Comparative studies of two AA10 family lytic polysaccharide monooxygenases from Bacillus thuringiensis

Sequence and structure analysis

The sequences of BtLPMO10A (GenBank ID: AJP62637) and BtLPMO10B (GenBank ID: ALP73598) can be accessed in the Genbank database and the crystal structure of BtLPMO10A was obtained from the Protein Data Bank database with accession code 5WSZ (Zhang et al., 2020). The three-dimensional structure of the catalytic domain of BtLPMO10B (BtLPMO10B-CD) was generated by homology modeling using Modeller 9.19 (Webb & Sali, 2016) with the crystal structure of BaLPMO10A from Bacillus amyloliquefaciens (PDB ID: 2YOX) (Hemsworth et al., 2013) as the template since they share the highest sequence identity (66%) (Zhang & Madden, 1997). After been further validated by DOPE score, a structure-based sequence alignment of BtLPMO10A and BtLPMO10B was conducted using Mega 7.0 (Kumar, Stecher & Tamura, 2016) and ESPript 3.0 (Robert & Gouet, 2014).

Cloning of BtLPMO10B and its catalytic domain BtLPMO10B-CD

We produced three recombinant LPMOs from B. thuringiensis kurstaki ACCC10066 in E. coli BL21 (DE3), including the previously reported BtLPMO10A which stored in the lab. The gene encoding BtLPMO10B was amplified from the genomic DNA of B. thuringiensis kurstaki ACCC10066 using a forward primer F1: 5′-GGAATTCCATATGCACGGTTTTGTTGAAAAGCCCGGTA-3′ encoding a restriction site for NdeI and a reverse primer R1: 5′-CCGCTCGAGCACTGTTTTCCATAATGATAATGCA- 3′ with a restriction site for XhoI. The amplified gene was then subcloned into the pET23b vector through double digestion with the two restriction enzymes. The catalytic domain of BtLPMO10B (BtLPMO10B-CD) was synthesized and cloned into the same vector by the Taihe Biotechnology Co., Ltd (Beijing, China). After verification by sequencing, three recombinant plasmids were transformed into Escherichia coli BL21 (DE3) competent cells, respectively, for protein expression.

Protein expression and purification

The recombinant E. coli BL21 (DE3) cells were cultivated in 1 L Luria-Bertani (LB) medium at 37 °C with constant shaking at the speed of 180 rpm. When the OD₆₀₀ of the culture reached 0.6, a final concentration of 0.05 mM IPTG and 0.2 mM CuSO₄ were added and the cultivation was continued for an additional 4 h at 30 °C. Afterword, the cells were harvest by centrifugation at 4 °C for 10 min with the speed of 8,000 × g, and then resuspended in 100 mL of hypertonic solution containing 100 mM Tris–HCl pH 8.0, 20% sucrose and 0.5 mM EDTA. This step was performed two times. Finally, the precipitated cells obtained by centrifugation were resuspended in 100 mL hypotonic solution (1 mM MgCl₂) and incubated on ice for 10 min. After 10 min of centrifugation at 8,000 × g, the supernatant was collected for further purification.

For the purification of BtLPMO10A, a chitin beads affinity chromatography method was performed as described previously (Zhang et al., 2015). For the BtLPMO10B, a similar method was adopted with some modifications. The loading buffer was changed to 20 mM Tris–HCl (pH 8.0) and 0.15M (NH₄)₂SO₄, and the protein was eluted by 20 mM acetic acid. For the purification of BtLPMO10B-CD, an ion exchange chromatography with HiTrap Q column (GE Healthcare, USA) was performed. The protein solution was loaded onto the column equilibrated with 20 mM Tris–HCl buffer (pH 7.5) and eluted with a linear salt gradient using 1 M NaCl (pH 7.5). The obtained fractions were pooled and concentrated using the Amicon 8400 stirred cell (Millipore, Burlington, MA, USA) installed with a 3kDa cut-off membrane. Samples purity was analyzed by SDS-PAGE and the protein concentrations were measured by Bradford, using bovine serum albumin as a standard.

Substrate binding assays

The reactions were conducted in 20 mM Tris–HCl (pH 8.0) buffer containing 1 µM enzyme and 5 mg mL⁻¹ α-chitin, β-chitin and colloidal chitin, respectively, prepared according to the procedure described previously (Zhang et al., 2015). The mixture was incubated 6 h at 25 °C with constant shaking at 800rpm using Thermo block (Eppendorf, Hamburg, Germany). After been separated from the mixture by filtration through a 0.22 µm membrane, the concentrations of the free proteins measured using the Quick Start™ Bradford assay (Bio-Rad, Hercules, CA, USA). The mixtures without substrate were treated in the same way and used as the basis for calculating the percentage of free and bound protein.

H₂O₂ generation assays

The reactions were conducted in 20 mM Tris–HCl (pH 8.0) buffer containing 1 µM enzyme, 1 mM ascorbic acid and 5 mg mL⁻¹ α-chitin, β-chitin and colloidal chitin, respectively. The mixture was incubated 2 h at 30 °C with constant shaking at 800 rpm using Thermo block (Eppendorf, USA). After been separated from the reaction mixture by filtration through a 0.22 µm membrane, the concentrations of H₂O₂ in the supernatant were measured using the Fluorimetric Hydrogen Peroxide Assay Kit (Sigma, St. Louis, MO, USA). The reactions without substrate were set as the control.

Enzymatic reactions

Enzymatic reaction was performed in a 500 µL reaction mixture containing 5 mg mL⁻¹ substrate, 20 mM Tris–HCl (pH 8.0), 1 µM enzyme, and 1 mM ascorbic acid. For the reaction using both BtLPMO10A and BtLPMO10B, 0.5 µM BtLPMO10A and BtLPMO10B was added. The reaction was last for 16 h at 30 °C with constant shaking at the speed of 800 rpm. After been separated from the reaction mixture by filtration through a 0.22 µm membrane, the generated oligosaccharides were analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and high-performance liquid chromatography (HPLC) with protocols described previously (Zhang et al., 2015; Zhang et al., 2020). Since the content of produced DP4_ox is a feasible parameter to evaluate the efficiency of LPMO reactions (Zhang et al., 2020), the peak areas of DP4_ox, DP5_ox, and DP6_ox were calculated for comparative analysis.

Synergetic assays

The chitinase synergetic experiments were carried out in 20 mM PBS buffer (pH 6.0) containing 5 mg mL⁻¹ α-chitin (Sigma, USA), 1 µM SmChiB (anexo-type chitinase from Serratia marcescens), 1 µM LPMO (BtLPMO10A or BtLPMO10B) and 1 mM ascorbic acid mixed in 500 µL reaction. The mixtures were incubated at 30 °C for 4, 8, 12, 24, 48, 72 h with an 800 rpm shaking. After separated the products from the reaction mixture by filtration through a 0.22 µm membrane, an equal volume of acetonitrile was added into the product solution. For BtLPMO10A and BtLPMO10B synergy studies, 0.5 µM BtLPMO10A and BtLPMO10B was added into the mixture. The reaction without the presence of LPMO was used as the control. The (GlcNAc)₂ released from the reactions was analyzed by HPLC equipped with an X-Amide column and a UV detector at 195 nm. The concentration of (GlcNAc)₂ in samples was calculated using commercial (GlcNAc)₂ as a standard. All experiments were performed in triplicates.

Structure and sequence analysis of BtLPMO10A and BtLPMO10B

BtLPMO10A and BtLPMO10B shares a sequence identity of 61%, and both enzymes contain a typical active site of AA10 family LPMOs that are comprised of a type II copper ion coordinated with two fully conserved histidines (Figs. 1A and 1B). A Phenylalanine residue was identified to be the axial residue of the copper ion. Furthermore, the residues in the substrate binding surface, as shown in Fig. 1B, were also highly conserved, which may involve in substrate binding similar as described in Sm CBP21A (Vaaje-Kolstad et al., 2005). Structure-based sequence alignment indicated that BtLPMO10A and BtLPMO10B possess similar loop regions surrounding the catalytic center (Fig. 1C).

Substrate binding ability and activity of BtLPMO10A and BtLPMO10B

The substrate-binding ability of BtLPMO10A and BtLPMO10B on α-, β-chitin, and colloidal chitin were assessed. As shown in Fig. 2B, BtLPMO10A exhibits comparable binding ability to all three types of chitins. In contrast, for BtLPMO10B, 80% of the enzyme protein was found bound to the colloidal chitin, which is significantly higher than the percentages of protein bound to the crystalline α- and β-chitin. Deletion of the extra domains of BtLPMO10B significantly decreased its binding ability towards all three kinds of chitins tested (Fig. 2B).

Product analysis of BtLPMO10A, BtLPMO10B, and BtLPMO10B-CD

The enzymatic activity of BtLPMO10B towards various types of chitins, including α-, β-chitin, colloidal chitin, and chitin oligosaccharides were investigated using MALDI-TOF MS. As shown in Figs. 3A and 3B, BtLPMO10B and BtLPMO10B-CD can act on α-, β-chitin, and colloidal chitin and generate a product profile with even-numbered oxidized oligosaccharides as the dominant products. The products of BtLPMO10A, BtLPMO10B and BtLPMO10B-CD from different kinds of chitins were further analyzed by HPLC (Table 1, Fig. 3D). The results showed that the DP4_ox and DP6_ox were the main oxidation products for BtLPMO10A alone or in combination with BtLPMO10B on almost all tested chitins. Differently, the DP4_ox was the predominant product for BtLPMO10B reactions and the highest production of the DP4_ox was obtained when β-chitin was used as the substrate. The deletion of the CBM5 from BtLPMO10B significantly reduced the production of DP4_ox, while relative mild reduction of observed for DP5_ox and DP6 _ox (Table 1). Moreover, although the binding ability of BtLPMO10B-CD toward colloidal chitin was significantly lower than the full-length enzyme (Fig. 2B), the amount of oxidized products (DP4_ox, DP5_ox, and DP6_ox) generated by BtLPMO10B-CD were similar as compared to BtLPMO10B. The combination of BtLPMO10A and BtLPMO10B led to similar product profiles on both α-chitin and colloidal chitin as compared to BtLPMO10B. In contrast, the product profile on β-chitin with the combined BtLPMO10A and BtLPMO10B was similar to that of BtLPMO10A. These results suggest that BtLPMO10B can affect the activity of BtLPMO10A on α-chitin and colloidal chitin.

H₂O₂ production of BtLPMO10A, BtLPMO10B and BtLPMO10B-CD

When the substrate was absent, H₂O₂ produced by BtLPMO10A was much higher than that produced by BtLPMO10B and BtLPMO10B-CD (Figs. 4A and 4B). Accordingly, a much stronger suppression of H₂O₂ generation in BtLPMO10A was observed as the substrate, especially α-chitin or colloidal chitin, has been provided. Similarly, a mild suppression of H₂O₂ production by substrate had been observed in the BtLPMO10B, in which all three substrates showed comparable effect. As for the BtLPMO10B-CD, similar inhibition in H₂O₂ production could be observed when colloidal chitin has been added, while negligible effect on the H₂O₂ production was recorded when provided with α-chitin or β-chitin.

The synergy in chitin degradation

The synergetic effects of BtLPMO10A or BtLPMO10B with SmChiB in α-chitin degradation were performed in the present study (Fig. 5). SmChiB from Serratia marcescens is a model GH18 exo-chitinase which degrades the polymer chains from their non-reducing ends and dominantly produces (GlcNAc)₂ (Chen et al., 2020; Van Aalten et al., 2000). The synergy experiment results showed that when only SmChiB was present, the concentration of generated chitobiose reached its plateau (0.336 ± 0.0187 mg/ml) after 12 h of reaction. In contrast, the additional supply of BtLPMO10A or BtLPMO10B can both significantly boost the accumulation of chitobiose, which has reached 1.464 mg ml⁻¹ for BtLPMO10A and 1.232 mg ml⁻¹ for BtLPMO10B, respectively, after 72 h of incubation. Moreover, when both BtLPMO10A and BtLPMO10B were provided, the curve of the concentration of GlcNAc₂ over time was similar to that observed when only BtLPMO10B was provided.