Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002

Materials

We obtained DNA polymerases, BamHI and XhoI restriction endonucleases and T₄ ligase from TaKaRa (Dalian, China). The TIANamp Bacteria DNA Kit, TIANprep Mini Plasmid Kit, and Universal DNA Purification Kit were all purchased from Tiangen Biotech CO., Ltd. (Beijing, China). EasyPure Quick Gel Extraction Kit was purchased from TransGen Biotech CO., Ltd. (Beijing, China). His-Tagged Protein Purification Kit (Soluble Protein) was obtained from ComWin Biotech Co., Ltd. (Beijing, China). Host cell strains, E. coli TOP10 and Rosetta-gami B (DE3), were obtained from TransGen Biotech CO., Ltd. and Sangon Biotech Co., Ltd. (Shanghai, China) and the pET-22b (+) was purchased from Sangon Biotech Co., Ltd. Chitin powder (from crab shells), chitosan oligosaccharide degree of deacetylation (DDA) >90%, carboxymethyl cellulose sodium salt (CMC-Na), and carboxymethyl chitosan (DDA ≥ 90%) were obtained from Solarbio (Beijing, China), Dalian Zhongke Glake Biotechnology Co., Ltd. (Dalian, China), Damao Chemical Reagent Factory (Tianjin, China) and Hefei Bomei Biotechnology Co., Ltd. (Hefei, China), respectively. All other chemicals used in this study were of analytical grade. The P. chitinolyticus strain UMBR 0002 was reserved in the Guangdong culture collection center under the number GDMCC 60710.

Strains and culture conditions

Paenibacillus chitinolyticus strain UMBR 0002 was grown in sterile chitin medium at pH 7.0 containing (in g L⁻¹): colloidal chitin 3.0, tryptone 5.0, inorganic salt mixture 10.0 and agar powder 15.0 (for solid culture medium). The inorganic salt mixture contained (in g L⁻¹): KNO₃ 100.0, K₂HPO₄ 50.0, NH₄NO₃ 10.0, MgSO₄·7H₂O 50.0, NaCl 50.0, FeSO₄ 1.0, MnCl₂·H₂O 0.1 and ZnSO₄·7H₂O 0.1.

The E. coli TOP10 and Rosetta-gami B (DE3) strains were cultivated in sterile Luria-Bertani (LB) medium at pH 7.4 ± 0.2, containing (in g L⁻¹): tryptone 10.0, yeast extract 5.0, NaCl 10.0, and agar powder 15.0 (for solid culture medium).

Preparation of colloidal chitin

Colloidal chitin was prepared according to the method of Laribi-Habchi et al. (2015) with minor modifications. Briefly, 10.0 g of chitin powder dissolved in 176 mL of concentrated hydrochloric acid (12 M), stirred well and stored at 4 °C for 24 h. Thereafter, 1.0 L of distilled water was added to the chitin solution and the mixture incubated for another 24 h at 4 °C. The precipitate was harvested by centrifugation at 8,000 rpm for 10 min and washed repeatedly with sterile distilled water until the pH reached 7.0. Finally, the precipitate was dissolved in sterile distilled water to a final concentration of 25.0 g L⁻¹. The solution was stored at 4 °C for subsequent use.

Activity assay

The specific activity (mU mg⁻¹ protein) of purified recombinant CHI and crude protein extracts solution toward colloidal chitin were determined according to the method described by Miller (1959). Diluted recombinant protein solution (100 μL) in phosphate buffer was mixed with colloidal chitin (at a final concentration of 0.5%), and incubated for 1 h at 45 °C along with appropriate blanks. The reaction was stopped by the addition of 300 μL 3,5-dinitrosalicylic acid (DNS) followed by heating for 10 min in a boiling water bath. After the reaction system was cooled to room temperature, 300 μL of deionized water was added to the mixture, and the resulting solution was centrifuged at 8,000 rpm for 10 min. The optical density (OD) of the supernatant was measured at 540 nm using a BioTek spectrophotometer (Epoch, Winooski, VT, USA). Chitinase activity was determined by colorimetry, based on the amount of GlcNAc released from colloidal chitin. This was calculated by comparing OD₅₄₀ data with a standard curve prepared from serial dilutions of GlcNAc (from 0.1 to 1 mg mL⁻¹) (Mohamed et al., 2019). One unit of chitinase activity was defined as the amount of enzyme that releases 1 μmoL of GlcNAc from colloidal chitin per min under the specified assay conditions.

Plasmids and cloning of the chitinase gene

Oligonucleotide primers used for the cloning of the chitinase (CHI) gene from P. chitinolyticus strain UMBR 0002 were designed from the genomic database of P. chitinolyticus NBRC 15660 (GenBank accession number NZ_BBJT00000000.1). The nucleotide sequence of CHI gene was deposited in GenBank with the accession number MN121846. A DNA extraction kit was used to obtain DNA, which was then used as the template for polymerase chain reaction (PCR) amplification. The gene encoding CHI was amplified using Pfu DNA polymerase and the following primer pairs: 5′-GGATCCCGAACCGGCCAAAATCGTCGG-3′ and 5′-CTCGAGGCTGCCTGTTACCACAATATTCG-3′ (underlining indicates the added BamHI and XhoI sites). The PCR program was as follows: initial denaturation at 98 °C for 8 min, followed by 30 cycles of 98 °C for 30 s, 55 °C for 45 s and 72 °C for 2 min 30 s, with a final extension step at 72 °C for 10 min. The PCR samples were purified using a Universal DNA Purification Kit, digested with BamHI and XhoI and then ligated into a BamHI- and XhoI-digested vector pET-22b (+) to generate pET-22b (+)-CHI. The recombinant plasmid was transformed into E. coli TOP10 and verified using automated DNA sequencing (Sangon, Shanghai, China).

Bioinformatic analyses

The open reading frame (ORF) and encoded amino acid sequence of CHI were analyzed using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/) followed by translation of the nucleotide sequence using DNAMAN software. Domain structure was analyzed at the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and the three-dimensional structure of CHI was predicted using SWISS-MODEL (https://swissmodel.expasy.org/) (Jiang et al., 2017). A phylogenetic neighbor-joining tree was constructed using MEGA 5.0 software, and ClustalW was used for sequence alignment (Liu et al., 2018; Noby et al., 2018).

Expression and purification of chitinase

For the expression of recombinant CHI, pET-22b (+)-CHI was transformed into E. coli Rosetta-gami B (DE3) cells. Cells were cultured at 37 °C in liquid LB medium containing 100 μg mL⁻¹ ampicillin. Expression was induced by the addition of 0.5 mM isopropyl thio-β-D-galactoside (IPTG) when the OD₆₀₀ reached 0.6–1.0, after which cells were incubated at 37 °C for a further 8 h. Protein expression was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10% separation gel, 4% concentrated gel). The cell pellet was collected by centrifugation at 8,000 rpm for 10 min and stored at 4 °C. The freshly-prepared cell pellet was resuspended in lysis buffer (0.1 M Tris-HCl, pH 8.1, containing 0.3 M NaCl), then lysed on ice using an ultrasonic cell disrupter (JY92-IIN; Ningbo Scientz Biotechnology Co., LTD., Ningbo, China) followed by centrifugation (8,000 rpm, 15 min, 4 °C) to remove cell debris and unbroken cells. The supernatant, comprising crude protein extracts containing CHI, was immediately applied to a 5 mL Ni-NTA affinity chromatography column pre-equilibrated with binding buffer (0.1 M Tris-HCl, pH 8.1, 0.3 M NaCl and 10 mM imidazole) at 4 °C. The column was washed with 50 mL of wash buffers (0.1 M Tris-HCl, pH 8.1, 0.3 M NaCl and 20 mM imidazole, then 0.1 M Tris-HCl, pH 8.1, 0.3 M NaCl and 60 mM imidazole). Chitinase was eluted with 40 mL buffer containing 0.1 M Tris-HCl (pH 8.1), 0.3 M NaCl, and 150 mM and 250 mM imidazole, respectively. Purity of the recombinant protein was analyzed using 10% SDS-PAGE with Coomassie Brilliant Blue R-250 staining. The molecular mass of the purified enzyme was determined by comparison with protein molecular weight markers (Product #: 26616, Thermo Scientific, Beijing, China). The eluted fractions which contained target protein were concentrated and exchanged into a 50 mM phosphate buffer, pH 7.0, using 30 kDa centrifugal filter units (Millipore, Beijing, China). Protein concentrations were determined using the Bradford method (Bradford, 1976), and enzyme preparations were stored at 4 °C until use.

Biochemical characterization of chitinase

The optimum temperature for CHI activity was determined by incubating enzyme preparations with colloidal chitin at various temperatures between 25 °C and 80 °C in 50 mM phosphate buffer at pH 7.0. Thermostability was tested by preincubating the enzyme at various temperatures from 25 °C to 70 °C without substrate for 60 or 90 min. The optimum pH was determined by carrying out the reaction at pH 2.0–11.0 with 0.2 M phosphate (pH 2.0–8.0) or 0.2 M glycine-NaOH (pH 8.0–11.0) buffers. Chitinase was preincubated at pH 2.0–11.0 in the appropriate buffer at 4 °C for 5 h prior to the activity assay. The residual enzyme activity was tested at the optimum pH. The effects of metal ions and chemical reagents (Ca²⁺, Mg²⁺, Zn²⁺, Na⁺, K⁺, Al³⁺, NH₄⁺, Co²⁺, Ba²⁺, Cu²⁺, Fe³⁺, Mn²⁺, Ag⁺, Cd²⁺, Tris, SDS), ethylenediaminetetraacetic acid (EDTA), and urea) on CHI activity at final concentrations of 10 and 50 mM were also investigated, as were the effects of organic solvents and reagents (methanol, ethanol, isopropanol, glycerin, isoamyl alcohol, Tween 80, acetic acid, chloroform, dimethylsulfoxide (DMSO), and β-mercaptoethanol) at final concentrations of 10% and 20% (v/v). The effect of NaCl on CHI activity was determined by incubating the enzyme with NaCl at final concentrations of 0.5%, 1.5%, 3%, 4.5%, 6%, 7.5%, 9%, or 12.5% at 4 °C for 1 h. Residual CHI activity was measured in standard assay conditions. Appropriate controls were used.

Substrate specificity

The specificity of recombinant CHI was tested toward various substrates such as colloidal chitin, chitin powder, chitosan oligosaccharide, carboxymethyl cellulose sodium salt (CMC-Na) and carboxymethyl chitosan by adding the compounds to the assay mixture individually at a final concentration of 0.5% (w/v). Relative enzyme activity was then determined in standard conditions with the highest activity being taken as 100%.

Product analysis

The major products of colloidal chitin hydrolysis by CHI was identified using electrospray ionization-mass spectrometry (ESI-MS; AB Sciex, Framingham, Ma, USA) analysis. The reaction mixture containing 100 μL colloidal chitin (1%) and 0.25 mg CHI in 50 mM 100 μL phosphate buffer (pH 7.0) was incubated at 45 °C for 0.5 h, 1 h, 6 h and 14 h. After incubation, the reaction mixture was boiled for 5 min and centrifuged at 8,000 rpm for 10 min at 4 °C. The supernatant was analyzed by ESI-MS.

Expression and purification of chitinase

The gene encoding chitinase was successfully isolated from P. chitinolyticus strain UMBR 0002 and recombinant CHI was heterologously expressed in E. coli Rosetta-gami B (DE3) and purified. The molecular weight was estimated to be between 70 and 100 kDa by SDS-PAGE (Fig. 1), which is consistent with the expected weight of approximately 80 kDa according to the translated nucleotide sequence. In our study, recombinant CHI was purified with 13.36-fold enrichment and a recovery yield of 72.20%. The purified enzyme had a specific activity of 750.64 mU mg⁻¹ (Table 1).

Bioinformatic analysis

The gene encoding CHI included a complete ORF encoding a protein of 765 amino acids. Domain structure analysis revealed CHI to contain a chitin-binding domain (CBD) and two fibronectin-type-III domains (FN3). The enzyme was identified to belong to glycoside hydrolase family 18 (Fig. 2A). Sequence alignment using BLASTP software (NCBI) revealed that CHI exhibits 98.26% sequence identity and 97% sequence coverage with chitinase from P. chitinolyticus (WP_042235807.1), followed by chitinase Chi80 from P. ehimensis (BAC76694.1) (97.58% identity and 97% sequence coverage), chitinase from Paenibacillus sp.UNC499MF (WP_103996460.1) (95.49% identity and 95% sequence coverage), chitinase from Paenibacillus sp. FJAT-26967 (WP_068776920.1) (79.59% identity and 94% sequence coverage), and chitinase from Bacillus sp. EAC (WP_088042647.1) (62.62% identity and 94% sequence coverage). Multiple sequence alignment showed that, although the coding amino acid sequences of different chitinases vary greatly, the key amino acids within the catalytic domain are highly conserved. In this study, two conserved motifs with the sequences KxxxxxGGW and FDGxDLDWEYP were identified in CHI (Fig. 2B). Within these conserved sequences, CHI has two conserved modules typical of the glycoside hydrolase family 18, SxGG and DxxDxDxE, which are responsible for substrate binding and catalytic degradation, respectively. Glutamic acid (E) represents a general acid/base (catalytic acid/base) for the protonation of oxygen atoms from sugar molecules. Phylogenetic analysis revealed that CHI shares close evolutionary relationships with chitinases from P. ehimensis and P. chitinolyticus (Fig. 3A). Comparisons using the Protein Data Bank (PDB) revealed the homology of CHI and Chitinase ChiA 74 from B. thuringiensis to be 62.40%, with high matching accuracy. The results of homologous modeling are illustrated in Fig. 3B.

Effect of temperature and pH on enzyme activity and stability

The determined temperature-activity profile of CHI is shown in Fig. 4A. Chitinase activity increased gradually with increasing temperature from 25 to 45 °C; maximal activity was observed at 45 °C. Relative enzyme activity of >90% was retained at 50 °C. Above 50 °C, the activity of CHI decreased markedly. The thermal stability of CHI is illustrated in Fig. 4B. The enzyme was generally stable at 25–45 °C for both periods of incubation.

The effect of pH on the activity of CHI is shown in Fig. 4C. The highest activity was observed within the pH range 4.0–8.0, with maximal activity recorded at pH 5.0 (relative activities at pH 4.0 and 7.0 were 53.16% and 71.02% respectively). Activity at pH 8.0 was higher in glycine-NaOH than phosphate buffer. The effect of pH on CHI stability is shown in Fig. 4D. The optimum pH for stability of this chitinase was 8.0, but the enzyme was very stable within the pH range 4.0–10.0 (with relative activity of >70% observed after incubation at 4 °C for 5 h).

Effect of chemical reagents, metal ions, and solvents on enzyme activity and stability

The influence of metal ions and chemical reagents on the activity of purified CHI is shown in Fig. 5. The presence of Mn²⁺_, Ca²⁺ and urea significantly increased the activity of CHI. We found that the activity of CHI was not significantly affected by Mg²⁺, Na⁺, K⁺, or NH₄⁺ in the present study. When mixed with 10 mM Co²⁺_, Tris, or Cd²⁺, the residual activity of CHI was high, but decreased moderately when these reagents were present at 50 mM. Relatively high activity (>100%) was maintained in the presence of 10 mM Ba²⁺, although the activity dropped sharply when the concentration of Ba²⁺ was increased to 50 mM. We found CHI activity to be strongly inhibited at both tested concentrations of Zn²⁺, Al³⁺, Ag⁺ and SDS (relative activity of <60% in each case). However, the presence of Cu²⁺ and Fe³⁺ obviously inhibited the activity of CHI. We also found that 10 mM EDTA enhanced activity, while 50 mM EDTA significantly reduced activity.

The influence of various organic solvents on the stability of purified CHI is shown in Fig. 6; the activity obviously increased in the presence of Tween 80. In addition, CHI activity was moderately increased (relative activity >100%) in the presence of 10% chloroform, although activity decreased sharply when 20% chloroform was used. Most of the organic solvents tested in the present study; including methanol, ethanol, isopropanol, glycerin and DMSO; caused CHI activity to decrease somewhat when used at 10% final concentration, while 20% organic solvent markedly suppressed activity. However, β-mercaptoethanol, isoamyl alcohol and acetic acid strongly inhibited CHI activity (by >50%) at both concentrations.

Effect of NaCl concentration on enzyme activity

As Fig. 7 shows, the optimum salt concentration was found to be 1.5% NaCl. At higher concentrations, chitinase activity decreased, reaching <60% relative activity at 12.5% NaCl.

Substrate specificity

The specific activities of purified recombinant CHI toward different substrates are shown in Table 2. The highest specific activity was toward colloidal chitin (567.20 mU mg⁻¹). The next highest activity was observed when chitin powder was used as a substrate (288.69 mU mg⁻¹); in this case, the relative activity was 50.90% ± 3.04% compared with colloidal chitin (100%). No activity was detected toward chitosan oligosaccharide (DDA > 90%), carboxymethyl chitosan (DDA ≥ 90%) or CMC-Na.

Mass spectrometry analysis of the hydrolysis product

We used ESI-MS in positive ion mode to analyze the major products of colloidal chitin hydrolysis by purified CHI and the degradation mode of chitinase was inferred. As shown in Figs. 8 and 9, peaks with m/z of 244/260 (GlcNAc), 447/463 (GlcNAc)₂ and 650 (GlcNAc)₃ were attributed to sodium, potassium, or hydrogen adducts of oligosaccharides with different degrees of polymerization. ESI-MS analyses showed the major hydrolysis product of colloidal chitin to be GlcNAc, with some (GlcNAc)₂ produced (Fig. 9).