Introduction
The Ser-Gly-Asn-His (SGNH) hydrolase family was first reported by Upton and Buckley [27] through the identification of conserved domain characteristics 20 years ago. The hydrolases of this superfamily did not contain the classical pentapeptide motif GXSXG, which is different from most esterases and lipases, illustrating the SGNH hydrolase family was a new subfamily of hydrolytic/lipolytic enzymes [20]. All of the SGNH hydrolases have a consensus sequence that is divided into four conserved sequence blocks (I–V). Block I has the characteristic of a GDS sequence motif; block II has a Gly as the only conserved residue in the members of this family; GXND is the consensus sequence in block III; and block V has the DXXHP conserved sequence. Furthermore, SGNH hydrolases employ a catalytic triad consisting of Ser in block I, Asp, and His in block V which is different from that of common α/β hydrolases [16,17].
The SGNH superfamily hydrolases are widespread in eukaryotic and prokaryotic organisms, display broad substrate specificities, and have a diverse range of hydrolytic functions such as thioesterase, protease, lysophospholipase [17], lipase [4], arylesterase [12], carbohydrate esterase [8,18,23], and acyltransferase [14] activities. An assumption is that the broad substrate specificities are because of the flexibility of the active site of the enzymes, according to the experimental data of protease I/thioesterase I/lysophospholipase L1 from Escherichia coli (TAP) [17], but there are no information about the substrate specificities of other SGNH hydrolases [1]. For function, several SGNH family lipases/esterases from plants have been identified [28], which play important roles in plant development and morphogenesis, plant tolerance to environmental stresses, defense, and the metabolism of cutin and wax [21,22,30]. However, limited reports on the characterization of the SGNH superfamily hydrolases from bacteria are available. Esterases from the SGNH superfamily have been reported to have deacetylase activity. Deacetyl cephalosporins are a highly valuable starting material for producing semisynthetic β-lactam antibiotics. These compounds can be derived from 7-aminocephalosporanic acid (7-ACA) deacetylation at position C-3 [6]. Until now, only two SGNH hydrolases (BH1115 and cephalosporin C deacetylase) from Bacillus C-125 and Bacillus sp. KCCM10143 have been reported to be involved in the deacetylation of 7-ACA [6,19]. Besides this, an O-acetylpeptidoglycan esterase from Neisseria gonorrhoeae belongs to the SGNH family and is reported to have the function to hydrolyze O-acetylated peptidoglycan and release O-acetyl groups from the C-6 position, thereby permitting the continued metabolism of this essential cell wall heteropolymer [23]. Moreover, the acetylxylan esterase Axe2 from Geobacillus stearothermophilus also belongs to the SGNH family and can hydrolyze the ester linkages of acetyl groups at positions 2 and/or 3 of the xylose moieties in xylan, playing an important role in accelerating the further degradation of the xylan backbone [2]. No other esterases that belong to the SGNH family have been characterized and functions explored. This prompted us to explore the characterizations and functions of SGNH hydrolases from bacteria.
In this study, a new SGNH family hydrolase (Est19) from Bacillus sp. K91 was cloned, purified, and characterized. Catalytic properties of Est19 were characterized, including substrate specificity, optimal pH, optimal temperature, pH stability, thermostability, and effects of metal ions, chemical reagents, and mutations of the catalytic triads on esterase activity. A 3D structural model was also built and compared with other reported structures.
Materials and Methods
Chemicals and Materials
Bacillus sp. K91 was isolated from a hot spring in Tengchong, Yunnan, China, by our team. Genomic DNA and plasmid isolation kits were purchased from TianGen (Beijing, China). DNA polymerase, dNTPs, ampicillin, and isopropyl thio-β-ᴅ-galactopyranoside (IPTG) were purchased from TaKaRa (Dalian, China). The expression vector pEASY-E2 kit, monoclonal His-tag antibody (IgG2b), peroxidase-conjugated goat anti-mouse IgG (H+L), and Fast Mutagenesis System were purchased from TransGen Biotech (Beijing, China). Nickel-NTA agarose was purchased from Qiagen (Valencia, CA, USA). p-Nitrophenyl acetate (pNPC2), p-nitrophenyl butyrate (pNPC4), p-nitrophenyl caproate (pNPC6), p-nitrophenyl caprylate (pNPC8), p-nitrophenyl caprate (pNPC10), p-nitrophenyl laurate (pNPC12), p-nitrophenyl myristate (pNPC14), p-nitrophenyl palmitate (pNPC16), and α-, β-naphthyl acetate were from Sigma-Aldrich (USA) or TCI (Tokyo, Japan). All other reagents and solvents used in this study were of analytical grade.
Sequence Analysis of Est19
Genomic DNA of Bacillus sp. K91 was extracted using a TianGen genomic DNA isolation kit from cells grown overnight at 55℃. Genomic sequencing was performed by Beijing Genomics Institute (Guangzhou, China) using a Solexa Genome Analyzer, and a partial genomic sequence was obtained. Oligonucleotide primers were synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). The full-length esterase gene est19 was revealed based on the prediction of ORFs from the partial genomic sequence by the GeneMark.hmm online tool (http://exon.gatech.edu/GeneMark/gmhmmp.cgi). Putative functions were inferred using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST). Protein similarity searchers and alignment were performed using the data from Clustal W [26]. ESPript output was used to render the analysis of multiple sequence alignments [11]. The theoretical molecular mass of the deduced EstD1 protein sequence was calculated using the Compute pI/Mw tool on the ExPASy proteomics server (available at http://expasy.org/tools/pi_tool. html)
Cloning, Expression, and Purification of Est19
The gene est19 was amplified via PCR using the genomic DNA of Bacillus sp. K91 as template with primers est19-Forward (5’-AAGCTGCGCATTTTTTCAATC-3’) and est19-Reverse (5’-CTCTTTCGGTAATCCTTCT-3’). The PCR products were ligated into the pEASY-E2 expression vector according to the manufacturer’s instructions at 25℃ for 10 min. The recombinant plasmid was transformed into E. coli BL21 (DE3) for esterase gene est19 heterologous expression. The recombinant E. coli strains were grown on LB medium containing 100 μg/ml of ampicillin at 37℃ until the OD600 reached 0.6. Then IPTG was added to the culture to a final concentration of 0.7 mM and shaken at 20℃ for 20 h to induce the Est19 protein expression. The cells were harvested by centrifugation at 8,000 rpm for 10 min, and resuspended in 50 mM citrate-Na2HPO4 buffer (pH 7.0). The cells were disrupted by ultrasonication for 5 min in cycles of 3 sec on/5 sec off, on ice, and then the mixture was centrifuged at 12,000 rpm for 10 min at 4℃. The supernatant, which was referred to as the crude extract, was purified using a column of Ni-NTA agarose. The purified protein was examined by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. Protein concentration was estimated using the Bradford procedure employing BSA as the standard [5]. Detection of the His-tagged Est19 was carried out by western blot analysis (transfer buffer: 192 mM glycine, 25 mM Tris base, and 20% methanol, pH 8.0). Monoclonal His-tag antibody (IgG2b), peroxidase-conjugated goat anti-mouse IgG (H+L), and a Super Signal West Pico kit from Thermo Scientific Pierce (Grand Island, NY, USA) were used, and procedures were conducted according to the methods reported by Yang et al. [29].
Enzyme Assays
Est19 activity was measured using p-NP esters as described previously with some changes [15]. Briefly, the standard assay consisted of activity measurements with 0.6 mM p-NPC2 as substrate in 50 mM Tris-HCl buffer (pH 9.0) at 60℃. Absorbance was measured at 405 nm for the appearance of released p-NP. One unit of activity (U) was defined as the amount of enzyme required to release 1 μM p-NP from the substrate per minute. Kinetic parameters were determined by direct fitting of the data, obtained from multiple measurements, to the Michaelis–Menten curve. p-NPC2 substrate with a concentration of 0.1 to 1.2 mM was used to determine the Km and Vmax. The deacetylase activity of Est19 toward 7-ACA was determined using an acetic acid detection kit (K-ACET) from Megazyme (Dublin, Ireland).
Different lengths of p-NP esters, namely pNPC2, pNPC4, pNPC6, pNPC8, pNPC10, pNPC12, pNPC14, and pNPC16, were used to determine the substrate specificity.
The effect of pH on esterase activity was studied in the pH range from 5.0 to 12.0. Reactions were performed in 50 mM citrate-phosphate (pH 5.0–8.0), 50 mM Tris-HCl (pH 8.0–9.0), and 50 mM sodium carbonate-sodium bicarbonate (pH 9.0–12.0) buffers. The same buffers were used to test the pH stability of Est19. The effect of temperature on Est19 activity was determined using 0.6 mM pNPC2 as substrate in the temperature range of 20–90℃. The enzyme was incubated at 37℃, 50℃, and 65℃ for various time intervals to study the enzyme thermostability. Residual activity was determined in the standard assay.
Different potential inhibitors or activators (metal ions and chemical agents) were added individually to the reaction assay in 50 mM Tris-HCl (pH 9.0) at 60℃ for 5 min to study their effects to the activity of Est19. AlCl3, ZnSO4, FeCl3, CoSO4, CuSO4, MnSO4, NiSO4, KCl, NaCl, MgSO4, HgCl2, CaCl2, EDTA, SDS or Tween 80, TritonX-100, and β-mercaptoethanol were determined with a final concentration of 1 mM or 1% (v/v), and the residual activity was measured using the standard assay. The activity of the enzyme without inhibitor or activator was defined as 100%.
Structural Modeling
Model building was developed by the SWISS-MODEL and Swiss-Pdb Viewer programs (http://www.expasy.ch/spdbv) [3,25] using phospholipase A1 from S. albidoflavus NA297 (PDB code 4HYQ) (belonging to the SGNH hydrolase family) as template, which has a sequence identity of 30% to Est19.
Site-Directed Mutagenesis
Site-directed mutagenesis was measured by a fast mutagenesis system kit using the wild-type recombinant plasmid pEASY-E2/est19 as template, with the following primers (modified codons underlined): 5’-GCGGCTGTCGGAGATGCGCTGACAGAAGG-3’ and 5’-CGCATCTCCGACAGCCGCTATGACAAT-3’ for the S49A mutant, 5’-GAATTTCTGAGGAAGCTGATTTTCAT-3’ and 5’-AGCTTCCTCAGAAATTCGGCTGCTGT-3’ for the D227A mutant, and 5’-GAGGAAGATGATTTTGCTCCTAATGGT-3’ and 5’-AGCAAAATCATCTTCCTCAGAAATTCG-3’ for the H230A mutant. Manipulations were performed according to the manufacturer’s instructions. All mutation sites were confirmed by DNA sequencing. For expression, the mutant plasmids were transformed into E. coli BL21 (DE3) cells, respectively. The enzymatic activity of the mutant esterases was detected as described above.
Nucleotide Sequence Accession Numbers
The nucleotide sequences of the 16S rRNA and est19 genes were deposited in GenBank under the accession numbers KJ131181 and KR534852, respectively.
Results and Discussion
Sequence Analysis of Enzyme Est19
The thermophilic bacterium Bacillus sp. K91 growing in the range of 30-75℃ with 55℃ as the optimum growth temperature was isolated from a hot spring in Tengchong, Yunnan, China. Bacillus sp. K91 was a B. subtilis strain as reported before [10]. In the genome sequence data of Bacillus sp. K91, a gene annotated as “SGNH_hydrolase_YpmR_like” with a 768-long ORF that encoded a 255-amino-acid protein was found. We named this protein as Est19. Est19 is an intracellular enzyme and has no predicted signal sequence, using SignalP 4.0 server for sequence analysis. Amino acid comparison of Est19 with those deposited in the GenBank database showed that it had 30%, 99%, 93%, 76%, 38%, and 38% identity to the GDSL-like lipase from Streptomyces albidoflavus NA297 (PDB: 4HYQ), GDSL-like lipase from Streptococcus pneumoniae (GenBank: CON19522.1), hypothetical protein from B. vallismortis (WP_010327977.1), lipase/acylhydrolase from B. atrophaeus (WP_010788983.1), hypothetical protein from B. alveayuensis (WP_044896322.1), and hypothetical protein from Halobacillus halophilus (WP_014645042.1), respectively. As shown in Fig. 1, a putative Ser-containing active GDSL-like motif (located in block I) close to the N-terminus, and the presence of four highly conserved blocks (blocks I–V) in the amino acid sequence of Est19 were shared with SGNH proteins as previously reported [27]. Furthermore, the catalytic triad of Est19 (consisted of the Ser49, Asp227 and His230 residues) was assigned by comparison with other SGNH hydrolases, which appeared in corresponding positions to those in other SGNH hydrolases. However, alignments of Est19 with those deposited in GenBank (BLASTp) showed that most of the proteins are annotated as “hypothetical protein.” Moreover, none of these proteins have been characterized before, suggesting that Est19 was a new SGNH hydrolase and may have a new function that has never been reported before.
Fig. 1.Amino acid sequence alignment analysis of Est19. Sequences retrieved from the NCBI server were aligned in Clustal W and rendered using ESPript output. Sequences are grouped according to similarity. 4HYQ from S. albidoflavus NA297; Est19 from Bacillus sp. K91; CON19522.1 from S. pneumoniae; WP_010327977.1 from B. vallismortis; WP_010788983.1 from B. atrophaeus; WP_044896322.1 from B. alveayuensis; and WP_014645042.1 from H. halophilus. Conserved motifs are highlighted. Residues strictly conserved among groups are shown in white font on a red background. Four conserved sequence blocks (blocks I–V, SGNH) found in all GDSL lipases are bracketed. Block I has the characteristic GDS sequence motif; block II has Gly as the only conserved residue in the members of this family; GXND is the consensus sequence in block III. Finally, block V has the DXXHP conserved sequence. Green triangles at the top of the alignment represent the locations of the SGNH. The possible catalytic triads (serine (S), aspartic acid (D), histidine (H)) are shown at the bottom of the alignment with blue asterisks. Symbols above blocks of sequences represent the secondary structure, springs represent helices, and arrows represent β-strands.
Cloning, Overexpression, and Purification of Est19
The est19 gene was cloned from Bacillus sp. K91 genomic DNA and expressed in the pEASY-E2 vector. Expression of the protein was induced with 0.7 mM IPTG at 20℃ for about 20 h. The crude enzyme extracted from recombinant E. coli BL21(DE3) cells was purified by Ni2+-NTA metal-chelating affinity chromatography. Cell-free extracts of induced E. coli BL21(DE3) with blank vector pEASY-E2 (lane 1), cell-free extracts of induced E. coli BL21(DE3) with recombinant pEASY-E2-est19 (lane 2), and purified Est19 (lane 3) were separated by SDS-PAGE, respectively. However, the theoretical 29.73 kDa target protein was not present clearly on the SDS-PAGE gel (lanes 2 and 3) (Fig. 2A). In order to check whether the target protein has been heterologously expressed, we detected the corresponding protein by western blot analysis with anti-His antibody. As shown in Fig. 2B, one corresponding band was detected in lane 2 and lane 3, respectively. The assay indicated that the target protein had been expressed, and could be purified by Ni2+-NTA metal-chelating affinity chromatography.
Fig. 2.Purification and western blot detection of Est19. (A) SDS-PAGE. M, marker proteins (kDa); 1, cell-free extracts of induced E. coli BL21 (DE3) with blank vector pEASY-E2; 2, cell-free extracts of induced E. coli BL21 (DE3) harboring plasmid pEASY-E2-est19; 3, purified Est19. (B) Western blotting was used to detect Est19 protein in lanes 1, 2, and 3 in subpanel (A), using anti-His antibody.
Substrate Specificity
The substrate specificity of Est19 was determined using pNPC2–pNPC16 and α-, β-naphthyl acetate as substrates at pH 9.0 or pH 7.3, 60℃ (Table 1). α-Naphthyl acetate or β-naphthyl acetate is unstable when the pH of reaction buffer is higher than 7.5. The reaction buffer becomes darker and the value can not be determined in such conditions. Therefore, pH 7.3 was used to determine the activity of Est19 when α-, β-naphthyl acetate were used as substrates. To pNP esters, Est19 only showed the hydrolytic activity toward short-length acyl chains. The catalytic efficiency (kcat/Km) toward pNPC2 was approximately 18-fold and 1,130-fold higher than toward pNPC4 and pNPC6, respectively. However, Est19 displayed no hydrolytic activity for the substrates with a chain length >C6, indicating that Est19 is an esterase, not a lipase. To α-, β-naphthyl acetate, the catalytic efficiency (kcat/Km) toward β-naphthyl acetate was approximately 2-fold higher than toward α-naphthyl acetate, documenting that the configuration of the substrate has influence on the catalytic activity of Est19.
Table 1.aReactions were detected in triplicates at 60℃, pH 9.0 in 50 mM Tris-HCl buffer, by the standard assay. bThe experiment was conducted at pH 7.3 in Na2HPO3/citrate buffer, owing to the substrate’s chemical instability.
Biochemical Characterization of Est19
The effect of pH on Est19 activity was investigated using pNPC2 as a substrate, in 50 mM buffer ranging from pH 5.0 to pH 12.0, at 37℃. As shown in Fig. 3A, Est19 was sensitive to pH, showing maximum activity at pH 9.0. At pH below 6.0 or above 10.0, Est19 lost most of activity (Fig. 3A). Est19 retained more than 80% of its maximal activity at alkaline pH 8.5 to 9.0, but at pH above 10.0 or below 6.0, there was substantial loss of activity after pre-incubation at that pH range for 1 h at 37℃ (Fig. 3B). The enzyme showed maximum activity at a pH of approximately 9.0, and lost most of its activity when the pH was below 6.0, which is similar to most SGNH hydrolases reported. For example, BH115 from B. halodurans C-125 showed maximum activity at pH 8.0 [19], FNE from Fervidobacterium nodosum Rt17-B1 at pH 8.5 [29], and Sm23 from Sinorhizobium meliloti 1021 at pH 8.0 [12], and their activity was almost zero when the pH was below 6.0.
Fig. 3.Biochemical characterization of Est19. (A) Effect of pH on Est19 activity. Reactions were performed in citrate-phosphate (pH 5.0 to 8.0), Tris-HCl (pH 8.0 to 9.0), and boric acid (pH 9.0 to 12.0) buffers with identical ionic strength (50 mM) at 37℃, respectively. (B) pH dependence of Est19 stability. The enzyme was incubated for 1 h in buffers of various pH values (pH 2.0–12.0) at 37℃. (C) Effect of temperature on Est19 activity was determined using pNPC2 as substrate in the temperature range of 20℃ to 90℃ in Tris-HCl buffer (50 mM, pH 9.0). (D) Thermostability of Est19. The enzyme was incubated in Tris-HCl buffer (50 mM, pH 9.0) at 37℃, 50℃, and 65℃ for 1 h, respectively.
The effect of temperature on Est19 activity was determined using pNPC2 as a substrate in 50 mM Tris-HCl buffer at pH 9.0, with temperature that ranged 20–90℃. Est19 showed the highest hydrolytic activity at around 60℃ (Fig. 3C), and no activity was observed when the temperature was lower than 20℃ or higher than 90℃. In order to assess the thermostability of Est19, the enzyme was separately incubated at 37℃, 50℃, and 60℃ for 1 h, respectively, and the residual activities were determined. As shown in Fig. 3D, Est19 was stable and no enzyme activity loss was detected when incubated at 37℃ for 1 h. However, Est19 was sensitive when the temperature was above 50℃, with a half-life of less than 30 min and 10 min at 50℃ and 60℃, respectively. The optimal temperature for Est19 (60℃) was higher than that of the SGNH hydrolase from B. halodurans C-125 [19], Aspergillus aculeatus [13], and Anaerovibrio lipolyticus 5ST, which have an optimal temperature of 40℃ [24], and lower than that of the SGNH hydrolase from F. nodosum Rt17-B1, which has an optimum temperature of 75℃ [31].
The effect of different metal ions and chemical reagents on Est19 activity was examined using pNPC2 as a substrate in 50 mM Tris-HCl buffer (pH 9.0), at 60℃. As shown in Table 2, 1 mM Ni+, K+, and EDTA had no significant effect on the enzyme activity; the presence of 1 mM Al3+, Zn2+, Fe3+, Co2+, Cu2+, and Mn2+ activated the activity to 16–66% more than the control; and the addition of 1 mM Na+, Mg2+, Hg2+, Ca2+, SDS, and 1% (v/v) Tween-80, Triton X-100, and β-mercaptoethanol inhibited the activity, with a decrease of 15-40%.
Table 2.Effects of metal ions and chemical reagents on Est19 activity.
Structural Modeling and Site-Directed Mutagenesis
SWISS-MODEL was used to generate a 3D structural model to obtain structural insights into Est19 [3]. The phospholipase A1 from S. albidoflavus NA297 (PDB code 4HYQ), belonging to the SGNH hydrolase family with 30% identity to Est19, was used to build the Est19 model (Fig. 4). The predicted 3D structural of Est19 adopted an α/β-hydrolase fold, and the putative catalytic triad Ser49, Asp227, and His230 were located in a pocket of the enzyme to serve as the active center, which was the same as the sequence alignment result in Fig. 1. In the modeled structure of Est19, the position of the nucleophilic residue Ser49 was similar to those of solved SGNH hydrolases, which do not contain a nucleophile elbow [18].
Fig. 4.Predicted 3D structure of Est19. (A) Overview of the whole modeled structure. (B) Representations of the catalytic triad. The proposed active-site residues Ser49, Asp227, and His230 are shown in stick representations. These figures were rendered using SWISS-MODEL and Swiss-Pdb viewer.
To confirm that the predicted catalytic triad was the actual activity site, site-directed mutagenesis was used to substitute these three amino acid residues of the catalytic triad (Ser49, Asp227, and His230) by alanine. The engineered proteins was overexpressed in E. coli BL21 (DE3) cells, and purified by Ni2+-NTA metal-chelating affinity chromatography. Western blot assay was also used to detect the purification of the three mutants (Fig. 5). The enzyme activities of mutants Ser49Ala, Asp227Ala, and His230Ala were assayed using the method as previously described; none of the mutants had any detected activity. These results suggest that Ser49, Asp227, and His230 were the critical catalytic residues of Est19. This is different from the Est8 we reported before of which the catalytic residues Asp and His were more critical than Ser. Est8 had detectable activity after mutation of Ser, Asp, and His [10]. Moreover, if the 7-ACA acetate can be hydrolyzed at the C-3 position to form deacetyl-7-ACA by Est19, then the released acetic acid can be detected by the K-ACET kit. Unfortunately, the deacetylase activity of Est19 toward 7-ACA was quite low and could not be detected in our test (data not shown). Est19 may be an attractive candidate for the production of new semisynthetic antibiotics owing to its high temperature, pH, and organic solvent stability, although the activity of Est19 toward 7-ACA is not high. Currently, an abundance of available methods have been developed in order to improve enzyme activity and function, such as directed evolution [9,32], which only add to its potential utility. For example, an AXE from Penicillium purpurogenum can catalyze several fatty acid esters of up to 14 carbons in length by the method of site-directed mutagenesis, compared with its wild-type preference for acetate [7]. Thus, Est19 can also be engineered to improve its activity toward 7-ACA during the future further study. With the improvement of activity of Est19 toward 7-ACA, all of these characteristics would endow Est19 with great potential for industrial application. In this work, the study of Est19 can enrich the knowledge about the SGNH superfamily enzymes.
Fig. 5.Western blot detection of Est19 derivative products after 20 h of induction with 0.7 mM IPTG at 20℃ using anti- His antibody. Lane 1, cell-free extracts of induced E. coli BL21(DE3) with blank vector pEASY-E2; Lane 2, purified Ser49Ala mutant of Est19 after Ni2+ affinity chromatography; Lane 3, cell-free extracts of induced E. coli BL21(DE3) with blank vector pEASY-E2; Lane 4, purified Asp227Ala mutant of Est19 after Ni2+ affinity chromatography; Lane 5, cell-free extracts of induced E. coli BL21(DE3) with blank vector pEASY-E2; Lane 6, purified His230Ala mutant of Est19 after Ni2+ affinity chromatography.
In conclusion, in this work, we cloned and characterized a new esterase, Est19, that belongs to the SGNH hydrolase subfamily, which has never been characterized before. The enzyme was active toward short-length pNP esters, with high stability under alkaline pH, a temperature of 60℃, denaturant agents, and detergents, which are important characteristics required for applications in detergent formulation and biotransformation. The biochemical functions (structure-function relationships) of many SGNH superfamily enzymes remain unknown and require further investigation.
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