A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities

Background N-Acetyl glucosamine (GlcNAc) and N-Acetyl chitooligosaccharides (N-Acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N-Acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)4–(GlcNAc)7) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity toward natural killer cells. Thus, it is of great significance to discover a β-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize 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 (12–35%) with the corresponding domain of most well-characterized NAGases. The CmNAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified CmNAGase toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) was 4878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40 °C. The Vmax, Km, Kcat, and Kcat/Km of CmNAGase for pNP-GlcNAc were 16,666.67 μmol min−1 mg−1, 0.50 μmol mL−1, 25,555.56 s−1, and 51,111.12 mL μmol−1 s−1, respectively. Analysis of the hydrolysis products of N-Acetyl COSs and colloidal chitin revealed that CmNAGase is a typical exo-acting NAGase. Particularly, CmNAGase can synthesize higher N-Acetyl COSs ((GlcNAc)3–(GlcNAc)7) from (GlcNAc)2–(GlcNAc)6, respectively, showed that it possesses transglycosylation activity. In addition, CmNAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. Conclusions The observations recorded in this study that CmNAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggest a possible application in the production of GlcNAc or higher N-Acetyl COSs.

exoskeletons [1]. Comprehensive utilization of these chitin biomasses may have economic and ecological benefits [2]. However, there is no satisfactory method to utilize them to date. A vast majority of the chitin biomasses are directly disposed of or landfilled without utilization, which leads to serious pollution and wasted resources [3]. Therefore, it would be beneficial to utilize the numerous chitin resources to produce value-added chemicals and materials.
Chitin can be used to produce biomaterials such as films, adhesives, preservative coatings, and antibacterial/anticancer materials [4][5][6]. Besides the direct utilization of chitin mentioned above, chitin can be used as a substrate for producing nitrogen-containing chemicals like GlcNAc, N-Acetyl chitooligosaccharides (N-Acetyl COSs), ethanolamine, N-containing furan derivatives and so on [2]. Among these chemicals, GlcNAc and N-Acetyl COSs are considered promising platform molecules, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries [7,8]. Especially, higher N-Acetyl COSs with the degree of polymerization from 4 to 7 ((GlcNAc) 4 -(GlcNAc) 7 ) exhibit many biological activities. For example, (GlcNAc) 4 was found to have strong stimulating activity toward natural killer cells [9]. (GlcNAc) 5 is an important building block for NOD factor synthesis [10]. (GlcNAc) 6 and (GlcNAc) 7 show antitumor activity against mice sarcoma 180 [11] and antimicrobial activity against fungal pathogens [12].
Chitin is traditionally chemically degraded to GlcNAc and N-Acetyl COSs using acid, which leads to toxicity and risks associated with serious pollution during the production process [13]. With increased environmental awareness, increasing attention has been paid to developing enzymatic hydrolysis of chitin using chitinolytic enzymes as catalysts because they are environmentally friendly and result in products with high bioactivity compared to classical chemical routes [14].
In our previous study, a chitinolytic bacterium Chitinolyticbacter meiyuanensis SYBC-H1 with a good ability to degrade chitin was isolated from soil [37]. In this study, a gene encoding NAGase 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 and catalytic characteristics of the purified recombinant NAGase were described. Furthermore, its transglycosylation and reverse hydrolysis activities were also investigated.

Results and discussion
Purification of wild-type NAGase from C. meiyuanensis

SYBC-H1
Purification of the NAGase from C. meiyuanensis SYBC-H1 was performed. 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 and 100 kDa (Fig. 1).
The protein strip in the stained gel was excised for peptide mass fingerprinting (PMF) analysis using matrixassisted laser desorption ionization-time-of-flight (MALDI-TOF MS/MS), and the results of PMF were interpreted by referencing the Mascot database [38]. 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 WNQFANRLGQRELARLDGFLGGY GYR VPV, 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 expected, a PCR product of 2.5 kb was obtained. Analysis of the PCR product showed that the CmNAGase gene was 2508 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 the strain SYBC-H1. Furthermore, most reported NAGases are secretory proteins to date [21][22][23]. These results show that the prediction of CmNAGase location may be wrong.

Expression of CmNAGase gene and purification of recombinant CmNAGase
The full-length of CmNAGase gene was successfully expressed in E. coli BL 21 (DE3) at a high expression level (~ 50% of total protein). The recombinant CmNA-Gase with a C-terminal His6-tag was purified by Ni-NTA affinity chromatography with a yield of 80.5%. The reason for the high purification yield may be that the C-terminal His6-tag in the recombinant CmNAGase was well exposed, which led to the higher affinity with Ni-NTA resin. The specific activity of recombinant CmNA-Gase increased 1.5-fold from 3156.5 U/mg to 4878.6 U/ mg after purification (Additional file 1: 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 His6-tag (Additional file 1: Fig. S3).

Effect of metal ions on activity of recombinant 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 not metaldependent. 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 A. caviae is strongly inhibited by Cu 2+ and Zn 2+ [42]; the GH20 NAGase from Paenibacillus sp. is strongly inhibited by Zn 2+ [48], and the GH20 NAGase from T. reesei is partially inhibited by Fe 3+ [51]. Mn 2+ enhanced the activity of CmNAGase, which is different from the GH20 NAGase from A. caviae (strongly inhibited by Mn 2+ ) [42]. However, the specific activated mechanism of Mn 2+ is unclear and the in-depth study is needed in the future.

The substrate specificity of recombinant CmNAGase
The specific activities of the recombinant CmNAGase against various substrates were investigated using standard assay conditions. As shown in Table 2, CmNAGase can hydrolyze pNP-GlcNAc, 4-MU-GlcNAc, and (Glc-NAc) 2 -(GlcNAc) 6 . No activity was observed when pNPglucose, pNP-acetyl galactosaminide, and cellobiose were used as the substrates. These results showed that CmNA-Gase represents the typical NAGase activity with strict substrate specificity. The specific activity of CmNAGase toward pNP-GlcNAc can reach 4878.6 U/mg, which is higher than most reported NAGases (< 2000 U/mg) [17,21,27,48,56].
In addition, the kinetic parameters for CmNAGase were also measured with pNP-GlcNAc as the substrate.

Table 1 Effect of different metal ions on CmNAGase
Samples were preincubated with various mental ions (10 mM) at pH 7.0 (Tris-HCl buffer) and 4 °C for 30 min. The remaining activity was measured with pNP-GlcNAc at pH 7.0 (Tris-HCl buffer) and 40 °C for 10 min. Activity in the absence of any additives was taken as 100%

Hydrolysis reaction of recombinant CmNAGase toward colloidal chitin
As shown in Fig. 4a, hydrolysis of colloidal chitin resulted in GlcNAc as the only product, and its concentration increased with the increase of hydrolysis time. Konno et al. [58] reported that the NAGase LeHex20A from L. edodes hydrolyzes colloidal chitin to various N-acetyl COSs at the start of the reaction, and these N-Acetyl COSs convert to GlcNAc after 3 h, which is different from CmNAGase.
In addition, the products ((GlcNAc) 3 -(GlcNAc) 7 ) from transglycosylation disappeared soon with the increase of reaction time (Fig. 4b-f ). This phenomenon may be because the NAGase activity of CmNAGase outweighs its transglycosylation activity, which leads to the transient existence of (GlcNAc) 3 -(GlcNAc) 7 .

Reverse hydrolysis activity of recombinant CmNAGase toward GlcNAc
In view of the transglycosylation activity toward (Glc-NAc) 2 -(GlcNAc) 6 , the reverse hydrolysis activity of CmNAGase was also investigated using GlcNAc as the substrate. As shown in Fig. 5, 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 GlcNAc-(GlcNAc) 6 . 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) (Additional file 1: Fig. S5). 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 GH20 NAGases. Analysis of the hydrolysis products from N-Acetyl COSs and colloidal chitin revealed that CmNAGase exhibited exo-acting activity. Interestingly, CmNAGase possesses transglycosylation activity toward (GlcNAc) 2 -(GlcNAc) 6 , which respectively leads to synthesis of (GlcNAc) 3 -(GlcNAc) 7 . In addition, CmNAGase also have reverse hydrolysis activity toward GlcNAc, which can produce its dimers with different linked. This is first report of a bacterial NAGase, which can produce GlcNAc dimers from GlcNAc via reverse hydrolysis activity.

Strains, culture conditions, and plasmids
The C. meiyuanensis strain SYBC-H1 (ATCC BAA-2140) used in this study was isolated previously [37]. SYBC-H1 was cultivated according to our previous study [67]. The supernatant was collected as crude enzyme by centrifugation at 6000×g at 4 °C and used for NAGase purification.
The strains, plasmids, and primers used in this study are listed in Additional file 1: Table S1. E. coli strains were routinely cultivated aerobically at 37 °C 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-type 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 4 °C. 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 12,000g 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 using a fast protein liquid chromatography (FPLC) system (AKTA Pure 150; GE healthcare Co., Fairfield, USA) with a DEAE Sepharose ™ anion exchange 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 to 0.5 M. The purified NAGase was concentrated and collected using an ultrafiltration tube (10 kDa, Millipore, USA) at 4 °C. 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 [68].
After electrophoresis, the gel was sliced vertically into two parts for staining and zymogram analysis. One 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. The other part was incubated with 2.5% (vol/vol) Triton X-100 for 30 min twice to refold, then was sprayed with 50 mM PBS (pH 7.0) containing 1 mM 4-MU-GlcNAc and incubated at 37 °C for 30 min; the NAGase strip became visible as fluorescence at 340 nm. Two parts were compared to determine the position of the CmNAGase, and the corresponding band was sliced for peptide mass fingerprinting (PMF) analysis.

Peptide mass fingerprinting of the enzyme
The gel sliced above was analyzed using the electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF MS/MS) technique (PROTTECH, Inc., Suzhou, China). These masses were then compared to theoretical mass values in the Mascot website databases (http://www.matri xscie nce.com) to reveal the amino acid sequences of the peptide fragments. The peptide sequence was then aligned with the genome of C. meiyuanensis SYBC-H1 (GenBank Accession number, CP041335) to find the NAGase and its coding gene.

Cloning of the CmNAGase gene and sequence analysis
The genomic DNA of C. meiyuanensis SYBC-H1 was used as the template for polymerase chain reaction (PCR) amplification. According to the results of PMF and the complete genome of C. meiyuanensis SYBC-H1, the coding region of the CmNAGase gene was amplified by PCR with the primer pair CmNAGase-F-5′-GAA TTC CAT ATG ATG AGC CGT CCC GCC GGA TC-3′ and CmNAGase-R-5′-TCC GCT CGA GTC AGG CGC CCA CCT GCA CCG -3′. The PCR conditions were as follows: 5 min at 94 °C, followed by 30 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 10 min. The amplified PCR product was purified by gel electrophoresis, digested with restriction enzymes NdeI and XhoI, and then ligated into pMD19-T Simple vector and sequenced by Invitrogen Corporation (Shanghai, China). The positive recombinant plasmids were digested with NdeI and XhoI, and the gene was inserted into the pET-28a(+) vector expression plasmid with a C-terminal His6-tag to generate the pET-28a(+)-CmNAGase.

Expression of the CmNAGase gene in E. coli BL21(DE3) and purification of the recombinant enzyme
The recombinant plasmid pET-28a(+)-CmNAGase above was transformed into competent E. coli BL21(DE3) for protein expression. The E. coli BL21(DE3) harboring the pET-28a(+)-CmNAGase plasmid were cultured in LB medium (containing 50 μg/mL kanamycin) at 37 °C in a shaker with a rotation speed of 200 rpm. When the optical density (OD 600 ) of the culture medium reached 0.6-0.8, isopropyl β-d-thiogalactoside (IPTG) was added at a final concentration of 1 mM for protein induction, and the culture was further grown at 25 °C for 12 h.
The cells were harvested by centrifugation at 6000g and 4 °C for 10 min, after which the cells were re-suspended with His6-tag binding buffer (20 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole [pH 7.0]) and lysed by JY92-IIN ultrasonication (Ningbo Xinzhi Biotechnology, Ltd., Ningbo, China). Cell debris was removed by centrifugation at 6000g for 10 min at 4 °C and the supernatant was retained as crude enzyme. The recombinant CmNAGase were purified using an FPLC system (AKTA Pure 150; GE healthcare Co., Fairfield, USA)) with a Ni-nitrilotriacetic acid affinity chromatography (Ni-NTA) column (His Trap ™ FF 5 mL). The target protein was eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole [pH 7.0]). The eluted fractions were passed through an ultrafiltration tube of 10 kDa (Millipore, USA) to remove the imidazole with sodium phosphate buffer (pH7.0) and concentrate the enzyme solution.

Determination of protein concentration and molecular weight
Concentration of protein was quantified using the Bradford method [76]. Bovine serum albumin (BSA) was used to construct a standard calibration curve.

Determination of enzymatic activity
The NAGase activity for CmNAGase used pNP-GlcNAc as the substrate [77]. A total of 20 μL of the enzyme solution (0.1 g/L) was added to 0.98 mL pNP-GlcNAc (0. 25 mM) in 50 mM sodium citrate buffer (pH 5.4) 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 NAGase 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 using different buffers: 50 mM citrate buffer (pH 3.0-6.0), 50 mM phosphate buffer (pH 6.0-7.5), and Tris-HCl buffer (pH 7.0-9.0) at 40 °C for 30 min. To measure the pH stability, enzyme was incubated at 35 °C for 96 h in the different buffers and the residual activities were determined against 1 mM pNP-GlcNAc.
The optimum temperature of CmNAGase activity was measured, and the reaction solutions were incubated at temperatures that ranged from 25 to 55 °C for 30 min. Enzyme thermostability was determined by measuring the residual activities after pre-incubation of the purified enzyme in 50 mM sodium citrate buffer (pH 5.4) at 20-40 °C without substrate for 12 h. The residual activities were performed at pH 5.4 and 40 °C according to enzymatic activity assay above.
The substrate specificity of CmNAGase was determined by measuring the activity of enzyme toward colloidal chitin, N-acetyl COSs ((GlcNAc) 2 -(GlcNAc) 6 ), cellobiose, 4-MU-GlcNAc, and pNP-glycosides as substrates) at a concentration of 10 mg/mL. Reaction mixture (1 mL) containing enzyme (3 μg) and various substrates (10 g/L) was incubated at 40 °C and pH 5.4 for 10 min. The amount of reducing sugars from colloidal chitin and (GlcNAc) 2 -(GlcNAc) 6 was quantified with HPLC. The amount of pNP released in the reaction mixture was determined by measuring the absorbance at 405 nm. Hydrolytic activity of the enzyme against N-acetyl COSs was assayed by measuring the amount of GlcNAc released during the enzymatic reaction. After incubation, the enzymatic reaction was stopped by heating the mixture in a boiling water bath for 5 min. One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 μmol of pNP or GlcNAc per minute under the assay conditions. Kinetics experiments were performed using pNP-GlcNAc as the substrate. The initial velocities were determined by incubating 17 ng purified CmNAGase with pNP-GlcNAc concentrations ranging from 50 to 1000 μM at 40 °C in a 1 mL reaction system (50 mM sodium citrate buffer, pH 5.4) for 5 min. 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 [78].

Hydrolysis reaction of the recombinant CmNAGase toward colloidal chitin
A 1 mL reaction system (50 mM sodium citrate buffer, pH 5.4) containing colloidal chitin (10 g/L) and purified CmNAGase (3 μg) was conducted at 35 °C for various time intervals. Boiling (5 min) was used to stop the reaction.

Hydrolysis and transglycosylation reactions of the recombinant CmNAGase toward N-acetyl COSs
A 100 μL volume (50 mM sodium citrate buffer, pH 5.4) with 10 g/L N-Acetyl COSs ((GlcNAc) 2 -(GlcNAc) 6 ) and 0.3 μg purified CmNAGase was conducted at 35 °C for various time intervals. The enzyme reactions were stopped by boiling at 100 °C for 5 min.