Improving thermostability of salt-tolerant xylanase from Hortaea werneckii EXF-12619 by N-terminal replacement and C-terminal carbohydrate binding module fusion

doi:10.21203/rs.3.rs-4454065/v1

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Improving thermostability of salt-tolerant xylanase from Hortaea werneckii EXF-12619 by N-terminal replacement and C-terminal carbohydrate binding module fusion

https://doi.org/10.21203/rs.3.rs-4454065/v1

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Xylanase from glycoside hydrolases family 11 (GH 11) is widely applied because of their substrate selectivity and broad optimal pH. However, higher thermostability is always preferred in addition to other stress tolerance of the enzyme. Therefore, we first expressed and characterized a novel GH 11 xylanase from Hortaea werneckii (HWxyn11) tolerant to NaCl and extreme pH conditions although still unsatisfied thermostability. To achieve better thermostability, we then constructed mutants by applying N-terminal replacement or C-terminal carbohydrate binding module (CBM) fusion, considering that thermostability of GH 11 xylanase could be affected by structural differences between N-terminus and C-terminus significantly. The combined mutant HWxA31-CBM 6 had increased thermostability than that of HWxyn11. Molecular dynamics simulation analysis reveals that the N-terminal replacement or C-terminal CBM fusion resulted in declined flexibility, indicating the improvement of structural rigidity and thermostability. Structural analysis confirms that the removal of N-terminal propeptide and the fusion of CBM contributed to improved thermostability. Our findings prove that the combination of replacing N-terminal sequences and fusing with CBMs from thermophilic xylanases contributed to declined structural flexibility, providing a practicable method to improve the thermostability of GH 11 xylanases.

Carbohydrate binding module

N-terminal replacement

Thermostability

Xylanase

HWxyn11 is a halophilic xylanase with remarkable pH stability.
A combined mutant HWxA31-CBM 6 with better thermostability was obtained.
Removal of propeptide and CBM fusion facilitate thermostability of GH 11 xylanases.

Xylan, a complex heteropolysaccharide, is the primary hemicellulose component in the cell wall of plants (> 30% of the dry weight) and ranks second after cellulose in terms of its global abundance (Saravanan et al. 2022). The transformation of xylan to xylooligosaccharides is essential for recycling of natural resources. For this transformation, endo-ß-1,4-xylanase (EC 3.2.1.8) completes depolymerization of xylan through cleaving its ß-1,4-xylosidic linkage randomly and converts xylans to xylooligosaccharides. Thus, xylanase is successfully applied in the industries of food, paper bleaching, animal feed and biofuels(Walia et al. 2017; Kaushal et al. 2021; Fernandes de Souza et al. 2022; Chaudhary et al. 2023). Among glycoside hydrolase families (GH), xylanases belonging to GH 11 are attractive for industrial use because of high substrate selectivity and broad optimal pH.

Currently used xylanases of GH 11 have poor thermostability that hampers the wide industrial application. Structural differences are the reason of this poor thermostability (Ishida et al. 2020; Kim et al. 2023). Most structure of GH 11 xylanase is consisted of α-helixs and twisted antiparallel ß-sheets, and the whole structure is shaped like a partially closed right hand (Madan and Lee 2018). Molecular dynamics simulations show that N-terminal loop is the main flexible region influencing the stability of GH 11 xylanase (Yu and Huang 2014). Replacing part or all the N-terminal sequence of thermophilic xylanase can improve the thermostability of GH 11 xylanases successfully (Gao et al. 2013; Tian et al. 2022; Wu et al. 2023a). Besides, carbohydrate-binding module (CBM), an essential part of thermophilic glycosidase, also plays important role in thermostability (Li et al. 2023). The fusion or removal of CBM also affects the thermostability of GH 11 xylanases(Yang et al. 2018; Miao et al. 2022). Therefore, rational design of N-terminus or C-terminus is a feasible strategy to improve the thermostability of xylanase. However, the combined effect of these two strategies has remained unclear. Studying the combination of N-terminal replacement and C-terminal CBM fusion will expand our understanding of the relationship between thermostability and structure-function of GH 11 xylanases.

Therefore, we first expressed and characterized a GH 11 xylanase HWxyn11 from Hortaea werneckii EXF-12619To further improve the thermostability of HWxyn11, we constructed new mutants using N-terminal replacement with a fragment of different lengths from rxyn11A, and C-terminal fusion with CBMs from thermophilic xylanases. Our findings prove that both N-terminal replacement and C-terminal CBM fusion contributed to better thermostability of xylanases.

Gene, strains and substrates

A xylanase gene from Hortaea werneckii EXF-12619 (HWxyn11, GenBank Accession number: KAI6862253.1) was identified according to amino acid sequences, structural domain compositions and 3-dimensional structures of reported xylanases. The whole gene synthesis of HWxyn11 was constructed into pPICZαA vector with 6×His tag added to C-terminus for protein purification by Sangon Biotech (Shanghai, China). Escherichia coli Top10 competent cells for cloning were purchased from TIANGEN Biotech (Beijing, China). Pichia pastoris X33 cells preserved in the laboratory were used for protein expression. Prime STAR Max Polymerase were obtained from Takara (Shiga, Japan). Zeocin was obtained from Solarbio (Beijing, China). Yeast extract and peptone were obtained from OXOID (Basingstoke, Hampshire, UK), yeast nitrogen base without amino acids was purchased from Sangon Biotech (Beijing, China). Beechwood xylan was obtained from Yuanye (Shanghai, China). All other chemicals used were of reagent grade obtained from standard sources.

Sequence and structure analysis

The searches and analysis of xylanase nucleotide and amino acid sequence were done by NCBI BLAST (https://blast.ncbi.nlm.nih.gov). Signal peptide of HWxyn11 was predicted by SignalP 5.0 website (https://services.healthtech.dtu.dk/services/SignalP-5.0/). Conserved domain analyses were accomplished by NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and Interpro (https://www.ebi.ac.uk/interpro). Multiple sequence alignments of xylanases were conducted and drawn by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript). Protein structure prediction was done by AlphaFold 2.3.2 and PyMoL 3.0 was used for structure visualization.

Construction of mutants

To obtain mutants of N-terminal replacement, pPICZαA-HWxyn11 and pPICZαA-rxyn11A plasmids were used as templates. Based on sequence alignment, mutants HWxA6, HWxA15, HWxA26, HWxA31 and HWxA40 were constructed by sequentially replacing fragments of 6, 15, 26, 31 and 40 amino acid residues in length from rxyn11A to the corresponding fragments in HWxyn11. The mutant construction was carried out using overlap extension PCR and seamless cloning. To obtain mutants of C-terminal CBM fusion, 5 CBMs were fused at the C-terminal end of the HWxyn11 sequence, including CBM 2, RMCBM 4, TNCBM 4, CBM 6 and CBM 9. The sources and sequence information of the above CBMs are shown in Table S1. The temperature characteristics were analyzed to determine the best mutants obtained by the two above strategies and combined mutant was constructed using similar methods as N-terminal replacement.

PCR products were ligated into pPICZαA and transformed into Escherichia coli Top10 competent cells. All mutants were verified through sequencing of entire genes by Sangon Biotech (Shanghai, China). The primers used in this study are listed in Table S2.

Expression and purification

All plasmids were linearized by restriction digestion with Sac I, linearized DNA were transformed into Pichia pastoris X-33 by electroporation using MicroPulser (Bio-Rad, Hercules, CA). Transformants were selected on the yeast extract peptone dextrose medium agar plates (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, 20 g/L agar) containing 100 µg/mL Zeocin after cultivating at 30 ℃ for 2–3 days. A single colony was inoculated into 50-mL buffered glycerol-complex medium (10 g/L yeast extract, 20 g/L peptone, 100 mM potassium phosphate (pH 6.0), 13.4 g/L yeast nitrogen base without amino acids, 4×10^− 4 g/L biotin, 1% (v/v) glycerol) in a 500-mL baffled flask and shaken (250 rpm) at 29 ℃ for 20 hours. Cells were harvested and resuspended to an OD₆₀₀ of 1.0 in 500-mL buffered methanol-complex medium (10 g/L yeast extract, 20 g/L peptone, 100 mM potassium phosphate (pH 6.0), 13.4 g/L yeast nitrogen base without amino acids, 4×10^− 4 g/L biotin, 0.5% (v/v) methanol) in a 2-L baffled flask, shaken at 29 ℃ and added 0.5% (v/v) methanol every 24 h for inducing 108 h.

The culture supernatant was collected by centrifuging at 12,000×g for 25 min at 4 ℃ and concentrated by Vivaflow 200 ultrafiltration membrane package (Sartorius, Göttingen, Germany). The AKTA avant 25 chromatography system (GE Healthcare, Little Chalfont, UK) and Ni²⁺-column were used for purification. Unwanted proteins were removed with 20 mM Tris-HCl buffer (pH 7.4) containing 20 mM imidazole and target proteins were eluted using 20 mM Tris-HCl buffer (pH 7.4) containing 500 mM imidazole. Purified proteins were concentrated by ultrafiltration with an Amicon Ultra-15 10 kDa centrifugal filter unit (Merck Millipore, MA). The purity of xylanase was assessed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein concentrations were determined by BCA method using bovine serum albumin as standard.

Xylanase activity assay

The xylanase activity was measured by calculating the amount of reducing sugar released from beechwood by 3,5-dinitrosalicylic acid method using xylose as standard(Miller 1959). The reaction mixture containing 180 µL of 1% (w/v) beechwood solution in 50 mM citric acid buffer (pH 5.0) and 20 µL of appropriately diluted xylanase enzyme solution was incubated at 45 ℃ for 5 min. The reaction was terminated by adding 200 µL 3,5-dinitrosalicylic acid to mixtures. Samples were cooled to room temperature after boiling for 10 min. The absorption was measured at 540 nm using iMark Microplate Reader (Bio-Rad, Hercules, CA). One unit of xylanase activity was defined as the amount of xylanase that produced 1 µmol of reducing sugar equivalent xylose per minute. All xylanase activity values were averages obtained from triplicate measurements and an inactivated-enzyme mixture was used as negative control.

Biochemical characteristics of xylanases

The optimal pH of xylanase was measured at optimal temperature with a pH range of 3.0–11.0 using 50 mM citrate buffer (pH 3.0–5.0), 50 mM phosphate buffer (pH 6.0–8.0) or 50 mM Glycine-NaOH sodium buffer (pH 9.0–11.0). To test the pH stability, enzymes were incubated in the pH range of 3.0–11.0 at 4 ℃ for 24 h and residual enzyme activities were determined under standard assay.

The optimal temperature of xylanase was determined at optimal pH from 30–60 ℃. The thermostability of xylanase was measured by calculating residual enzyme activity after incubation at different temperatures for 60 min. The half-life of enzymes was determined by incubating for different time at 55 ℃ and calculated using the formula y = Ae^− kt, where y is relative enzyme activity, A is initial enzyme activity (100%), t is time and k is slope.

The effect of metal ions on the activity of xylanase was determined by incubating the enzyme in 50 mM phosphate buffer (pH 5.0) with various metal ions (final concentration of 5 mM and 10 mM) at 4 ℃ for 24 h. The residual enzyme activities were determined under standard assay.

The NaCl stability of xylanase was determined by incubating the enzyme in different NaCl concentrations ranging from 0.0 to 5.0 M at 4 ℃ for 24 h, and the residual enzyme activities were determined under standard assay.

Molecular dynamics simulations

To analyze the differences of heat fluctuations between wild-type and mutants, molecular dynamics simulations were done using GROMACS 2021.5. The structure of proteins was solvated in SPC water box using amber14sb force field. Molecular dynamics simulations were conducted at 328 K (55 ℃) for 100 ns with the pressure of 1 bar after energy minimization in vacuum with 100 ps NVT and NPT equilibration. The root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) of simulated proteins were analyzed using software in GROMACS 2021.5. PyMoL 3.0 is used to display the results of B-factor.

Sequence and structure analysis of HWxyn11

The sequence of HWxyn11 gene from Hortaea werneckii EXF-12619 has one open reading frame of 993 bp encoding 331 amino acids with a putative signal peptide of 20 amino acids (Fig. 1). The signal peptide was removed in cloning and expression subsequently. Based on the BLAST analysis, xylanase HWxyn11 shows the highest similarity to GH 11 family and exhibits the highest amino acid identity of 58.08% with xylanase rXyn11A from Thermobifida fusca (PDB codes: 3zse.1) (Zhao et al. 2015). The sequence alignment and conserved domain prediction show that HWxyn11 possesses only one catalytic domain of GH 11 and two glutamate residues are important in the catalytic reaction as same as other GH 11 xylanases.

The structure of HWxyn11 is a classical right-handed palm consisting of α-helixs and ß-sheets, involving two glutamates acting as catalytic residues as other xylanases belonging to GH 11 (Fig. 2). The hydrophobic faces are packed against each other to form the hydrophobic core of protein. Exceptionally, HWxyn11 contains a propeptide in front of protein catalytic domain at N-terminus and an unfolded random curly with a length of 109 amino acids at C-terminus.

Expression and purification of HWxyn11

After inducing with 0.5% (v/v) methanol at 29 ℃ for 108 h, the mature HWxyn11 without signal peptide was successfully expressed in Pichia pastoris X-33. The protein was purified to apparent homogeneity by Ni-NTA affinity chromatography. The specific activity of HWxyn11 was 99.94 U/mg. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that recombinant HWxyn11 was approximately 60 kDa and significantly larger than the predicted value of 33.78 kDa. However, the molecular weight of HWxyn11 did not change after deglycosylation treatment with endoglycosidase F1 to remove N-glycosylation (Fig. 3). The results indicated that increased molecular weight of HWxyn11 was due to extensive O-glycosylation modifications.

Biochemical characteristics of HWxyn11

As shown in Fig. 4a, HWxyn11 revealed its peak activity at 45°C. HWxyn11 is a mesophilic xylanase, maintaining more than 80% relative enzyme activity with a wide range from 35 ℃ to 60 ℃. Results of thermostability tests indicated that HWxyn11 showed high stability at lower temperature, preserving nearly 88% of its initial activity at 45 ℃ after 60 min. However, the enzyme activity was only 19.34% after incubating at 55 ℃ for 60 min (Fig. 4b). The half-life of HWxyn11 at 55 ℃ is 22.95 min.

Consistent with most reported xylanases of GH 11, HWxyn11 exhibited highest activity under weakly acidic conditions at pH 5.0(Luong et al. 2023; Kim et al. 2023; Zhang et al. 2023). HWxyn11 maintained more than 75% of its maximal enzyme activity in a range of pH 4.0–7.0 (Fig. 4c). Furthermore, HWxyn11 showed exceptional stability between pH 3.0–11.0, retaining over 87% of its residual enzyme activity after 24 h incubation at 4 ℃ (Fig. 4d).

Figure 4e shows the effects of metal ions on the enzyme activity of HWxyn11. Fe²⁺, Al³⁺ had inhibitory effect on recombinant HWxyn11. However, Na⁺ improved the enzyme activity to 1.16 times at a concentration of 10 mM. Other metal ions such as K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Cu²⁺, Zn²⁺, Co⁺, Ni²⁺, Mn²⁺ and Li⁺ all showed little influence on the enzyme activity of HWxyn11, and residual enzyme activity is all above 78% after incubation at 4 ℃ for 24 h.

As shown in Fig. 4f, HWxyn11 is a halophilic xylanase. The stability of HWxyn11 was barely affected in 0–5.0 M concentration of NaCl after incubating at 4 ℃ for 24 h. The enzyme activity retained about 83% of when hydrolyzing beechwood xylan at a final concentration of 5.0 M NaCl.

Construction of mutants based on predicted 3-dimensional structure and molecular dynamic simulation

As shown in Fig. 5, the RMSF values and B-factor for N-terminal and C-terminal regions of HWxyn11 are significantly higher than those in other areas. Generally, the higher the RMSF values and B-factor, the more flexible the amino acid residues are(Sun et al. 2019). Both regions mentioned above exhibit greater structural flexibility and lower thermostability.

By sequence comparison and structural domain analysis, there is a relatively low similarity of N-terminal and C-terminal sequences between HWxyn11 and thermophilic xylanase rXyn11A. Moreover, rXyn11A is composed of a GH 11 xylanase structural domain and a CBM 2. However, HWxyn11 contains a GH 11 xylanase structural domain and a long flexible loop region at C-terminus instead of CBM (Fig. 6). It is inferred that significant differences in N-terminal and C-terminal sequences and structural domain composition of HWxyn11 may be important factors affecting its thermostability.

The structure of wild-type HWxyn11 was superimposed with the structure of rXyn11A with results shown in Fig. 7. HWxyn11 contains a short peptide that spatially distances itself from adjacent ß-sheet. Additionally, its length and rotation angle of ß-sheet positioned similarly in N-terminus of rXyn11A are different. Molecular dynamics simulations analysis also showed greater fluctuations in the N-terminus of HWxyn11, suggesting that the presence of this peptide could impact dynamic stability of protein conformation. The substantial N-terminal structural differences caused by this peptide may be the reason for the accelerated unfolding and inferior thermostability of HWxyn11 under high temperatures.

Therefore, we chose to modify N-terminal and C-terminal regions of HWxyn11 for constructing mutants. Two types of mutants were constructed. HWxAs were composed of HWxyn11 amino acid sequence with different lengths of N-terminal sequence of rXyn11A (Fig. 6a). HWxyn11-CBMs were constructed by fusing different CBMs from thermophilic bacteria at C-terminus.

Temperature characteristics of mutants

All mutants were successfully expressed and purified to apparent homogeneity by Ni-NTA affinity chromatography (Fig. 8). The effect of temperature on enzyme activity of mutants are presented in Fig. 9.

The optimal temperature for both HWxyn11 and HWxAs was 45 ℃ (Fig. 9a). HWxA31 showed over 60% relative enzyme activity and HWxA40 had 30% after incubation at 55 ℃ for 60 min. The wild-type HWxyn11 had 19% residual enzyme activity. In contrast, HWxA6, HWxA15 and HWxA26 showed lower thermostability compared with wild-type HWxyn11 (Fig. 9b). The half-life of HWxA31 (77.9 min) was approximately 3.4-fold higher than that of HWxyn11 (23.0 min), indicating that HWxA31 was more stable than HWxyn11 at 55 ℃.

The optimal temperature for both HWxyn11 and HWxyn11-CBMs was also 45 ℃ (Fig. 9c). And the residual enzyme activity of HWxyn11-CBM 6 exhibited over 50% at 55 ℃ for 60 min, other CBMs contributed to thermostability of HWxyn11 with varying degrees (Fig. 9d). The half-life of HWxyn11-CBM 6 was 65.4 min.

The combined mutant HWxA31-CBM 6, constructed by both N-terminal replacement and C-terminal fusion strategies, showed an optimal reaction temperature of 50 ℃ (Fig. 9e). Its residual enzyme activity was 65% after incubation at same condition as above xylanases, proving the thermostability of combined mutant is better than that of other mutants (Fig. 9f). Additionally, the half-life of HWxA31-CBM 6 (111.8 min) represented an 4.8-fold higher than that of HWxyn11 (23.0 min), showing that HWxA31-CBM 6 has better stability than other mutants at 55 ℃.

Molecular dynamics simulations analysis of mutants

To obtain deep insight into the influence of mutants on structure, molecular dynamics simulations of HWxA31, HWxyn11-CBM 6 and HWxA31-CBM 6 were done at 328 K (55 ℃) (Fig. 10). RMSF represents the influence of fluctuation of single amino acid residue on the whole kinetics of protein and RMSD value refers to root mean square deviation of each atom between protein conformation. Initial conformation under simulated high temperature can be used to evaluate thermostability of protein(Malau and Sianturi 2017). Both RMSF and RMSD are the parameters to characterize structural stability of protein. The lower values of RMSF and RMSD, the stronger structural rigidity and the higher heat resistance of protein. As shown in Fig. 10, RMSD and RMSF of mutants were lower than HWxyn11 at 55 ℃, especially combined mutant of HWxA31-CBM 6, indicating that declined flexibility of these areas. Molecular dynamics simulations analysis showed that the thermostability of mutants by N-terminal replacement and C-terminal CBM fusion were better than wild-type HWxyn11. Our findings were consistent with previous thermostability analysis.

Xylanase HWxyn11 consists of one catalytic domain of GH 11, and exhibited optimal enzyme activity under weakly acidic and medium temperature conditions. HWxyn11 was stable below the optimum temperature and would be quickly inactivated above 45 ℃ and had a remarkable pH stability under weakly acidic to alkaline conditions. HWxyn11 showed good metal ion tolerance and was tolerant at high salt concentrations, offering potential applications in the degradation of xylan substrates in complex industrial application environments. Compared with terrestrial-derived xylanases, marine-derived xylanase has its unique salt tolerance and cold tolerance because the influence of low temperature, barren and high salt environment(Wu et al. 2018; Yu et al. 2019; Yi et al. 2020).

The exploration of thermostable xylanases is essential to fulfill practical industry demands. To investigate the strategy of improving thermostability of HWxyn11, the substitution of N-terminus was used in this xylanase according to the sequence alignment to thermophilic xylanase rXyn11A. Moreover, we fused different CBMs from thermophilic bacteria at C-terminus. Both methods resulted in mutants with improved thermostability, and the combination of these two strategies can further improve the thermostability of HWxyn11. By molecular dynamics simulations, N-terminus and CBMs are decisive to the mechanism for improved thermostability of HWxyn11.

Previous study had reported that N-terminal residues accounted for the great importance of thermostability because N-terminus is the beginning of fold(Xiong et al. 2019; He et al. 2020). The thermostability of mesophilic xylanase SoxB achieved a significant increase by replacing 33 N-terminal amino acid residues with the corresponding residues of thermophilic xylanase TfxA(Zhang et al. 2010). The thermostability, acid tolerance as well as catalytic efficiency of XynAR also significantly improved by substituting the N-terminal of GH 11 xylanases(Wu et al. 2023a). The optimal temperature of CFXyl3 was improved from 55 ℃ to 85 ℃ by replacing its N-terminus with corresponding area of SyXyn11P that belongs to hyperthermostable GH 11 family(Tian et al. 2022). In this work, we adopted N-terminal replacement based on sequence alignment and molecular dynamics simulations. After replacing corresponding sequences, the propeptide of HWxyn11 is removed, resulting in significantly improved overall stability. Research indicates that high surface net charge can promote reversible refolding of proteins by preventing aggregation of xylanases(Wu et al. 2023b). After N-terminal replacement, the surface electrostatic potential of N-terminal region underwent a considerable change, with the surface net charge (at pH 7.4) shifting from − 14.814 to -15.772 (Fig. S1). The significant change occurred in electrostatic potential on the surface of HWxyn11, suggesting that surface net charge may influence its thermal unfolding and refolding. This phenomenon indicates that N-terminal replacement can increase surface net charge by altering distribution of protein surface charges to improve thermostability of xylanases. In summary, it’s hard to inductive which amino acid was decisive to the thermostability of xylanase, but it’s determined that N-terminus is important for GH 11 xylanases.

Consistent with previous studies, CBMs have great influence on the stability of xylanases(Liu et al. 2021). Removing CBM 22 of Xyn10B from Clostridium stercorarium resulted the optimum temperature of enzyme reduced from 75 ℃ to 65 ℃(ZHAO et al. 2005). To explore the biochemical properties and the role of associated CBMs in catalysis of endoglucanase AtGH9C, different combination with CBM 3A and CBM 3B were constructed, finding that associated CBMs imparted thermostability to its catalytic module(Mandal et al. 2023, p. 27405). CBM 9 − 2 was fused to both N-terminus and C-terminus of Xyn to construct a sandwich enzyme, resulting in a 7.9-fold and 24.8-fold increase in catalytic efficiency and half-life at 50 ℃(Yang et al. 2018). Generally, the structure of heterologously expressed protein will be destroyed and denatured easily at high temperature, resulting in aggregation and precipitate of protein and then lose its catalytic activity. Our study found that the thermostability of HWxyn11 was improved by fusing CBM from thermophilic bacteria at C-terminus. It was speculated that the thermal denaturation of xylanase was reversible after CBM fusion as previous studies(Davoodi et al. 1998; Stavropoulos et al. 2018). Besides, predicted 3-dimensional structure of HWxyn11 showed that there was an unfolded flexible polypeptide chain of C-terminal which contains more amino acids such as Pro, Thr and Ser. Compared to wild-type xylanase HWxyn11, the C-terminal flexibility of mutant HWxyn11-CBM6 after fusion with a CBM was significantly reduced, suggesting that thermostable CBM contributes to the stabilization of the overall structure of protein. Meanwhile, the polypeptide chain can form a flexible hinge region, acting as a flexible linker to separate different domains from each other in space and form an flexible spatial conformation(Li et al. 2022). It’s speculated that HWxyn11 and CBM connected with the enzyme by linker will not be destroyed at the same time after incubation at high temperature for a period of time. The depolymerized HWxyn11 can refold because thermostable CBM may impede the process of enzyme’s aggregation and precipitation through the connection with HWxyn11. Renaturated xylanase can be restored to its natural conformation and still has normal catalytic activity after denaturalization factors are removed.

Overall, the reasons for improved thermostability of HWxyn11 may involve the removal of the N-terminal propeptide of HWxyn11 which leads to the changes in interaction force and net charge, and the reversible thermal denaturation attributed to CBM fusion.

In this study, we conducted biochemical characterization and molecular modification of thermostability of a salt-adapted xylanase HWxyn11 originated from Hortaea werneckii. HWxA31 and HWxyn11-CBM 6 showed improved thermostability compared with HWxyn11 by constructing the mutants of N-terminal replacement and C-terminal CBM fusion. Besides, the phenomenon of "1 + 1 > 2" will appear by taking measures of combining HWxA31 and HWxyn11-CBM 6. Molecular dynamics simulation of mutants implied that lessened flexibility of N-terminal and C-terminal contributed to the synergistic impact on improving the thermostability of HWxyn11. Removal of the N-terminal propeptide of HWxyn11 which leads to changes in interaction force and net charge, and the reversible thermal denaturation attributed to the fusion of CBM may contribute to the improved thermostability. These findings offer new insights for improving the thermostability of xylanases from GH 11.

Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Funding

This work was supported by National Key R&D Program of China [Grant numbers 2022YFC2805105].

Author Contributions

All authors contributed to the study conception and design. HFZ: Writing-Original draft preparation, investigation, methodology, data curation and formal analysis. QZC: Writing- Reviewing & Editing. YTL, JJL and SYP: Writing-Original draft preparation. YHW: Supervision, funding acquisition and resources.

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Improving thermostability of salt-tolerant xylanase from Hortaea werneckii EXF-12619 by N-terminal replacement and C-terminal carbohydrate binding module fusion

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