A novel β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation activity and its use in generating long-chain N-acetyl chitooligosaccharides


 Background: N-acetyl glucosamine (GlcNAc) and N-acetyl chitooligosaccharides (N-acetyl COSs) exhibit antitumor and antimicrobial activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Thus, it is crucial to discover a NAGase that can both synthesize GlcNAc and N-acetyl COSs. Results: The gene encoding the novel β-N-acetyl glucosaminidase, designated CmNAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of CmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity with the corresponding domain of the well-characterized NAGases. CmNAGase gene was highly expressed with soluble form in Escherichia coli BL21 (DE3) cells, whereupon it had a specific activity of 4,878.6 U/mg of protein toward p-nitrophenyl-N-acetyl glucosaminide. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40ºC. The Vmax, Km, and Kcat of CmNAGase were 833.33 μmol·L-1 ·min-1, 10.9 mmol, and 6.37 ´ 108 mM·mg-1, respectively. Analysis of the hydrolysis products of N-acetyl chitooligosaccharides and colloidal chitin revealed that CmNAGase exhibits exo-acting activities. Particularly, it possesses transglycosylation activity, which can synthesize (GlcNAc)n+1 from (GlcNAc)n (n=1−6), respectively. In addition, CmNAGase also can catalyze GlcNAc to its dimers with various linked forms. Conclusions: The observations recorded in this study that CmNAGase is an exo NAGase with unique transglycosylation activity, suggests a possible application in the production of long-chain N-acetyl CHOs. This is first report of a bacterial NAGase, which can produce long-chain N-acetyl COSs via transglycosylation activity.

In our previous study, Chitinolyticbacter meiyuanensis SYBC-H1 with a good ability to degrade chitin, was isolated from soil [19]. Herein, a gene encoding NAGase (with transglycosylation activity) was cloned from the SYBC-H1 strain, based on the results of peptide mass fingerprinting and complete genome sequencing, and heterologously expressed in Escherichia coli BL21(DE3). The phylogenetic relationships, catalytic characteristics, and hydrolysis mode of purified recombinant NAGase are described. Furthermore, its transglycosylation activity toward GlcNAc and N-acetyl COSs was also investigated.

Results And Discussion
Purification of wild NAGase from C. meiyuanensis SYBC-H1 Purification of the NAGase from C. meiyuanensis SYBC-H1 was performed as described above.
Following ammonium sulfate precipitation, anion exchange chromatography, and SDS-PAGE, a protein band with NAGase activity was obtained, as shown by zymogram analysis. The band with NAGase activity (named CmNAGase) had a molecular mass of between 75 kDa and 100 kDa (Fig. 1).
The protein strip in the stained gel was excised for peptide mass fingerprinting (PMF) analysis using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF MS/MS), and the results of PMF were interpreted by referencing the Mascot database [20]. Proteins receiving the highest molecular weight search scores (MOWSE) were selected as the peptide fragments of purified protein. Peptide fragments of purified protein were mainly detected with the amino acid sequences of YDGDTFLARLTLTNH, AMNVRYERLVKAGK, and WNQFANRLGQRELARLDGFLGGYGYRVPV, which showed 100% identity to the peptides from an annotated NAGase in the complete genome of C. meiyuanensis SYBC-H1.
Cloning of the CmNAGase gene and sequence analysis. The CmNAGase gene was cloned as described above. As expected, a PCR product of 2.5 kb was obtained. Analysis of the PCR product showed that the CmNAGase gene was 2,508 bp, encoding for a protein of 836 amino acids. The calculated molecular mass of CmNAGase was 91.6 kDa, and the isoelectric point (pI) was predicted to be 5.48.
The sequence analysis suggested no putative signal peptide in the sequence of CmNAGase, which suggested that CmNAGase should be a non-secretory protein. The prediction using Gneg-mPLoc 2.0 also showed that the location of CmNAGase was in the periplasm. However, CmNAGase could be purified from the fermentation broth of SYBC-H1. This phenomenon shows that SYBC-H1 cells rupture during a long period of fermentation, which leads to the release of CmNAGase. To date, most reported NAGases are secretory proteins [21][22][23]. The results showed that the transport of N-acetyl COSs into the intracellular compartments of strain SYBC-H1 could be achieved by specific membrane transporters, then further hydrolyzed to GlcNAc by NAGase for cell growth, which is similar with other reports [24,25].

Expression of CmNAGase gene and purification of recombinant CmNAGase
The full-length CmNAGase gene was soluble expressed in E. coli BL 21 (DE3) at a high expression level (~ 50% of total protein). The recombinant CmNAGase with a C-terminal His 6 tag was purified by Ni-NTA affinity chromatography with a yield of 80.5%, which is higher than that of other recombinant proteins with a His 6 tag [31,32]. The reason may be that C-terminal His 6 in the recombinant CmNAGase was well exposed, which led to the higher affinity with Ni-NTA resin. The specific activity of recombinant CmNAGase increased 1.5-fold from 3,156.5 U/mg to 4,878.6 U/mg after purification (Table S2). The SDS-PAGE analysis showed that purified recombinant CmNAGase possesses a high purity with an approximate molecular weight of 92 kDa, which agrees with 92,571 kDa calculated from the amino acid sequence containing the His 6 -tag (Fig. 4).
Effects of pH and temperature on activity and stability of recombinant CmNAGase The pH and temperature profile of CmNAGase activity are shown in Fig. 5. Typically, the optimal pH of reported GH20 NAGases are in the range of pH 5.0 to pH 8.0. CmNAGase exhibited a high level of activity at pH 4.0 − 7.0 with the optimal pH of 5.4, which is different from that of NAGases from Aeromonas sp. 10S-24 (7.0) [27], Paenibacillus sp. TS12 (6.0) [33], Salmonella enterica (4.0) [34], and Cellulomonas fimi (7.3 − 8.7) [35]. In addition, CmNAGase presented good activity after being stored at pH 4.0 − 8.5 for more than 84 h, which suggested that the CmNAGase possesses good pH stability compared with other reported NAGases [22,36].

Effect of metal ions on activity of CmNAGase
The effects of metal ions on CmNAGase were investigated. All counter-ions of the used metal ions were Cl − . As shown in Table 1, EDTA did not inhibit the enzymatic activity, which indicates that CmNAGase is non-metal dependent. CmNAGase activity is completely inhibited by Zn 2+ , Cu 2+ , and Al 3+ , severely inhibited by Ba 2+ , Fe 3+ , and Cr 3+ . To date, many studies have shown that Zn 2+ , Cu 2+ , Fe 3+ , and Al 3+ inhibit the activity of NAGases. For example, the GH20 NAGase from Aeromonas caviae is strongly inhibited by Cu 2+ and Zn 2+ [40]; the GH20 NAGase from Paenibacillus sp. is strongly inhibited by Zn 2+ [33], and the GH20 NAGase from T. reesei is partially inhibited by Fe 3+ [36]. Ni 2+ and Mn 2+ enhanced the activity of CmNAGase. The GH20 NAGase from Paenibacillus is strongly inhibited by Ni 2+ [33], and the GH20 NAGase from A. caviae is strongly inhibited by Mn 2+ [40]; these reports are different from CmNAGase. Samples were preincubated with various mental ions (10 mM) for 10 min at 37 °C. The remaining activity was measured using standard assays. Activity in the absence of any additives was taken as 100%.
The kinetic parameters for CmNAGase were measured with pNP-GlcNAc as a substrate. The V m , K m , and k cat values for CmNAGase were determined to be 833.33 µmol·L − 1 ·min − 1 ,10.9 mmol, and 6.37 × 10 8 mmol·mg − 1 , respectively.
Hydrolysis pattern of recombinant CmNAGase.
To evaluate the hydrolysis mode of CmNAGase, colloidal chitin and (GlcNAc) 2 −(GlcNAc) 6 were used as substrates. Hydrolysis of colloidal chitin resulted in GlcNAc as the only product, and its concentration increased with the increase of hydrolysis time (Fig. 6a). Konno et al. [21] reported that the NAGase LeHex20A from L. edodes hydrolyzes colloidal chitin to various oligomers at the start of the reaction, and these oligomers convert to GlcNAc after 3 h, which is different from CmNAGase.
To date, some chitinases have been used to synthesize longer N-acetyl COSs from shorter substrates.
These results suggest that CmNAGase is a novel bacteria-derived multi-functional NAGase, which has both exo NAGase activity and transglycosylation activity. Furthermore, transglycosylation activity of CmNAGase did not need the activated glycoside donor.

Transglycosylation activity of CmNAGase toward GlcNAc
In view of the activity toward (GlcNAc) 2 −(GlcNAc) 6 , the transglycosylation activity of CmNAGase was also investigated using GlcNAc as the substrate. As shown in Fig. 7, two new peaks at 8.2 min (peak 1) and 10.9 min (peak 2) were detected by HPLC. Of these, peak 1 was (GlcNAc) 2 compared with the standard of N-acetyl COSs. However, the retention time of peak 2 was between that of (GlcNAc) 2 (8.2 min) and (GlcNAc) 3 (12.3 min). To further identify peak 2, mass spectrum analysis was conducted. The m/z value of peak 2 was at 447.1586, which corresponds to (GlcNAc) 2 (425.1766 Da) with a sodium adduct (22.9898 Da) (Fig. S3). The result showed that peak 2 was also GlcNAc dimer.

Conclusions
This study reports the isolation, cloning, and recombinant expression of the gene encoding CmNAGase from C. meiyuanensis SYBC-H1. CmNAGase contains a GH20 family catalytic module and exhibits low similarity with reported NAGases. Analysis of the hydrolysis products from N-acetyl COSs and colloidal chitin revealed that CmNAGase exhibited exo-acting activity. Interestingly, CmNAGase also possesses transglycosylation activity toward GlcNAc−(GlcNAc) 6 , which respectively leads to synthesis of (GlcNAc) 2 −(GlcNAc) 7 . Among, CmNAGase can produce different isomers of GlcNAc dimer from GlcNAc. This is first report of a bacterial NAGase, which can produce (GlcNAc) n+1 from (GlcNAc) n Colloidal chitin was prepared as described by Gao et al. [9]. Other chemicals and solvents used in this study were purchased from local suppliers and were of analytical grade.
SYBC-H1 was cultivated according to our previous study [51]. The supernatant was collected as crude enzyme by centrifugation at 6,000 × g at 4ºC and used for NAGase purification.
The strains, plasmids, and primers used in this study are listed in Table S1. E. coli strains were routinely cultivated aerobically at 37ºC in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl). E. coli transformants were grown in LB medium or on agar plates containing 50 μg/mL kanamycin.
Purification of wild NAGase from C. meiyuanensis SYBC-H1 NAGase was purified by saturation with ammonium sulfate, followed by anion exchange chromatography, and all purification procedures were carried out at 4ºC. The supernatant of the culture was used as a crude enzyme preparation and then fractionated at 40% to 60% saturation with ammonium sulfate. The precipitate was centrifuged at 12,000 g for 30 min and dissolved in a suitable volume of 50 mM PBS (pH 7.0). The enzyme obtained in the previous step was further purified by anion exchange chromatography (GE health care chromatography) with a DEAE Sepharose TM column.
The column was equilibrated with 50 mM Tris-HCl at pH 8.0, then protein was separated by gradient elution with NaCl solutions from 0.05 M to 0.5 M. The purified NAGase was concentrated and collected using an ultrafiltration tube (10 kDa, Millipore, USA) at 4ºC. Then the purified enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 3% stacking gel and a 10% separating gel, according to the method described by Laemmli [52].
After electrophoresis, the gel was sliced vertically into two parts for staining and zymogram analysis.
The other part was stained using 0.1% Coomassie brilliant blue R-250 and then decolorized with a mixture of 10% acetic acid, 30% methanol, and 60% water. One part was incubated with 2.5% Determination of protein concentration and molecular weight Concentration of protein was quantified using the Bradford method [53]. Bovine serum albumin (BSA) was used to construct a standard calibration curve.
Reductive SDS-PAGE with a 3% stacking gel and 10% separating gel was performed to determine the molecular weight of purified recombinant protein according to purification part of wild NAGase above.

Determination of enzymatic activity
The enzymatic activity for CmNAGase used pNP-GlcNAc as the substrate [32]. A total of 20 μL of the enzyme solution was added to 0.98 mL pNP-GlcNAc (0.25 mM) in 50 mM sodium phosphate buffer (pH 7.0) and incubated at 40°C for 10 min. The reaction was terminated by adding 2 mL NaOH (0.5 M).
The absorbance was measured at 405 nm to determine the amount of pNP produced using a standard curve. One unit of chitinase activity was defined as the amount of enzyme required to release 1 μmol pNP from the substrate per minute at 40°C.

Characterization of recombinant CmNAGase
With 1 mM pNP-GlcNAc as the substrate, the optimum pH for activity of CmNAGase was determined Kinetics experiments were performed using pNP-GlcNAc as the substrate. The initial velocities were determined by incubating 3 μg purified CmNAGase with pNP-GlcNAc concentrations ranging from 50 μM to 1000 μM at 40°C in a 1 mL reaction system (50 mM sodium citrate buffer, pH 5.4) for 5 min.
The assay was performed in triplicate, and the values of V max , K m , and K cat were estimated by linear regression from double-reciprocal plots according to the method of Lineweaver [54].

Analysis of hydrolysis products from N-acetyl COSs and GlcNAc
To evaluate the hydrolysis mode of CmNAGase toward N-acetyl COSs and GlcNAc. The hydrolysis reactions of N-acetyl COSs and GlcNAc (10 g/L) were performed with purified CmNAGase (0.3 μg) in a 100 μL volume at 35°C for various time intervals. Boiling (5 min) was used to stop the reaction. The resulting reaction products were analyzed with an Agilent 1260 series HPLC system according to our previous report [32]. The molecular mass of the product was analyzed using electrospray ionization mass spectrometry (ESI-MS, API 2000).
Nucleotide sequence accession number Carbohydrate-Active enZYmes.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
All authors have seen and approved the manuscript before submission to Biotechnology for Biofuels.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare no conflicts of interest.   Multiple alignments of the catalytic domain in CmNAGase with other GH20 NAGases. Similar

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