Cloning of the NAGaseA gene and sequence analysis.
Based on the gene function prediction of the complete genome of Chitinbacter sp. GC72, ORF 159 was annotated as a potential β-N-acetylglucosaminidase (NAGaseA) gene. The total length of NAGaseA gene is 2, 535 bp, encoding 844 amino acids. After PCR, the same nucleic acid sequence was obtained, which indicated that NAGaseA gene was successfully cloned. Besides, the predicted molecular weight and theoretical pI of NAGaseA were 92.4 kDa and 5.24, respectively.
According to result of BLASTP analysis of the amino acid sequence, NAGaseA belonged to glycoside hydrolase (GH) family 20 (GH20) and shared the highest identity of 94.43% with the putative GH20 NAGase from Chitinibacter fontanus (WP_180317904), followed by 88.27% with GH20 NAGase from Chitinibacter sp. ZOR0017 (WP_047394852). However, the relative enzymatic characterization of these proteins has not been reported. Among the studied NAGases, NAGaseA displayed the highest identity (68.84%) with GH20 NAGase from Aeromonas sp. 10S-24 (accession no. BAA92145) [24], followed by 67.65% with GH20 NAGase from C. meiyuanensis (accession no. WP_148716590) [20], 32.83% with GH20 NAGase from Serratia marcescens (PDB 1QBA) [25], 30.79% with GH20 NAGase from Vibrio harveyi (PDB 6EZR) [26], 27.05% with GH20 NAGase from Microbacterium sp. HJ5 (PDB 7BWG) [27]. A phylogenetic tree of NAGaseA with some putative and verified GH20 family NAGases was further constructed base on their sequence similarities. The results suggested that NAGaseA shared low sequence similarity with most experimentally characterized GH20 NAGases (Fig. S1).
The result of multiple alignment of NAGaseA with other GH20 NAGases was shown in Fig. S2. The typical acidic pairs D512-E513 in NAGaseA are completely aligned with many other functionally characterized GH20 NAGases, which probably functioned as the catalytic residues. In addition, other highly conserved residues among NAGase species including R319, H426, V467, Q468, W562, W600, Y625, D627, L628, Y639, W641, W698, and E700 were also observed, which may play an important role in binding the GlcNAc ligand [28]. Furthermore, the consensus H-X-G-G motif before the catalytic residue in NAGaseA is highly conserved among the catalytic domain of GH20 NAGases. Based on the analysis of secondary structure, NAGaseA possesses 20 α-helices and 30 β-sheets with the typical (β/α)8 barrel fold in the GH20 catalytic domain, which is highly consistent with various GH20 NAGases from different sources [29].
The structure feature of NAGaseA was shown in Fig. 1 (A). The predicted protein structure consisted of four domains as follows: domain I (CHB_HEX domain of residues 2-153); domain II (Glyco_hydro_20b domain of residues 174−287); domain III (Glyco_hydro_20 domain residues 308−726, the catalytic domain containing a TIM barrel fold); domain IV (CHB_HEX_C domain of residues 758−841). As presented in Fig. 1 (B), the model of NAGaseA was predicted on the basis of the crystal structure of GH20 NAGase from Serratia marcescens (PDB 1QBA) with a protein identity of 34.22% [25]. The active pocket formed by R319, H426, V467, Q468, D512, E513, W562, W600, Y625, D627, L628, Y639, W641, W698, and E700 were labeled in the 3D structure model of NAGaseA (Fig. 1 (C)).
Expression of NAGaseA gene and purification of recombinant NAGaseA
The gene encoding NAGaseA was successfully expressed as soluble protein in E. coli BL21(DE3). The SDS-PAGE analysis (Fig. 2) showed that a single target protein band was obtained with a molecular weight of ~ 92 kDa after Ni-NTA resin affinity purification, which was consistent with the 92,379 Da calculated from the amino acid sequence containing the His6-tag. This is different from that of some GH20 NAGases from Microbacterium sp. HJ5 (55.9 kDa) [27], Paenibacillus sp. (57.5 kDa), V. harveyi 650 (73 kDa) [26]and S. thermoviolaceus (60 kDa) [31]. However, the Mw of NAGaseA is similar with the previously reported GH20 NAGase from C. meiyuanensis with a molecular mass of 92,571Da [32]. The specific activity of recombinant NAGaseA exhibited a 1.39-fold increase from 270.17 U/mg to 373.29 U/mg with a protein recovery of 78.6% yield after purification (Table S1).
Effects of temperature and pH on the enzymatic activity and stability of purified recombinant NAGaseA
The temperature and pH profiles of recombinant NAGaseA were investigated in Fig 3. As shown in Fig. 3(A), the recombinant NAGaseA displayed the optimal temperature at 40℃, which was consisted with NAGase from C. Meiyuanensis SYBC-H1 (40℃) [32], but different from NAGases from S. marcescens (52°C) [33], Microbacterium sp. HJ5 (45℃) [27], Streptomyces sp. F-3 (60℃) [34], and P. hydrolytica S66 (50°C) [35]. As for the thermostability profile, the activity dropped rapidly after incubation at temperatures above 40ºC, suggesting the poor thermostability of NAGaseA, which were similar with that of GH20 GlcNAGases from C. meiyuanensis [32], Microbacterium sp. HJ5 [27] and Aeromonas sp. 10S-24[24].
Regarding the effect of pH, NAGaseA exhibited the optimum pH at 6.5 (Figure 3 (B)). The optimal pH value of NAGaseA was higher than some reported NAGases, such as NAGase from C. meiyuanensis (5.4) [18], Salmonella enterica (4.0) [36] and Lactobacillus casei (5.0) [33]. In addition, NGAaseA retained excellent activity after incubation under the corresponding buffers of pH 6.5–9.5, indicating that NAGaseA possessed a good pH stability compared to other reported NAGases [37-39].
Effects of metal ions on activity of recombinant NAGaseA
Many reports have shown that metal ions affected the enzymatic activity. Thus, the effects of various metal ions on NAGaseA activity were also investigated. As shown in Table 1, the enzyme retained approximately 96% of its initial activity after incubation in 10 mM EDTA, suggesting that EDTA did not inhibit the enzymatic activity and NAGaseA is non-metal dependent. Cu2+ showed greatly inhibition effect on activity of NAGaseA, which was similar with that of NAGases from A. caviae [40] and C. meiyuanensis [20]. Besides, NAGaseA activity was partially inhibited by Fe3+ and Co2+, NAGases from R. miehei and Streptomyces alfalfa shared the same profile as reported [13, 41].
Table 1. Effects of metal ions on the activity of NAGaseA
Metal ions
|
Chemicals
|
Concentration (mM)
|
Relative activity (%)
|
No addition
|
-
|
0
|
100
|
Cu2+
|
CuCl2
|
10
|
24.27 ± 2.42
|
Fe3+
|
FeCl3
|
10
|
45.19 ± 2.25
|
Co2+
|
CoCl2
|
10
|
78.55 ± 5.49
|
Ni2+
|
NiCl2
|
10
|
92.95 ± 7.43
|
Ca2+
|
CaCl2
|
10
|
94.01 ± 1.88
|
Al3+
|
AlCl3·6H2O
|
10
|
91.97 ± 3.67
|
Mg2+
|
MgCl2
|
10
|
90.19 ± 6.31
|
Zn2+
|
ZnCl2
|
10
|
93.07 ± 4.65
|
Mn2+
|
MnCl2
|
10
|
98.98 ± 2.96
|
EDTA
|
EDTA
|
10
|
95.71 ± 4.78
|
Substrate specificity of NAGaseA
The substrate specificity of NAGaseA was measured using standard assay conditions. As depicted in Table 2, NAGaseA exhibited the highest specific activity toward pNP-GlcNAc, with specific activity of 333.33 U/mg. Among (GlcNAc)2-6, NAGaseA showed the highest activity toward (GlcNAc)2, followed by (GlcNAc)3, (GlcNAc)4, (GlcNAc)5 and (GlcNAc)6, which showed that the specific activity toward N-acetyl COSs decreased with increasing degree of polymerization [42]. Besides, little activity (0.0037 U/mg) was detected using colloid chitin as substrate, which was agreed with other reported GH20 NAGases that capable of degrading chitin to some extent without the addition of other chitinases [27]. Moreover, no activity was observed when chitosan, chitin power, CMC was used as the substrates. These results indicated that NAGaseA possessed the typical NAGase activity with strict substrate specificity.
In addition, the kinetic parameters for NAGaseA were also measured with pNP-GlcNAc as the substrate. The results showed that the Vmax, Km, Kcat, and Kcat/Km for NAGaseA were 3333.33 μmol min-1 L-1, 39.99 μM, 4667.07 s-1, 116.71 mL μmol -1 s-1, respectively.
Table 2. Substrate specificity of NAGaseA
Substrates
|
Specific activity (U/mg of protein)
|
Colloidal chitin
|
0.0037 ± 0.00047
|
Chitosan
|
0
|
Chitin power
|
0
|
pNP-GlcNAc
|
333.33±19.21
|
(GlcNAc)2
|
201.68 ± 11.69
|
(GlcNAc)3
|
152.84 ± 7.18
|
(GlcNAc)4
|
81.34 ± 5.49
|
(GlcNAc)5
|
55.52 ± 2.11
|
(GlcNAc)6
|
23.59 ± 1.13
|
Hydrolysis mechanism of NAGaseA toward colloid chitin and N-acetyl COSs
The hydrolysis patterns of colloid chitin and N-acetyl COSs by NAGaseA were measured (Fig. 4). As shown in Fig. 4(A), GlcNAc was the sole product hydrolyzed by colloid chitin, with its concentration raised as hydrolysis time increased. In the hydrolysis process of NAGaseA, (GlcNAc)2 was converted to GlcNAc as the sole product (Figure 4 (B)), (GlcNAc)3 to (GlcNAc)2 and GlcNAc (Figure 4 (C)), (GlcNAc)4 to (GlcNAc)3, (GlcNAc)2 and GlcNAc (Fig.4 (D)), (GlcNAc)5 to (GlcNAc)4, (GlcNAc)3, (GlcNAc)2 and GlcNAc (Fig.4 (E)), and (GlcNAc)6 was converted to (GlcNAc)5, (GlcNAc)4, (GlcNAc)3, (GlcNAc)2 and GlcNAc (Fig.4 (F)) at the initial incubation within 5 min. Furthermore, NAGaseA could hydrolyze (GlcNAc)2−(GlcNAc)6 into pure GlcNAc after an incubation time of 15 min to 180 min, respectively. The overall rates of hydrolysis were in the order: (GlcNAc)2 > (GlcNAc)3 > (GlcNAc)4 > (GlcNAc)5 > (GlcNAc)6, which was in accordance with the results of substrate specificity. These results indicated that NAGaseA is a typical exo-NAGase.
In addition, minor (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, (GlcNAc)6, were also produced from (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5 in short reaction times. These results indicated that NAGaseA capable of producing higher N-acetyl COSs ((GlcNAc)3−(GlcNAc)6) from (GlcNAc)2−(GlcNAc)5, which exhibited trans glycosylation activity. Our previous study also reported that CmNAGase from Chitinolyticbacter meiyuanensis SYBC-H1 could produce higher N-acetyl COSs (GlcNAc)3−(GlcNAc)7 from (GlcNAc)2−(GlcNAc)6, respectively [32]. However, unlike CmNAGase, no new peak presumed as (GlcNAc)7 generated when using (GlcNAc)6 as the substrate, which could be attributed to the lower reverse hydrolysis activity of NAGaseA.
Synergistic action between NAGaseA and chitinases on chitin degradation
To investigate the potential application of NAGaseA in GlcNAc production, the synergistic action between NAGaseA and other chitinases on chitin degradation was studied. As illustrated in Fig 5, the released reducing sugar concentrations from cooperation of NAGaseA with purified chitinase chiA, crude enzyme from C. meiyuanensis SYBC-H1 and crude enzyme from Chitinibacter sp. GC72 were 0.759 g/L, 0.481 g/L and 0.986 g/L, which were 1.6-, 2.36-, and 2.69- fold that of concentration of the two enzyme accumulated, respectively. Among, NAGaseA behaved the best improve efficiency with the crude enzyme from GC72, which could be attributed to the better synergistic effect with other chitinases from Chitinibacter sp. GC72. Zhou et al reported a combination of commercial chitinase CtnSg and NAGase rHJ5Nag used for chitin degradation, with an improvement rate of 2.02- fold [27]. Chenyin Lv et. al also investigated the synergistic action between commercial chitinase SgCtn and NAGase SaHEX, which obtained higher production of reducing sugars than the single enzyme for SgCtn (4.3-fold) and SaHEX (8.1-fold) [13]. In our study, NAGaseA can not only combine with purified chitinase but also crude chitinases in the production of GlcNAc from chitin. Moreover, it was worth noted that GlcNAc purity of 96% was obtained and little other N-acetyl COSs were detected in the final reaction mixture, indicating that NAGaseA has great potential in the production of GlcNAc in the multi-enzyme combination system.