Biochemical Characterization and Structural Analysis of a β- N-Acetylglucosaminidase from Paenibacillus barengoltzii for Efficient Production of N-Acetyl- d -glucosamine

Article abstract of DOI:10.1021/acs.jafc.9b08085

Bioproduction of N-acetyl-d-glucosamine (GlcNAc) from chitin, the second most abundant natural renewable polymer on earth, is of great value in which chitinolytic enzymes play key roles. In this study, a novel glycoside hydrolase family-18 β-N-acetylglucosaminidase (PbNag39) from Paenibacillus barengoltzii suitable for GlcNAc production was identified and biochemically characterized. It possessed a unique shallow catalytic groove (5.8 ?) as well as a smaller C-terminal domain (solvent-accessible surface area, 5.1 × 10³ ?²) and exhibited strict substrate specificity toward N-acetyl chitooligosaccharides (COS) with GlcNAc as the sole product, showing a typical manner of action of β-N-acetylglucosaminidases. Thus, an environmentally friendly bioprocess for GlcNAc production from ball-milled powdery chitin by an enzyme cocktail reaction was further developed. By using the new route, the powdery chitin conversion rate increased from 23.3% (v/v) to 75.3% with a final GlcNAc content of 22.6 mg mL^-1. The efficient and environmentally friendly bioprocess may have great application potential in GlcNAc production.

Full text of DOI:10.1021/acs.jafc.9b08085

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Biotechnology and Biological Transformations
Biochemical characterization and structural analysis of a #-
N-acetylglucosaminidase from Paenibacillus barengoltzii
for efficient production of N-acetyl-D-glucosamine
Yihao Liu, Zhengqiang Jiang, Junwen Ma, Shuai Ma, Qiaojuan Yan, and Shaoqing Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b08085 • Publication Date (Web): 26 Apr 2020
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Biochemical characterization and structural analysis of a β-N-acetylglucosaminidase
from Paenibacillus barengoltzii for efficient production of N-acetyl-D-glucosamine
Yihao Liu,† Zhengqiang Jiang,‡ Junwen Ma,‡ Shuai Ma,‡ Qiaojuan Yan,† and Shaoqing Yang*,‡
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† Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of
Engineering, China Agricultural University, No.17 Qinghua East Road, Haidian District,
Beijing 100083, China.
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‡ College of Food Science and Nutritional Engineering, China Agricultural University, No.17
Qinghua East Road, Haidian District, Beijing 100083, China.
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* Corresponding authors: ysq@cau.edu.cn.
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ABSTRACT
Bio-production of N-acetyl-D-glucosamine (GlcNAc) from chitin, the second most abundant
natural renewable polymer on earth is of great value, in which chitinolytic enzymes play key
roles. In this study, a novel glycoside hydrolase family 18 β-N-acetylglucosaminidase
(PbNag39) from Paenibacillus barengoltzii suitable for GlcNAc production was identified
and biochemically characterized. It possessed a unique shallow catalytic groove (5.8Å) as
well as a smaller C-terminal domain (solvent accessible surface area, 5.1×103Å2) and
exhibited strict substrate specificity towards N-acetyl chitooligosaccharides (COS), with
GlcNAc as the sole product, showing typical action manner of β-N-acetylglucosaminidases.
Thus, an environmental friendly bioprocess for GlcNAc production from ball milled powdery
chitin by an enzyme cocktail reaction was further developed. By using the new route, the
powdery chitin conversion rate increased from 23.3% (v/v) to 75.3%, with a final GlcNAc
content of 22.6 mg mL-1. The efficient and environmental friendly bioprocess may have great
application potential in GlcNAc production.
KEYWORDS:
β-N-acetylglucosaminidase,
N-acetyl-D-glucosamine
powdery
chitin,
crystal
structure,
ball
milling,
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INTRODUCTION
Chitin, a linear β-1,4-linked biopolymer of N-acetyl-D-glucosamine (GlcNAc), is the second
most abundant renewable polymer in nature after cellulose, as a structural constituent in the
exoskeletons of arthropods and crustaceans and fungal cell walls.1,2 More than 80,000 tons of
chitin have been produced annually worldwide without utilization, the discarding of which in
nature not only results in environmental problems, but also waste of biomass.3,4 Hence,
development of efficient ways for the conversion of chitin to value added products is highly
desired.
GlcNAc has attracted considerable attention in recent years due to its biological activities,
such as antimicrobial, anti-osteoarthritis and anti-gastritis activities, and has been widely used
in pharmaceutical, cosmetic, food and biofuel industries.5-7 Thus, production of GlcNAc is a
promising way for the disposal and recycling of enormous waste chitin materials. Currently,
the commercial GlcNAc is mainly produced by concentrated acid hydrolysis of chitin,
suffering from serious associated environmental pollution.3 Consequently, the mild enzymatic
approach for the production of GlcNAc is highly preferred owing to product consistency,
environmental protection and sustainable development.7,8
Generally, the enzymatic conversion of chitin to GlcNAc needs the synergistic and
consecutive action of two kinds of chitinolytic enzymes, viz. chitinases (EC 3.2.1.14) and
β-N-acetylglucosaminidases (GlcNAcase, EC 3.2.1.52), where chitinases catalyze the
cleavage of β-1,4-linkages in chitin to release N-acetyl chitooligosaccharides (COS), and
GlcNAcases further hydrolyze N-acetyl COSs to yield GlcNAc.4 To date, a large number of
chitinolytic enzymes have been identified, characterized and applied with the aim of
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improving the hydrolysis efficiency of chitin.5,9 However, most of them are endochitinases.10
The GlcNAcases, especially thermostable GlcNAcases are still less. Moreover, most reported
GlcNAcases have been classified into GH families 3, 20, 84 and 116 according to their
structure and sequence similarities, while no GlcNAcase from GH family 18 has been
identified (http://www.cazy.org/).
Despite many efforts have been performed to produce GlcNAc by enzymatic hydrolysis
from chitin, the conversion rate still remains too low to be scaled up.3,7,11 This may be due to
the relatively high crystalline structure of the powdery chitin mainly owing to the hydrogen
bonding between chains, which restricts the enzymatic activity.12 Several pretreatment
methods have been well investigated to increase the solubility or reduce crystallinity of
lignocellulosic biomass, thereby improving the accessibility of the enzyme.13 Among them,
ball milling (BM) is a relatively efficient pretreatment method, which can reduce the degree
of crystallinity of lignocellulosic biomass significantly by physical friction.14
Paenibacillus barengoltzii CAU904, a thermophilic marine bacterium, has been reported
to secrete multiple kinds of chitinolytic enzymes in our previous studies.9,10 Here, we reported
biochemical characterization and crystal structure of a GlcNAcase from P. barengoltzii
CAU904, which is a novel GlcNAcase in GH family 18. Moreover, the enzymatic production
of GlcNAc from ball milled powdery chitin, followed by a binary enzyme reaction was
further investigated.
MATERIALS AND METHODS
Materials. Restriction endonucleases and T4 DNA ligase were purchased from New England
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Biolabs (Ipswich, MA). Ex Taq and Pfu DNA polymerase were obtained from Promega
(Madison, WI). The pMD18-T simple vector system was purchased from Takara Co. (Shiga,
Japan). pET28a (+) expression vector was purchased from Novagen (Madison, WI). E. coli
DH5α (used for plasmid amplification) and E. coli BL21 (DE3) (used as host for expression)
were purchased from Invitrogen (Carlsbad, CA).
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Powdery chitin (from crab shells) was provided by Laizhou Haili Biological Product Co.,
Ltd. (China). Choline chloride, betaine, p-nitrophenyl N-acetyl-β-D-glucosaminide
(pNP-NAG) and p-nitrophenyl N-acetyl-β-D-galactosaminide (pNP-GalNAc) were purchased
from Sigma Chemical Co. (USA). Glycol chitin was obtained from Wako Pure Chemistry
Industries, Ltd. (Japan). N-acetyl chitobiose ((GlcNAc)2) was prepared according to the
method described in our previous study.10 N-acetyl chitooligosaccharides (N-acetyl COSs)
with degrees of polymerization (DP) 3-5 were purchased from Megazyme (Ireland).
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Gene cloning and sequence analysis of a GlcNAcase gene. Paenibacillus barengoltzii
CAU904 used in this study has been deposited in the China General Microbiological Culture
Center (CGMCC, accession number 9530). Genomic DNA of P. barengoltzii CAU904 was
isolated by an Easy-pure Bacteria DNA kit (Beijing, China, TransGen Biotech) and used as
template for polymerase chain reaction (PCR) amplification. To clone GlcNAcase gene,
specific primers PbNag39-F: (5’CCGGAATTCATGAGCCGGAACTATGTGGTGG3’) and
PbNag39-R:
(5’GAATGCGGCCGCTCACGATGATTGTAACCCTTGGTAC3’)
were
o
designed. The PCR conditions were as follows: 2 min at 95 C, followed by 35 cycles of 95
oC for 20 s, 58 oC for 20 s and 72 oC for 30 s, with a final extension at 72 oC for 5 min. The
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PCR product was purified, ligated to vector pMD19-T (Shiga, Japan, TaKaRa) and confirmed
by sequencing.
The
signal
peptide
was
predicted
at
the
SignalP
4.1
server
(http://www.cbs.dtu.dk/services/SignalP/). BLAST analysis for DNA and protein sequence
alignments were performed at NCBI server (http://www.ncbi.nlm.nih.gov/BLAST/).
Phylogenetic tree was constructed by MEGA 5.0 using the method of neighbor joining (NJ)
with the bootstrap value of 1000.
Expression of a GlcNAcase gene in E. coli. The GlcNAcase gene (PbNag39) products were
digested with Eco RI and Not I. The resulting fragments were cloned into EcoRI/NotI-digested
SUMO-pET28a(+) expression vector, and then transformed into E. coli BL21(DE3) for
protein expression. The positive clones were directly screened by colony PCR, inoculated in
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10 mL LB medium (containing 50 μg mL-1 of kanamycin) and then cultured at 37 C in a
shaker (200 rpm). After the optical density (OD600) of the culture broth reached up to 0.6,
isopropyl-β-D-thio-galactopyranoside (IPTG) was added to a final concentration of 1 mM for
induction, and the culture was further grown at 30 oC for 12 h.
Purification of PbNag39. The crude enzyme solution was loaded onto a Ni Sepharose
column (1 × 5 cm, GE Healthcare, USA) pre-equilibrated with buffer A (50 mM Tris-HCl
buffer pH 8.0 containing 20 mM imidazole and 300 mM NaCl). After the impurities were
washed with 5 column volumes of buffer B (50 mM Tris-HCl buffer pH 8.0 containing 50
mM imidazole and 300 mM NaCl), the bonded proteins were eluted by buffer C (50 mM
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Tris-HCl buffer pH 8.0 containing 200 mM imidazole and 300 mM NaCl). The flow rate was
fixed at 1 mL min-1. The eluted fractions showing GlcNAcase activity were collected and
checked for purity by SDS-PAGE. SUMO protease (Invitrogen™) was added into the purified
fractions and incubated at 4 oC for 12 h to remove SUMO tag. The digested sample was then
loaded onto the Ni Sepharose column pre-equilibrated with buffer A again, and the
flow-through fractions were collected, concentrated and buffer exchanged with 20 mM
acetate buffer pH 5.5 by ultrafiltration.
Enzyme assay, protein determination and Molecular Mass Determination. GlcNAcase
activity was determined by measuring the releasing amount of GlcNAc from (GlcNAc)2.
Briefly, a 200 μL mixture containing 100 μL of 1% (w/v) (GlcNAc)2 and 100 μL properly
o
diluted enzyme solution in 50 mM acetate buffer pH 5.5 was incubated at 75 C for 10 min,
and then boiled for 5 min to terminate the reaction. The amount of released GlcNAc was
determined by HPLC (Agilent 1260 infinity II, Agilent Technologies, USA) with BP-800
Pb++ (Benson Polymeric, Reno, NE, 7.8×300 mm, USA) equipped with a refractive index
(RI) detector. One unit of enzyme activity was defined as the amount of enzyme required to
liberate 1 μmol GlcNAc per min under the assay conditions. For HPLC analysis, the oven
temperature was set at 80 oC and deionized water was used as mobile phase at a flow rate of
0.8 mL min-1.
Protein content was determined by the method of Lowry et al.15 using bovine serum
albumin (BSA) as the standard. Specific activity was expressed as units per milligram protein.
The native molecular mass of the purified PbNag39 was determined by gel filtration on a
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Sephacryl S-100 (GE Life Science) HR column (1.0 × 50 cm). The protein samples were
loaded onto the column and washed with 20 mM phosphate buffer pH 7.0 containing 150 mM
NaCl at a flow rate of 0.3 mL min-1. The standard proteins used were phosphorylase b (from
rabbit muscle, 97.2 kDa), albumin bovine V (66.2 kDa), ovalbumin (from chicken egg white,
45.0 kDa), chymotrypsinogen A (type II from bovine pancreas, 25.6 kDa) and lysozyme (14.4
kDa).
Effects of pH and temperature on the activity of PbNag39. The optimal pH of purified
PbNag39 was determined by measuring the enzyme activity in different buffers (50 mM), viz.
citrate (pH 3.5-6.0), acetate (pH 4.0-5.5), Mcllvaine (pH 4.0-7.0), phosphate (pH 6.0-8.0),
Tris-HCl (pH 7.0-9.0) and glycine-NaOH (pH 8.5-10.0). To determine the pH stability, the
o
enzyme was incubated in the above-mentioned buffers at 50 C for 30 min, and the residual
activities were then measured by the standard assay.
The optimal temperature of PbNag39 was determined by measuring the enzyme activity
in the temperature range of 40-85 oC in 50 mM acetate buffer pH 5.5. To determine the
thermostability, the enzyme was incubated in 50 mM acetate buffer pH 5.5 at different
temperatures (40-85 oC) for 30 min, and the residual activities were then measured by the
standard assay. The thermal inactivation of PbNag39 was evaluated by incubating PbNag39 in
50 mM acetate buffer pH 5.5 at different temperatures (60, 65, 70 and 75 oC) for 4 h and the
residual activities were then measured by the standard assay.
Substrate specificity of PbNag39. The substrate specificity of PbNag39 was determined by
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measuring the enzyme activity using different substrates including N-acetyl COSs (DP 2-5),
colloidal chitin, glycol chitin, pNP-NAG and pNP-GalNAc in 50 mM acetate buffer pH 5.5 at
75 oC. For N-acetyl COSs (DP 3-5), the enzyme activity was determined by calculating the
GlcNAc-releasing rate from N-acetyl COSs (DP 3-5) according to the method of Liu et al.16
Briefly, a 200 μL mixture containing 100 μL of 1% (w/v) N-acetyl COSs (DP 3-5) and 100 μL
o
properly diluted enzyme solution in 50 mM acetate buffer pH 5.5 was incubated at 75 C for
10 min, and then boiled for 5 min to terminate the reaction. The amount of GlcNAc released
at the beginning was determined by HPLC mentioned above. One unit of enzyme activity was
defined as the amount of enzyme required to liberate 1 μmol GlcNAc per min under the assay
conditions. For colloidal chitin and glycol chitin, the enzyme activity was determined by
3,5-dinitrosalicylic acid (DNS) method,17 using 1% (w/v) of colloidal chitin or glycol chitin
as the substrate, and the enzyme activity was defined as the amount of enzyme required to
liberate 1 μmol GlcNAc equivalent reducing sugars per min under the assay conditions. For
pNP-NAG, 200 μL mixture containing 100 μL pNP-NAG (2 mM) and 100 μL suitable diluted
enzyme solution was incubated for 10 min, and the reaction was terminated by the addition of
200 μL NaOH solution (500 mM). The amount of pNP released was determined by measuring
the absorbance at 410 nm. One unit of enzyme activity was defined as the amount of enzyme
required to liberate 1 μmol pNP per min under the assay conditions.
Hydrolysis properties of PbNag39. The hydrolysis properties of PbNag39 towards N-acetyl
COSs (DP 2-5) were evaluated by incubation 1% (w/v) of substrates in 50 mM acetate buffer
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pH 5.5 in the presence of 1 U mL-1 enzyme at 60 C for 1 h. For chitin hydrolysis, 3% (w/v)
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of ball milled powdery chitin in 50 mL 20 mM citrate buffer pH 5.5 were hydrolyzed by 1.0 U
mL-1 of PbNag39, 5.0 U mL-1 of PbChi70 (a chitinase reported previously10) and 1.0 U mL-1
o
of PbNag39 combined with 5.0 U mL-1 of PbChi70 at 55 C for 36 h, respectively. Samples
were taken at different time intervals, and boiled for 5 min to terminate the enzyme reaction
immediately. After centrifugation at 10,000 g for 10 min, the supernatant was collected and
analyzed by thin layer chromatography (TLC). For TLC analysis, the samples were spotted
onto a silica gel plate (Merck, Germany) and developed twice in a solvent containing
butanol/methanol/ammonia/water = 5:4:2:1 (v/v/v/v). The sugars separated on the TLC plate
were visualized by spraying with sulfuric acid/methanol (95:5, v/v), followed by heating at
130 oC for few seconds. HPLC analysis was performed as mentioned above.
Crystallization and data collection. The purified enzyme was concentrated to 10 mg mL-1
and screened at 20 °C by crystallization solution kits (Hampton Research, USA). The
crystallization experiments were performed in 48-well plates using the sitting-drop
vapor-diffusion method, where 2 μL protein was mixed with 1 μL reservoir solution (700 mM
sodium acetate trihydrate, 5.6% (w/v) polyethylene glycol 4000 and 30% (v/v) glycerol). For
X-ray diffraction experiments, the single crystal was fished out from the crystallization drop
and transferred to reservoir solution, and immediately flash-frozen in liquid nitrogen. The
X-ray diffraction data of PbNag39 were collected in a cold nitrogen gas stream (100 K) by
beamline BL17U at the Shanghai Synchrotron Research Facility (SSRF, China). The data
were indexed, integrated and scaled by the HKL-2000 package.
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Structure determination, refinement and site-directed mutagenesis. The phases of
PbNag39 were determined by the molecular replacement method using the searching model
of Chiw (identity 44%; PDB: 5GZU). The procedure for automatic model building was
performed with Phenix.autobuild.18 All of the structures were completed via iterative rounds
of manual building with Coot19 and refinement with phenix.refine. The final model was
analyzed by MolProbity.20 Structure illustrations were prepared in PyMOL (v.1.3;
Schrödinger LLC).
Mutants Y261A, Y261N, Y261T and Y261S were performed in PbNag39 using the Fast
Mutagenesis System site-directed mutagenesis kit (TransGen Biotech, China) with the
primers (Table S1). All transformants were confirmed by DNA sequencing.
Preparation of ball milled powdery chitin. The powdery chitin samples were pretreated by
ball milling before enzymatic hydrolysis. 100 g powdery chitin samples and zirconia beads
(10 mm diameter) were mixed in a 2 L Zirconia tank (with a sample to ball ratio of 1: 2) with
rotation speed of 380 rpm for 4 h using a CJM-SY-B vibration ball milling (Qinhuangdao
Taiji Ring Nano Ltd., China).
SEM, X-ray diffraction and FT-IR analysis of powdery chitin. Scanning electron
microscopy (SEM) was performed according to the method of Husson et al.11 The samples
were coated with gold and then observed by a Hitachi SU3500 (Hitachi, Japan) scanning
electron microscope at 25 kV. X-ray diffraction (XRD) measurements were performed
according to the method of Wei et al. with minor modifications.3 Patterns were collected using
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a Bruker D4 Advance diffractometer (Bruker, Germany) with an incident Cu Kα radiation
(operating at 40 kV-40 mA) and wavelength of λ= 1.5406 Å in the 2θ range of 5-40, with a
step size of 0.02. Crystallinity indexes (CrI) were determined by the equation: CrI (%) =
(I110-Iam)/I110×100, in which the I110 and Iam were at around 2θ = 20 and 16, respectively.
Fourier transform infrared spectroscopy (FT-IR) was performed according to the method
of Wei et al.3 FT-IR spectra were obtained in KBr disks in the range of 4000-400 cm-1 on a
Nicolet IS10 FT-IR instrument (Nicolet, USA).
Conversion of chitin to GlcNAc. 1.5 g colloidal chitin (3%) were dissolved in 50 mL 20 mM
citrate buffer pH 5.5, and hydrolyzed by 1.0 U mL-1 of PbNag39 combined with 5.0 U mL-1 of
o
PbChi70 at 55 C for 48 h. Samples were taken at different time intervals, and boiled for 5
min to terminate the enzyme reaction immediately. After centrifugation at 10,000 g for 10 min,
the supernatant was collected and analyzed by HPLC as mentioned above. The chitin
conversion rate was calculated as follows: Chitin conversion rate (%) = GlcNAc (g) released/
chitin (g) × 100%.
1.5 g (or 60 g) pretreated powdery chitin (3%) were dissolved in 50 mL (or 2 L) 20 mM
citrate buffer pH 5.5, and hydrolyzed by 1.0 U mL-1 of PbNag39 combined with 5.0 U mL-1 of
o
PbChi70 at 55 C for 48 h. Samples were taken at different time intervals, and boiled for 5
min to terminate the enzyme reaction immediately. After centrifugation at 10,000 g for 10 min,
the supernatant was collected and analyzed by HPLC as mentioned above. The chitin
conversion rate was calculated as mentioned above.
1
The product was further identified by H nuclear magnetic resonance (NMR)
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spectroscopy. The samples pretreated by D2O were dissolved in
5
mM
1
4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS). The 1D H NMR spectroscopy was
determined by an Agilent DD2 NMR spectrometer at 500 MHz (Agilent Technologies, USA).
RESULTS
Sequence analysis and biochemical characterization of a novel GlcNAcase. A GlcNAcase
gene, designated as PbNag39 (NCBI accession number MH797015) was obtained. The
deduced amino acid sequence of PbNag39 shared the highest identity of 49% with a chitinase
from Bacillus circulans (BAA13974) (Fig. S1), but no similarity with other reported
GlcNAcases. A phylogenetic tree of PbNag39 with other GH family 18 chitinases was further
constructed on the basis of their sequence similarities. PbNag39 composed a single branched
cluster with some hypothetical protein sequences (Fig. S2). After expression in E. coli, the
recombinant GlcNAcase (PbNag39) was purified and determined to be a homodimer by
Sephacryl S-100 HR column (Fig. S3), with a denatured molecular mass of 39 kDa on
SDS-PAGE (Fig. S4). The optimal pH of PbNag39 was found to be pH 5.5, and it was stable
o
within pH 4.5-8.0 (Fig. 1A and 1B). The optimal temperature of PbNag39 was 75 C, and it
was stable up to 65 oC (Fig. 1C and 1D). The thermal denaturing half-lives of PbNag39 at 60,
65, 70 and 75 oC were determined to be 995, 249, 151 and 14 min, respectively (Fig. 1E).
Substrate specificity and hydrolysis properties of PbNag39. PbNag39 showed the highest
specific activity towards N-acetyl chitotetraose ((GlcNAc)4, 254.7 U mg-1), followed by
N-acetyl chitopentaose ((GlcNAc)5, 230.8 U mg-1), N-acetyl chitotriose ((GlcNAc)3, 219.8 U
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mg-1) and N-acetyl chitobiose ((GlcNAc)2, 28.3 U mg-1). However, it exhibited relatively low
activity towards p-nitrophenyl N-acetyl-β-D-glucosaminide (pNP-NAG) (1.02 U mg-1) and
p-nitrophenyl N-acetyl-β-D-galactosaminide (pNP-GalNAc) (0.18 U mg-1), and no activity
towards colloidal chitin and glycol chitin (Table 1). PbNag39 hydrolyzed N-acetyl COSs with
DP 2-5 to yield GlcNAc as sole end product with the extension of incubation time (Fig. 2A
and 2B). But it could not hydrolyze chitin (Fig. 2C). However, the co-action of PbNag39 and
a previously reported chitinase (PbChi70) efficiently converted powdery chitin to GlcNAc
(Fig. 2C).
Crystal structure, structure comparison and site-directed mutants of PbNag39. The
crystal structure of PbNag39 was determined at 1.43Å in space group C2. The Rwork and Rfree
of this model were 17.4% and 19.3% (Table S2). The crystal contained a monomer in the
asymmetric unit (Fig. 3A). The model of PbNag39 comprises 345 amino acid residues
(Met1-Ser345), and the 97.6% and 2.4% of residues were in the favored and allowed regions,
respectively (Table S2). The molecule contains two domains: the N-terminal domain adopts a
typical (α/β)8-TIM barrel fold with eight parallel β-sheets (β1, β2, β5-β9 and β15) surrounded
by eight α-helices (α1-α6, α8 and α9). While the C-terminal domain shows a β-barrel structure
with a five-stranded β-sheet (β10-β14) and an α-helix (α7). The two catalytic residues (D107
and E111) were located β6-sheets and the loop between β6 and α4 on the (α/β)8-TIM barrel
domain, respectively (Fig. 3B).
Although PbNag39 contained a conserved sequence motif found in GH family 18, the
function of PbNag39 is different from GH18 chitinases. The structure of PbNag39 shared
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relative high similarities to that of several chitinases, with the highest homologous to 5GZU
(RMSD = 1.6 Å), followed by 1ITC (RMSD = 1.6 Å), 3G6L (RMSD = 1.6 Å), 5WVP
(RMSD = 1.8 Å) and 4PTM (RMSD = 1.8 Å). However, PbNag39 does not display chitinase
activity
but
showed
high
β-N-acetylglucosaminidases
activity.
The
β-N-acetylglucosaminidases in GH family 3, 20 and 84 displayed typical (α/β)8-TIM barrel
and the substrate-binding pocket in the (β/α)8-barrel. Catalytic sites are located on the pocket,
which was different from the PbNag39.
To explain its higher GlcNAcase activity but no chitinase activity, four mutants
(Y261A, Y261N, Y261T and Y261S) were successfully expressed in E. coli BL21 (DE3).
The GlcNAcase activities of all mutants were reduced. However, mutants of Y261A, Y261N
and Y261T showed chitinase activity, the specific activities were 0.06, 0.12 and 0.09 U mg-1,
respectively (Table S3).
Effect of pretreatments on the structure of powdery chitin. The powdery chitin had large
particles with rough and compacted structure (Fig. 4A). After pretreatment by ball milling, the
particle sizes of powdery chitin were decreased drastically (Fig. 4B). Moreover, ball milling
pretreatment significantly reduced the crystallinity indexes (CrI) from 86.9% to 52.2% (Fig.
5). FT-IR analysis showed that the overall spectra of powdery chitin and pretreated powdery
chitin were similar (Fig. S5). The major difference is that the small absorption peak at 1 660
cm-1 observed in powdery chitin disappeared after the pretreatment (Fig. S5), indicating that
the hydrogen bonds between powdery chitin molecules were destructed.
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351
352
Conversion of powdery chitin to GlcNAc. Time course profile for GlcNAc production from
colloidal chitin by PbNag39 combined with the chitinase (PbChi70) was performed. The
highest conversion rate of 97.0% with GlcNAc content of 29.1 mg mL-1 was achieved after 24
h hydrolysis (Fig. 6A).
The effects of ball milling pretreatment on the enzymatic conversion (PbNag39 coupled
with the chitinase) of powdery chitin to GlcNAc were investigated. Ball milling pretreatment
significantly increased the chitin conversion rate from 23.3% to 75.3%. Time course profile
for GlcNAc production from ball milled powdery chitin by PbNag39 combined with the
chitinase (PbChi70) was performed. The highest conversion rate of 75.3% with GlcNAc
content of 22.6 mg mL-1 was achieved after 24 h hydrolysis (Fig. 6B). To assess the feasibility
of scale-up for GlcNAc production, the bioprocess was conducted in a 5-L fermentor. The
conversion rate of 72.7% with GlcNAc content of 21.8 mg mL-1 was obtained after 24 h
incubation, which was slightly lower than that in the shake flask (Table S4). HPLC analysis
showed that GlcNAc was the sole end product in the hydrolysate, with the purity high up to
1
95% (Table S5). The obtained product was further confirmed to be GlcNAc by H NMR
spectroscopy (Fig. S6).
DISCUSSION
GlcNAcase has been widely used in many industrial applications due to its various
biochemical properties.21 Currently, most reported GlcNAcases have been classified into GH
families 3, 20, 84 and 116, while no GlcNAcase from GH family 18 has been identified. In
this study, a novel GH family 18 β-N-acetylglucosaminidase (PbNag39) from Paenibacillus
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barengoltzii suitable for GlcNAc production was identified, structurally elucidated and
biochemically characterized.
It is worth noting that PbNag39 is a GlcNAcase, but it exhibited relatively high
similarities with other reported chitinases in GH family 18, which is distinct from other
reported GlcNAcases.22-25 The majority of reported GlcNAcases are monomers, having
molecular masses in a wide range of 10-313 kDa.26,27 However, PbNag39 is a homodimer,
which is similar to that of the GlcNAcase from Spodoptera frugiperda (Sfhex).22 Generally,
GlcNAcases have a wide range of pH optima from pH 3.0 to 10.0.21,23 PbNag39 was an acidic
GlcNAcase with an optimal pH of 5.5, which is similar to that of the GlcNAcases from
Bifidobacterium longum subsp. longum (pH 5.5)28 and Streptomyces cerradoensis (pH 5.0).29
In addition, PbNag39 was stable in a relatively wider pH range (pH 4.5-8.0) when compared
with most other reported GlcNAcases, such as the GlcNAcases from Streptococcus gordonii
(GcnA, pH 5.2-7.2)30 and Pseudoalteromonas sp. (W-Chi22718, pH 6.0-9.0).31 PbNag39 had
o
an optimal temperature of 75 C, which is the same as that of the GlcNAcases from T.
o
o
neapolitana (75 C)32 and T. emersonii (75 C),33 but obviously higher than those of most
other GlcNAcases,23,24,32,34,35 representing the highest optimal temperature for GlcNAcases
ever reported. The excellent enzymatic properties may enable the enzyme great potential in
industrial applications.
PbNag39 exhibited strict substrate specificity towards N-acetyl COSs, but trace (pNP
derivates) or no activity towards other tested substrates, which is in accordance with that of
most other reported GlcNAcases.4,36,37 PbNag39 showed relatively low activity towards
pNP-NAG (1.02 U mg-1), which is similar to that of the GlcNAcase from Penicillium
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396
chrysogenum (1.2 U mg-1),38 but lower than that of the GlcNAcase from Ostrinia furnacalis
(OfHex1, 23.9 U mg-1).39 However, PbNag39 exhibited relatively high activity towards
(GlcNAc)2 (28.3 U mg-1), which is much higher than those of the GlcNAcases from
Rhizomucor miehei (RmNag, 1.1 U mg-1)24 and Sphingobacterium sp. (rNag3HWLB1, 0.3 U
mg-1),40 but lower than that of the GlcNAcase from Shinella sp. (rJB10Nag, 35.4 U mg-1).4
GlcNAcases reported to date possess two major types of substrate specificity. The first type of
GlcNAcases prefers to hydrolyze (GlcNAc)2, while the other type of GlcNAcases favors
N-acetyl COSs with DP 3-6 over (GlcNAc)2.24 PbNag39 belongs to the second type
GlcNAcases from this classification aspect, as it preferred to hydrolyze (GlcNAc)4 followed
by (GlcNAc)5, (GlcNAc)3 and (GlcNAc)2. The GlcNAcase from Vibrio harveyi also belongs
to the second type GlcNAcases, but it hydrolyzed N-acetyl COSs in the order of (GlcNAc)4 >
(GlcNAc)3 > (GlcNAc)2 > (GlcNAc)5.36 While most other reported GlcNAcases belong to the
first type GlcNAcases, preferring to hydrolyze N-acetyl COSs in the order of (GlcNAc)2 >
(GlcNAc)3 > (GlcNAc)4 > (GlcNAc)5.24,30,41 Thus, PbNag39 displayed unique hydrolysis
properties in comparison to all the other reported GlcNAcases.
So far, many chitinases have been characterized, in which 32 crystal structures have been
solved in GH family 18. PbNag39 showed the sequence identity of 44% with the chitinase
(Chiw) from the Paenibacillus sp. FPU-7 (PDB: 5GZU) and 41% with chitinase (ChiA) from
Bacillus circulans WL-12 (PDB: 1ITX) (Fig. 3). Although PbNag39 contained a GH family
18 motif and showed high sequence identities with chitinases, it was confirmed to be a
β-N-acetylglucosaminidase by its substrate specificity. PbNag39 consists of an N-terminal
(α/β)8-TIM barrel domain and a C-terminal β-barrel domain. D107 and E111 are located in
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the (α/β)8-TIM barrel domain as the general acid/base. Structural comparison of PbNag39
with other GH family 18 chitinases revealed some similarities in the active sites and catalytic
grooves. D107 and E111 of PbNag39 occupied the positions corresponding to the conserved
aspartic acid and glutamic acid in the GH family 18 members, such as D154/E158 in Chiw
(PDB: 5GZU), D168/E1721 in ChiA(PDB: 1ITX) and D321/E322 in ChiB (PDB: 1E15).
PbNag39 showed a catalytic groove between N-terminal domain and C-terminal domain,
which is similar to GH family 18 chitinases. However, the catalytic groove of PbNag39 is
relatively narrow, is 5.8Å, while it is 11.4Å and 8.6Å in Chiw (PDB: 5GZU) and ChiB (PDB:
1E15) (Fig. 3C, 3D and 3E), respectively. Besides, Y261 of PbNag39 forms a steric hindrance
in the catalytic groove entrance to hinder the substrate access to it, which may cause the loss
of the catalytic activity on colloidal chitin. Mutations of Y261A, Y261N, Y261T and Y261S
resulted in decreasing of β-N-acetylglucosaminidase activities but increasing of chitinase
activities (except for Y261S), which confirmed the speculation mentioned above. Although
PbNag39 shared a N-terminal (α/β)8-TIM barrel and C-terminal β-sheets, which is similar to
other β-N-acetylglucosaminidases, including GH family 3, 20 and 84 (Fig. 7). However, many
differences were observed in the conserved catalytic sites. Structural comparison of PbNag39
to GH family 3, 20 and 84 β-N-acetylglucosaminidases suggested that their catalytic acid/base
residues were different, such as Asp232/His234 in BsNagZ (PDB: 3BMX), Asp360/Glu361 in
GH20C (PDB：5A69), Asp 297/Asp 298 in CpNagJ (PDB: 2CBI) and Asp 107/Glu111 in
PbNag39, respectively (Fig 7). Moreover, the C-terminal domain of GH family 3, 20 and 84
β-N-acetylglucosaminidases were relatively large and complex. Their solvent accessible
surface area was 1.14×104Å2, 1.42×104Å2, 9.8×103Å2 in BsNagZ (PDB: 3BMX), GH20C
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438
439
440
(PDB: 5A69), CpNagJ (PDB: 2CBI), respectively, while it was 5.1×103Å2 in PbNag39 (Fig.
8). Therefore, PbNag39 possesses the unique structure features, which are different from
chitinases in GH family 18 and β-N-acetylglucosaminidases in GH families 3, 20 and 84.
The effect of ball milling pretreatment on chitin is similar to that in the previous study.11
The loose and porous morphology significantly increased the surface area of powdery chitin,
thus may increase the accessibility of enzyme molecule to the substrate. The CrI of ball milled
chitin was decreased dramatically, meaning that the intermolecular and intramolecular
hydrogen bonds networks were broken up.11 Meanwhile, the absorption peak at 1 660 cm-1
disappeared after the pretreatment, suggesting that partial hydrogen bonds between powdery
chitin molecules were destructed. However, the constituents of the powdery chitin before and
after pretreatments were not changed as indicated by the FT-IR spectra, which is in
accordance with the result of Wei et al.3
Enzymatic productions of GlcNAc from colloidal chitin by the co-action of GlcNAcases
and chitinases have been reported in previous studies,9,31 and a nearly 100% conversion rate
of the colloidal chitin has been achieved in this study. However, the preparation of colloidal
chitin before enzymatic hydrolysis not only brought environmental pollution due to
concentrated hydrochloric acid, but also consumed a large amount of water.6 Hence,
development of environmental friendly and efficient bioprocesses for GlcNAc production
from chitin is of great value from an industrial point of view. Pretreatment before enzymatic
conversion is crucial for chitin due to its insolubility and high crystallinity, the characteristics
of which impeded the binding between enzyme and substrate.1 Balling milling pretreatment
significantly enhanced the conversion rate by more than 3 folds. This may be due to that the
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drastic physical friction destroyed the crystal structure of chitin by weakening intermolecular
hydrogen bonding network, thus improved the accessibility of enzyme molecule to the
glycoside bonds in chitin.14 The similar results were observed in the ball milling pretreatment
of cellulose for glucose production42-44 and pretreatment of chitin for N-acetyl COS.14 Hence,
it was used to pretreat powdery chitin for GlcNAc production in the present study. The
highest chitin conversion rate of 75.3% was obtained after pretreatment by ball milling,
followed by an enzyme cocktail hydrolysis (Fig. 6B). The results suggesting that the
bioprocess using pretreatment of ball milling, followed by two-enzyme hydrolysis, is a highly
efficient way for GlcNAc production from powdery chitin. Some other routes for GlcNAc
production have been reported (Table 2). For example, The chitin from crayfish shell was
pretreated by high pressure homogenization, and then hydrolyzed by the chitinase from
Serratia proteamaculans NJ303, with a GlcNAc yield of 3.9 mg mL-1.3 Crystalline α-chitin
was hydrolyzed by the β-N-acetylhexosaminidase from Fusarium oxysporum CFR directly,
with a GlcNAc yield of 2.4 mg mL-1.7 Powdery chitin was pretreated by ionic liquid, followed
by an enzymatic hydrolysis of commercial chitinase and GlcNAcase, with a GlcNAc yield of
7.6 mg mL-1.11 Powdery chitin was pretreated by bacterial fermentation for 12 h prior to an
enzymatic hydrolysis of a chitinase, with a GlcNAc yield of 19.2 mg mL-1.45 Therefore, the
novel efficient route may possess great application potential in GlcNAc production from
powdery chitin.
ASSOCIATED CONTENT
Supplementary material
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Primers used in the study; Data-collection and refinement statistics; Chitinase activities of
PbNag39 and its mutants; Scale up of the bioprocess for GlcNAc production; GlcNAc purity;
Sequence alignment; Phylogenetic analysis; Native molecular mass determination;
Purification; IR spectra analysis; Product identification.
AUTHOR INFORMATION
Corresponding authors
*E-mail for S.Q. Y.: ysq@cau.edu.cn.
Notes
The authors declare that there are no conflict of interest in the manuscript.
ACKNOWLEDGEMENTS
This work was supported in part by the National Science Fund for Excellent Young Scholars
(No. 31822037) and National Natural Science Foundation of China (No. 21576283). We
would like to thank Prof. Mikami from Kyoto University for revising the structure part of the
manuscript.
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