Open Access

Distinctive molecular and biochemical characteristics of a glycoside hydrolase family 20 β-N-acetylglucosaminidase and salt tolerance

Contributed equally
BMC Biotechnology201717:37

DOI: 10.1186/s12896-017-0358-1

Received: 20 December 2016

Accepted: 4 April 2017

Published: 11 April 2017

Abstract

Background

Enzymatic degradation of chitin has attracted substantial attention because chitin is an abundant renewable natural resource, second only to lignocellulose, and because of the promising applications of N-acetylglucosamine in the bioethanol, food and pharmaceutical industries. However, the low activity and poor tolerance to salts and N-acetylglucosamine of most reported β-N-acetylglucosaminidases limit their applications. Mining for novel enzymes from new microorganisms is one way to address this problem.

Results

A glycoside hydrolase family 20 (GH 20) β-N-acetylglucosaminidase (GlcNAcase) was identified from Microbacterium sp. HJ5 harboured in the saline soil of an abandoned salt mine and was expressed in Escherichia coli. The purified recombinant enzyme showed specific activities of 1773.1 ± 1.1 and 481.4 ± 2.3 μmol min−1 mg−1 towards p-nitrophenyl β-N-acetylglucosaminide and N,N'-diacetyl chitobiose, respectively, a V max of 3097 ± 124 μmol min−1 mg−1 towards p-nitrophenyl β-N-acetylglucosaminide and a K i of 14.59 mM for N-acetylglucosamine inhibition. Most metal ions and chemical reagents at final concentrations of 1.0 and 10.0 mM or 0.5 and 1.0% (v/v) had little or no effect (retaining 84.5 − 131.5% activity) on the enzyme activity. The enzyme can retain more than 53.6% activity and good stability in 3.0–20.0% (w/v) NaCl. Compared with most GlcNAcases, the activity of the enzyme is considerably higher and the tolerance to salts and N-acetylglucosamine is much better. Furthermore, the enzyme had higher proportions of aspartic acid, glutamic acid, alanine, glycine, random coils and negatively charged surfaces but lower proportions of cysteine, lysine, α-helices and positively charged surfaces than its homologs. These molecular characteristics were hypothesised as potential factors in the adaptation for salt tolerance and high activity of the GH 20 GlcNAcase.

Conclusions

Biochemical characterization revealed that the GlcNAcase had novel salt–GlcNAc tolerance and high activity. These characteristics suggest that the enzyme has versatile potential in biotechnological applications, such as bioconversion of chitin waste and the processing of marine materials and saline foods. Molecular characterization provided an understanding of the molecular–function relationships for the salt tolerance and high activity of the GH 20 GlcNAcase.

Keywords

β-N-Acetylglucosaminidase Activity N-Acetylglucosaminide tolerance Salt tolerance Microbacterium

Background

β-N-acetylglucosaminidases (GlcNAcases, EC 3.2.1.52) are glycoside hydrolases (GHs) that cut the O-glycosidic bonds formed by N-acetylglucosamine (GlcNAc) residues from the non-reducing terminals of oligosaccharides, such as chitooligosaccharides and muropeptides [1, 2]. Chitooligosaccharides can be produced from the degradation of chitin by hydrochloric acid [3] or chitinases (EC 3.2.1.14) [2]. Chitin is second only to lignocellulose in natural abundance, but more than 80,000 tons of chitin from marine sources per year is unutilized chitinous waste [2]. GlcNAcases hydrolyse chitooligosaccharides to produce GlcNAc, which can be further used for the production of bioethanol [4] and single cell protein [5], the treatment of ulcerative colitis and other gastrointestinal inflammation disorders [6] and pharmaceutical therapy for osteoarthritis [7] and tumours [8]. Muropeptides can be derived from the peptidoglycan of the cell wall [1] and flagella [9]. The hydrolysis of muropeptides by GlcNAcases indicates that the enzymes function in cell wall recycling [1] and flagellum assembly of bacteria [9]. Furthermore, GlcNAcases also exhibit many additional important biological functions and wide range of industrial applications, such as catabolism of ganglioside storage in Tay-Sachs disease [10], induction of chitinolytic enzymes [11] and synthesis of biologically important oligosaccharides [12, 13].

According to a homology comparison of amino acid sequences, GlcNAcases have been classified into GH 3, 20, 73 and 84 (http://www.cazy.org/). With the rapid development of genome sequencing technology, many GlcNAcases have been identified in the genomes of animal tissues, insects, plants, bacteria and fungi in recent years. GH 20 GlcNAcases are mainly found in microorganisms, especially bacteria (http://www.cazy.org/GH20.html). However, the molecular and biochemical characteristics of the majority of these GH 20 GlcNAcases have not been reported (http://www.cazy.org/GH20.html).

Yunnan Province is known as “the Kingdom of Nonferrous Metals”, “the Kingdom of Plants” and “the Kingdom of Animals” in China. In these natural habitats containing metal ions in high concentrations or special products from plants or animals, novel microorganisms and their enzymes may have to evolve, indicating biotechnological potential. Heijing is called the “town of salt” in Yunnan and has produced salts since ancient China. We previously sampled saline soil from an old mine in the town and reported novel enzymes from bacteria isolated from the soil [1417]. One example is the GH 32 invertase InvHJ14, showing novel low-temperature and alkaline activity and sucrose tolerance [15]; another is the GH 27 α-galactosidase AgaAHJ8, showing novel salt − protease tolerance and transglycosylation activity, from a potential novel species of Pontibacter [16].

In this study, a novel GH 20 GlcNAcase, called HJ5Nag, was mined from the genome of Microbacterium sp. HJ5—a new isolate from the saline soil. The purified recombinant HJ5Nag (rHJ5Nag), which was expressed in E. coli, showed better tolerance to salts and GlcNAc and higher activity than most GlcNAcases reported in the literature, including the GH 3 GlcNAcase we previously reported [18]. Potential molecular adaptations for salt tolerance and the high activity of the enzyme were presumed.

Methods

Strains, vectors and reagents

Strain HJ5 was isolated from the saline soil of an old salt mine located in Heijing Town. The details of the strain isolation and identification were described in our previous study [14]. The pure culture was deposited in the Strains Collection of the Yunnan Institute of Microbiology under registration no. YMF 4.00007. E. coli BL21 (DE3) was purchased from TransGen (Beijing, China) and was used for gene expression.

The following vectors and reagents were used: plasmid isolation and genomic DNA kits (Tiangen, Beijing, China); dNTPs and DNA polymerases (TaKaRa, Otsu, Japan); isopropyl-β-d-1-thiogalactopyranoside (IPTG; Amresco, Solon, OH, USA); Ni2+-NTA agarose (Qiagen, Valencia, CA, USA); Qubit protein assay kit (Invitrogen, Carlsbad, CA, USA); pEASY-E2 vector (TransGen); N,N'-diacetyl chitobiose (GlcNAc2), N,N',N'',N'''-tetraacetyl chitotetraose (GlcNAc4), p-nitrophenyl β-N-acetylgalactosaminide (pNPGalNAc), and p-nitrophenyl β-d-glucopyranoside (pNPGlc) (J&K Scientific Ltd., Beijing, China); peptidoglycan from Bacillus subtilis (Ekear, Shanghai, China); p-nitrophenol (pNP), p-nitrophenyl-β-d-xylopyranoside (pNPXyl), p-nitrophenyl α-d-galactopyranoside (pNPGal), p-nitrophenyl-α-l-arabinofuranoside (pNPAra), p-nitrophenyl β-N-acetylglucosaminide (pNPGlcNAc), mutanolysin from Streptomyces globisporus ATCC 21553, chitin and chitosan (Sigma − Aldrich, St. Louis, MO, USA); and silica gel G plates (Haiyang, Qingdao, China). All other chemicals were of analytical grade.

Gene cloning and sequence analysis

The GlcNAcase-encoding gene, designated hj5Nag, was obtained by genome sequencing performed on a Miseq sequencer (Illumina) in our lab. The HJ5 genome sequencing details were described in our previous study [19].

Open reading frames from the draft genome of HJ5 were predicted using the tools described in our previous study [17]. BLASTN and BLASTP (http://www.ncbi.nlm.nih.gov/BLAST/) online tools were used to search homologous sequences and to calculate the identity values. SignalP (http://www.cbs.dtu.dk/services/SignalP/) and InterPro (http://www.ebi.ac.uk/interpro/) online tools were used to predict the signal peptide and protein domain, respectively. Vector NTI 10.3 (InforMax, Gaithersburg, MD, USA) software was used to calculate frequencies of the amino acid residues. Multiple sequences and structures alignments were generated by ESPript [20].

Structure analysis

The homology model of HJ5Nag was predicted using the SwissModel platform (http://swissmodel.expasy.org/). The proportions of secondary structures were analysed by VADAR [21]. The charge distributions on the surfaces of various GlcNAcases were calculated using Discovery Studio 2.5 software (Accelrys, San Diego, CA, USA).

Heterologous expression of HJ5Nag

The coding sequence of HJ5Nag was amplified by PCR using the primer set rhj5NagEF (5'-GGGTGCAGCCCCGCCGC-3') and rhj5NagER (5'-CTCGGTGGCCCAGTCGATCTCGC-3'). The resulting PCR product was ligated to pEASY-E2 vector, which has a single 3′-T overhang at the insertion site according to the manufacturer. E. coli BL21 (DE3) competent cells were transformed with the plasmid for recombinant enzyme expression. A positive transformant harbouring the recombinant plasmid was confirmed by DNA sequencing performed by Tsingke (Beijing, China). A seed culture of the positive transformant was grown overnight at 37 °C and then inoculated in 1:100 dilutions into fresh Luria–Bertani medium with the addition of 100 μg mL−1 ampicillin. Upon reaching an OD600 nm of approximately 0.7, IPTG at a final concentration of 0.25 mM was added to the culture to induce enzyme expression at 20 °C for approximately 20 h.

Purification and identification of recombinant GlcNAcase

Cultures containing positive transformant cells were centrifuged and resuspended in ice-cold buffer A containing 20 mM Tris–HCl, 0.5 M NaCl, and 10% (w/v) glycerol (pH 7.2). The cells were disrupted by ultrasonication on ice with 100 short bursts of 4 s each at a power output of 150 W. After removing cell debris by centrifugation, the supernatant was loaded onto Ni2+-NTA agarose gel columns to bond the recombinant enzyme. The target recombinant enzyme was eluted with a linear imidazole gradient of 20–500 mM in buffer A. The protein concentration was determined using a Qubit protein assay kit using a Qubit 2.0 fluorometer (Invitrogen).

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed to analyse the expression of the recombinant enzyme and the purity of the eluted fractions. The molecular masses of internal peptides from the single band present in the SDS–PAGE gel were analysed via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) and were compared with the molecular masses of the internal peptides from rHJ5Nag.

Enzyme assay and substrate specificity

An enzyme assay of purified rHJ5Nag towards various substrates was performed spectrophotometrically using the pNP and 3,5-dinitrosalicylic acid (DNS) methods, as described in our previous study [18]. The pNP method was applied to determine the activity of rHJ5Nag towards substrates pNPXyl, pNPGal, pNPGlc, pNPAra, pNPGlcNAc and pNPGalNAc. The DNS method was used for the substrates GlcNAc2, GlcNAc4, colloidal chitin, chitosan, peptidoglycan and muropeptides (degradation products of peptidoglycan by mutanolysin treatment).

Biochemical characterization

The pNP method using pNPGlcNAc as substrate was applied to determine the biochemical characteristics of purified rHJ5Nag, unless otherwise noted. McIlvaine buffer was used for pH 5.0–8.0, and 0.1 M glycine–NaOH was used for pH 9.0–12.0.

The pH-dependent activity of rHJ5Nag was determined at 37 °C, with pH values ranging from 5.0 to 9.0. The pH-dependent stability of rHJ5Nag was determined by measuring the residual enzyme activity after incubating the enzyme at pH 5.0–12.0 for 1 h.

The temperature-dependent activity of rHJ5Nag was examined at pH 6.0 and temperatures from 0 to 60 °C. The thermostability of rHJ5Nag was determined by measuring the residual activity of the enzyme after pre-incubation at 30, 45 and 50 °C and pH 6.0 for 1–60 min.

To determine the effects of various metal ions and chemical reagents on purified rHJ5Nag, the enzyme activities were measured after the addition of different metal ions and chemical reagents to the reaction mixture. The metal ions and chemical reagents included 1.0 or 10.0 mM (final concentration) KCl, AgNO3, CaCl2, CoCl2, HgCl2, Pb(CH3COO)2, MgSO4, NiSO4, CuSO4, MnSO4, ZnSO4, FeCl3, EDTA, β-mercaptoethanol and SDS, and 0.5 or 1.0% (v/v) Tween 80 and Triton X-100. The absorption was measured after removing the precipitate by centrifugation when AgNO3, HgCl2 or SDS was added to the reaction mixture. Furthermore, the enzyme activity in 1.0–30.0% (w/v) NaCl was also examined, and the enzyme stability in NaCl was determined by measuring the residual enzyme activity after pre-incubating the enzyme in 3.0–30.0% (w/v) NaCl for 1 h.

The K m, V max and k cat values of purified rHJ5Nag were analysed using 0.1–1.2 mM pNPGlcNAc as substrate at 45 °C and pH 6.0. A nonlinear regression Michaelis − Menten fit was applied to analyse the data using GraphPad Prism (GraphPad Software, San Diego, CA, USA). The extent of GlcNAc inhibition on rHJ5Nag activity was determined adding 5.0 and 10.0 mM GlcNAc to the reaction solution. Then, the β-N-acetylglucosaminidase activity was determined according to the aforementioned kinetic assay. The inhibition constant (K i) was calculated using GraphPad Prism using the nonlinear mixed-model inhibition equation described detailedly in a previous paper [22].

Hydrolytic property of purified rHJ5Nag

The hydrolysis mixture included 160 μL of 0.5% (w/v) GlcNAc2 or GlcNAc4 and approximately 0.1 units of purified rHJ5Nag. After incubating the mixture in McIlvaine buffer (pH 6.0) at 37 °C for 6 h, the hydrolysis products were analysed by thin-layer chromatography (TLC), as previously described [17].

Synergistic action

Commercial chitinase from Streptomyces griseus, designated CtnSg, was purchased from YuanYe Bio-Technology (Shanghai, China). The reaction mixture contained colloidal chitin (0.5%, w/v) and either CtnSg (0.01 U mL−1 reaction system), rHJ5Nag (0.5 U mL−1 reaction system) or both enzymes and was incubated for 120 min. In the time-course study, the reaction mixture containing 0.5% (w/v) colloidal chitin and CtnSg alone was incubated for 30, 60 or 90 min, rHJ5Nag was then added to the reaction and incubated for 90, 60 or 30 min, respectively. According to the manufacturer’s instructions for CtnSg and the biochemical characteristics of rHJ5Nag, degradation of colloidal chitin was performed at 25 °C and pH 6.0 (McIlvaine buffer). The DNS method was used to measure the amount of reducing sugars released from the colloidal chitin. A reaction containing heat-inactivated enzyme (incubating in a boiling water bath for 10 min) was used as a control. The degree of synergy was calculated using the following equations:
$$ D S=\frac{R{S}_{rHJ5 Nag CtnSg}}{R{S}_{rHJ5 Nag}+ R{S}_{CtnSg}} $$
where DS is the degree of synergy, RS rHJ5NagCtnSg is the amount of reducing sugars released from simultaneous or sequential enzyme reactions, and RS rHJ5Nag and RS CtnSg are the amounts of reducing sugars released from individual enzyme reactions.

Accession numbers

The accession numbers of hj5Nag and Microbacterium sp. HJ5 16S rDNA in GenBank are KX400857 and KX400858, respectively.

Results

Strain identification

Based on the results of a BLASTN search, the nucleotide identity was 98.8% between the partial 16S rDNA sequence from HJ5 (1375 bp) and the 16S rDNA sequences from Microbacterium flavescens (accession no. HQ530520), Microbacterium takaoensis (AB201047) and Microbacterium trichothecenolyticum (KU179351). Phylogenetic analysis also placed HJ5 in the Microbacterium cluster but not in the clusters grouped by the species of other genera of Microbacteriaceae (Additional file 1: Figure S1). Therefore, HJ5 belonged to the genus Microbacterium.

Gene cloning and sequence analysis

Sequence data of approximately 606.6 Mbp was generated by genome sequencing for HJ5, and these data yielded a draft genomic DNA sequence of approximately 3.6 Mbp after sequence assembly. The analysis of the draft genome predicted an open reading frame designated gene hj5Nag. hj5Nag has a length of 1608 bp and encodes the 535-residue GlcNAcase HJ5Nag, with a predicted molecular mass of 55.9 kDa.

The alanine (A) and glycine (G) frequencies of the GlcNAcase were 16.3 and 10.8%, respectively, both of which are the highest values among the GlcNAcases shown in Table 1. The frequency of acidic aspartic acid (D) and glutamic acid (E) of the GlcNAcase was 13.5%, which was the third highest (Table 1). However, frequencies of cysteine (C) and lysine (K) in the GlcNAcase were only 0.2 and 1.1%, respectively, both of which were the second lowest (Table 1). Therefore, the ratio of the total frequency of D, E, A and G to the total frequency of C and K of HJ5Nag (31.2) was the highest among the GlcNAcases shown in Table 1.
Table 1

Amino acid residue frequencies of the experimentally characterized GH 20 GlcNAcases

GlcNAcase (PDB ID or accession no.)

Frequency (%)

Organism

D & E

A

G

C

K

DEAG/CK

ADJ68332

14.3

7.7

6.9

0.0

4.7

6.1

Vibrio harveyi [71]

AAC44672

13.7

8.2

5.2

1.6

3.8

5.0

Vibrio furnissii [37]

KX400857

13.5

16.3

10.8

0.2

1.1

31.2

Microbacterium sp. (This study)

ADJ68333

13.5

7.6

5.6

1.9

4.5

4.2

V. harveyi [71]

3RCN (WP_014923268)

13.4

11.2

10.1

0.2

1.7

18.3

Arthrobacter aurescens (Unpublished)

3NSM (ABI81756)

13.3

7.5

5.9

2.0

6.1

3.3

Ostrinia furnacalis [72]

ABA27426

13.0

4.5

6.5

1.3

5.1

3.8

Spodoptera frugiperda [70]

ABA27427

13.0

4.5

6.5

1.3

5.1

3.8

S. frugiperda [70]

4PYS

12.9

6.1

7.5

1.6

6.1

3.4

Bacteroides fragilis (Unpublished)

BAD00143

12.9

9.5

7.6

0.7

7.1

3.8

Aeromonas hydrophila [73]

CAH55822

12.9

9.7

7.6

0.7

7.1

3.9

Aeromonas caviae [48]

1QBA (AAB03808)

12.8

9.9

8.4

0.7

6.9

4.1

Serratia marcescens [23]

BAC76622

12.8

14.4

8.7

0.5

0.7

29.9

Streptomyces thermoviolaceus [42]

AAQ05800

12.7

12.1

8.9

0.0

1.2

28.1

Cellulomonas fimi [45]

1 M01 (AAC38798)

12.5

11.9

10.5

0.4

3.6

8.7

Streptomyces plicatus [74]

BAC41255

12.5

8.0

5.7

1.2

3.0

6.2

Aspergillus oryzae [12]

AKC34129

12.3

9.7

7.1

0.4

7.1

3.9

Uncultured organism [44]

BAA92145

12.2

10.1

9.7

0.7

4.3

6.4

Aeromonas sp. [75]

2GJX (AAD13932)

11.9

4.9

6.4

1.5

3.6

4.5

Homo sapiens [24]

BAF76001

11.8

9.1

8.3

1.8

2.4

7.0

A. hydrophila [76]

CAD10500

11.5

6.9

5.7

1.8

6.2

3.0

Entamoeba histolytica [77]

BAM42836

11.2

11.2

6.5

1.1

1.6

10.7

Lentinula edodes [78]

AKC34128

10.7

14.4

9.1

0.4

1.6

17.1

Uncultured organism [44]

3GH4 (BAI63641)

10.5

8.0

8.8

0.4

5.1

5.0

Paenibacillus sp. [25]

BAA88762

10.4

12.9

10.0

0.4

2.4

11.9

S. thermoviolaceus [46]

BAA06136

10.4

10.0

5.3

1.2

6.0

3.6

Pseudoalteromonas piscicida (previous Alteromonas sp.) [79]

BAB84321

10.4

8.3

5.1

0.4

4.9

4.5

P. piscicida (previous Alteromonas sp.) [80]

CAE46968

10.3

4.3

5.1

1.4

6.4

2.5

E. histolytica [77]

BAA76352

10.1

15.2

5.5

0.0

7.2

4.3

Lactobacillus casei [41]

EGR50812

8.9

10.0

6.4

1.0

3.4

5.8

Trichoderma reesei [49]

The predicted signal peptide of HJ5Nag is cleaved between A25 and A26 to yield a mature polypeptide consisting of the typical N-terminal domain of bacterial GlcNAcases (from V14 to R153; domain signature: IPR015882) and the catalytic domain of GH 20 GlcNAcases (from F154 to V510; IPR015883; Fig. 1, Additional file 1: Figure S2). A BLASTP search for HJ5Nag showed that the enzyme was most similar to multiple putative GH 20 GlcNAcases annotated based on genome analysis. Among these putative GH 20 members, the enzyme from Microbacterium sp. Root180 (accession no. WP_056119055) shared the highest identity of 78.9% with HJ5Nag. Based on the alignment of HJ5Nag with other experimentally characterized GlcNAcases (Fig. 1) [2325], residue D334 probably functioned as the catalytic nucleophile/base, and E335 probably functioned as the catalytic proton donor/acceptor. In addition, residues D162, R165, D194, D195, W365, W430 and W465 might play an important role in binding the GlcNAcase ligand (Fig. 1) [2325].
Fig. 1

Partial amino acid sequences and structures alignments of HJ5Nag with GH 20 GlcNAcases. Sequence names are shown with PDB IDs (except HJ5Nag) as follows: 1QBA (accession no. AAB03808), the GlcNAcase from S. marcescens [23]; 2GJX (accession no. AAD13932), the GlcNAcase from H. sapiens [24]; and 3GH4 (accession no. BAI63641), the GlcNAcase from Paenibacillus sp. [25]. Asterisks and number signs show the putative catalytic residues and ligand-binding residues, respectively. Dotted purple line shows the N-terminal domain of bacterial GlcNAcases (domain signature: IPR015882). Solid purple line shows the catalytic domain of GH 20 GlcNAcases (domain signature: IPR015883)

Structure analysis

The model of HJ5Nag showed a GMQE score of 0.72 and a sequence identity of 48.9% when the template was the crystal structure of the GH 20 GlcNAcase from Streptomyces plicatus (PDB ID: 1 M01). The homology model of HJ5Nag and the crystal structures of 7 GlcNAcases were used to calculate the proportions of secondary structures and the charge distributions on the surfaces. Among these GlcNAcases, the proportions of amino acids used to build the α-helix and β-sheet structures of HJ5Nag were the second lowest, while the proportion of amino acids used to build random coil structures and the ratio of random coils to α-helices of HJ5Nag were both the highest (Table 2). As shown in Fig. 2, almost the whole surface of HJ5Nag was negatively charged. Compared with other GlcNAcases, HJ5Nag had a much larger negatively charged surface (Fig. 2).
Table 2

Proportions of secondary structures of GH 20 GlcNAcases

GlcNAcase (PDB ID)

α-Helix

β-Sheet

Coil

Coil/α-Helix

2GJX [24]

176 (35.8%)

133 (27.0%)

183 (37.2%)

1.04

3RCN (Unpublished)

185 (35.2%)

146 (27.8%)

194 (37.0%)

1.05

1 M01 [74]

170 (34.1%)

121 (24.2%)

208 (41.7%)

1.22

3NSM [72]

193 (33.7%)

162 (28.3%)

217 (37.9%)

1.12

4PYS (Unpublished)

165 (33.5%)

134 (27.2%)

193 (39.2%)

1.17

3GH4 [25]

165 (32.5%)

137 (27.0%)

205 (40.4%)

1.24

KX400857 (This study)

152 (30.2%)

126 (25.0%)

226 (44.8%)

1.49

1QBA [23]

232 (27.0%)

282 (32.9%)

344 (40.1%)

1.48

Fig. 2

Charge distributions on the surfaces of the GH 20 GlcNAcases. The A-chain of GlcNAcase is used. PDB IDs are presented as the names of GlcNAcases (except HJ5Nag) as follows: 1 M01, the GlcNAcase from S. plicatus [74]; 1QBA, the GlcNAcase from S. marcescens [23]; 2GJX, the GlcNAcase from H. sapiens [24]; 3GH4, the GlcNAcase from Paenibacillus sp. [25]; 3NSM, the GlcNAcase from O. furnacalis [72]; 3RCN, the GlcNAcase from A. aurescens; 4PYS, the GlcNAcase from B. fragilis. The charge distribution on the surface was calculated at pH 7.0. Positive charges are depicted in blue and negative charges in red

Expression, purification and identification of rHJ5Nag

As shown by an SDS–PAGE gel (Additional file 1: Figure S3), the recombinant GlcNAcase was successfully expressed in E. coli BL21 (DE3) and purified by Ni2+-NTA affinity chromatography. The MALDI–TOF MS spectrum of the purified band further confirmed that the purified product was rHJ5Nag (Additional file 1: Figure S4).

Biochemical characterization

At pH 6.0 and 45 °C, purified rHJ5Nag showed specific activities of 1773.1 ± 1.1 (Table 3), 146.0 ± 3.1, 481.4 ± 2.3 and 445.0 ± 1.7 μmol min−1 mg−1 towards substrates of pNPGlcNAc, pNPGalNAc, GlcNAc2 and GlcNAc4, respectively. However, no rHJ5Nag activity was detected towards substrates of pNPXyl, pNPAra, pNPGlc, pNPGal, peptidoglycan, muropeptides, colloidal chitin or chitosan. Thus, rHJ5Nag is a GlcNAcase that can hydrolyse chitooligosaccharides but cannot participate in cell wall turnover or recycling of bacteria.
Table 3

Activities of the experimentally characterized GlcNAcases

GlcNAcase

GH Family

Specific activitya

V max a

Organism

-

84

1.2

212

Penicillium chrysogenum [81]

NagA

20

1926.0

-

A. oryzae [12]

HJ5Nag

20

1773.1

3097

Microbacterium sp. (This study)

-

20

913.6

-

P. piscicida (previous Alteromonas sp.) [79]

Nag1

20

319.9

-

T. reesei [49]

ExoI

20

200

270

V. furnissii [37]

-

20

73.3

-

L. casei [41]

Sfhex

20

 

68.4

S. frugiperda [70]

NagC

20

44.7

-

S. thermoviolaceus [42]

NAG20A

20

30.0

115

A. hydrophila [73]

VhNag2

20

19.4

-

V. harveyi [82]

-

20

3.8

3.8

E. histolytica [77]

Hex1

20

-

212

Paenibacillus sp. [25]

Hex2

20

-

150

Paenibacillus sp. [25]

NagA

3

64.4

24.8

S. thermoviolaceus [83]

RmNag

3

21.2

49.3

Rhizomucor miehei [38]

Nag3HWLB1

3

19.4

19.9

Sphingobacterium sp. [18]

NahA

3

8.8

-

Symbiobacterium thermophilum [47]

ExoII

3

0.7

1.1

V. furnissii [68]

NagA

3

-

0.3

Thermotoga maritima [84]

CbsA

3

-

0.3

Thermotoga neapolitana [84]

-

-

521.5

-

Enterobacter sp. [85]

-

-

67.2

-

Vibrio alginolyticus [65]

-

-

40.5

-

Talaromyces emersonii [86]

-

-

27

2.0

Streptomyces cerradoensis [40]

NagA

-

5.3

245

Pseudomonas fluorescens [3]

 

-

0.03

-

Trichinella spiralis [64]

aThese values (μmol min−1 mg−1) were determined using pNPGlcNAc as substrate

Purified rHJ5Nag had activity in the range of pH 5.5–9.0 and 10 − 50 °C, with apparent optima at pH 6.0 and 45 °C (Fig. 3a, c). The enzyme exhibited good stability at 30 °C and maintained more than 55% of its initial activity after incubation in buffers ranging from pH 6.0 to 10.0 for 1 h (Fig. 3b, d).
Fig. 3

Enzymatic properties of purified rHJ5Nag. a pH-dependent activity profiles. b pH-dependent stability profiles. c Temperature-dependent activity profiles. d Thermostability assay. e Effect of NaCl on rHJ5Nag activity. f Stability of rHJ5Nag in NaCl. Error bars represent the means ± SD (n = 3)

Purified rHJ5Nag exhibited good salt tolerance. Although the activity of purified rHJ5Nag was completely inhibited by AgNO3, HgCl2 and SDS, the addition of most metal ions and chemical reagents at final concentrations of 1.0 and 10.0 mM or 0.5 and 1.0% (v/v) showed little to no effect (retaining 84.5 − 131.5% activity) on the enzyme activity (Table 4). Furthermore, the enzyme retained more than 80% activity in the presence of 1.0–10.0% (w/v) NaCl and even 53.6% activity in 20.0% (w/v) NaCl (Fig. 3e). The enzyme retained approximately 100% of its initial activity after incubation in 3.0–20.0% (w/v) NaCl for 60 min (Fig. 3f).
Table 4

Effects of metal ions and chemical reagents on the activity of purified rHJ5Nag

Reagent

Relative activity (%)a

Reagent

Relative activity (%)a

1.0 mM

10.0 mM

1.0 mM

10.0 mM

None

100.0 ± 0.4

100.0 ± 2.7

MnSO4

100.9 ± 2.6

99.6 ± 2.4

FeCl3

117.4 ± 1.8

103.1 ± 0.0

ZnSO4

96.8 ± 3.1

84.5 ± 9.9

MgSO4

113.8 ± 5.8

99.3 ± 3.4

AgNO3

0.0

0.0

CaCl2

108.7 ± 5.4

96.6 ± 3.5

HgCl2

0.0

0.0

KCl

105.4 ± 4.8

101.4 ± 1.3

Tween 80

131.5 ± 1.7b

108.1 ± 2.1c

CoCl2

103.7 ± 3.1

100.4 ± 1.7

EDTA

106.7 ± 4.5

99.5 ± 2.1

NiSO4

101.7 ± 8.7

95.0 ± 0.7

β-Mercaptoethanol

106.1 ± 1.2

100.1 ± 0.6

CuSO4

101.4 ± 4.8

88.6 ± 5.5

Triton X-100

100.5 ± 4.7b

93.9 ± 5.2c

Pb(CH3COO)2

101.0 ± 5.8

106.7 ± 3.7

SDS

0.0

0.0

aValues represent the means ± SD (n = 3) relative to the untreated control sample

bFinal concentration: 0.5% (w/v)

cFinal concentration: 1.0% (w/v)

Without the addition of GlcNAc, the K m, V max, k cat and k cat/K m of purified rHJ5Nag towards pNPGlcNAc were 0.52 ± 0.05 mM, 3097 ± 124 μmol min−1 mg−1, 2886 ± 116 s−1 and 5550 ± 223 mM−1 s−1, respectively (Table 3; Fig. 4). When 5.0 and 10.0 mM GlcNAc was added to the reaction solution, the V max of purified rHJ5Nag towards pNPGlcNAc decreased to 2732 ± 91 and 2606 ± 87 μmol min−1 mg−1, respectively, the k cat decreased to 2546 ± 84 and 2429 ± 81 s−1, respectively, and the k cat/K m decreased to 4106 ± 135 and 3327 ± 111 mM−1 s−1, respectively (Fig. 4). However, the K m of purified rHJ5Nag towards pNPGlcNAc increased to 0.62 ± 0.04 and 0.73 ± 0.05 mM in the presence of 5.0 and 10.0 mM GlcNAc, respectively (Fig. 4). Furthermore, the value of the parameter Alpha in the mixed-model inhibition equation was 3.412 based on the analysis of kinetic data using GraphPad Prism. These results indicate that GlcNAc can inhibit the activity of rHJ5Nag via a mixture of competitive and noncompetitive inhibition. As a result, the enzyme was inhibited by GlcNAc, with a K i of 14.59 mM.
Fig. 4

Kinetic characterization of purified rHJ5Nag. Error bars represent the means ± SD (n = 3)

Hydrolytic properties of purified rHJ5Nag

As shown in Fig. 5, GlcNAc was the end product of GlcNAc2 and GlcNAc4 hydrolysed by purified rHJ5Nag.
Fig. 5

TLC analysis of the hydrolytic product. Lanes: M, 0.5% (w/v) GlcNAc; CK2 and CK4, inactivated rHJ5Nag (incubating in a boiling water bath for 10 min) with 0.5% (w/v) GlcNAc2 and GlcNAc4, respectively; S2 and S4, hydrolysis of 0.5% (w/v) GlcNAc2 and GlcNAc4 by purified rHJ5Nag, respectively

Synergistic action

Although purified rHJ5Nag cannot hydrolyse colloidal chitin alone, simultaneous addition of CtnSg and rHJ5Nag improved the degradation of colloidal chitin by 2.02-fold (Table 5). Sequential addition of the two enzymes improved the degradation of colloidal chitin by 1.98-fold, 1.72-fold and 1.29-fold when rHJ5Nag was added and incubated for 90, 60 and 30 min, respectively (Table 5). Furthermore, these results indicate that the synergistic effect benefits from the earlier addition of rHJ5Nag.
Table 5

Synergy between the commercial chitinase CtnSg and rHJ5Nag for the degradation of colloidal chitin

Order of enzyme addition and reaction time

Reducing sugars (μmol)

Degree of synergya

First enzyme

Time (min)

Second enzyme

Time (min)

CtnSg

120

No

0

0.28

1.00

rHJ5Nag

120

No

0

NDb

-

CtnSg & rHJ5Nag

120

No

0

0.57

2.02

CtnSg

30

rHJ5Nag

90

0.55

1.98

CtnSg

60

rHJ5Nag

60

0.48

1.72

CtnSg

90

rHJ5Nag

30

0.36

1.29

aThe degree of synergy was calculated using the following equations:

\( D S=\frac{R{S}_{rHJ5 Nag CtnSg}}{R{S}_{rHJ5 Nag}+ R{S}_{CtnSg}} \)

where DS is the degree of synergy, RS rHJ5NagCtnSg is the amount of reducing sugars released from simultaneous or sequential enzyme reactions, and RS rHJ5Nag and RS CtnSg are the amounts of reducing sugars released from individual enzyme reactions

bNot detected

Discussion

Microorganisms isolated from extremophilic locations are of great interest and have attracted substantial attention. Microbacterium strains have been found in various extremophilic locations, including Antarctic habitats [26], the Atacama Desert [27], sediment of the Indian Ocean [28], uranium-rich soil [29] and salty soil [30]. To be active in these extremophilic locations, enzymes from local Microbacterium strains may have to evolve. To the best of our knowledge, although some pioneering studies have revealed the important functions of some enzymes from Microbacterium strains in recent years [3136], few of these enzymes were found to have adapted to extremophilic locations [32]. Furthermore, we found that all the GlcNAcases from Microbacterium strains were annotated based on in silico analysis without biochemical characterization. Therefore, the study may be the first to report an experimentally characterized GlcNAcase from Microbacterium.

Drastic inhibition by Ag+ and Hg2+ appears to be the common property of GlcNAcases, including HJ5Nag, the GH 20 GlcNAcases from Paenibacillus [25] and V. furnissii [37], the GH 3 GlcNAcases from R. miehei [38] and Sphingobacterium sp. [18], the GH 84 GlcNAcase from C. paraputrificum [39] and the GlcNAcase from S. cerradoensis [40]. Ag+ and Hg2+ have irreversible effects on sulfhydryl groups, and Hg2+ can also oxidize indole rings. Therefore, cysteine or tryptophan residues may be involved in the catalytic activity of GH 20 GlcNAcases. Sequence analysis revealed that W365, W430 and W465 of HJ5Nag might affect ligand binding. As shown in Fig. 1, the conserved W491 may also be essential for the activity of HJ5Nag. SDS is an inhibitor of GlcNAcases, in addition to Ag+ and Hg2+. All the GlcNAcases from Microbacterium sp. in this study, as well as R. miehei [38], Sphingobacterium sp. [18] and L. casei [41], are strongly inhibited by SDS. Purified rHJ5Nag showed peak activity at pH 6.0 and 45 °C. These properties are the same or similar to those of many GlcNAcases. For example, the GH 20 GlcNAcase from S. thermoviolaceus presents maximum activity at pH 6.0 and 50 °C [42], the GH 20 GlcNAcase from H. latex presents maximum activity at pH 6.0 and 45–50 °C [43], the GH 3 GlcNAcase from R. miehei presents maximum activity at pH 6.5 and 50 °C [38], and the GH 84 GlcNAcase from Clostridium paraputrificum presents maximum activity at pH 6.5 and 50 °C [39]. Furthermore, the molecular mass of HJ5Nag (55.9 kDa) is similar to that of some GlcNAcases, such as the GH 20 GlcNAcases from a soil-derived uncultured organism (53.4 kDa) [44], C. fimi (54.2 kDa) [45], Paenibacillus sp. (57.5 kDa) [25] and S. thermoviolaceus (58.3 kDa) [46], and the GH 3 GlcNAcase from S. thermophilum (55.9 kDa) [47].

Purified rHJ5Nag exhibited good salt tolerance, as the addition of most salts had little to no effect on the enzyme activity, especially CuSO4 and NaCl. Most GlcNAcases reported to date are strongly inhibited by Cu2+, such as the GH 20 GlcNAcases from Paenibacillus [25], A. caviae [48] and L. casei [41], the GH 3 GlcNAcase from R. miehei [38], the GH 84 GlcNAcase from C. paraputrificum [39] and the GlcNAcase from S. cerradoensis [40]. Furthermore, the GH 20 GlcNAcases from Paenibacillus are strongly inhibited by Ni2+ [25]; the GH20 GlcNAcase from A. caviae is strongly inhibited by Mn2+ [48]; the GH20 GlcNAcase from T. reesei is inhibited by Fe3+ [49]; the GH 84 GlcNAcase from C. paraputrificum is inhibited by Ni2+, Co2+, Mn2+, Zn2+ and Fe3+ [39]; and the GH 3 GlcNAcase from R. miehei is inhibited by Ni2+, Co2+, Zn2+ and EDTA [38]. Salt tolerance has attracted considerable attention because various salts are present or used in industries [14, 16, 18, 5060], such as various marine materials and foods in China, with NaCl contents of 3.5–20.0% (w/w) [16, 18]. In addition, material processing and fermentation with a high concentration of NaCl is helpful to reduce the overall cost because sterilization is unnecessary [61].

To reveal the potential catalytic adaptations of rHJ5Nag to high concentrations of salt, sequence and structure comparisons to its homologs were performed. (I). Similar to the results from other salt-tolerant enzymes [50, 51, 5359], statistical analysis of the amino acid residues showed that HJ5Nag presented higher proportions of acidic D and E along with A and G but lower proportions of C and K than its homologs (Table 1). Carboxyl groups from acidic amino acid residues can bind more water molecules and cations than other groups from other amino acid residues, especially K, and thus enable the protein to form a particle stabilized by cooperative hydrated ion networks [57, 58]. Hydrophobicity must be finely balanced to maintain a protein’s flexibility and stability [62]. High NaCl concentrations strengthen hydrophobic interactions, which can affect protein folding [62]. Salt-tolerant proteins are thought to reduce the size of hydrophobic residues to compensate for the increased hydrophobic effect caused by the high salt concentration [58]. The high proportion of A and G, which are small hydrophobic residues [57], is compatible with that function. In addition, a low abundance of C might give a protein more flexibility in high salt environments due to the decrease in rigid disulphide bridges [56]. (II). At the level of the secondary structure, a higher percentage of random coil structures at the expense of α-helices is observed for salt-tolerant proteins [56, 58], including HJ5Nag (Table 2). Greater protein rigidity is usually accompanied by a higher percentage of α-helix structures, while the opposite is observed for a higher percentage of random coils. (III). At the level of the tertiary structure, an excess of negatively charged surfaces is a typical property of salt-tolerant proteins [50, 51, 53, 5659, 62, 63]. The increase in negative charge on the surface can counteract the lower dielectric constant at high salinity and improve the ability of enzymes to bind water and salt ions, thus improving protein solubility [5658].

Most characterized GlcNAcases, including GH 3, 20 and 84 members, show specific activities or V max of lower than 1000 μmol min−1 mg−1 towards pNPGlcNAc, as summarized in Table 3. In this study, rHJ5Nag exhibited a specificity activity of 1773.1 ± 1.1 μmol min−1 mg−1 and a V max of 3097 ± 124 μmol min−1 mg−1 towards pNPGlcNAc. Furthermore, rHJ5Nag showed a specificity activity of 481.4 ± 2.3 μmol min−1 mg−1 towards GlcNAc2, which is much higher than the values from other GlcNAcases recorded in the literature, such as the values from Sphingobacterium sp. (0.3 μmol min−1 mg−1) [18], T. spiralis (0.79 nmol min−1 mg−1) [64], R. miehei (1.06 μmol min−1 mg−1) [38], V. alginolyticus (18.1 μmol min−1 mg−1) [65] and S. thermophilum (76.8 μmol min−1 mg−1) [47]. As discussed above, the structure of HJ5Nag may be more flexible because it has higher proportions of A, G and random coil structures but lower proportions of C and α-helix structures than its homologs. Furthermore, HJ5Nag has a higher proportion of D and E, which are not only negatively charged but also flexible residues. High B-factor values, which are believed to provide high structural flexibility, are usually observed for D and E. The high flexibility is presumed to be a factor in the high activity of the GH 20 GlcNAcase rHJ5Nag.

Although the synergistic action of chitinases and GlcNAcases for complete enzymatic degradation of chitin is attractive, there are few reports [18, 66, 67]. This study may be the first to report a synergistic effect between a chitinase and a GH 20 GlcNAcase. Significant synergy was observed between the commercial CtnSg and rHJ5Nag for chitin degradation, indicating that rHJ5Nag has great potential in the bioconversion of chitin waste. The tolerance of rHJ5Nag to GlcNAc may be one contributor to the significant synergy because GlcNAc can inhibit GlcNAcases and result in the accumulation of chitooligosaccharides, which can inhibit chitinases [38]. The GlcNAcases from V. furnissii, Trichoderma harzianum, A. oryzae, S. frugiperda, R. miehei and T. spiralis were competitively or noncompetitively inhibited by GlcNAc, with K i values of 0.21 [68], 0.21 [69], 1.6 [13], 2.0 [70], 9.68 [38] and 15.75 mM [64], respectively. In this study, rHJ5Nag showed relatively high GlcNAc tolerance, with a K i of 14.59 mM.

Conclusions

A GH 20 GlcNAcase was isolated from Microbacterium sp. HJ5 harboured in the saline soil of an abandoned salt mine. Biochemical characterization revealed that the GlcNAcase had novel salt–GlcNAc tolerance and high activity. These characteristics suggest that the enzyme has versatile potential in biotechnological applications, such as bioconversion of chitin waste and processing of marine materials and saline foods. Molecular characterization provides an understanding of the molecular–function relationships for the salt tolerance and the high activity of the GH 20 GlcNAcase.

Abbreviations

A: 

Alanine

Buffer A: 

20 mM Tris–HCl, 0.5 M NaCl, 10% (w/v) glycerol, pH 7.2

C: 

Cysteine

D: 

Aspartic acid

DNS: 

3,5-dinitrosalicylic acid

DS

Degree of synergy

E: 

Glutamic acid

G: 

Glycine

GH: 

Glycoside hydrolase

GlcNAc: 

N-acetylglucosamine

GlcNAc2

N,N'-diacetyl chitobiose

GlcNAc4

N,N',N'',N'''-tetraacetyl chitotetraose

GlcNAcases: 

β-N-acetylglucosaminidases

HJ5Nag: 

The GlcNAcase from Microbacterium sp. HJ5

hj5Nag

The GlcNAcase-encoding gene from Microbacterium sp. HJ5

IPTG: 

Isopropyl-β-d-1-thiogalactopyranoside

K: 

Lysine

MALDI-TOF MS: 

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

pNP: 

p-nitrophenol

pNPAra: 

p-nitrophenyl-α-l-arabinofuranoside

pNPGal: 

p-nitrophenyl α-d-galactopyranoside

pNPGalNAc: 

p-nitrophenyl β-N-acetylgalactosaminide

pNPGlc: 

p-nitrophenyl β-d-glucopyranoside

pNPGlcNAc: 

p-nitrophenyl β-N-acetylglucosaminide

pNPXyl: 

p-nitrophenyl-β-d-xylopyranoside

rHJ5Nag: 

The recombinant HJ5Nag

RS CtnSg

The amount of reducing sugars released by CtnSg from colloidal chitin without the addition of rHJ5Nag

RS rHJ5Nag

The amount of reducing sugars released by rHJ5Nag from colloidal chitin without the addition of CtnSg

RS rHJ5NagCtnSg

The amount of reducing sugars released from simultaneous or sequential enzyme reactions when determining the synergistic action

SDS-PAGE: 

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

TLC: 

Thin-layer chromatography

Declarations

Acknowledgements

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (Grant no. 31260215, 31660445), the Yunling Scholars (Grant no. 2015 56), the Yunling Industry Leading Talents (Grant no. 2014 1782), the Reserve Talents Project for Young and Middle-Aged Academic and Technical Leaders of Yunnan Province (Grant no. 2015HB033) and the Applied and Basic Research Foundation of Yunnan Province (Grant no. 201401PC00224).

Authors’ contributions

JZ conceived of the idea, designed the experiments, performed data analysis and wrote the manuscript. ZS and RL performed the experiments. RZ helped perform data analysis and write the manuscript. QW, JL, XT, BX, JD and NH coordinated the study. ZH conceived of the idea, designed the experiments and coordinated the study. All authors read and approved the final manuscript.

Availability of data and materials

The data supporting our findings can be found in the main paper and supplemental material.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University
(2)
College of Life Sciences, Yunnan Normal University
(3)
Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment
(4)
Key Laboratory of Enzyme Engineering, Yunnan Normal University

References

  1. Litzinger S, Duckworth A, Nitzsche K, Risinger C, Wittmann V, Mayer C. Muropeptide rescue in Bacillus subtilis involves sequential hydrolysis by β-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase. J Bacteriol. 2010;192(12):3132–43.View ArticleGoogle Scholar
  2. Patil RS, Ghormade V, Deshpande MV. Chitinolytic enzymes: an exploration. Enzyme Microb Tech. 2000;26(7):473–83.View ArticleGoogle Scholar
  3. Park JK, Kim WJ, Park YI. Purification and characterization of an exo-type β-N-acetylglucosaminidase from Pseudomonas fluorescens JK-0412. J Appl Microbiol. 2011;110(1):277–86.View ArticleGoogle Scholar
  4. Cody RM, Davis ND, Lin J, Shaw D. Screening microorganisms for chitin hydrolysis and production of ethanol from amino sugars. Biomass. 1990;21(4):285–95.View ArticleGoogle Scholar
  5. Vyas P, Deshpande M. Enzymatic hydrolysis of chitin by Myrothecium verrucaria chitinase complex and its utilization to produce SCP. J Gen Appl Microbiol. 1991;37(3):267–75.View ArticleGoogle Scholar
  6. Salvatore S, Heuschkel R, Tomlin S, Davies SE, Edwards S, Walker-Smith JA, et al. A pilot study of N-acetyl glucosamine, a nutritional substrate for glycosaminoglycan synthesis, in paediatric chronic inflammatory bowel disease. Aliment Pharm Ther. 2000;14(12):1567–79.View ArticleGoogle Scholar
  7. Shikhman AR, Amiel D, D’Lima D, Hwang SB, Hu C, Xu A, et al. Chondroprotective activity of N-acetylglucosamine in rabbits with experimental osteoarthritis. Ann Rheum Dis. 2005;64(1):89–94.View ArticleGoogle Scholar
  8. Suzuki K, Mikami T, Okawa Y, Tokoro A, Suzuki S, Suzuki M. Antitumor effect of hexa-N-acetylchitohexaose and chitohexaose. Carbohyd Res. 1986;151:403–8.View ArticleGoogle Scholar
  9. Herlihey FA, Moynihan PJ, Clarke AJ. The essential protein for bacterial flagella formation FlgJ functions as a β-N-acetylglucosaminidase. J Biol Chem. 2014;289(45):31029–42.View ArticleGoogle Scholar
  10. Okada S, Obrien JS. Tay–Sachs disease: generalized absence of a β-D-N-acetylhexosaminidase component. Science. 1969;165(3894):698–700.View ArticleGoogle Scholar
  11. Li HZ, Morimoto K, Katagiri N, Kimura T, Sakka K, Lun S, et al. A novel β-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose. Appl Microbiol Biot. 2002;60(4):420–7.View ArticleGoogle Scholar
  12. Matsuo I, Kim S, Yamamoto Y, Ajisaka K, Maruyama J, Nakajima H, et al. Cloning and overexpression of β-N-acetylglucosaminidase encoding gene nagA from Aspergillus oryzae and enzyme-catalyzed synthesis of human milk oligosaccharide. Biosci Biotech Bioch. 2003;67(3):646–50.View ArticleGoogle Scholar
  13. Rajnochova E, Dvorakova J, Hunkova Z, Kren V. Reverse hydrolysis catalysed by β-N-acetylhexosaminidase from Aspergillus oryzae. Biotechnol Lett. 1997;19(9):869–72.View ArticleGoogle Scholar
  14. Zhou JP, Gao YJ, Dong YY, Tang XH, Li JJ, Xu B, et al. A novel xylanase with tolerance to ethanol, salt, protease, SDS, heat, and alkali from actinomycete Lechevalieria sp. HJ3. J Ind Microbiol Biot. 2012;39(7):965–75.View ArticleGoogle Scholar
  15. Zhou JP, He LM, Gao YJ, Han NY, Zhang R, Wu Q, et al. Characterization of a novel low-temperature-active, alkaline and sucrose-tolerant invertase. Sci Rep. 2016;6:32081.View ArticleGoogle Scholar
  16. Zhou JP, Liu Y, Lu Q, Zhang R, Wu Q, Li CY, et al. Characterization of a glycoside hydrolase family 27 α-galactosidase from Pontibacter reveals its novel salt–protease tolerance and transglycosylation activity. J Agr Food Chem. 2016;64(11):2315–24.View ArticleGoogle Scholar
  17. Zhou JP, Lu Q, Zhang R, Wang YY, Wu Q, Li JJ, et al. Characterization of two glycoside hydrolase family 36 α-galactosidases: novel transglycosylation activity, lead–zinc tolerance, alkaline and multiple pH optima, and low-temperature activity. Food Chem. 2016;194:156–66.View ArticleGoogle Scholar
  18. Zhou JP, Song ZF, Zhang R, Ding LM, Wu Q, Li JJ, et al. Characterization of a NaCl-tolerant β-N-acetylglucosaminidase from Sphingobacterium sp. HWLB1. Extremophiles. 2016;20(4):547–57.View ArticleGoogle Scholar
  19. Zhou JP, Shen JD, Zhang R, Tang XH, Li JJ, Xu B, et al. Molecular and biochemical characterization of a novel multidomain xylanase from Arthrobacter sp. GN16 isolated from the feces of Grus nigricollis. Appl Biochem Biotech. 2015;175(1):573–88.View ArticleGoogle Scholar
  20. Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014;42(W1):W320–W4.View ArticleGoogle Scholar
  21. Willard L, Ranjan A, Zhang HY, Monzavi H, Boyko RF, Sykes BD, et al. VADAR: a web server for quantitative evaluation of protein structure quality. Nucleic Acids Res. 2003;31(13):3316–9.View ArticleGoogle Scholar
  22. Meux E, Morel M, Lamant T, Gerardin P, Jacquot JP, Dumarcay S, et al. New substrates and activity of Phanerochaete chrysosporium Omega glutathione transferases. Biochimie. 2013;95(2):336–46.View ArticleGoogle Scholar
  23. Tews I, Perrakis A, Oppenheim A, Dauter Z, Wilson KS, Vorgias CE. Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay–Sachs disease. Nat Struct Mol Biol. 1996;3(7):638–48.View ArticleGoogle Scholar
  24. Lemieux MJ, Mark BL, Cherney MM, Withers SG, Mahuran DJ, James MNG. Crystallographic structure of human β-hexosaminidase A: interpretation of Tay–Sachs mutations and loss of G(M2) ganglioside hydrolysis. J Mol Biol. 2006;359(4):913–29.View ArticleGoogle Scholar
  25. Sumida T, Ishii R, Yanagisawa T, Yokoyama S, Ito M. Molecular cloning and crystal structural analysis of a novel β-N-acetylhexosaminidase from Paenibacillus sp. TS12 capable of degrading glycosphingolipids. J Mol Biol. 2009;392(1):87–99.View ArticleGoogle Scholar
  26. Han SR, Kim KH, Ahn DH, Park H, Oh TJ. Complete genome sequence of carotenoid-producing Microbacterium sp. strain PAMC28756 isolated from an Antarctic lichen. J Biotechnol. 2016;226:18–9.View ArticleGoogle Scholar
  27. Mandakovic D, Cabrera P, Pulgar R, Maldonado J, Aravena P, Latorre M, et al. Complete genome sequence of Microbacterium sp. CGR1, bacterium tolerant to wide abiotic conditions isolated from the Atacama Desert. J Biotechnol. 2015;216:149–50.View ArticleGoogle Scholar
  28. Zhang YB, Ren HH, Zhang GY. Microbacterium hydrothermale sp. nov., an actinobacterium isolated from hydrothermal sediment. Int J Syst Evol Micr. 2014;64:3508–12.View ArticleGoogle Scholar
  29. Mondani L, Piette L, Christen R, Bachar D, Berthomieu C, Chapon V. Microbacterium lemovicicum sp. nov., a bacterium isolated from a natural uranium-rich soil. Int J Syst Evol Micr. 2013;63:2600–6.View ArticleGoogle Scholar
  30. Kook M, Son HM, Yi TH. Microbacterium kyungheense sp. nov. and Microbacterium jejuense sp. nov., isolated from salty soil. Int J Syst Evol Micr. 2014;64:2267–73.View ArticleGoogle Scholar
  31. Valk V, Eeuwema W, Sarian FD, van der Kaaij RM, Dijkhuizen L. Degradation of granular starch by the bacterium Microbacterium aurum strain B8.A involves a modular α-amylase enzyme system with FNIII and CBM25 domains. Appl Environ Microb. 2015;81(19):6610–20.View ArticleGoogle Scholar
  32. Lu J, Wu XD, Jiang YL, Cai XF, Huang LY, Yang YB, et al. An extremophile Microbacterium strain and its protease production under alkaline conditions. J Basic Microbiol. 2014;54(5):378–85.View ArticleGoogle Scholar
  33. Isotani K, Kurokawa J, Suzuki F, Nomoto S, Negishi T, Matsuda M, et al. Gene cloning and characterization of two NADH-dependent 3-quinuclidinone reductases from Microbacterium luteolum JCM 9174. Appl Environ Microb. 2013;79(4):1378–84.View ArticleGoogle Scholar
  34. Kim DW, Feng JH, Chen HZ, Kweon O, Gao Y, Yu LR, et al. Identification of the enzyme responsible for N-acetylation of norfloxacin by Microbacterium sp. strain 4 N2-2. Appl Environ Microb. 2013;79(1):314–21.View ArticleGoogle Scholar
  35. Ohta Y, Hatada Y, Hidaka Y, Shimane Y, Usui K, Ito T, et al. Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 β-fructofuranosidase. Appl Microbiol Biot. 2014;98(15):6667–77.View ArticleGoogle Scholar
  36. Wang JJ, Zhu YX, Zhao GG, Zhu JG, Wu S. Characterization of a recombinant (+)-γ-lactamase from Microbacterium hydrocarbonoxydans which provides evidence that two enantiocomplementary γ-lactamases are in the strain. Appl Microbiol Biot. 2015;99(7):3069–80.View ArticleGoogle Scholar
  37. Keyhani NO, Roseman S. The chitin catabolic cascade in the marine bacterium Vibrio furnissii—molecular cloning, isolation, and characterization of a periplasmic β-N-acetylglucosaminidase. J Biol Chem. 1996;271(52):33425–32.View ArticleGoogle Scholar
  38. Yang SQ, Song S, Yan QJ, Fu X, Jiang ZQ, Yang XB. Biochemical characterization of the first fungal glycoside hydrolyase family 3 β-N-acetylglucosaminidase from Rhizomucor miehei. J Agr Food Chem. 2014;62(22):5181–90.View ArticleGoogle Scholar
  39. Li HZ, Morimoto K, Kimura T, Sakka K, Ohmiya K. A new type of β-N-acetylglucosaminidase from hydrogen-producing Clostridium paraputrificum M-21. J Biosci Bioeng. 2003;96(3):268–74.View ArticleGoogle Scholar
  40. da Silva Junior Sobrinho I, Bataus LAM, Maitan VR, Ulhoa CJ. Purification and properties of an N-acetylglucosaminidase from Streptomyces cerradoensis. Biotechnol Lett. 2005;27(17):1273–6.View ArticleGoogle Scholar
  41. Senba M, Kashige N, Nakashima K, Miake F, Watanabe K. Cloning of the gene of β-N-acetylglucosaminidase from Lactobacillus casei ATCC 27092 and characterization of the enzyme espressed in Escherichia coli. Biol Pharm Bull. 2000;23(5):527–31.View ArticleGoogle Scholar
  42. Kubota T, Miyamoto K, Yasuda M, Inamori Y, Tsujibo H. Molecular characterization of an intracellular β-N-acetylglucosaminidase involved in the chitin degradation system of Streptomyces thermoviolaceus OPC-520. Biosci Biotech Bioch. 2004;68(6):1306–14.View ArticleGoogle Scholar
  43. Sukprasirt P, Wititsuwannakul R. A chitinolytic endochitinase and β-N-acetylglucosaminidase-based system from Hevea latex in generating N-acetylglucosamine from chitin. Phytochemistry. 2014;104:5–11.View ArticleGoogle Scholar
  44. Nyffenegger C, Nordvang RT, Zeuner B, Lezyk M, Difilippo E, Logtenberg MJ, et al. Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β-N-acetylhexosaminidases. Appl Microbiol Biot. 2015;99(19):7997–8009.View ArticleGoogle Scholar
  45. Mayer C, Vocadlo DJ, Mah M, Rupitz K, Stoll D, Warren RAJ, et al. Characterization of a β-N-acetylhexosaminidase and a β-N-acetylglucosaminidase/β-glucosidase from Cellulomonas fimi. FEBS J. 2006;273(13):2929–41.View ArticleGoogle Scholar
  46. Tsujibo H, Hatano N, Mikami T, Izumizawa Y, Miyamoto K, Inamori Y. Cloning, characterization and expression of β-N-acetylglucosaminidase gene from Streptomyces thermoviolaceus OPC-520. BBA-Gen Subjects. 1998;1425(2):437–40.View ArticleGoogle Scholar
  47. Ogawa M, Kitagawa M, Tanaka H, Ueda K, Watsuji T, Beppu T, et al. A β-N-acetylhexosaminidase from Symbiobacterium thermophilum; gene cloning, overexpression, purification and characterization. Enzyme Microb Tech. 2006;38(3–4):457–64.View ArticleGoogle Scholar
  48. Lin H, Xiao X, Zeng X, Wang FP. Expression, characterization and mutagenesis of the gene encoding β-N-acetylglucosaminidase from Aeromonas caviae CB101. Enzyme Microb Tech. 2006;38(6):765–71.View ArticleGoogle Scholar
  49. Chen F, Chen XZ, Qin LN, Tao Y, Dong ZY. Characterization and homologous overexpression of an N-acetylglucosaminidase Nag1 from Trichoderma reesei. Biochem Bioph Res Co. 2015;459(2):184–8.View ArticleGoogle Scholar
  50. Liu XS, Huang ZQ, Zhang XN, Shao ZZ, Liu ZD. Cloning, expression and characterization of a novel cold-active and halophilic xylanase from Zunongwangia profunda. Extremophiles. 2014;18(2):441–50.View ArticleGoogle Scholar
  51. Yamaguchi R, Tokunaga H, Ishibashi M, Arakawa T, Tokunaga M. Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium. Kocuria varians. Appl Microbiol Biot. 2011;89(3):673–84.View ArticleGoogle Scholar
  52. Wang GZ, Wang QH, Lin XJ, Ng TB, Yan RX, Lin J, et al. A novel cold-adapted and highly salt-tolerant esterase from Alkalibacterium sp. SL3 from the sediment of a soda lake. Sci Rep. 2016;6:19494.View ArticleGoogle Scholar
  53. Premkumar L, Greenblatt HM, Bageshwar UK, Savchenko T, Gokhman I, Sussman JL, et al. Three-dimensional structure of a halotolerant algal carbonic anhydrase predicts halotolerance of a mammalian homolog. Proc Natl Acad Sci U S A. 2005;102(21):7493–8.View ArticleGoogle Scholar
  54. Shen JD, Zhang R, Li JJ, Tang XH, Li RX, Wang M, et al. Characterization of an exo-inulinase from Arthrobacter: a novel NaCl-tolerant exo-inulinase with high molecular mass. Bioengineered. 2015;6(2):99–105.View ArticleGoogle Scholar
  55. Zhou JP, Peng MZ, Zhang R, Li JJ, Tang XH, Xu B, et al. Characterization of Sphingomonas sp. JB13 exo-inulinase: a novel detergent-, salt-, and protease-tolerant exo-inulinase. Extremophiles. 2015;19(2):383–93.View ArticleGoogle Scholar
  56. Paul S, Bag SK, Das S, Harvill ET, Dutta C. Molecular signature of hypersaline adaptation: insights from genome and proteome composition of halophilic prokaryotes. Genome Biol. 2008;9(4):R70.View ArticleGoogle Scholar
  57. Madern D, Ebel C, Zaccai G. Halophilic adaptation of enzymes. Extremophiles. 2000;4(2):91–8.View ArticleGoogle Scholar
  58. Warden AC, Williams M, Peat TS, Seabrook SA, Newman J, Dojchinov G, et al. Rational engineering of a mesohalophilic carbonic anhydrase to an extreme halotolerant biocatalyst. Nat Commun. 2015;6:10278.View ArticleGoogle Scholar
  59. Qin YJ, Huang ZQ, Liu ZD. A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: molecular cloning, heterologous expression and biochemical characterization. Extremophiles. 2014;18(2):271–81.View ArticleGoogle Scholar
  60. Shi RR, Li ZM, Ye Q, Xu JH, Liu Y. Heterologous expression and characterization of a novel thermo-halotolerant endoglucanase Cel5H from Dictyoglomus thermophilum. Bioresource Technol. 2013;142:338–44.View ArticleGoogle Scholar
  61. Margesin R, Schinner F. Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles. 2001;5(2):73–83.View ArticleGoogle Scholar
  62. Siglioccolo A, Paiardini A, Piscitelli M, Pascarella S. Structural adaptation of extreme halophilic proteins through decrease of conserved hydrophobic contact surface. BMC Struct Biol. 2011;11:50.View ArticleGoogle Scholar
  63. Wu GJ, Wu GB, Zhan T, Shao ZZ, Liu ZD. Characterization of a cold-adapted and salt-tolerant esterase from a psychrotrophic bacterium Psychrobacter pacificensis. Extremophiles. 2013;17(5):809–19.View ArticleGoogle Scholar
  64. Bruce AF, Gounaris K. Characterisation of a secreted N-acetyl-β-hexosaminidase from Trichinella spiralis. Mol Biochem Parasit. 2006;145(1):84–93.View ArticleGoogle Scholar
  65. Ohishi K, Murase K, Etoh H. Purification and properties of β-N-acetylglucosaminidase from Vibrio alginolyticus H-8. J Biosci Bioeng. 1999;88(1):98–9.View ArticleGoogle Scholar
  66. Fukamizo T, Kramer KJ. Mechanism of chitin oligosaccharide hydrolysis by the binary enzyme chitinase system in insect moulting fluid. Insect Biochem. 1985;15(1):1–7.View ArticleGoogle Scholar
  67. Suzuki K, Sugawara N, Suzuki M, Uchiyama T, Katouno F, Nikaidou N, et al. Chitinases A, B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: enzymatic properties and synergism on chitin degradation. Biosci Biotech Bioch. 2002;66(5):1075–83.View ArticleGoogle Scholar
  68. Chitlaru E, Roseman S. Molecular cloning and characterization of a novel β-N-acetyl-D-glucosaminidase from Vibrio furnissii. J Biol Chem. 1996;271(52):33433–9.View ArticleGoogle Scholar
  69. Nieder V, Kutzer M, Kren V, Gallego RG, Kamerling JP, Elling L. Screening and characterization of β-N-acetylhexosaminidases for the synthesis of nucleotide-activated disaccharides. Enzyme Microb Tech. 2004;34(5):407–14.View ArticleGoogle Scholar
  70. Tomiya N, Narang S, Park J, Abdul-Rahman B, Choi O, Singh S, et al. Purification, characterization, and cloning of a Spodoptera frugiperda Sf9 β-N-acetylhexosaminidase that hydrolyzes terminal N-acetylglucosamine on the N-glycan core. J Biol Chem. 2006;281(28):19545–60.View ArticleGoogle Scholar
  71. Suginta W, Chuenark D, Mizuhara M, Fukamizo T. Novel β-N-acetylglucosaminidases from Vibrio harveyi 650: cloning, expression, enzymatic properties, and subsite identification. BMC Biochem. 2010;11:40.View ArticleGoogle Scholar
  72. Liu TA, Zhang HT, Liu FY, Wu QY, Shen X, Yang Q. Structural determinants of an insect β-N-acetyl-D-hexosaminidase specialized as a chitinolytic enzyme. J Biol Chem. 2011;286(6):4049–58.View ArticleGoogle Scholar
  73. Lan XQ, Ozawa N, Nishiwaki N, Kodaira R, Okazaki M, Shimosaka M. Purification, cloning, and sequence analysis of β-N-acetylglucosaminidase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9. Biosci Biotech Bioch. 2004;68(5):1082–90.View ArticleGoogle Scholar
  74. Mark BL, Wasney GA, Salo TJS, Khan AR, Cao ZM, Robbins PW, et al. Structural and functional characterization of Streptomyces plicatus β-N-acetylhexosaminidase by comparative molecular modeling and site-directed mutagenesis. J Biol Chem. 1998;273(31):19618–24.View ArticleGoogle Scholar
  75. Ueda M, Fujita Y, Kawaguchi T, Arai M. Cloning, nucleotide sequence and expression of the β-N-acetylglucosaminidase gene from Aeromonas sp. no. 10S-24. J Biosci Bioeng. 2000;89(2):164–9.View ArticleGoogle Scholar
  76. Lan XQ, Zhang X, Kodaira R, Zhou Z, Shimosaka M. Gene cloning, expression, and characterization of a second β-N-acetylglucosaminidase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9. Biosci Biotech Bioch. 2008;72(2):492–8.View ArticleGoogle Scholar
  77. Riekenberg S, Flockenhaus B, Vahrmann A, Muller MCM, Leippe M, Kiess M, et al. The β-N-acetylhexosaminidase of Entamoeba histolytica is composed of two homologous chains and has been localized to cytoplasmic granules. Mol Biochem Parasit. 2004;138(2):217–25.View ArticleGoogle Scholar
  78. Konno N, Takahashi H, Nakajima M, Takeda T, Sakamoto Y. Characterization of β-N-acetylhexosaminidase (LeHex20A), a member of glycoside hydrolase family 20, from Lentinula edodes (shiitake mushroom). AMB Express. 2012;2:29.View ArticleGoogle Scholar
  79. Tsujibo H, Fujimoto K, Tanno H, Miyamoto K, Kimura Y, Imada C, et al. Molecular cloning of the gene which encodes β-N-acetylglucosaminidase from a marine bacterium, Alteromonas sp. strain O-7. Appl Environ Microb. 1995;61(2):804–6.Google Scholar
  80. Tsujibo H, Miyamoto K, Yoshimura M, Takata M, Miyamoto J, Inamori Y. Molecular cloning of the gene encoding a novel β-N-acetylhexosaminidase from a marine bacterium, Alteromonas sp. strain O-7, and characterization of the cloned enzyme. Biosci Biotech Bioch. 2002;66(2):471–5.View ArticleGoogle Scholar
  81. Slamova K, Kulik N, Fiala M, Krejzova-Hofmeisterova J, Ettrich R, Kren V. Expression, characterization and homology modeling of a novel eukaryotic GH84 β-N-acetylglucosaminidase from Penicillium chrysogenum. Protein Expres Purif. 2014;95:204–10.View ArticleGoogle Scholar
  82. Meekrathok P, Suginta W. Probing the catalytic mechanism of Vibrio harveyi GH20 β-N-acetylglucosaminidase by chemical rescue. PLoS One. 2016;11(2):e0149228.View ArticleGoogle Scholar
  83. Tsujibo H, Hatano N, Mikami T, Hirasawa A, Miyamoto K, Inamori Y. A novel β-N-acetylglucosaminidase from Streptomyces thermoviolaceus OPC-520: gene cloning, expression, and assignment to family 3 of the glycosyl hydrolases. Appl Environ Microb. 1998;64(8):2920–4.Google Scholar
  84. Choi KH, Seo JY, Park KM, Park CS, Cha J. Characterization of glycosyl hydrolase family 3 β-N-acetylglucosaminidases from Thermotoga maritima and Thermotoga neapolitana. J Biosci Bioeng. 2009;108(6):455–9.View ArticleGoogle Scholar
  85. Matsuo Y, Kurita M, Park JK, Tanaka K, Nakagawa T, Kawamukai M, et al. Purification, characterization and gene analysis of N-acetylglucosaminidase from Enterobacter sp. G-1. Biosci Biotech Bioch. 1999;63(7):1261–8.View ArticleGoogle Scholar
  86. O’Connell E, Murray P, Piggott C, Hennequart F, Tuohy M. Purification and characterization of a N-acetylglucosaminidase produced by Talaromyces emersonii during growth on algal fucoidan. J Appl Phycol. 2008;20(5):557–65.View ArticleGoogle Scholar

Copyright

© The Author(s). 2017

Advertisement