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.
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.
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.
Chitin, a linear polysaccharide of 1, 4-β-linked N-Acetyl glucosamine (GlcNAc), is the second most abundant renewable source in nature behind cellulose and mainly exists in crustacean shells, fungal cell walls, and insect exoskeletons . Comprehensive utilization of these chitin biomasses may have economic and ecological benefits . 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 . 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 . 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 . (GlcNAc)5 is an important building block for NOD factor synthesis . (GlcNAc)6 and (GlcNAc)7 show antitumor activity against mice sarcoma 180  and antimicrobial activity against fungal pathogens .
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 . 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 .
Chitinolytic enzymes, the essential enzymes involved in catabolism of chitin, primarily include chitinase [mainly belonging to glycoside hydrolase (GH) families 18 and 19, hydrolyzes chitin to N-Acetyl COSs] , β-N-acetyl glucosaminidase (GH families 3, 20, 73, 84, and 85, hydrolyzes N-Acetyl COSs to GlcNAc) [16, 17], and lytic polysaccharide monooxygenase [Auxiliary Activity (AA) families 10 and 11, cleavage of chitin chains with oxidation to enhance the hydrolysis of chitin] . Of these, β-N-acetyl glucosaminidase (NAGase), an indispensable member of the chitinolytic system, has a significant physiological role depending on its origin. With the cooperative action of NAGase and chitinase, chitin can be hydrolyzed into GlcNAc. Moreover, some GH20 NAGases can also be used to synthesize high-value GlcNAc-containing products and N-Acetyl COSs by transglycosylation and reverse hydrolytic reactions [19,20,21,22,23,24]. These excellent features make GH20 NAGases receive increased attention. GH20 NAGases can be found in a wide variety of organisms including bacteria, fungal, insects, plants, and animals [24,25,26,27]. However, studies about the GH20 NAGases with transglycosylation and reverse hydrolysis activities are mainly derived from fungal sources [28,29,30,31,32,33,34]. There are few reports about the bacterial NAGases possessing transglycosylation and reverse hydrolysis activities [19, 21, 35, 36].
In our previous study, a chitinolytic bacterium Chitinolyticbacter meiyuanensis SYBC-H1 with a good ability to degrade chitin was isolated from soil . 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 matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF MS/MS), and the results of PMF were interpreted by referencing the Mascot database . 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 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.
According to the Carbohydrate-Active enZYmes (CAZy) database (http://www.cazy.org/), NAGases can be classified as part of the glycoside hydrolase (GH) families 3, 20, 73, 84, and 85 based on amino acid sequence homology. BLASTP analysis showed that CmNAGase belonged to GH family 20 (GH20) and shared the highest identity (81.68%) with the GH20 NAGase ChiI from Chitiniphilus shinanonensis (WP_018749679) , followed by GH20 NAGase (81.44%) from Chitiniphilus sp. HX-2-15 (WP_136772659). However, these coding genes have not been expressed and studied. Among characterized GH20 NAGases, CmNAGase showed the highest identity (77.58%) with GH20 NAGase from Aeromonas sp. 10S-24 (Accession no. BAA92145) , following by the GH20 NAGase (34.02%) from Serratia marcescens (PDB 1QBA) , GH20 NAGase (30.44%) from Aeromonas caviae CB101 (Accession no. CAH55822) , GH20 NAGase Nag2 (30.04%) from Vibrio harveyi (PDB 6EZR) , GH20 NAGase (29.01%) from Enterobacter sp. G-1 (Accession no. BAA74506) , and GH20 NAGase (24.64%) from Aeromonas hydrophila SUWA-9 (Accession no. BAF76001) , GH20 NAGase Nag1 (12.26%) from V. harveyi (Accession no. ADJ68332) , and GH20 NAGase (12.24%) from Arthrobacter sp. TAD20 (Accession no. CAB72127) . The putative GH20 NAGases with the highest similarity of CmNAGase and verified GH20 NAGases were performed to construct the phylogenic tree, which also showed that CmNAGase exhibited a low sequence identity (12–35%) with most of functionally characterized bacterial GH20 NAGases (Fig. 2).
GH20 NAGases employ the retaining mechanism for catalysis. The enzymes carry out substrate-assisted catalysis, in which a Glu (E509 in CmNAGase) acts as the general acid/base residue for protonation, while Asp (D508 in CmNAGase) acts to orient the C2-acetamido group into position for correct nucleophilic attack by a water molecule, and subsequently provides the negatively charged carboxylate groups to stabilize the positively charged oxazolinium ion intermediate . Multiple alignments of the catalytic domain in CmNAGase with other GH20 NAGases from different sources indicated the substrate-binding residues (R315, H422, V463, Q464, W558, W594, Y621, D623, L624, Y635, W637, W693, and E695) and catalytic residues (D508 and E509) in CmNAGase, which are highly conserved among GH20 members (Additional file 1: Fig. S1). Moreover, sequence 504H/N-X-A/C/G/M-D-E-A/I/L/V510 in CmNAGase is the highly conserved amino acid sequence in the catalytic domain of GH20 NAGases from bacteria, fungi, and archaea . The analysis of secondary structure showed that CmNAGase possesses 21 α-helices and 31 β-sheets with the typical TIM-barrel(β/α)8 fold in the GH20 catalytic domain (Additional file 1: Fig. S1), which is consistent with various GH20 NAGases from different sources .
Domain structure prediction revealed that CmNAGase contains four domains: CHB_HEX domain of residues 7–155 (putative carbohydrate binding domain); Glyco_hydro_20b domain of residues 174–300 (N-terminal domain of the beta-hexosaminidases); Glyco_hydro_20 domain of residues 304–719 (catalytic domain); and CHB_HEX C_1 domain of residues 753–828 (unknown function) (Additional file 1: Fig. S2a). As shown in Additional file 1: Fig. S2b, the 3D structure of CmNAGase was predicted based on the structure model of 1QBA (34.02%) . The active sites (R315, H422, V463, Q464, D508, E509, W558, W594, Y621, D623, L624, Y635, W637, W693, and E695) were also shown in the 3D structure of CmNAGase (Additional file 1: Fig. S2c), and form an active pocket .
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 CmNAGase 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 CmNAGase 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).
Effects of pH and temperature on activity and stability of recombinant CmNAGase
The pH and temperature profile of CmNAGase activity are shown in Fig. 3. Typically, the optimal pH of reported GH20 NAGases is 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 (Fig. 3a), which is different from that of NAGases from Aeromonas sp. 10S-24 (7.0) , Paenibacillus sp. TS12 (6.0) , Vibrio harveyi 650 (7.5) , Enterobacter sp. G-1 (6.0) , Salmonella enterica (4.0) , Paraglaciecola hydrolytica S66 (6.0) , and Cellulomonas fimi (7.3–8.7) . In addition, CmNAGase presented good activity after being stored at pH 4.0–8.5 for more than 84 h (Fig. 3b), which suggested that the CmNAGase possesses a good pH stability compared with other reported NAGases [45, 51, 52].
As shown in Fig. 3c, the effect of temperature on enzymatic activity showed that the optimal temperature of CmNAGase was 40 °C, which is different from that of NAGases from Shinella sp. (50 °C) , Trichoderma reesei (60 °C) , P. hydrolytica S66 (50 °C) , Enterobacter sp. G-1 (45 °C) , and S. marcescens (52 °C) . The CmNAGase was unstable at temperatures ˃ 40 °C (Fig. 3d). Half-lives of CmNAGase toward 30 °C, 35 °C, 40 °C, and 45 °C were 13.0, 9.5, 6.3, and 0.6 h, respectively (Additional file 1: Table S3), which were similar with that of GH20 NAGases from Lactobacillus casei , Sphingobacterium sp. , and Aeromonas sp. 10S-24 . These results suggested that CmNAGase is a mesophilic and acidic enzyme.
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 metal-dependent. CmNAGase activity is completely inhibited by Zn2+, Cu2+, and Al3+, severely inhibited by Ba2+, Fe3+, and Cr3+. To date, many studies have shown that Zn2+, Cu2+, Fe3+, and Al3+ inhibit the activity of NAGases. For example, the GH20 NAGase from A. caviae is strongly inhibited by Cu2+ and Zn2+ ; the GH20 NAGase from Paenibacillus sp. is strongly inhibited by Zn2+ , and the GH20 NAGase from T. reesei is partially inhibited by Fe3+ . Mn2+ enhanced the activity of CmNAGase, which is different from the GH20 NAGase from A. caviae (strongly inhibited by Mn2+) . However, the specific activated mechanism of Mn2+ 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 (GlcNAc)2–(GlcNAc)6. No activity was observed when pNP-glucose, pNP-acetyl galactosaminide, and cellobiose were used as the substrates. These results showed that CmNAGase 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].
Most of the GH20 NAGases have the highest catalytic efficiency for (GlcNAc)2 among natural substrates, and do not hydrolyze N-acetyl COSs and chitin polymer [22, 56]. However, CmNAGase showed good activities toward (GlcNAc)2–(GlcNAc)6 with the highest activity for (GlcNAc)2, followed by (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6. These results show that specific activity of CmNAGase toward N-Acetyl COSs decreased when the degree of polymerization increased, which is similar with other reports . The catalytic efficiency of GH20 LeHex20A from Lentinula edodes for (GlcNAc)6 was greater than for (GlcNAc)2 . The GH20 NAGase VhNag2 from V. harveyi showed the highest activity against (GlcNAc)4, while the lowest activity was observed with (GlcNAc)2 . These NAGases are different from CmNAGase. Furthermore, CmNAGase showed some activity (0.02 U/mg) toward colloidal chitin. The result is similar with the GH20 NAGases from V. harveyi  and the fungal NAGases from Myceliopthora thermophila , L. edodes , and M. anisopliae , which were reported to degrade chitin to some extent without the cooperation of chitinase.
In addition, the kinetic parameters for CmNAGase were also measured with pNP-GlcNAc as the substrate. The results showed that the Vmax, Km, Kcat, and Kcat/Km for CmNAGase were 16,666.67 μmol min−1 mg−1, 0.5 μmol mL−1, 25,555.56 s−1, and 51,111.12 mL μmol−1 s−1, respectively.
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.  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.
Hydrolysis and transglycosylation reactions of recombinant CmNAGase toward N-Acetyl COSs
To evaluate the hydrolysis and transglycosylation activities of CmNAGase, (GlcNAc)2–(GlcNAc)6 were used as substrates. The overall rates of hydrolysis were in the order: (GlcNAc)2 > (GlcNAc)3 > (GlcNAc)4 > (GlcNAc)5 > (GlcNAc)6, which is consistent with the results of substrate specificities above. (GlcNAc)2 was degraded by CmNAGase to GlcNAc (Fig. 4b). GlcNAc and (GlcNAc)2 are produced from (GlcNAc)3 (Fig. 4c). When using (GlcNAc)4 as a substrate, GlcNAc, (GlcNAc)2, and (GlcNAc)3 were produced (Fig. 4d). GlcNAc, (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4 were obtained from (GlcNAc)5 (Fig. 4e). When using (GlcNAc)6 as the substrate, GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5 were found (Fig. 4f). Finally, (GlcNAc)2–(GlcNAc)6 were both hydrolyzed to GlcNAc as the final product with the increase of hydrolysis time. Similar results were reported for GH20 NAGase (VhNag2) from V. harveyi  and LeHex20A from L. edodes . However, the hydrolysis rates of VhNag2 and LeHex20A toward N-Acetyl COSs were in the order: (GlcNAc)4 > (GlcNAc)3 > (GlcNAc)5 > (GlcNAc)6 > (GlcNAc)2. Based on these results, we conclude that CmNAGase is a typical exo-acting NAGase.
In addition, minor (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, (GlcNAc)6, and new peak generated after the peak of (GlcNAc)6 were also produced from (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6 in short reaction times, respectively (Fig. 4b–f). The mass spectrum analysis of the new peak is at m/z values of 1440.5756 and 1441.5743, which respectively correspond to (GlcNAc)7 and (GlcNAc)7 with a hydrogen adduct (Additional file 1: Fig. S4). These results showed that CmNAGase can produce higher N-Acetyl COSs (GlcNAc)3–(GlcNAc)7 from (GlcNAc)2–(GlcNAc)6, respectively. To date, most scholars all believe that production of (GlcNAc)n+1 from (GlcNAc)n (n=2–7) is a transglycosylation reaction [28, 29, 34]. However, it may also be a reverse hydrolysis reaction. For example, (GlcNAc)2 and (GlcNAc)n may form (GlcNAc)n+1 by transglycosylation. Meanwhile, GlcNAc and (GlcNAc)n could also form (GlcNAc)n+1 through reverse hydrolysis. It is very hard to tell the two reactions apart in this setup. Thus, we call this reaction transglycosylation for the time being.
To date, some chitinases have been used to synthesize higher N-Acetyl COSs from shorter N-Acetyl COSs substrates via transglycosylation activity. For example, a chitinase from C. shinanonensis generated (GlcNAc)5 and (GlcNAc)6 when incubated with (GlcNAc)4 . A chitinase from T. reesei KDR-11 was shown to convert (GlcNAc)4 into (GlcNAc)6 . An endochitinase of Flavobacterium johnsoniae synthesized (GlcNAc)6–(GlcNAc)8 and (GlcNAc)7–(GlcNAc)9 from (GlcNAc)5 and (GlcNAc)6, respectively . A chitinase from Microbulbifer thermotolerans DAU221 produced (GlcNAc)4 from (GlcNAc)3 .
For GH20 NAGases, production of higher N-Acetyl COSs from shorter N-Acetyl COSs was often catalyzed by an auto-condensation reaction (a special case of transglycosylation, which involves only one substrate (acts both donor and acceptor) . For example, a GH20 NAGase from A. oryzae was used to catalyze the formation of (GlcNAc)3 and (GlcNAc)4 from (GlcNAc)2 . Singh et al.  reported that the GH20 NAGase from A. oryzae produces (GlcNAc)4–(GlcNAc)6 and (GlcNAc)5–(GlcNAc)6 from (GlcNAc)3 and (GlcNAc)4, respectively. GH20 NAGases SmHex from S. marcescens YS-1  and NoHex from N. orientalis IFO12806  were shown to convert (GlcNAc)2 to (GlcNAc)3. GH20 NAGases AoHex from A. oryzae CCF1066 produce the mixture of (GlcNAc)2–(GlcNAc)8 from the mixture of GlcNAc–(GlcNAc)7 . In comparison with these reports, CmNAGase is a novel bacteria-derived NAGase, which possesses transglycosylation activity toward (GlcNAc)2–(GlcNAc)6.
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 (GlcNAc)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.
The reverse hydrolysis reaction of GH20 NAGases toward GlcNAc can form many connection configurations of the GlcNAc dimer, for example by β(1 → 3), β(1 → 4), and β(1 → 6) glycosidic bonds, which is mainly determined by the regioselectivity of glycosidase . To date, studies on the synthesis of GlcNAc dimer from GlcNAc were mainly reported by Rauvolfová et al., which produce β-GlcNAc-(1 → 3)-GlcNAc, β-GlcNAc-(1 → 4)-GlcNAc, and β-GlcNAc-(1 → 6)-GlcNAc using the library of fungal GH20 NAGases . For example, the GH20 NAGases from Acremonium persicinum CCF 1850, A. oryzae CCF 1066, Aspergillus flavipes CCF 2026, A. flavus CCF 1129, P. oxalicum CCF 2315, and A. terreus CCF 2539, which only produced β-GlcNAc-(1 → 6)-GlcNAc from GlcNAc [30, 31]. The GH20 NAGases from P. funiculosum CCF 1994, P. funiculosum CCF 2325, P. chrysogenum CCF 1269, and Trichoderma harzianum CCF 2687 synthesized β-GlcNAc-(1 → 3)-GlcNAc, β-GlcNAc-(1 → 4)-GlcNAc, and β-GlcNAc-(1 → 6)-GlcNAc from GlcNAc . The GH20 NAGases from A. fumigatus CCF 1059, P. pittii CCF 2277, and A. sojae CCF 3060 synthesized β-GlcNAc-(1 → 4)-GlcNAc and β-GlcNAc-(1 → 6)-GlcNAc from GlcNAc. Among these fungal NAGases, β-GlcNAc-(1 → 6)-GlcNAc is always the main product. Thus, the peak 1 in Fig. 5 was β-GlcNAc-(1 → 4)-GlcNAc ((GlcNAc)2), and main peak 2 should be β-GlcNAc-(1 → 6)-GlcNAc. These results suggested that CmNAGase has reverse hydrolysis activity toward GlcNAc.
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.
Chitin, 4-Methylumbelliferyl N-Acetyl glucosaminide (4-MU-GlcNAc), p-Nitrophenyl N-Acetyl glucosaminide (pNP-GlcNAc), pNP-glucose, and pNP-acetyl galactosaminide were purchased from Aladdin reagent Co., Ltd (Shanghai, China). The standards of N-Acetyl chitooligosaccharides (N-Acetyl COSs) (purity: ≥ 95%) with degree of polymerization between 2 and 6 were acquired from Qingdao Bozhi Biotechnology Co., Ltd (Qingdao, China). Peptone and yeast extract were purchased from the Oxoid Co., Ltd. (Beijing, China). All molecular reagents were purchased from TaKaRa (Dalian, China). Colloidal chitin was prepared as described by Gao et al. . Other chemicals and solvents used in this study were purchased from local suppliers and were of analytical grade.
Strains, culture conditions, and plasmids
The C. meiyuanensis strain SYBC-H1 (ATCC BAA-2140) used in this study was isolated previously . SYBC-H1 was cultivated according to our previous study . 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 .
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.matrixscience.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′-GAATTCCATATGATGAGCCGTCCCGCCGGATC-3′ and CmNAGase-R-5′-TCCGCTCGAGTCAGGCGCCCACCTGCACCG-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 (OD600) 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 . 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-type NAGase above. A premixed protein marker (Takara Biotechnology Co., Ltd., Nanjing, China) containing 180-, 140-, 100-, 75-, 60-, 45-, 35-, 25-, 15-, and 10-kDa bands was used as the molecular mass standard.
Determination of enzymatic activity
The NAGase activity for CmNAGase used pNP-GlcNAc as the substrate . 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 effects of metal ions on the activity were also determined. Purified recombinant CmNAGase was treated with 10 mM EDTA for 4 h at 4 °C and then dialyzed against 50 mM Tris–HCl buffer (pH 7.0) to remove the EDTA. For reactivation, the metal-free enzyme was incubated with various metal salts containing Cr3+ (CrCl3), Fe3+ (FeCl3), Fe2+ (FeCl2), Ca2+ (CaCl2), Cu2+ (CuCl2), Mg2+ (MgCl2), Zn2+ (ZnCl2), Mn2+ (MnCl2), Ni2+ (NiCl2), Co2+ (CoCl2), K+ (KCl), or Na+ (NaCl) at final concentrations of 10 mM for 30 min, and the residual activities were then measured with pNP-GlcNAc at 40 °C and pH 7.0 (Tris–HCl buffer) for 10 min. The activity without addition of metal ions was used as the control (100%).
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 Vmax, Km, and Kcat were estimated by linear regression from double-reciprocal plots according to the method of Lineweaver .
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.
Reverse hydrolysis reaction of the recombinant CmNAGase toward GlcNAc
The determination of reverse hydrolysis activity was performed with 10 g/L GlcNAc and 0.3 μg purified CmNAGase in a 100 μL volume at pH 5.4 and 35 °C for 1 h. The reactions were stopped by boiling at 100 °C for 5 min.
Analysis of products
The resulting reaction products were analyzed with an Agilent 1260 series HPLC system according to our previous report . The molecular mass of the product was analyzed using electrospray ionization mass spectrometry (ESI-MS, API 2000) with the ESI positive mode. The quadrupole scan mode was used under a capillary voltage of 2.8 kV, cone voltage of 30 V, desolvation gas temperature of 350 °C and source temperature of 120 °C.
Nucleotide sequence accession number
The sequence for the gene encoding CmNAGase cloned from strain SYBC‑H1 was deposited in GenBank under Accession number no. MN548778.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
4-Methylumbelliferyl N-Acetyl glucosaminide
p-Nitrophenyl N-Acetyl glucosaminide
Peptide mass fingerprinting
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
Polymerase chain reaction
Fast protein liquid chromatography
Molecular weight search scores
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The authors thank professor Hongzhi, Tang from Shanghai Jiaotong University for valuable discussions.
This work was supported by the National Key Research and Development Program (2016YFA0204300), the National Natural Science Foundation of China (31700092), the National Nature Science Foundation for Young Scientists of China (Grant No. 21908101, 21576134), and the China Postdoctoral Science Foundation (2018M642237).
Authors and Affiliations
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211800, People’s Republic of China
Alei Zhang, Xiaofang Mo, Ning Zhou, Yingying Wang, Guoguang Wei, Jie Chen, Kequan Chen & Pingkai Ouyang
AZ, KC, and PO designed the work. XM, GW, NZ, YW, and JC constructed the clone of CmNAGase gene, purified the recombinant protein, performed enzyme characterization, and HPLC studies. AZ conducted bioinformatic studies. The data were analyzed by AZ, XM, NZ, and JC. The manuscript was written by AZ by taking critical inputs from KC. All authors read and approved the final manuscript.
Strains, plasmids, and primers used in this study. Table S2. Purification of recombinant CmNAGase. Table S3. Half-lives of recombinant CmNAGase. Fig. S1. Multiple alignments of the catalytic domain in CmNAGase with other GH20 NAGases. Fig. S2. The domain and structure prediction of CmNAGase. a) The conserved domain of CmNAGase. b) The prediction of the 3D structure of CmNAGase. c) The active site of CmNAGase. Fig. S3. SDS-PAGE analysis of recombinant CmNAGase. Fig. S4. Mass spectrum of new peak (~ 16.0 min) after (GlcNAc)6 in HPLC spectra. Fig. S5. Mass spectrum of peak 2 (~ 10.9 min) in HPLC spectra.
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Zhang, A., Mo, X., Zhou, N. et al. A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities.
Biotechnol Biofuels13, 115 (2020). https://doi.org/10.1186/s13068-020-01754-4