Recombinant acetylxylan esterase of Halalkalibacterium halodurans NAH-Egypt: molecular and biochemical study

More research is needed to determine the precise nature of microbial AXEs in terms of amino acid sequence, function, structure, biochemical characteristics, and substrate selectivity. In this context, the AXE gene from the locally obtained unique Halalkalibacterium halodurans strain NAH-Egypt was cloned, heterologously expressed, characterized, and in silico sequenced at the protein level. At NCBI, there are just two whole genome sequence (WGS) records for A. halodurans strains, C-125 and LB-1. Despite the putative gene of AXE, it was only found in the genome of A. halodurans C-125, not the LB-1 strain. Furthermore, there is only one report on A. halodurans C-125 recombinantly expressed AXE (BhAXE) in E. coli (Min-Jeong et al., 2020). Kim’s study did not cover either thorough biochemical characterization of recombinantly expressed BhAXE in E. coli nor in silico sequence analysis at the protein level (Min-Jeong et al., 2020). As a result, it was worthwhile to compare the properties of recombinantly expressed AXE (AXE-HAS10) from a locally obtained unique strain designated A. halodurans NAH-Egypt with those of recombinant BhAXE from A. halodurans C-125 previously produced in E. coli (Min-Jeong et al., 2020).

In silico sequence analysis of AXE-HAS10 revealed its affiliation to family CE7. So far, there are seven members of microbial AXEs in CE7 that are well characterized with determined crystal structures deposited in the Protein Databank (PDB) under the accession numbers: 3FVR_A, 1L7A, 7CUZ, 3FCY, 1LVQ, 6AGQ, and 6FKX from B. pumilus, B. subtilis, Lactococcus lactis, Thermoanaerobacter saccharolyticum, Thermotoga maritima, Paenibacillus sp., and a metagenome library, respectively. According to the classification of CAZy Server (Brandi et al. 2009), the previously reported signature motifs of CE7 (–RGQ–, –GxSxG– and HE) were localized at amino acid positions (118–120), (185–189), and (303–304), respectively, in the primary structure of AxeB (AHG97602.1), isolated from the termite hindgut metagenome (Mokoena et al. 2015). The signature- motifs of –RGQ–, –GXSQG–, and HE of CE7 were found in B. pumilus AXE (3FVR_A) at amino acids positions 118–120, 179–183, and 298–299, respectively (Degrassi et al. 2000).

It has been reported that the serine residue involved in the signature motif G-x-S-x-G is the catalytic serine. This serine residue helps attack the carbonyl carbon atom of the ester bond (Bornscheuer 2002; Mokoena et al. 2013). Based on the multiple sequences analysis, it was deduced that Ser183, Asp273, and His 302, in AXE-HAS10 did form the catalytic triad residues. Similarly, the catalytic triad residues in AXeA (AHG97601.1), AXeB (AHG97602.1), and AXE (AGF25253.1) from termite hindgut metagenome library (Mokoena et al. 2015), termite hindgut metagenome library (Mokoena et al. 2015)d subtilis CICC 20,034 (Tian et al. 2014) were localized as follows: Ser182-Asp272-His301, Ser187-Asp273-His303, and Ser181-Asp269-His298, respectively.

Likewise, AXE-HAS10 and AlAXEase from Arcticibacterium luteifluviistationis SM1504T were severely inhibited by PMSF but at a much higher concentration (10 mM) of PMSF (Zhang et al. 2021). This would in turn suggest that AXE-HAS10 is a serine hydrolase.

With the online programme SignalP 5.0, no signal peptide was detected in the AXE-HAS10 primary amino acid sequence. It was reported that AXEs lacking signal peptides could be secreted due to internal signal peptide. Contrary to this speculation, AXEs lacking signal peptides could be intracellular enzymes rather than secretory enzymes with internal signal peptides (Degrassi et al. 2000; Walter et al., 1997). The recombinant AXE-HAS10’s experimentally measured molecular mass (41.39 kDa) was somewhat higher than the theoretical molecular mass (36.5 kDa). An additional sequence of 42 amino acids produced from the pET-28a (+) vector could account for the extra 4.6 kDa added to the molecular mass of the expressed recombinant AXE-HAS10. The 4.6 kDa sequence (42 amino acids produced from the pET-28a (+) vector) had 33 amino acids flanking the AXE-HAS10 fragment on the N-terminal side and 9 amino acids flanking the AXE-HAS10 fragment on the C-terminal side (Tham et al. 2020; Abady et al. 2022). The experimentally determined molecular mass of BhAXE from B. halodurans C-125, on the other hand, was completely consistent with that calculated theoretically (36 kDa). This could be due to the different expression vectors employed in the two studies: pHCXHD in Kim’s work (Min-Jeong et al., 2020) and pET-28a (+) in this study. The experimental molecular weight of AXE-HAS10 was in good agreement with the range of molecular weight of AXE(s) previously reported (27.0–41.0 kDa) (Table 2).

As a general principle, determining an enzyme’s physico-biochemical properties is a critical essential factor in the agenda of prospective industrialization for an enzyme’s ultimate and efficient exploitation.

In this study, AXE-HAS10 showed the greatest activity at pH 8.5 and a temperature of 40 oC, respectively. Previously characterized AXEs showed a discrepancy in their optimal pH, temperature, and stability profiles as well. BhAXE exhibited its optimal activity at a pH and temperature of 8 and 50 oC, respectively. After 15 h of incubation, AXE- HAS10 retained 100% of its activity at pHs 7.0–9.0. After 2 h of incubation, the retained activity of AXE-HAS10 at 35 and 40 oC was ~ 80 and ~ 50%, respectively (Fig. 4D). For BhAXE, there was no obvious information in the study of Kim et al. about its thermal and pH profile stability (Min-Jeong et al., 2020).

Obviously, AXE-HAS10 is a cold-adapted AXE as it could display substantial activity (20, 30, 40, ~ 42, 50, 70, 100, 60, and ~ 0.0%) over a wide range of temperatures, 5, 10, 15, 20, 25, 30–35, 40, 45, and 55 oC, respectively (Fig. 4 C). Moreover, the poor thermostability pattern of AXE-HAS10 (Fig. 4D) would greatly underpin its cold-adept nature. The cold-adapted nature of (A) halodurans NAH-Egypt was previously reported for estHIJ (Noby et al. 2020). The pH stability profile of AXE-HAS10 revealed its alkali-stability property. The difference between BhAXE and AXE-HAS10 could be attributed to the enzyme source strain difference and the ecological niche of the two producer strains: (B) halodurans C-125 (previously isolated from deep-see sediment) and A. halodurans NAH-Egypt (previously isolated from Natroun Valley, Egypt) (Noby 2020). Upon comparing the pH-temperature profile of other previously reported AXE(s) (Table 2) with that of AXE-HAS10, the pH and temperature optima spanned from 5.5 to 8.5 and 40–90 oC, respectively.

Table 2 Comparison between some reported AXE(s) and AXE-HAS10 regarding biochemical properties

The ratio of flexible residues and their arrangement around the active site or across the protein structure could explain the differences in thermal stability (Marx et al. 2007). The ratio and distribution of Arg, Lys, Met, and Gly have been shown to play an important role in imposing cold-adeptness on cold-adapted enzymes (Marx et al. 2007; Fu et al. 2013). There are no reports on cold-adapted AXEs accessible, except for two that mention AlAXEase: AXE from Arcticibacterium luteifluviistationis SM1504T (Zhang et al. 2021) and PbAcE (PDB: 6AGQ) from Paenibacillus sp. R4 (Mavromatis et al., 2018). As a result of the scarcity of publications on cold-adapted AXEs, our cold-adapted AXE-HAS10 would be compared to AlAXEase, a new CE family member, in terms of the content of both enzymes from Arg, Lys, Met, and Gly. AXE-HAS10 has a Met and Gly content of 3.13 and 7.2%, respectively, whereas AlAXEase has a Met and Gly content of 2.7 and 7.2%, respectively. As shown, the Met and Gly ratios in AXE-HAS10 and AlAXEase are almost quite similar throughout the whole protein structure. Conversely, AXE-HAS10 showed a greater ratio of Arg/Arg + Lys (0.58) when compared to that of AlAXEase (0.275). Despite this difference in Arg/Arg + Lys ratio between AXE-HAS10 and AlAXEase being almost quite similar, full residual activity was traced for both enzymes at 5 oC after 1 h. In comparison to that of AXE-HAS10, cold-adapted PbAcE (PDB: 6AGQ) from Paenibacillus sp. R4 had a slightly higher content of Met (3.1%) and Gly (8.39%) and a slightly higher content of Arg/Arg + Lys (0.607) (Park et al. 2018). However, the cold-adpation mechanism of proteins is case-by-case and should be explored for each protein by site-directed mutagenesis.

Reportedly, AXEs of microbial origin exhibit a special metal ion preference that would stimulate their activity. Unlike AXE-HAS10, AXE of O. pacifca was inhibited by 5 mM of Zn2+, Mn2+, and Ca2+ (Hettiarachchi et al., 2013). AXE from B. subtilis CICC 20,034 (Tian et al. 2014) was strongly inhibited by a very small concentration (1 mM) of Mn2+, Fe3+, Ca2+, and Zn2+. Like AXE-HAS10, an AXE from P. chrysogenum P33 (Hettiarachchi et al., 2017), was relatively stable in the presence of the following metal ions at high concentration (10 mM):Mn2+, Fe3+, Ca2+, and Zn2+. Despite the partial inhibitory effect (almost 60% retained activity) of some metal ions (i.e., Cu2+, Mg2+, and Mo2+) at 10 mM on AXE-HAS10, it was considered more metal ion stable than other AXEs from O. pacifca and B. subtilis CICC 20,034 that were inhibited by 1 and 5 mM of these metal ions. Although we do not yet have a strong explanation for the increased AXE-HAS10 activity in the presence of Mn2+ and Fe3+, prospective research of the Fe3+ and Mn2+: AXE-HAS10 ratio in this complex formation would be beneficial. It’s possible that the extraordinary increased activity in the presence of Fe3+ and Mn2+ is due to their binding to the amino acids that contribute to the active site. To test this hypothesis, a future study would need to co-crystallize AXE-HAS10 with these two ligands, Fe3+ and Mn2+. In conclusion, when compared to previously identified AXEs, AXE-HAS10 could tolerate a wide range of metal ions at relatively high concentrations (10 mM). This suggests that AXE-HAS10 is appropriate and efficient for usage in industrial settings with high metal ion loads.

AXE-HAS10 showed a promising profile regarding stability in the presence of a wide range of detergents (i.e., non-ionic, ionic, and cationic detergents). Incubation of AXE-HAS10 for 30 min with Triton x-100 at both concentrations of 0.01 and 0.02% exhibited a significant stimulatory effect with retained activity of 150 and 130%, respectively. The increased AXE-HAS10 activity in the presence of Triton-X100 in both concentrations could be owing to increased substrate availability to the active center associated with surfactant hydrophobic binding (Rao et al. 2013; Hua et al., 2013; Mitaku et al., 2002). Despite the inhibitory effect of both cationic and anionic detergents on AXE-HAS10, almost 85% of the activity was retained. Unfortunately, no data on the impact of detergents on the activity of CE7 AXEs could be described in the literature. The current data would strongly support the usage of AXE-HAS10 in industrial applications with a substantial detergent load. Regarding organic solvents, AXE-HAS10 demonstrated a relatively moderate stable profile towards hexane, glycerol, isopropanol, DMSO, methanol, and ethanol at 10% (v/v) after incubation for 30 min. Only one available report regarding the organic solvent stability of AXEs of CE7 was assigned to AXE from B. subtilis CICC 20,034 (Tian et al. 2014). In the study of Tian et al., the AXE showed robust stability (80–100%) towards 30% (v/v) of the organic solvents DMSO, methanol, acetone, isopropanol, benzene, toluene, and n-hexane after incubation for 24 h (Tian et al. 2014).

When compared to other previously reported AXEs, AXE-EST1051(Xu et al. 2021) and AXE of O. pacifca (Hettiarachchi et al. 2019), AXE-HAS10 appears to be more stable in the presence of high NaCl concentrations. AXE-EST1051 could retain 65% of its activity after pre-incubation for 20 min with 3 M NaCl (Xu et al. 2021). However, after 12 h of pre-incubation with 0.5 M NaCl, O. pacifca AXE could retain nearly 60% of its activity (Hettiarachchi et al. 2019). The current research clearly implies that AXE-HAS10 could be used in industrial environments with high salinity loading.

With regard to the effect of EDTA, the present findings reveal that AXE-HAS10 is a metallo-hydrolase similar to AXE from Ochrovirga pacifica (Hettiarachchi et al. 2019). Present data is in disagreement with the non-metallo acetyl xylan esterases AlAXEase from Arcticibacterium luteifluviistationis SM1504T (Zhang et al. 2021) and FjoAcXE (Razeq et al. 2018).

AXE-HAS10 displayed very high activity toward p-NPA (C2) and had dramatically reduced activity towards p-nitrophenyl esters with carbon atoms from C2-C8 (Fig. 6B). Conversely, AxE of B. subtilis CICC 20,034 (Tian et al. 2014), AXE of T. maritima (Levisson et al. 2009), and AXE-EST1051 from a metagenomic library (Marx et al. 2007) demonstrated specific activity toward p-nitrophenyl esters with carbon atoms from C2-C4, C2-C12, and C2-C16, respectively. AxeA and AxeB activity from the termite hindgut metagenome (Mokoena et al. 2015) on p-nitrophenyl esters with carbon atoms from C2-C8 was found to be nearly identical to that of AXE-HAS10. A fold increase of 1.04, 1.25, and 2.39 in the Km value of AxeA from T. maritima (Drzewiecki et al. 2010), AxeA and AxeB from a termite hindgut metagenome (Mokoena et al. 2015), on p-NPA-C2 when compared to that of AXE-HAS10. Hence, it would indicate the superior substrate specificity of AXE-HAS10 toward p-NPA (C2) relative to the aforementioned AXEs. In this perspective, AXE-HAS10 showed a superior kcat (s-1) of 63.06 s-1 on p-NPA (C2) when compared to those of AxeA and AxeB from a termite hindgut metagenome (Mokoena et al. 2015), with 4.5 × 10–11 and 1.82 × 10–11 s-1, respectively. Conversely, the kcat of AXE-HAS10 was comparable to those of Axe from T. maritima (69.9 s-1) (Drzewiecki et al. 2010) and PbAcE from Paenibacillus sp. R4 (53.3 s-1) (Park et al. 2018).

The synergistic effects of some AXEs on the xylan polymer degradation were previously investigated with β-xylanase. Likewise, Kim and co-workers reported a fold enhancement of 1.44 upon simultaneous treatment of beech wood xylan with LaAXE (AXE from L. antri) and TnXNB. The AXE from Volvariella volvacea did succeed in enhancing the hydrolysis of xylan polymer by 1.4-fold when compared to the efficiency of xylan hydrolysis elicited by β-xylanases alone (Zheng et al. 2013). Similarly, the AXE from O. pacifica exhibited a 1.4-fold enhancement in the hydrolysis of beech wood xylan with a commercial β-xylanase (Hettiarachchi et al. 2019).

This is the first study to look at Halalkalibacterium halodurans strain NAH-cold-adapted Egypt’s AXE. Cold sensitivity, as well as detergent, metal ion, and halo-tolerance, would put AXE-HAS10 ahead of previously identified orthologous AXEs from other species. When combined with β-xylanase from P. chyrysogenum Strain A3 DSM105774, AXE-HAS10 produced synergistic xylan hydrolysis. Recombinant AXE-HAS10’s robust characteristics would support its potential in industrial applications. However, AXE-HAS10 should be subjected to a prospective study in order to highlight certain issues such as x-ray crystallography, site-directed evolution, and amino acid sequencing to unveil the structural-functional relationship encountered in this enzyme.

留言 (0)

沒有登入
gif