Characterization of a New Multifunctional GH20 β-N-Acetylglucosaminidase From Chitinibacter sp. GC72 and Its Application in Converting Chitin Into N-Acetyl Glucosamine

In this study, a gene encoding β-N-acetylglucosaminidase, designated NAGaseA, was cloned from Chitinibacter sp. GC72 and subsequently functional expressed in Escherichia coli BL21 (DE3). NAGaseA contains a glycoside hydrolase family 20 catalytic domain that shows low identity with the corresponding domain of the well-characterized NAGases. The recombinant NAGaseA had a molecular mass of 92 kDa. Biochemical characterization of the purified NAGaseA revealed that the optimal reaction condition was at 40°C and pH 6.5, and exhibited great pH stability in the range of pH 6.5–9.5. The Vmax, Km, kcat, and kcat/Km of NAGaseA toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) were 3333.33 μmol min–1 l–1, 39.99 μmol l–1, 4667.07 s–1, and 116.71 ml μmol–1 s–1, respectively. Analysis of the hydrolysis products of N-acetyl chitin oligosaccharides (N-Acetyl COSs) indicated that NAGaseA was capable of converting N-acetyl COSs ((GlcNAc)2–(GlcNAc)6) into GlcNAc with hydrolysis ability order: (GlcNAc)2 > (GlcNAc)3 > (GlcNAc)4 > (GlcNAc)5 > (GlcNAc)6. Moreover, NAGaseA could generate (GlcNAc)3–(GlcNAc)6 from (GlcNAc)2–(GlcNAc)5, respectively. These results showed that NAGaseA is a multifunctional NAGase with transglycosylation activity. In addition, significantly synergistic action was observed between NAGaseA and other sources of chitinases during hydrolysis of colloid chitin. Finally, 0.759, 0.481, and 0.986 g/l of GlcNAc with a purity of 96% were obtained using three different chitinase combinations, which were 1.61-, 2.36-, and 2.69-fold that of the GlcNAc production using the single chitinase. This observation indicated that NAGaseA could be a potential candidate enzyme in commercial GlcNAc production.


INTRODUCTION
Chitin is the second most abundant polysaccharide on earth after cellulose, it is mainly derived from fungal cell walls, insect exoskeletons, and the crab and shrimp shells. An estimated 10 10 -10 11 tons of chitin are produced per year (Anitha et al., 2014). However, 35-45% of chitin biomass is discarded as waste due to a lack of efficient refinery protocols, which leads to waste of resources and severe environmental problems (Wei et al., 2017;Zhou et al., 2017a). N-acetyl glucosamine (GlcNAc), the monomeric unit of chitin, possesses many specific bioactivities and has been widely used in biomedical, food, and chemical industries (Bhattacharya et al., 2007;Suresh and Kumar, 2012;Kisiel and Kepczynska, 2017). Therefore, it is of economic and environmental value to realize the efficient production of GlcNAc from abundant chitin resources (Gao et al., 2018).
Commercial GlcNAc was often produced via acid hydrolysis of chitin. However, this protocol is difficult to directly obtain GlcNAc owing to the deacetylation of the N-acetyl group of products (Aam et al., 2010). In this case, chitin is first hydrolyzed to GlcN, and then chemical acetylated to form GlcNAc. This multistep process not only results in low yield, high cost, and poor biological activity of products but also leads to numerous environmental issues (Chen et al., 2010;Kim et al., 2017). Alternatively, enzymatic hydrolysis of chitin into GlcNAc using chitinolytic enzymes was shown to be a more attractive approach in recent years, because of the green process and the excellent bioactivity of the product (Park et al., 2011).
Chitinolytic enzymes are complex enzyme systems with a good synergistic effect, which could be classified into three types: endo-acting chitinases that cut randomly chitin chains to generate N-acetyl chitin oligosaccharides (N-acetyl COSs); progressive exo-acting chitinases that release GlcNAc dimer from the non-reducing or reducing end of chitin chains; NAGases that hydrolyze N-acetyl COSs or GlcNAc dimer into GlcNAc (Zhou et al., 2017a;Lv et al., 2019;Liu et al., 2020). Among, NAGase plays a key role in the control of the ratio and yield of GlcNAc during the hydrolysis process of chitin. Thus, it is of great significance to excavate NAGase with high activity for efficiently converting N-acetyl COSs into GlcNAc.
In our previous study, chitinolytic enzymes were derived from the bacterium Chitinibacter sp. GC72 isolated from pond mud in Nanjing were capable of hydrolyzing chitin into GlcNAc as the sole product (Gao et al., 2015). Moreover, only one gene encoding NAGase, named NAGaseA, was found in strain GC72 via complete genome sequencing and analysis (Zhang et al., 2020a). In this study, the NAGaseA gene was cloned from the genome of strain GC72 and heterologously expressed in Escherichia coli (BL21). The enzymatic properties and hydrolysis mode of the recombinant NAGaseA were investigated. Furthermore, the synergetic effect between NAGaseA and various chitinases in converting chitin to produce GlcNAc was also studied. This study provided a possible application in the enzymatic production of GlcNAc.

Chemicals, Strains, and Plasmids
Chitin powder, 4-methylumbelliferyl N-acetyl glucosaminide (4-MU-GlcNAc), and pNP-acetyl galactosaminide (pNP-GlcNAc) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). N-acetyl chitooligosaccharides (N-acetyl-COSs) standards ranging from dimer to hexamer were purchased from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China). The molecular reagents were purchased from Takara Bio Inc. (Dalian, China). All chemicals used in this study were of analytical grade or higher purity. Colloidal chitin was prepared from chitin powder according to the methods described by Gao et al. (2015).

Molecular Cloning and Sequence Analysis
The genomic DNA of strain GC72 was extracted using a bacteria genome extraction Kit (TIANGEN, China) and was used as the PCR template. Two primers used to amplify the NAGaseA were synthesized by Genscript Biotech (Nanjing, China) and the sequences were as followed: forward primer 5 -GTGCCGCGCGGCAGCCATATGAACAAGCCAGCAGGT-3 ; reserve primer 5 -GTGGTGGTGGTGCTCGAGCACCGCAAC CACCCGGCT-3 . The PCR system and conditions were as follows: 94 • C for 5 min, followed by 30 cycles of 95 • C for 30 s, 55 • C for 30 s, and 72 • C for 1 min, and a final extension at 72 • C for 10 min. The NAGaseA gene generated from PCR and the plasmid pET-28a (+) was double digested with NdeI and XhoI, followed by a ligation using the ClonExpressTM II/One Step Cloning Kit (Vazyme, China). The recombinant plasmid was transformed into E. coli Trans-T1 competent cells and sequenced by Genscript Biotech (Nanjing, China).
Nucleotide and amino acid sequences were analyzed using Snap Gene TM 1.1.3 software 1 and the ExPASy protparam tool. 2 The DNA and protein sequence alignments were performed via the NCBI server with the programs BLASTN and BLASTP, 3 respectively. Phylogenetic trees were inferred using the neighborjoining algorithm in MEGA 7. The conserved domains and the GH family classification were identified via the website. 4 The signal peptide was predicted in the SignalP 4.1 server. 5 Protein homologous sequences alignment was carried out using ClustalX 2.1 software and ESPript 3.0. 6 The structure of NAGaseA was predicted with RaptorX. 7

Expression and Purification of Recombinant NAGaesA
The recombinant plasmid pET-28a (+) harboring NAGaesA gene was transformed into E. coli BL21(DE3), incubated in LB liquid medium (containing 50 µg/ml kanamycin), and then cultured at 37 • C with shaking at 200 rpm. When the optical density (OD 600 ) of the culture medium was approximately 0.6, isopropyl β-Dthiogalactoside was added to a final concentration of 1 mM for protein induction, and the culture was incubated overnight at 18 • C with shaking at 200 rpm.
Cultures were harvested by centrifugation at 6,000 × g and 4 • C for 10 min, after which the pellet was gently resuspended in binding buffer A (50 mM phosphate buffer, 500 mM NaCl, 50 mM imidazole, pH 7.4) and lysed by JY92-IIN ultrasonication (Ningbo Xinzhi Biotechnology, Ltd., Ningbo, China). The cell debris was removed by centrifugation at 12,000 × g for 10 min at 4 • C and the supernatant was retained as a crude enzyme. The recombinant NAGaseA was purified using a fast protein liquid chromatography 448 system (GE AKTA Pure 150; General Electric Co., IA, America with a Ni-449 nitrilotriacetic acid affinity chromatography (Ni-NTA) column (His Trap TM FF 5 ml). The supernatant was filtered with a 0.22 µm membrane before being loaded onto a Ni Sepharose column. The NAGaseA protein was eluted with buffer B (50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, and pH 7.4) under a flow rate of 3 ml/min. The eluted protein was collected, concentrated, and exchanged with 20 mM phosphate buffer (pH 7.0) via ultrafiltration and stored at 4 • C before use (Zhang et al., 2020b).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to identify the target protein, and the protein concentration was determined using the Bradford method (Bradford, 1976).

Enzyme Assay and Substrate Specificity of Recombinant NAGaesA
NAGaseA activity was assayed using pNP-GlcNAc as substrate. A total of 1 ml reaction mixture containing 50 µl pNP-GlcNAc (10 mM) and 10 µl of purified NAGaseA in 50 mM phosphatebuffered saline (PBS) buffer (pH 7.0). The mixture was incubated at 40 • C for 10 min and then 1 ml NaOH (1 M) solution was added to terminate the reaction. The amount of pNP released was determined under the absorbance measured at 405 nm according to our previous reported (Zhang et al., 2018). One unit of NAGase activity was defined as the amount of enzyme required to release 1 µmol pNP per minute under the assay conditions. Chitinase activity was measured using colloid chitin as the substrate. The 1 ml reaction mixture was performed with 0.2 ml of enzyme and 0.3 ml colloid chitin (10 g/l) in 50 mM phosphate buffer (pH 7.0). The reaction was conducted at 37 • C for 30 min, and then 1 ml of 3,5-dinitrosalicylic acid (DNS) was added to the mixture followed by boiling at 100 • C for 5 min (Breuil and Saddler, 1985).

Enzymatic Characterization of Recombinant NAGaseA
The enzymatic characterization of recombinant NAGaseA was performed using pNP-GlcNAc as the substrate. To determine the optimum temperature of NAGaseA, the reaction was incubated under interval temperatures ranging from 30 • C to 80 • C in 50 mM PBS (pH 6.5). The thermostability of NAGaseA was determined by measuring the residual activity at pH 6.5 and 40 • C after the enzyme was treated in 50 mM sodium citrate (pH 6.5) for 12 h at different temperatures.
The kinetics parameters were determined by measuring the enzyme activity toward pNP-GlcNAc at 40 • C in 50 mM PBS (pH 6.5) for 10 min using different concentrations of substrate (50-2,500 µM) as the substrate. 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 (Price, 1985).

Hydrolytic Pattern of Recombinant NAGaseA
The reaction mixtures containing purified NAGaseA (60 ng) and various substrates ((GlcNAc) 2 -(GlcNAc) 5 ) at a final concentration of 10 g/l were incubated at 40 • C at various time intervals. In each case, the supernatant after hydrolysis was diluted with 50% acetonitrile and centrifuged at 8,000 × g for 10 min to remove the protein. The hydrolysis products were analyzed by Agilent 1260 series HPLC system according to our previous study (Zhang et al., 2018).

Cooperative Interaction Analysis of NAGaseA With Other Chitinases
The fermentation broth of strain GC72 and SYBC-H1 was centrifuged at 12,000 × g for 15 min at 4 • C, and the supernatant was collected as a crude enzyme before use. Exochitinase ChiA from Serratia proteamaculans (stored in our laboratory) was cloned, expressed, and purified as previously reported (Purushotham et al., 2012).
The cooperative interaction between NAGaseA and other sources of chitinases derived from strain Serratia proteamaculans (recombinant ChiA), strain SYBC (crude enzyme), and strain GC72 (crude enzyme) were determined using colloid chitin as the substrate. The reaction mixture (1 ml) contained colloidal chitin with a final concentration of 10 g/l and either 50 µl NAGaseA (4.8 U/ml reaction system), 50 µl ChiA (6.1 U/ml reaction system), 50 µl SYBC chitinase (2.8 U/ml reaction system), and 50 µl GC72 chitinase (5.2 U/ml reaction system) or both enzymes and was incubated at 40 • C in 50 mM PBS (pH 6.5) for 30 min. The amount of reducing sugars released was measured using the DNS method and HPLC mentioned above.

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.
The result of multiple alignments of NAGaseA with other GH20 NAGases was shown in Supplementary Figure 2. The typical acidic pairs D512-E513 in NAGaseA are completely aligned with many other functionally characterized GH20 To determine the temperature stability, the enzyme was incubated in 50 mM sodium citrate (pH 6.5) for 12 h at different temperatures, the residual activity was measured at pH 6.5 and 40 • C. (C) The optimal pH of the recombinant NAGaseA. The optimal pH was determined in 50 mM solutions of various buffers within the pH range 3-11. (G Citrate buffer (pH 3.0-6.0), NPhosphate buffer (pH (6.0-8.0), L Tris-HCl buffer (pH 7.0-9.0), I Glycine-NaOH buffer (pH 8.5-10.5)). (D) The pH stability of the recombinant NAGaseA. To determine the pH stability, the enzyme was incubated with various pH buffers, and the residual activities were measured (G Citrate buffer (pH 6.0), NPhosphate buffer (pH 6.5), L Phosphate buffer (pH 7.0), I Phosphate buffer (pH 8),NGlycine-NaOH buffer (pH 9), GGlycine-NaOH buffer (pH 10).

Expression of NAGaseA Gene and Purification of Recombinant NAGaseA
The gene encoding NAGaseA was successfully expressed as a soluble protein in E. coli BL21 (DE3). The SDS-PAGE analysis (Figure 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; Zhou et al., 2017b), Paenibacillus sp. (57.5 kDa), V. harveyi 650 (73 kDa; Suginta et al., 2010), and Streptomyces thermoviolaceus (60 kDa; Kubota et al., 2004). However, the M w of NAGaseA is similar to the previously reported GH20 NAGase from C. meiyuanensis with a molecular mass of 92,571 Da (Zhang et al., 2020b). The specific activity of recombinant NAGaseA exhibited a 1.39-fold increase from 270.17 to 373.29 U/mg with a protein recovery of 78.6% yield after purification (Supplementary Table 1).

Effects of Metal Ions on Activity of Recombinant NAGaseA
Many reports have shown that metal ions affected 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. Cu 2+ showed a great inhibition effect on the activity of NAGaseA, which was similar to that of NAGases from A. caviae (Cardozo et al., 2017) and C. meiyuanensis (Zhang et al., 2018). Besides, NAGaseA activity was partially inhibited by Fe 3+ and Co 2+ , NAGases from R. miehei and Streptomyces alfalfa shared the same profile as reported (Yang et al., 2014;Lv et al., 2019).

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 a 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 (Ogawa et al., 2006). Besides, little activity (0.0037 U/mg) was detected using colloid chitin as substrate, which was agreed with other reported GH20 NAGases that exhibited little hydrolysis activity toward chitin substrate without the cooperation with other chitinases (Zhou et al., 2017b). 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 V max , K m , k cat , and k cat /K m for NAGaseA were 3333.33 µmol min −1 l −1 , 39.99 µmol l −1 , 4667.07 s −1 , and 116.71 ml µmol −1 s −1 , respectively.

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 Figure 5, the released reducing sugar concentrations from the cooperation of NAGaseA with purified chitinase chiA, the crude enzyme from C. meiyuanensis SYBC-H1, and crude enzyme from Chitinibacter sp. GC72 were 0.759, 0.481, and 0.986 g/l, which were 1. 61-, 2. 36-, and 2.69-fold that of the concentration of the two enzymes accumulated, respectively. Among, NAGaseA behaved the best to 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 (Zhou et al., 2017b). 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; Lv et al., 2019). 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 noting that GlcNAc purity of 96% was obtained and few other N-acetyl COSs were detected in the final reaction mixture. Li et al. (2021) obtained the GlcNAc with a purity of 99.7% using colloidal chitin as the substrate under the co-action of two chitinases after 24 h of incubation, Du et al. (2019) reported the maximum GlcNAc yields of 87% using two enzyme combination during 2.25 h. These results indicate that NAGaseA has great potential in the production of GlcNAc in the multienzyme combination system.

CONCLUSION
In this study, the gene encoding a GH20 family β-Nacetylglucosaminidase NAGaseA from the chitinolytic bacterium Chitinibacter sp. GC72 was cloned and functionally expressed. The domain structure prediction showed that NAGaseA contains GH20 family catalytic domain and exhibited low similarity with reported GH20 NAGases. Analysis from the HPLC revealed that NAGaseA was a multifunctional NAGase exhibited the exo-acting activity and trans-glycosylation activity. Furthermore, NAGaseA also behaved with excellent synergistic performance with other chitinases during the degradation of colloidal chitin, and high purity of GlcNAc was obtained as the final product. These results indicated that NAGaseA has great potential in the bioconversion of chitin waste and behaved as an excellent candidate in GlcNAc production.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.