ApXyn11A cloning, expression and purification
A GH11 family xylanase gene (NCBI accession number: WP_179817163.1) in size of 696 bp, which was named apxyn11A, was predicted in the genome of Allostreptomyces psammosilenae. A signal peptide was predicted at the N-terminal (1–40) of this protein by signal peptide analysis, and a DNA fragment (576 bp) without the signal peptide was subjected to PCR amplification, cloning and heterologous expression of the gene. The amino acid sequence of ApXyn11A showed the highest sequence identity (89.58%) with GH11 xylanase obtained from Streptomyces taklimakanensis (NCBI accession number: WP_155070968.1). Phylogenetic analysis of the protein sequences showed that ApXyn11A clustered with S.taklimakanensis in one branch (Fig. 1). To our knowledge, A. psammosilena YIM DR4008T was recognized as a novel species of a new genus in the family Streptomycetaceae (Huang et al., 2017) and xylanase gene was obtained from this strain for the first time.
Recombinant protein was purified by Ni-chelating affinity column with His tags at the N-terminus. The theoretical isoelectric point (pI) and molecular weight (Mw) of the ApXyn11A gene encoding a 206 amino acid polypeptide from SDS without signal peptide were calculated to be 9.20 and 22.7 kDa, respectively. SDS-PAGE analysis indicated the molecular mass of the recombinant ApXyn11A protein (~ 23 kDa) was in good agreement with that calculated theoretically (Fig. 2A). Smaller enzymes can easily penetrate the fiber wall structure and improve pulp and paper quality more effectively (Seemakram et al., 2020; Yadav et al., 2018). This indicates that ApXyn11A has great potential application value in paper and pulp industry.
ApXyn11A was the most similar to crystal structure of GH11 xylanase (PDB: 5EJ3) from Streptomyces lividans. As in Fig. 2B, a model of ApXyn11A was obtained by homology modeling, which only contained the β-jellyroll catalytic domain (Gagné et al., 2016). This form could maintain an endo-acting activity profile on xylan substrates, generate a large number of oligo-edges with a degree of aggregation ranging from 1 to 8 xylose units (Hurtubise et al., 1995).
Substrate specificity of ApXyn11A
As shown in Table 1, ApXyn11A can hydrolyze a variety of substrates, including corn cob xylan, beechwood xylan, oat spelts xylan, bagasse xylan and different lignocelluloses (pine shavings, wheat core, straw core). Among them, ApXyn11A had the strongest relative hydrolysis ability for corn cob xylan (171.16 ± 7.61 U/mg). ApXyn11A also exhibited hydrolysis ability for beechwood xylan (158.72 ± 4.33 U/mg), oat spelts xylan (150.29 ± 3.98 U/mg), sugarcane bagasse xylan (102.58 ± 1.46 U/mg), pine shavings (11.32 ± 2.86 U/mg) and wheat core (5.46 ± 2.11 U/mg). In addition, ApXyn11A cannot hydrolyze straw core, poplar shavings, CMC, avicel and cellobiose. It was shown that ApXyn11A did not possess degradation of cellulose and cellobiose, which was similar to the hydrolytic properties of xylanases from Streptomyces sp.(Georis et al., 2000) and Caulobacter crescentus (Jacomini et al., 2020). Xylanase without cellulose degradation capacity can selectively remove hemicellulose compounds and reduce cellulose loss, and thus ApXyn11A can be better used in paper, textile, and food industries (Belfaquih et al., 2002). These indicated that ApXyn11A had a great application potential in the hydrolysis of lignocellulase into fermentable sugars.
Table 1
Substrate specificities of ApXyn11A.
Substrate | Special activity (U/mg) |
Corn cob xylan | 171.16 ± 7.61 |
Beechwood xylan | 158.72 ± 4.33 |
Oat spelts xylan | 150.29 ± 3.98 |
Begass xylan | 102.58 ± 1.46 |
Pine shavings | 11.32 ± 2.86 |
Wheat core | 5.46 ± 2.11 |
Straw core | 0 |
Poplar shavings | 0 |
CMC | 0 |
Avicel | 0 |
Cellobiose | 0 |
All substrates were tested at 1% (w/v). |
Effect of temperature and pH on the activity and stability of ApXyn11A
As shown in Fig. 3, the optimum reaction temperature and pH of ApXyn11A were 65°C and pH 5.6, respectively. The purified ApXyn11A exhibited more than 90% relative activity at 50–70°C and more than 65% relative activity at 75°C (Fig. 3A). It had a wide pH adaptation range, exhibiting more than 80% relative activity at pH 3.6–7.6 and still more than 50% relative activity at pH 3.0 and pH 8.0–9.0 (Fig. 3B). The effect of temperature on the stability of ApXyn11A was shown in Fig. 3C. The purified ApXyn11A retained more than 95% residual activity after incubation at 55°C for 120 min and more than 60% residual activity after incubation at 60°C for 100 min. The effect of pH on the stability of ApXyn11A was shown in Fig. 3D. The purified ApXyn11A had more than 80% residual activity after incubation in pH 4.0–8.0 for 12 h and more than 60% residual activity after incubation in pH 4.0–8.0 for 24 h.
The above results indicated that ApXyn11A was a thermophilic and pH tolerant xylanase. The optimal adaptive temperature range of 50–70°C, the better thermal stability, and the wide range of acidic pH adaptations identified by ApXyn11A can classify it as a natural thermophilic, acidic enzyme (Collins et al., 2005; Dey & Roy, 2018). However, most of the current commercial enzymes are from the mesophilic fungus Trichoderma reesei, which produces enzymes with an optimum reaction temperature less than 50°C (Kufner & Lipps, 2013). It is well known that xylanases play a very important role in food processing, animal feed preparation, biofuel production, brewing industry, etc (Collins et al., 2005; Yin et al., 2017).
Effect of salt concentration on the enzyme activity of ApXyn11A
The effect of salt (NaCl) concentration on the activity of ApXyn11A was shown in Fig. 4. ApXyn11A showed excellent tolerance to various salt concentrations and was stable at high salt concentrations. The enzyme still had more than 60% of its relative activity at 0.5 to 3.5 M NaCl. Its better salt tolerance may be related to the fact that strain A. psammosilenae YIM DR4008T can grow at up to 4% (w/v) NaCl (Huang et al., 2017). Previous studies have shown that salt-tolerant proteins have a high ratio of acidic and basic amino acids, as well as a lack of hydrophobic amino acids (Paul et al., 2008). However, amino acid sequence analysis revealed that ApXyn11A had a low ratio of acidic (4.3%) to basic (7.8%) amino acids. There was a high ratio of polar (46.6%) to non-polar amino acids (18.8%). These results were similar to GH11 xylanases from Phoma sp. MF13, which isolated from mangrove sediment (Wu et al., 2018) and marine bacterium Bacillus subtilis cho40 (Khandeparker et al., 2011). Furthermore, comparing with GH10 salt-tolerant xylanases, it was found that GH11 xylanases were much less acidic amino acids than GH10 xylanases in terms of amino acid ratios (Table 2). At present, the salt tolerance mechanism of salt-tolerant xylanase is still unknown, and subsequent studies through more in-depth structural level are needed.
Table 2
Amino Acid Composition Comparison of Salt-Tolerant Xylanases.
Parameters | ApXyn11A | XynMF13A | Xyl40 | XynAHJ14 | XynSL4 | XynA |
GH family | 11 | 11 | 11 | 10 | 10 | 10 |
acidic amino acids(%)a | 4.30 | 4.30 | 4.46 | 14.91 | 17.37 | 12.06 |
hydrophobic amino acids(%)b | 18.8 | 29.87 | 28.71 | 34.72 | 36.32 | 37.59 |
highest activity with NaCl(%) | 100 | 105 | 140 | 100 | 100 | 120 |
concentration of NaCl (M)c | 0 | 0.5 | 0.5 | 0 | 0 | 0.5 |
theoretical molecular weight(kDa) | 22.7 | 24.42 | 22.9 | 45.0 | 43.3 | 43 |
references | This study | (Wu et al., 2018) | (Khandeparker et al., 2011) | (Zhou et al., 2014) | (Huang et al., 2017) | (Guo et al., 2009) |
aIncluding amino acids: E D. |
bIncluding amino acids: A I L F W V. |
cThe highest activity of the enzyme when NaCl was added to the reaction system. |
Effect of different metal ions and inhibitors on ApXyn11A
The effects of different metal ions and inhibitors on ApXyn11A were shown in Fig. 5. In presence of 1 mM metal ions, there were no significant effect on the xylanase activity, except for Mn2+, which activated its activity to about 127% (Fig. 5A). In presence of 10 mM metal ions, most of them exhibited a weak inhibition, such as Ca2+ (81.72% ± 4.07%) and Cu2+ (81.68% ± 5.39%). However, 10 mM Ag+ showed significant inhibition of its xylanase activity (28.09%±1.54%). Among the inhibitors, 0.1% SDS showed significant inhibition (35.60% ± 3.39%) for ApXyn11A. In presence of 1% EDTA, ApXyn11A exhibited about 76% relative activity.
It is well known that most metal ions and chemical reagents can affect the stability of proteins. They not only promote enzyme activity, but also inhibit it (Qiu et al., 2017; Riordan, 1977; Sanghi et al., 2010). ApXyn11A exhibited higher relative activity at 1 mM Mn2+ than 10 mM Mn2+, which was similar to the properties of GH11 xylanase XynS1 from Streptomyces sp. strain J103 (Marasinghe et al., 2021). SDS is a strong denaturant for enzymes and can interfere with the hydrophobic region of enzymes, causing fluctuations in their three-dimensional structure and inactivating most enzymes (Zheng et al., 2012). Similarly, ApXyn11A was strongly inhibited by SDS. Furthermore, in the study by Kim et al. (2021), 1 mM Cu2+ could essentially completely inhibit GH10 xylanase activity of Duganella sp. PAMC 27433. In the presence of 10 mM Cu2+, Kumar and Shukla (2018) found that xylanase from Thermomyces lanuginosus VAPS24 was strongly inhibited by Cu2+. However, ApXyn11A was only weakly inhibited by Cu2+ at the same concentration. It was previously reported that Ca2+ and EDTA were enzyme promoters (Yin et al., 2017; G. Zhang et al., 2008). In the present study, Ca2+ and EDTA inhibited the enzymatic activity of ApXyn11A. In conclusion, ApXyn11A was resistant to most metal ions and chemical reagents, and it may have special industrial applications.
Determination of kinetic constants
Purified ApXyn11A showed the highest hydrolytic activity on corn cob xylan, so its kinetic constants were determined using corn cob xylan as substrate. The results indicated that ApXyn11A followed Michaelis-Menten kinetics in terms of corn cob xylan. Based on the Lineweaver-Burk plot, Km, Vmax and Kcat were calculated for ApXyn11A (Table 3). Compared to previously reported values of thermophilic xylanase (Marasinghe et al., 2021; Ouephanit et al., 2019; Yadav et al., 2018; Yin et al., 2017; Zhang et al., 2011), the present study provided lower Km and higher Vmax values. The Vmax values of ApXyn11A was as high as 2000 µmol/min/mg, which indicated that it favored the degradation of corn cob xylan. While Km values was as low as 0.2 mg/ml indicating purified ApXyn11A had high affinity for the substrate, which was important in the industrial application.
Table 3
Comparison of kinetic parameters between ApXyn11A and those of other thermophilic xylanases.
Xylanase | Opt. Tem. (°C) | Vmax | Km (mg/mL) | Kcat (S− 1) | Kcat/Km | References |
Nf Xyn11Aa | 80 | 351.6 | 6 | 136.9 | 22.8 | (Zhang et al., 2011) |
Native XylA b | 65 | 66.64 | 0.7 | / | / | (Yadav et al., 2018) |
rXynS1c | 55 | 898.2 | 51.4 | / | / | (Marasinghe et al., 2021) |
TrXyn10d | 55 | 29.6 | 47.7 | / | / | (Yin et al., 2017) |
Pc Xyn11Ae | 55 | 310.7 | 2.8 | 243 | 86.8 | (Ouephanit et al., 2019) |
ApXyn11A | 65 | 2000 | 0.2 | 755.09 | 3775.44 | This study |
a Nonomuraea flexuosa xylanase produced in Trichoderma reesei. |
b Native Anoxybacillus kamchatkensis Xylanase. |
c Streptomyces sp. xylanase produced in E. coli. |
d Thermoactinospora rubra xylanase produced in E. coli. |
e Recombinant Aspergillus niger xylanase produced in Pichia pastoris. |