15% of its original activity with 1.0% (v/v) Triton X-100. However, LmH lost most of its activity with 0.1% (w/v) SDS. LmH was almost inactive with the retention of ∼8% activity in 30% isopropanol (i-PrOH). Similar inhibition was also shown in 10 mM PMSF. To analyze the secondary structure of LmH, CD analysis was done at room temperature (Fig. 4C). The far-UV CD spectra of LmH showed strong negative bands at 210–220 nm (-sheet contents), and a positive band below 200 nm (␣-helical contents) [33]. The spectrum showed a minimum at 225 nm (−7000 deg cm2 dmol−1 ), and a maximum at 193 nm (20,500 deg cm2 dmol−1 ). The chemical stability of LmH was also investigated by monitoring the intrinsic fluorescence with urea addition [34]. In native state of LmH, the four tryptophan residues are well-buried with a maximum wavelength (max ) at 338 nm (Fig. 4D). However, there were noticeable decreases in the intensity with a red shift of max . A shift of max from 338 to 347 nm was observed with 4.0 M urea, implying that at this condition all tryptophan residues became largely exposed to the solvents.
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Using naphthyl derivatives as substrates, LmH exhibited activities for 1-NA, 1-NB, and 2-NA, respectively (Fig. 5A). The highest activities were observed when using 2-NA, followed by 1-NB and 1-NA. However, no activity against 1-NP was detected. For enantioselectivity analysis, a pH shift assay was employed with (R)- and (S)-methyl--hydroxyisobutyrate. Lipolytic activity was detected based on the color change in the phenol red indicator due to acid release [35]. After incubation with LmH, the color of mixture turned dark yellow in both (R)- and (S)-enantiomer containing solutions, which was also confirmed by spectral readings (Fig. 5B). An interesting feature of LmH is its ability to form hydrogels with Cu2+ ions (Fig. 5C). Although a large variety of compounds (10% EtOH, 1 M NaCl, 30% TFE, 5 mM DTT, 1% SDS, 2 M Urea, 2 M GdnHCl, and 1 mM Cu2+ ) were investigated, only Cu2+ ions resulted in the formation of a three-dimensional network of hydrogels. In SEM images, the surface morphology of hydrogels showed threedimensional networks of globular beads (Fig. 5D). 3.5. Aggregates formation and CLEA of LmH We investigated the propensities of aggregate formation of LmH using several chemical compounds. To investigate aggregates formation, LmH was incubated with several chemical compounds (10% 1-butyl-3-methylimidazolium trifluoromethanesulfonate, 1% SDS, 30% TFE, and 80% AMS), and images of light and fluorescence microscope were recorded (Fig. 6A–D). Thioflavin T, which specifically binds to intermolecular -sheets, was used for the characterization of aggregates [13,24,36]. With 10% 1-butyl-3-methylimidazolium trifluoromethanesulfonate, 1% SDS, or 30% TFE, small aggregates with low ThT fluorescence were observed (Fig. 6A-C). Similar behaviors were also observed for 1 M NaCl and 2 M GdnHCl (data not shown). However, in the presence of 80% AMS, aggregates with high ThT fluorescence intensities were observed (Fig. 6D). To investigate the efficiency of LmH immobilization, CLEAs of LmH were (CLEA-LmH) prepared by precipitating the LmH with
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80% AMS followed by chemical crosslinking with glutaraldehyde [37,38]. The scanning electron microscopic images of CLEA-LmH showed large fibrous structures of high surface density (Fig. 7A and B). The immobilized LmH showed negligible leaching from the crosslinked enzyme aggregates. In the recycling process, CLEALmH was reused for 18 successive cycles, retaining ∼80% of the initial activity (Fig. 7C). Furthermore, the effects of several chemical compounds on the activities of CLEA-LmH and free LmH were compared. As shown in Fig. 7D, CLEA-LmH showed more resistance against these chemical compounds (EtOH, SDS, Triton X-100, Tween 20, PMSF, and urea) compared to free LmH. Specifically, in the presence of 1.0% (v/v) Tween 20, the free LmH retained only ∼25% of its initial activity, as compared to ∼70% for CLEA-LmH. Moreover, although 10 mM PMSF almost completely inactivated free LmH, the residual activity of CLEA-LmH was as high as ∼85% of its initial one. Therefore, crosslinking of LmH (CLEA-LmH) could enable LmH to be used efficiently in industrial conditions. 4. Conclusions This study reports the molecular characteristics of LmH in pathogenic L. monocytogenes. LmH is a novel type of hydrolase produced by several Gram-positive bacterial species. Although several hydrolases are known to be involved in the pathogenesis by this enteric bacteria, molecular characterization of these proteins are largely unknown. Here biochemical characterization and enzymatic properties of LmH were extensively characterized. To the best of our knowledge, this is the first report of the functional and biochemical characterization of a novel type of hydrolase from L. monocytogenes. Acknowledgments This work was supported by National Research Foundation of Korea Grants funded by the Korean Government Q2 (NRF-2012S1A2A1A01028907, NRF-2013K2A1A2053659, NRF2012R1A1A2000910) to T.D.K. References [1] L. Casas-Godoy, S. Duquesne, F. Bordes, G. Sandoval, A. Marty, Methods Mol. Biol. 861 (2012) 3. [2] F. Hasan, A.A. Shah, A. Hameed, Biotechnol. Adv. 27 (2009) 782. [3] T. Tan, J. Lu, K. Nie, L. Deng, F. Wang, Biotechnol. Adv. 28 (2010) 628. [4] A. Robles-Medina, P.A. González-Moreno, L. Esteban-Cerdán, E. Molina-Grima, Biotechnol. Adv. 27 (2009) 398. [5] T. Kobayashi, Biotechnol. Lett. 33 (2011) 1911. [6] T. Hudlicky, J.W. Reed, Chem. Soc. Rev. 38 (2009) 3117. [7] U.T. Bornscheuer, FEMS Microbiol. Rev. 26 (2002) 73. [8] F. Niehaus, C. Bertoldo, M. Kahler, G. Antranikian, Appl. Microbiol. Biotechnol. 51 (1999) 711. [9] J.L. Arpigny, K.E. Jaeger, Biochem. J. 343 (1999) 177. [10] P.D. Carr, D.L. Ollis, Protein Pept. Lett. 16 (2009) 1137. [11] Z. Qian, C.J. Fields, Y. Yu, S. Lutz, Biotechnol. J. 2 (2007) 192. [12] L. Rao, Y. Xue, Y. Zheng, J.R. Lu, Y. Ma, PLOS ONE 8 (2013) e60645. [13] H. Ju, E. Jang, B.H. Ryu, T.D. Kim, Bioresour. Technol. 128 (2013) 81. [14] L. Rao, Y. Xue, C. Zhou, J. Tao, G. Li, J.R. Lu, Y. Ma, Biochim. Biophys. Acta 1814 (2011) 1695. [15] F. Ay, H. Karaoglu, K. Inan, S. Canakci, A.O. Belduz, Protein Expr. Purif. 80 (2011) 74. [16] S. Montoro-García, I. Martínez-Martínez, J. Navarro-Fernández, H. Takami, F. García-Carmona, A. Sánchez-Ferrer, J. Bacteriol. 191 (2009) 3076. [17] M. Levisson, J. van der Oost, S.W. Kengen, FEBS J. 274 (2007) 2832. [18] M.H. Lee, C.H. Lee, T.K. Oh, J.K. Song, J.H. Yoon, Appl. Environ. Microbiol. 72 (2006) 7406. [19] E.Y. Kim, K.H. Oh, M.H. Lee, C.H. Kang, T.K. Oh, J.H. Yoon, Appl. Environ. Microbiol. 75 (2009) 257. [20] H.E. Ewis, A.T. Abdelal, C.D. Lu, Gene 329 (2004) 187. [21] S.C. Corr, L.A. O’Neill, Cell. Microbiol. 11 (2009) 703. [22] M.J. Gray, N.E. Freitag, K.J. Boor, Infect. Immun. 74 (2006) 2505. [23] M. Hamon, H. Bierne, P. Cossart, Nat. Rev. Microbiol. 4 (2006) 423. [24] E. Jang, B.H. Ryu, H. Ju, T.D. Kim, Bioresour. Technol. 143 (2013) 691. [25] S.Y. Bae, B.H. Ryu, E. Jang, S. Kim, T.D. Kim, Appl. Microbiol. Biotechnol. 97 (2013) 1637.
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