Enhancing catalytic efficiency of a salt-induced Neurospora crassa xylose reductase for xylitol bioprocesses

Enhancing catalytic efficiency of a salt-induced Neurospora crassa xylose reductase for xylitol bioprocesses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancing catalytic efficiency of a salt-induced Neurospora crassa xylose reductase for xylitol bioprocesses M. J. Reyes-Rosas, J. Lopez-Miranda, J.A. Rojas-Contreras, M. Garcia-Curiel, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9316107/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Xylitol is a sugar alcohol widely used in the food industry due to its lower caloric value compared to sucrose. Industrial xylitol production relies primarily on chemical hydrogenation processes that require high energy input. Although biotechnological routes represent a more sustainable alternative, their industrial implementation is often limited by enzyme stability and cofactor costs. This study presents the design, expression, and biochemical analysis of recombinant xylose reductase from Neurospora crassa (rNcXR). The optimized gene was cloned into a salt-inducible T7-based expression system and expressed in Escherichia coli BL21-SI. The design incorporated an OmpT signal peptide to encourage secretion into the periplasm, and a 6×His tag at the C-terminus to simplify purification. The recombinant enzyme exhibited a specific activity of 13.93 U/mg, exceeding values previously reported for native and recombinant xylose reductases. rNcXR displayed high stability over a temperature range of 25–45°C and retained significant activity at elevated temperatures. These results demonstrate that the engineered rNcXR represents a robust and efficient biocatalyst with strong potential for enzymatic xylitol production. Neurospora crassa xylose reductase periplasmic expression xylitol biosynthesis salt-inducible Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1 Introduction Xylitol is a five-carbon polyol naturally present in plants, fungi, and yeasts [ 1 – 3 ]. It exhibits sweetness comparable to sucrose while providing a significantly lower caloric value (2.4 kcal/g for xylitol versus 4 kcal/g for sucrose). Owing to these properties, xylitol is commonly used as a sugar substitute, particularly in products intended for insulin-dependent individuals [ 4 ]. Beyond its nutritional advantages, xylitol has gained industrial relevance due to its applications in the food, pharmaceutical, and dental sectors [ 5 – 7 ]. Conventional industrial production involves the catalytic hydrogenation of xylose under high temperature and pressure, resulting in substantial energy consumption and environmental impact. Consequently, increasing attention has been directed toward biotechnological alternatives employing microorganisms or isolated enzymes [ 8 ]. Despite their advantages, enzymatic processes are often constrained by limited enzyme stability under industrial conditions, particularly at low pH and elevated temperatures [ 9 ]. Advances in recombinant DNA technology have enabled the heterologous expression of enzymes in suitable microbial hosts, allowing the production of recombinant biocatalysts with improved performance and tailored properties [ 10 ]. Recombinant enzyme production requires the insertion of the gene of interest into a plasmid, which acts as a vector to introduce the gene of interest into the host cell. Escherichia coli is a commonly used microbial host system because it has fast growth kinetics and lowers enzyme production costs. However, it is necessary to consider the availability of proper vectors to ensure protein expression [ 11 , 12 ]. To achieve this, two vector types are needed: a cloning vector, which provides multiple cloning sites to insert the gene but cannot express the protein of interest; and an expression vector, which shares some characteristics with cloning vectors. They differ because they are designed to have regulated sequences that function as enhancers and promoter regions that lead to efficient gene transcription conducted in the expression vector. The objective of this study was to design and evaluate the salt-inducible periplasmic expression of a Neurospora crassa xylose reductase, and to elucidate the role of its structural modifications in enhancing its catalytic efficiency and providing anchoring points for future immobilization in xylitol bioprocesses. 2 Materials and Methods 2.1 Design of the NcXR expression construct The original nucleotide sequence of the xylose reductase gene (XR) of Neurospora crassa (NCBI Reference Sequence: NC_026501.1) was expressed by the E. coli K12 strain using the codon optimization tool provided by Integrated DNA Technologies, Coralville, Iowa, USA. The OmpT leader, promoter, and terminator sequences of the T7 RNA polymerase were taken from the pET12a plasmid sequence deposited on the Addgene vector database. The final genetic construct was analyzed for correct assembly using GenSmart Design software. 2.2 Bacterial strain and growth media Escherichia coli BL21si was routinely grown at 37°C in Luria Bertani culture medium [ 13 ] without NaCl (LBON). Enzyme production and periplasm secretion were performed using LBON culture medium. 2.3 Transformation procedure and molecular techniques The procedure for making CaCl 2 competent cells and thermal shock transformation of E. coli were performed using standard techniques, as described previously [ 14 ]. Transformants were selected on solid LBON medium plates with 100 µg/mL ampicillin. The plates were incubated overnight at 37°C. The restriction endonuclease cutting assays were performed according to the instructions given by the supplier (Promega, Madison, Wisconsin, USA). 2.4 Xylose reductase expression assays The transformed strain ( E. coli BL21-SI) was grown overnight and diluted to an OD600 of 0.1 in two 50-mL of fresh LBON medium. The cultures were incubated until they reached an OD600 of 0.5. One of the flasks was supplemented with 0.3 M NaCl for 12h to induce enzyme production. The secreted proteins in the periplasm were recovered as follows: 1.5 mL of the culture medium with an OD600 of 2.0 was centrifuged at 10,000 × g for 2 min. The cell pack was suspended in 200 µL of osmotic shock buffer with 100 mM Tris-HCl, pH 7.4, 20% sucrose, 10 mM Ethylene diamine tetraacetic acid (EDTA), and kept on ice for 5 min. The suspension was centrifuged at 10,000 × g for 2 min. The cell pack was quickly suspended in 200 µL of Milli-Q water, vigorously shaken, and kept for 5 min in an ice bath [ 15 ]. The cell suspension was then centrifuged at 16,000 × g for 2 min. Aliquots (150 µL) of the supernatant (periplasmic proteins) were concentrated by precipitation (methanol, chloroform, water, 4:1:3) and analyzed using 12% SDS–polyacrylamide gel electrophoresis with a mini Protean Tetra Cell protein electrophoresis system (Bio-Rad). This was performed by applying a voltage of 100 V and a current intensity of 150 mA for 1.5 h. 2.5 Western blot analysis of rNcXR Proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane with a pore size of 0.45 mm (Bio-Rad) using a semi-dry transfer cell (Bio-Rad) at 40 V for 30 min. Subsequently, the membrane was blocked for 12 h with phosphate-buffered saline (PBS) having 5% nonfat milk and washed 3 times with PBS. Finally, the membrane was probed with 6xHis tag Monoclonal Antibody (HIS.H8) and HRP (Thermo Scientific) at a dilution of 1:1000 for 6 hours at 4°C on a rocking platform and washed in PBS. Detection was performed using a chemiluminescent substrate (Thermo Scientific). 2.6 Enzymatic activity The standard reaction mixture (1 mL) contained 100 mM sodium phosphate buffer (pH 6.5), 100 mM xylose, 0.2 mM NADH, and a suitable amount of purified enzyme 9.6 g/mL final concentration). The reaction was initiated by the addition of the substrate and monitored at 25°C for 3 min. One unit (1 U) of enzyme activity was defined as the amount of enzyme required to oxidize 1 µmol of NADH per minute under the assay conditions. Specific activity was expressed as units per milligram of protein (U/mg). All enzymatic assays, including kinetic and stability characterizations, were performed in independent biological triplicates. 2.7 Determination of rNcXR kinetic parameters. The Michaelis-Menten K M (substrate concentration necessary to obtain half the value of V max rate) and V max (enzyme max rate) parameters were calculated. Enzyme activity assays were performed using Lineweaver–Burk linearization, with xylose as the substrate and NADH as cofactor. The kinetic parameter K cat, or turnover number, represents the maximum number of substrate converted to product per active site per unit time. This value was calculated using Eq. 1 [ 16 ], where \(\:{\left[E\right]}_{t}\) represents the total molar concentration of the enzyme in the assay. $$\:{k}_{cat}\:=\:\frac{{V}_{max}}{{\left[E\right]}_{t}}$$ 1 2.8 Optimal pH determination The best pH for the rNcXR was found in protein aliquots tempered at 4°C for 12 h at pH values of 3, 4, 5, 6, 7, 8, 9, and 10. Buffers used included 0.05 M acetate buffer (pH 2–5), 0.05 M phosphate buffer (pH 6–7), and 0.05 M Tris-HCl buffer (pH 8–10). The enzymatic activity was evaluated at each pH value. 2.9 Determination of best temperature The best temperature for rNcXR was found by incubating the reaction mixtures for one hour at temperatures of 25, 35, 45, 55, and 65°C, and the enzymatic activity (U/mL) was measured at each temperature. 2.10 Stability of rNcXR at different pHs and temperatures Aliquots of 250 µL of the enzyme were tempered for 12h at 4°C, at pH values of 3, 4, 5, 6, 7, 8, 9 and 10, using 0.05 M acetate buffer (pH 2–5), 0.05 M phosphate buffer (pH 6–7), and 0.05 M Tris-HCl buffer (pH 8–10). The pH activity was measured by using the procedure conditions shown in section 2.6 . The stability of the enzyme activity concerning temperature was measured by heating aliquots of 250 µL of the enzyme to 10, 20, 30, 40, 50, and 60°C during a one-hour period. The enzymatic activity was evaluated every 10 min, as shown in section 2.6 . 2.11 Enzyme purification Purification of Recombinant rNcXR Purification was performed using Immobilized Metal Affinity Chromatography (IMAC). A 5 mL syringe was packed with 1 mL of Ni-NTA agarose resin. The column was equilibrated with 50 mL of 50 mM phosphate buffer to remove any contaminants. Subsequently, 6 mL of the periplasmic extract was loaded onto the Ni-NTA column. The column was washed with 50 mL of buffer containing 20 mM imidazole to remove non-specifically bound proteins. Finally, the recombinant rNcXR was eluted using 100 mM imidazole. Fractions containing the purified protein were collected and stored at 4°C for further analysis. 3 Results and Discussion 3.1 Design of the rNcXR expression construct To evaluate rNcXR production, a synthetic construct was engineered (Fig. 1). The construct measured 1296 bp in length and comprised an expression cassette featuring a 100-bp 5′ UTR region flanked by BglII and NdeI restriction sites; this region included the T7 RNA polymerase promoter as well as the ribosome binding site. A 66-bp segment encoded the ompT signal peptide sequence for periplasmic protein secretion, while a 969-bp sequence represented the optimized synthetic xylose reductase gene from N. crassa , bordered by BamHI and NotI restriction sites. Furthermore, the construct incorporated six CAT codons for 6XHis tag addition between NotI and HindIII restriction sites. Within the 3' UTR region, opal and ochre stop codons were introduced, and the final 43 bp contained the T7 RNA polymerase terminator sequence. Figure 1 . Schematic representation of the synthetic construct and alignment with the native sequence of the Neurospora crassa XR. Table 1 shows that the wild-type XR sequence changed according to the codon preference of Escherichia coli . Amino acid Wild gene XR Synthetic gene XR Codon usage in E. coli Glycine GGG, GGA, GGT GGC 40 Glutamate GAG GAA 69 Aspartate GAC GAT 63 Valine GTA, GTT, GTC GTG 37 Alanine GCA, GCT, GCC GCG 36 Arginine AGA, CGT CGC 40 Lysine AAG AAA 77 Asparagine AAT AAC 55 Isoleucine ATA, ATT, ATC ATT 51 Threonine ACG, ACA, ACT ACC 44 Cysteine TGT TGC 55 Tyrosine TAT, TAC TAT 57 Phenylalanine TTC TTT 57 Serine AGT, TCG, TCA, TCT, TCC AGC 28 Glutamine CAA CAG 31 Histidine CAC CAT 57 Leucine TTG, TTA, CTT CTG 50 Proline CCA, CCT, CCC CCG 52 The synthetic XR gene was introduced into the 2752-bp pUCIDT (AmpR) expression vector, designated as pITD02 (Fig. 2). Successful incorporation of the gene was confirmed by restriction enzyme analysis using BamHI and HindIII , as illustrated in Fig. 3. Figure 2. The plasmid map of pITD02 with the optimized gene for xylose reductase production flanked by BglII and HindIII restriction sites, the OmpT signal peptide for periplasmic export, a 6XHis tag for purification, and an ampicillin resistance cassette. Figure 3 . Restriction analysis of plasmid pITD02. Line 1 : DNA ladder 1 kb. Line 2 : Restriction digest with the Bgl II and Hind III enzymes. A band of approximately 2.8 kb (corresponding to the 2752 bp empty plasmid backbone) was observed just below the 3000 bp marker, and the smaller band corresponds to the rNcXR expression constructs of 1200 bp. Several factors affect recombinant protein expression. For example, codons that encode hydrophobic amino acids, such as phenylalanine (Phe), leucine (Leu), and isoleucine (Ile), are more often used than codons that encode hydrophilic amino acids, such as serine (Ser), threonine (Thr), and lysine (Lys); the translation machinery of E. coli adapts better to these codons, resulting in more efficient protein expression. 3.2 Expression and periplasm production of rNcXR The ability of the recombinant BL21-SI strain to regulate protein expression via the T7 promoter in the pITD02 plasmid was examined. As shown in Fig. 4a , a protein band of approximately 37 kDa corresponding to the calculated molecular weight of the mature rNcXR secreted to the periplasm was present even at the beginning of the assay in the non-induced culture. However, the intensity of this protein band increased significantly over time in the assay where NaCl was added as an inducer (Fig. 4b). The recombinant enzyme (rNcXR) displayed an apparent molecular mass of approximately 37 kDa in both SDS-PAGE and Western blot analysis (Fig. 4). While the native monomeric XR from N. crassa has a theoretical molecular weight of ~ 34 kDa, the observed shift is attributed to the presence of the N-terminal OmpT leader sequence (~ 2.5 kDa) and the C-terminal 6xHis-tag (~ 0.8 kDa) used for periplasmic secretion and affinity purification, respectively. The calculated theoretical mass for the fusion protein, including the translation of the additional vector-derived amino acids, is 37.3 kDa, which is in full agreement with the experimental migration observed in our electrophoresis assays. This confirms the successful expression of the full-length recombinant fusion protein. Figure 4 . Production kinetics of the rNcXR protein were conducted in LBON. a) Protein production assay without inducer addition; b) Protein production assay with 0.3 M NaCl added as an inducer. To confirm that the observed band corresponded to rNcXR and not a host E. coli protein, the identity was verified using a Western blot assay (Fig. 5a). A specific protein band of approximately 37 kDa was detected. Its intensity remained constant in the absence of the inducer (Fig. 6a) and increased markedly when NaCl was used (Fig. 5b). Figure 5. Western blot analysis using the 6xHis tag antibody. Line 1 : Unstained protein standard. Lines 2–8 (a) show the samples of the kinetic protein production over 6 h. Gels show a recombinant protein of 37 kDa marked with antibodies. Figure b shows the production kinetics of rNcXR without the supply of the inducer over 6 h. The resultant optimized gene showed 76% identity with the native gene. Codon optimization was not the only factor regulating protein expression. Elements such as the correct vector are strong inducible promoters with high affinity for polymerase enzymes, such as a T7 promoter, which can increase protein expression [ 17 ]. The observation of protein production without the use of an inducer suggests leaky regulation of the T7 promoter, allowing for basal expression [ 18 ]. Transcriptional regulation failures have been documented in other pET systems, in which the amount of T7 RNA polymerase (RNAP) produced after induction exceeds the inhibitory capacity of T7 lysozyme. Consequently, free T7 RNAP can engage in transcription, causing basal production of recombinant proteins [ 10 ]​. While basal expression can sometimes lead to toxicity and plasmid loss [ 19 ], no defects associated with toxicity were observed in this study. This confirms that the recombinant rNcXR is not toxic to the expression strain. Furthermore, the results support previous findings that the addition of a signal peptide (OmpT) and a 6xHis-tag can facilitate the solubility, extraction, and detection of the produced proteins [ 20 , 21 ]. 3.3 Kinetic characterization of rNcXR The kinetic characterization of rNcXR was performed at 25°C using xylose as a substrate. The specific activity of this enzyme was 13.93 U/mg, surpassing the highest activity reported for XR of N. crassa , which varies between 3.54 and 9.2 U/mg [ 22 – 24 ]. These results underscore the influence of culture conditions, measurement techniques, and microbial strains on enzyme activity. To further elucidate the structural basis for the kinetic parameters observed in our rNcXR ( K m = 4.53 mM, specific activity = 13.93 U/mg, and K cat = 51,979 min⁻¹), molecular docking simulations were performed (Fig. 10). These kinetic values contrast with those typically reported for the native N. crassa enzyme. We hypothesize that this functional divergence stems directly from the specific sequence extensions introduced by our expression vector. The incorporation of the OmpT signal peptide and the C-terminal 6xHis-tag successfully enhanced the structural protein stability and significantly improved its solubility, extraction, and purification [ 20 , 21 ]. Furthermore, as suggested by previous literature, the addition of a 6xHis-tag can alter secondary conformations, potentially increasing the overall stability of the recombinant protein [ 25 ]. Our docking model supports this hypothesis by revealing a highly stabilized cofactor-binding pocket. We postulate that the mechanical tension exerted by these terminal additions induces a subtle geometric shift in the C-terminal loops, which shortens the interaction distances between the Lys279/Arg280 pair and the NADH cofactor. Consequently, this engineered genetic design not only facilitates efficient downstream processing but also yields a highly stable and active biocatalyst. The optimization of enzyme performance is crucial for both industrial and biomedical research. The kinetic parameters of xylose reductase produced by different microorganisms (Table 2 ) and the values obtained in this study ( K m , 4.53 mM; V max , 13.36 mM/min; and K cat , 51,979 min⁻¹) fall within the typical range reported in the literature. However, upon comparing the models of both enzymes, differences were clear in all loops, including the rNcXR, suggesting a slight change in protein folding. This alteration significantly affected enzyme activity and stability, as shown in the present study. The catalytic activity, kinetic parameters, pH, and temperature improved enzyme stability, making rNcXR an attractive candidate for enzymatic xylitol production. Table 2 Kinetic parameters of xylose reductase enzymes from different microorganisms reported in the literature and in this study. Microorganism Specific activity (U/mg) Km (mM) Kcat (min − 1 ) Kcat/Km (min − 1 /mM − 1 ) Reference Spathaspora passalidarum (rXR) 0.81 12.8 N.R. N.R. [ 28 ] Pichia stipitis 12 40 N.R. N.R. [ 29 ] Neurospora crassa (rXR) 3.54 34.4 3600 104.6 [ 30 ] Neurospora crassa (rXR) 13.93 4.53 51979 11474 This work *rXR Recombinant Xilose Reductase Differences in expression systems, induction strategies, and tag removal protocols likely account for the observed discrepancies in kinetic parameters between previously reported recombinant xylose reductases and the rNcXR described in this study [ 30 ]. With some differences, our protein was expressed in the E. coli BL21-SI strain. They used IPTG as an inducer, while we used NaCl as an inducer, and we did not remove the histidine tag to perform kinetic analysis. These expression differences may explain the significant differences in the kinetic parameters (Table 2 ) between these recombinant proteins. A deeper analysis showed that the V max values follow a similar trend. The same could not be said for the K m values, in which an essential improvement in the affinity between NADH and rNcXR was seen in this study. This improvement could be due to the presence of phosphate ions in the buffer, which could alter the proteins’ activity. It is also essential to mention that the NADH and xylose concentrations used in these experiments differed, which may explain the differences in the kinetic parameters of both proteins. However, our rNcXR production occurred in the E. coli BL21-SI strain periplasm, in which the oxidizing conditions favored protein folding. Xylose reductase is usually produced in the cytoplasm of E. coli (DE3 strain), where the oxidizing properties may influence protein folding. Reducing the medium can promote the formation of inclusion bodies and protein misfolding, altering the catalytic activity of the expressed enzyme [ 29 ]. 3.4 Effect of pH on specific enzymatic activity The effect of pH on rNcXR stability was evaluated over time. As shown in Fig. 6, the enzyme exhibited maximum stability at pH 6.0. Interestingly, at this pH, the enzymatic activity did not decrease but rather showed a slight increase throughout the incubation period, maintaining values above 1.0 U/mL. In contrast, at pH values of 5.0 and 7.0, the enzyme retained approximately 80% of its initial activity after 60 min. Outside this range (pH 8.0), a significant loss of activity was observed, with residual activity falling below 80%. Regarding the catalytic conditions, Fig. 7 illustrates the dependence of rNcXR activity on the reaction pH. The data was fitted to a polynomial regression, revealing an optimal pH of approximately 6.5. This finding is consistent with values reported for other xylose reductases [ 30 , 31 ]. Notably, there is a distinct difference between the optimal catalytic pH (6.5) and the theoretical isoelectric point of the protein (pI 5.04). In our study, rNcXR exhibited a maximum activity at an optimum pH of 6.5, maintaining over 80% of its catalytic efficiency within a functional range of pH 6.0 to 7.5. This profile suggests that the enzyme is well-suited for bioprocesses where moderate acidity is required to prevent microbial contamination while maintaining high reaction rates, aligning favorably with industrial requirements. Figure 6. Stability profile of recombinant rNcXR incubated at different pH values over 60 min. Figure 7. Effect of reaction pH on the enzymatic activity of recombinant rNcXR. Our findings regarding stability align with those of Woodyer et al. (2005) [ 28 ], who noted that the addition of a 6xHis-tag to the N-terminus can increase enzyme activity compared to the native protein. However, kinetic parameters such as Km (4.54 mM) were lower than the reported value for Candida tenuis (34 mM). pH significantly influences enzyme structure and catalytic efficiency. At the optimal pH, amino acid residues adopt the appropriate conformation for efficient substrate-cofactor binding. At acidic pH values, the enzyme may undergo aggregation due to the exposure of hydrophobic regions, while at alkaline pH, alterations in the tertiary structure can lead to deactivation [ 32 ]. Furthermore, protein stability is also known to depend on factors such as the presence of polyols, which protect the native structure [ 35 ]. The negative charge at the optimal pH likely favors a more stable rNcXR conformation through electrostatic interactions. 3.5 Effect of Temperature on rNcXR Activity The thermal stability of rNcXR was assessed by incubating the enzyme at different temperatures over a period of 60 min (Fig. 8). The temperature dependence profile of the purified rNcXR was evaluated. As expected from standard thermodynamic behavior, the catalytic activity at 25°C was approximately twofold higher than the activity observed at 15°C (corresponding to a temperature coefficient, Q 10 ≈ 2). This indicates a typical Arrhenius-type increase in the reaction rate before reaching the enzyme's optimal temperature. However, this was followed by a sharp decline. In terms of sustained stability, the enzyme performed best at 45°C and 55°C. Conversely, temperatures above 60°C proved detrimental; at 65°C, the activity decreased rapidly, reaching near-zero levels after 60 min. Regarding the optimal reaction temperature, Fig. 9 shows a clear profile with maximum catalytic activity at 45°C (0.38 U/mL). Although activity decreased at 55°C, the enzyme still retained approximately 85% of its maximal activity. Figure 8. The thermal stability profile of recombinant rNcXR is over 60 min. Figure 9. Effect of temperature on the catalytic activity of recombinant rNcXR. The optimal temperature range observed aligns with findings for xylose reductases from various sources, which typically show optimal activity at 50°C [ 34 , 35 ]. The stability of recombinant rNcXR at temperatures up to 55°C suggests it can be effectively used in industrial processes without significant activity loss [ 36 ]. Table 3 Comparison of optimal temperature and thermal stability parameters of Xylose Reductase from various microorganisms. Microorganism T Optimal (°C) Denaturalization half-time (min) Denaturalization temperature (°C) Bibliographic Reference Lactobacillus brevis KU15006 50 30 60 [ 37 ] Candida mogii 55 9 60 [ 38 ] Candida tropicallis 65 40 60 [ 39 ] Neurospora crassa 50 30 65 This study As shown in Table 3 , the recombinant rNcXR exhibits competitive thermal properties. Protein stability at elevated temperatures is often attributed to subtle structural alterations and increased rigidity via hydrogen or ionic bonds [ 40 , 41 ]. However, stability depends on the source microorganism; for instance, Nair and Zhao (2008) [ 42 ] found that native XR from N. crassa retained > 50% activity between pH 4.5 and 8.5, consistent with our results. In the case of rNcXR, hydrophobic amino acids (such as Ala and Gly) influence protein folding via hydrophobic collapse, contributing to the stability of the structure against pH and temperature changes [ 43 , 44 ]. 3.6 Structural Analysis of XR recombinant enzyme Sequence alignment between the native N. crassa XR (Woodyer et al., 2005) and our recombinant rNcXR construct revealed strict conservation of the catalytic core. As observed in homologous enzymes, the catalytic residue Asp44 is crucial for xylose binding at the active site. During this process, Tyr49, a hydrophobic residue, plays a pivotal role in ensuring the efficient binding of the protein-ligand complex. In contrast, Lys78 facilitates the addition of NAD(P)H to the catalytic site through a precise interaction with the ribose phosphate, while His111 forms a catalytic duo with His27. Despite this active site conservation, specific sequence extensions were introduced by our cloning strategy. rNcXR is a fusion protein containing internal and N-terminal additions (e.g., Glu2, Thr3, Pro18, Gln19) resulting from signal peptide processing and restriction site scars. Most notably, rNcXR possesses a distinct C-terminal extension comprising a flexible linker (Ala333-Ala335) derived from the NotI restriction site, followed immediately by the 6xHis purification tag (His336-His341). Previous structural studies on aldo-keto reductases demonstrate that bulky sequence extensions at the termini can exert mechanical tension on them, likely inducing a conformational shift in the adjacent flexible loops that govern cofactor binding. To further elucidate the structural basis for the enhanced specific activity and improved kinetic parameters observed in our rNcXR compared to previously reported N. crassa enzymes, molecular docking simulations were performed (Fig. 10). The docking model reveals a highly stabilized cofactor-binding pocket. Specifically, the residues Lys279, Arg280, and Ser281 form a tight electrostatic interaction network with the ribose phosphate region of the cofactor. We postulate that the structural tension exerted by the before mentioned N-terminal additions and the C-terminal 6xHis-tag, combined with the oxidative folding environment of the periplasm, induces a subtle geometric shift in these C-terminal loops. This shift likely shortens the interaction distances between the Lys279/Arg280 pair and the cofactor, thereby enhancing binding affinity ( Km = 4.53 mM) and catalytic rate ( Vmax = 13.36 mM/min). Consequently, these structural divergences provide a mechanistic explanation for how our rNcXR creates a highly active biocatalyst (13.93 U/mg) distinct from the wild-type enzyme. Figure 10. Comparative 3D structural models of the native Neurospora crassa xylose reductase (Native XR) and the engineered recombinant enzyme (rNcXR). 4 Conclusions The present study confirms that modifying a plasmid with T7 regulatory sequences, an OmpT signal peptide, and a 6xHis-tag enables the highly efficient, salt-inducible production and periplasmic secretion of a recombinant xylose reductase (rNcXR) from Neurospora crassa in E. coli BL21-SI. The customized pITD02 expression vector successfully prevented inclusion body formation, allowing for the recovery of soluble, active protein without hindering high-yield production, despite the inherent basal expression of the T7 promoter. Biochemical characterization revealed that rNcXR possesses an optimal pH of 6.5, an optimal temperature range of 45–50°C, and robust thermal stability, retaining significant activity for up to 60 min at 55°C—traits that are critical for industrial operations. ​Beyond cost-effective expression and thermal stability, structural analysis via molecular docking highlighted the dual purpose of our genetic design. The specific sequence insertions at the termini, particularly the C-terminal flexible linker and the 6xHis-tag, not only optimize the NADH-binding pocket to enhance substrate affinity but also serve as highly reactive, accessible anchoring points for directed enzyme immobilization. Consequently, this engineered rNcXR stands out as an ideal biocatalyst for future integration onto solid matrices, such as nickel-chelated surfaces or carbon-based supports. This work not only provides a robust soluble enzyme for large-scale xylitol production but establishes the critical structural foundation for developing stable, reusable enzymatic systems tailored for continuous industrial production bioprocesses. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Use of Artificial Intelligence During the preparation of this work, the authors used Generative AI technologies (Gemini and Copilot) solely to improve the language, clarity, and readability of the manuscript. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. Funding The authors declare that no specific funding was received for this study other than the Ph.D. scholarship provided to M. J. Reyes-Rosas by CONACYT. Author Contribution M. J. Reyes-Rosas: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft. J. López-Miranda, J. A. Rojas-Contreras: Conceptualization, Resources, Writing - Review & Editing, Supervision, Project administration. M. García-Curiel, J. C. Aguilar-Cordero, D. Reyes-Jacquez, J. B. Páez-Lerma, N. O. Soto-Cruz: Methodology, Validation, Writing - Review & Editing. Acknowledgements M. J. Reyes-Rosas thanks the National Council of Science and Technology (CONACYT) for the grant (No. 775313) to conduct PhD studies. Data Availability All data generated or analyzed during this study are included in this published article. References Hernández-Pérez AF, Chaves-Villamil AC, de Arruda PV, dos Santos JC, Felipe MGA (2020) Sugarcane syrup improves xylitol bioproduction from sugarcane bagasse and straw hemicellulosic hydrolysate. 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Braz J Pharm Sci 42:251–257. https://doi.org/10.1590/S1516-93322006000200010 Zhang XF, Yang GY, Zhang Y, Xie Y, Withers SG, Feng Y (2016) A general and efficient strategy for generating stable enzymes. Sci Rep 6:33797. https://doi.org/10.1038/srep33797 Louie TM, Louie K, DenHartog S, Gopishetty S, Subramanian M, Arnold M, Das S (2021) Production of bio-xylitol from D-xylose by an engineered Pichia pastoris expressing a recombinant xylose reductase did not require any auxiliary substrate as an electron donor. Microb Cell Fact 20:50. https://doi.org/10.1186/s12934-021-01534-1 Carson M, Johnson DH, McDonald H, Brouillette C, DeLucas LJ (2007) His-tag impact on structure. Acta Crystallogr D Biol Crystallogr 63:295–301. https://doi.org/10.1107/S0907444906052024 Mouro A, dos Santos AA, Agnolo DD, Gubert GF, Bon EPS, Rosa CA, Fonseca C, Stambuk BU (2020) Combining Xylose Reductase from Spathaspora arborariae with Xylitol Dehydrogenase from Spathaspora passalidarum to Promote Xylose Consumption and Fermentation into Xylitol by Saccharomyces cerevisiae. Fermentation 6:72. https://doi.org/10.3390/fermentation6030072 Rizzi M, Erlemann P, Bui-Thanh NA, Dellweg H (1988) Xylose fermentation by yeast. 4. Purification and kinetic studies of xylose reductase from Pichia stipitis. Appl Microbiol Biotechnol 29(2–3):148–154 Woodyer R, Simurdiak M, Van Der Donk WA, Zhao H (2005) Heterologous expression, purification, and characterization of a highly active xylose reductase from Neurospora crassa. Appl Environ Microbiol 71:1642–1647. https://doi.org/10.1128/AEM.71.3.1642-1647.2005 Gopal GJ, Kumar A (2013) Strategies to produce recombinant protein in Escherichia coli. Protein J 32:419–425. https://doi.org/10.1007/s10930-013-9502-5 West TP (2021) Xylitol is produced by Candida species from hydrolyzates of agricultural residues and grasses. World J Microbiol Biotechnol 25:913–916. https://doi.org/10.1007/s11274-008-9947-4 Xu L, Liu L, Li S, Zheng W, Cui Y, Liu R, Sun W (2019) Xylitol production by Candida tropicalis 31949 from sugarcane bagasse hydrolysate. Sugar Tech 21:341–347. https://doi.org/10.1007/s12355-018-0650-y Malla S, Gummadi SN (2020) Counteraction of osmolytes on pH-induced unfolding of xylose reductase from Debaryomyces nepalensis NCYC 3413. Eur Biophys J 49:267–277. https://doi.org/10.1007/s00249-020-01432-1 Zheng T, Li J, Ji Y, Zhang W, Fang Y, Xin F, Dong W, Wei P, Ma J, Jiang M (2020) Progress and prospects of bioelectrochemical systems: Electron transfer and its applications in microbial metabolism. Front Bioeng Biotechnol 8:10. https://doi.org/10.3389/fbioe.2020.00010 Lugani Y, Puri M, Sooch BS (2021) Recent insights, applications, and prospects of xylose reductase: A futuristic enzyme for xylitol production. Eur Food Res Technol 247:921–946. https://doi.org/10.1007/S00217-020-03674-X Wang Y, Zhang J (2006) A novel hybrid process, enhanced by ultrasonication, for xylan extraction from corncobs and hydrolysis of xylan to xylose by xylanase. J Food Eng 77:140–145. https://doi.org/10.1016/j.jfoodeng.2005.06.056 Den Haan R, Van Zyl WH (2001) Differential expression of the Trichoderma reesei β-xylanase II (xyn2) gene in the xylose-fermenting yeast Pichia stipitis. Appl Microbiol Biotechnol 57:521–527. https://doi.org/10.1007/s002530100790 l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose Petra Staudigl, Dietmar Haltrich, and Clemens K. Peterbauer J Agricultural Food Chem 2014 62 (7), 1617–1624 10.1021/jf404785m Mayerhoff ZDVL, Roberto IC, Franco TT (2001) Activity of Xylose Reductase from Candida mogii Grown in Media Containing Different Concentrations of Rice Straw Hydrolysate. In: Davison, B.H., McMillan, J., Finkelstein, M. (eds) Twenty-Second Symposium on Biotechnology for Fuels and Chemicals. ABAB Symposium. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-4612-0217-2_61 Zhang F, Qiao D, Xu H et al (2009) Cloning, expression, and characterization of xylose reductase with higher activity from Candida tropicalis. J Microbiol 47:351–357. https://doi.org/10.1007/s12275-008-0225-9 Agarwal PK, Nuriso A, Cittaro D (2020) The role of electrostatics in protein–protein interactions. Curr Opin Struct Biol 64:154–161 Ali N, Aiman A, Shamsi A, Hassan I, Shahid M, Gaur NA, Islam A (2022) Identification of thermostable xylose reductase from Thermothelomyces thermophilus: A biochemical characterization approach to meet biofuel challenges. ACS Omega 7:44241–44250. https://doi.org/10.1021/acsomega.2c05690 Nair NU, Zhao H (2008) Evolution in reverse: Engineering a D-xylose-specific xylose reductase. ChemBioChem 9:1213–1215. https://doi.org/10.1002/cbic.200700765 Camilloni C, Bonetti D, Morrone A, Giri R, Dobson CM, Brunori M, Gianni S, Vendruscolo M (2016) Towards a structural biology of the hydrophobic effect in protein folding. Sci Rep 6:28285. https://doi.org/10.1038/srep28285 Sun X, Cui Q, Li R, Hao L, Liu H, Wang X, Xu N, Zhao X (2022) S4ructural and emulsifying properties of soybean protein isolate glycated with glucose based on pH treatment. J Sci Food Agric 102:4462–4472. https://doi.org/10.1002/JSFA.11800 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Apr, 2026 Reviews received at journal 22 Apr, 2026 Reviews received at journal 14 Apr, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviewers agreed at journal 09 Apr, 2026 Reviewers invited by journal 08 Apr, 2026 Editor assigned by journal 07 Apr, 2026 Submission checks completed at journal 06 Apr, 2026 First submitted to journal 03 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9316107","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":623194621,"identity":"8544123a-792d-4755-9d23-ecc3c82ea2ba","order_by":0,"name":"M. J. Reyes-Rosas","email":"","orcid":"","institution":"TecNM/Instituto Tecnologico de Durango","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"J.","lastName":"Reyes-Rosas","suffix":""},{"id":623194624,"identity":"5da45119-733c-4cc3-a659-5b4837bd9421","order_by":1,"name":"J. Lopez-Miranda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYBACxnYog5+ZgUECLsyDT0szlCHZzEykFgZmKG1wgFgtzM3Mxx58+HM42vg4/8HbvHvs8uTdGxgfvG1jiDY4gMthbOmGM3gO5247zMxszfMsudjwzAFmw7ltDLkzG3Bp4TGT5pEAa2GT5jnAnLhxRgKbNC9QSz8Oh4G1/DE4nLu5GaylHqSF/TdISxs+LQwJh3M3MIO1HE6cL5HAxozfFqBfeg6k5844zGxsOefA8cQNPAebJeeck8DpF8P25mMPfvyxzu3vP/jwxpsD1Ynz25sPfnhTZpO7AUeIGTYwsAGpZoSIwQFGkPES2NUDgTwDWEsdkggO94yCUTAKRsHIBQAPRFnoOWa40gAAAABJRU5ErkJggg==","orcid":"","institution":"TecNM/Instituto Tecnologico de Durango","correspondingAuthor":true,"prefix":"","firstName":"J.","middleName":"","lastName":"Lopez-Miranda","suffix":""},{"id":623194627,"identity":"3a764ec5-d829-44df-bb38-78ac0cfe9a20","order_by":2,"name":"J.A. 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Reyes-Jaquez","email":"","orcid":"","institution":"TecNM/Instituto Tecnologico de Durango","correspondingAuthor":false,"prefix":"","firstName":"D.","middleName":"","lastName":"Reyes-Jaquez","suffix":""},{"id":623194640,"identity":"d1148815-f79d-4b4d-a262-bf01e35563cc","order_by":6,"name":"J.B. Paez-Lerma","email":"","orcid":"","institution":"TecNM/Instituto Tecnologico de Durango","correspondingAuthor":false,"prefix":"","firstName":"J.B.","middleName":"","lastName":"Paez-Lerma","suffix":""},{"id":623194641,"identity":"7ca31f87-4747-4a04-b04b-95000ae53b57","order_by":7,"name":"N.O. Soto-Cruz","email":"","orcid":"","institution":"TecNM/Instituto Tecnologico de Durango","correspondingAuthor":false,"prefix":"","firstName":"N.O.","middleName":"","lastName":"Soto-Cruz","suffix":""}],"badges":[],"createdAt":"2026-04-03 20:53:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9316107/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9316107/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107481023,"identity":"be3eb628-3671-42aa-bebd-a5502a7dd2a8","added_by":"auto","created_at":"2026-04-22 02:15:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":195650,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the synthetic construct and alignment with the native sequence of the Neurospora crassa RXR.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9316107/v1/ba82efefef5cb3cb57e6a05d.png"},{"id":107123328,"identity":"877bd79d-be89-4ca1-96b6-fc64b6c33faa","added_by":"auto","created_at":"2026-04-17 05:14:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58153,"visible":true,"origin":"","legend":"\u003cp\u003eThe plasmid map of pITD02 with the optimized gene for xylose reductase production flanked by BglII and HindIII restriction sites, the OmpT signal peptide for periplasmic export, a 6XHis tag for purification, and an ampicillin resistance cassette.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9316107/v1/42764c6fc2ed82462a75f935.png"},{"id":107481351,"identity":"72ae0719-75a0-401f-afba-3aaf3f393f75","added_by":"auto","created_at":"2026-04-22 02:17:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28761,"visible":true,"origin":"","legend":"\u003cp\u003eRestriction analysis of plasmid pITD02. \u003cstrong\u003eLine 1\u003c/strong\u003e: DNA ladder 1 kb. \u003cstrong\u003eLine 2\u003c/strong\u003e: Restriction digest with the \u003cem\u003eBgl\u003c/em\u003eII and \u003cem\u003eHind\u003c/em\u003eIII enzymes. The 3000-bp bands correspond to the empty plasmid backbone, and the smaller band corresponds to the RXR expression constructs of 1200 bp.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9316107/v1/de16d31fe770962a839b82d0.png"},{"id":107481897,"identity":"c5b3b544-02c5-498e-8c88-f2e4acd5bbae","added_by":"auto","created_at":"2026-04-22 02:20:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68271,"visible":true,"origin":"","legend":"\u003cp\u003eProduction kinetics of the RXR protein conducted in LBON. a) Protein production assay without inductor addition; b) Protein production assay with 0.3 M NaCl added as an inducer.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9316107/v1/a1d489d978f9739f36851e21.png"},{"id":107481961,"identity":"b99efc69-12fc-4888-8f0a-af7653806338","added_by":"auto","created_at":"2026-04-22 02:21:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":87343,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot analysis using the 6xHis ag antibody. \u003cstrong\u003eLine 1:\u003c/strong\u003e Unstained protein standard. Lines 2–8 (a) show the samples of the kinetic protein production over 6 h. Gels offer a recombinant protein of 37 kDa marked with antibodies. Figure b shows the production kinetics of RXR without the supply of the inductor over 6 h.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9316107/v1/8c3005f286497f9acc087553.png"},{"id":107123331,"identity":"225dc6a2-e50a-41ba-b216-101ab52ed5e5","added_by":"auto","created_at":"2026-04-17 05:14:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":37516,"visible":true,"origin":"","legend":"\u003cp\u003eStability profile of recombinant RXR incubated at different pH values over 60 min.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9316107/v1/0973f6c4f7c7886886fde4ef.png"},{"id":107123332,"identity":"b91664cf-3c86-437f-8e39-e44d2900b1ff","added_by":"auto","created_at":"2026-04-17 05:14:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":16173,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of reaction pH on the enzymatic activity of recombinant RXR.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9316107/v1/6adf517f738b114a94d8bcfd.png"},{"id":107481916,"identity":"bacb6b12-2d3a-4c61-9d0c-f46cb4cf8405","added_by":"auto","created_at":"2026-04-22 02:20:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":51456,"visible":true,"origin":"","legend":"\u003cp\u003eThermal stability profile of recombinant RXR is over 60 min.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9316107/v1/d8cf3ab279371b9d9fa6d10f.png"},{"id":107123335,"identity":"badf27be-7c37-4b7d-afd8-6ef8f2dc2ea2","added_by":"auto","created_at":"2026-04-17 05:14:34","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":24100,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of temperature on the catalytic activity of recombinant RXR.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9316107/v1/9540138792b10a68c4496661.png"},{"id":107123336,"identity":"d959710f-28fc-451d-b1a3-18d3767efd55","added_by":"auto","created_at":"2026-04-17 05:14:34","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":3076053,"visible":true,"origin":"","legend":"\u003cp\u003eComparative 3D structural models of the native Neurospora crassa xylose reductase (Native XR) and the engineered recombinant enzyme (rNcXR).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-9316107/v1/0d89d78f6f8a1fbb00fc17ef.png"},{"id":107484621,"identity":"1b2ea6eb-4b7f-4d67-9436-3c1c3dffdef2","added_by":"auto","created_at":"2026-04-22 02:32:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5214510,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9316107/v1/6b961142-970d-46ae-85e9-017493d31af5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eEnhancing catalytic efficiency of a salt-induced \u003cem\u003eNeurospora crassa\u003c/em\u003e xylose reductase for xylitol bioprocesses\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eXylitol is a five-carbon polyol naturally present in plants, fungi, and yeasts [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It exhibits sweetness comparable to sucrose while providing a significantly lower caloric value (2.4 kcal/g for xylitol versus 4 kcal/g for sucrose). Owing to these properties, xylitol is commonly used as a sugar substitute, particularly in products intended for insulin-dependent individuals [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond its nutritional advantages, xylitol has gained industrial relevance due to its applications in the food, pharmaceutical, and dental sectors [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Conventional industrial production involves the catalytic hydrogenation of xylose under high temperature and pressure, resulting in substantial energy consumption and environmental impact. Consequently, increasing attention has been directed toward biotechnological alternatives employing microorganisms or isolated enzymes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite their advantages, enzymatic processes are often constrained by limited enzyme stability under industrial conditions, particularly at low pH and elevated temperatures [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Advances in recombinant DNA technology have enabled the heterologous expression of enzymes in suitable microbial hosts, allowing the production of recombinant biocatalysts with improved performance and tailored properties [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Recombinant enzyme production requires the insertion of the gene of interest into a plasmid, which acts as a vector to introduce the gene of interest into the host cell.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e is a commonly used microbial host system because it has fast growth kinetics and lowers enzyme production costs. However, it is necessary to consider the availability of proper vectors to ensure protein expression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To achieve this, two vector types are needed: a cloning vector, which provides multiple cloning sites to insert the gene but cannot express the protein of interest; and an expression vector, which shares some characteristics with cloning vectors. They differ because they are designed to have regulated sequences that function as enhancers and promoter regions that lead to efficient gene transcription conducted in the expression vector.\u003c/p\u003e \u003cp\u003eThe objective of this study was to design and evaluate the salt-inducible periplasmic expression of a \u003cem\u003eNeurospora crassa\u003c/em\u003e xylose reductase, and to elucidate the role of its structural modifications in enhancing its catalytic efficiency and providing anchoring points for future immobilization in xylitol bioprocesses.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Design of the NcXR expression construct\u003c/h2\u003e \u003cp\u003eThe original nucleotide sequence of the xylose reductase gene (XR) of \u003cem\u003eNeurospora crassa\u003c/em\u003e (NCBI Reference Sequence: NC_026501.1) was expressed by the \u003cem\u003eE. coli\u003c/em\u003e K12 strain using the codon optimization tool provided by Integrated DNA Technologies, Coralville, Iowa, USA. The OmpT leader, promoter, and terminator sequences of the T7 RNA polymerase were taken from the pET12a plasmid sequence deposited on the Addgene vector database. The final genetic construct was analyzed for correct assembly using GenSmart Design software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Bacterial strain and growth media\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e BL21si was routinely grown at 37\u0026deg;C in Luria Bertani culture medium [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] without NaCl (LBON). Enzyme production and periplasm secretion were performed using LBON culture medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Transformation procedure and molecular techniques\u003c/h2\u003e \u003cp\u003eThe procedure for making CaCl\u003csub\u003e2\u003c/sub\u003e competent cells and thermal shock transformation of \u003cem\u003eE. coli\u003c/em\u003e were performed using standard techniques, as described previously [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Transformants were selected on solid LBON medium plates with 100 \u0026micro;g/mL ampicillin. The plates were incubated overnight at 37\u0026deg;C. The restriction endonuclease cutting assays were performed according to the instructions given by the supplier (Promega, Madison, Wisconsin, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Xylose reductase expression assays\u003c/h2\u003e \u003cp\u003eThe transformed strain (\u003cem\u003eE. coli\u003c/em\u003e BL21-SI) was grown overnight and diluted to an OD600 of 0.1 in two 50-mL of fresh LBON medium. The cultures were incubated until they reached an OD600 of 0.5. One of the flasks was supplemented with 0.3 M NaCl for 12h to induce enzyme production. The secreted proteins in the periplasm were recovered as follows: 1.5 mL of the culture medium with an OD600 of 2.0 was centrifuged at 10,000 \u0026times; g for 2 min. The cell pack was suspended in 200 \u0026micro;L of osmotic shock buffer with 100 mM Tris-HCl, pH 7.4, 20% sucrose, 10 mM Ethylene diamine tetraacetic acid (EDTA), and kept on ice for 5 min. The suspension was centrifuged at 10,000 \u0026times; g for 2 min. The cell pack was quickly suspended in 200 \u0026micro;L of Milli-Q water, vigorously shaken, and kept for 5 min in an ice bath [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The cell suspension was then centrifuged at 16,000 \u0026times; g for 2 min. Aliquots (150 \u0026micro;L) of the supernatant (periplasmic proteins) were concentrated by precipitation (methanol, chloroform, water, 4:1:3) and analyzed using 12% SDS\u0026ndash;polyacrylamide gel electrophoresis with a mini Protean Tetra Cell protein electrophoresis system (Bio-Rad). This was performed by applying a voltage of 100 V and a current intensity of 150 mA for 1.5 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Western blot analysis of rNcXR\u003c/h2\u003e \u003cp\u003eProteins separated by SDS-PAGE were transferred to a nitrocellulose membrane with a pore size of 0.45 mm (Bio-Rad) using a semi-dry transfer cell (Bio-Rad) at 40 V for 30 min. Subsequently, the membrane was blocked for 12 h with phosphate-buffered saline (PBS) having 5% nonfat milk and washed 3 times with PBS. Finally, the membrane was probed with 6xHis tag Monoclonal Antibody (HIS.H8) and HRP (Thermo Scientific) at a dilution of 1:1000 for 6 hours at 4\u0026deg;C on a rocking platform and washed in PBS. Detection was performed using a chemiluminescent substrate (Thermo Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Enzymatic activity\u003c/h2\u003e \u003cp\u003eThe standard reaction mixture (1 mL) contained 100 mM sodium phosphate buffer (pH 6.5), 100 mM xylose, 0.2 mM NADH, and a suitable amount of purified enzyme 9.6 g/mL final concentration). The reaction was initiated by the addition of the substrate and monitored at 25\u0026deg;C for 3 min. One unit (1 U) of enzyme activity was defined as the amount of enzyme required to oxidize 1 \u0026micro;mol of NADH per minute under the assay conditions. Specific activity was expressed as units per milligram of protein (U/mg).\u003c/p\u003e \u003cp\u003eAll enzymatic assays, including kinetic and stability characterizations, were performed in independent biological triplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Determination of rNcXR kinetic parameters.\u003c/h2\u003e \u003cp\u003eThe Michaelis-Menten \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eM\u003c/em\u003e\u003c/sub\u003e (substrate concentration necessary to obtain half the value of \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e rate) and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e (enzyme max rate) parameters were calculated. Enzyme activity assays were performed using Lineweaver\u0026ndash;Burk linearization, with xylose as the substrate and NADH as cofactor. The kinetic parameter K\u003csub\u003ecat,\u003c/sub\u003e or turnover number, represents the maximum number of substrate converted to product per active site per unit time. This value was calculated using Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], where \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\left[E\\right]}_{t}\\)\u003c/span\u003e\u003c/span\u003e represents the total molar concentration of the enzyme in the assay.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{k}_{cat}\\:=\\:\\frac{{V}_{max}}{{\\left[E\\right]}_{t}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Optimal pH determination\u003c/h2\u003e \u003cp\u003eThe best pH for the rNcXR was found in protein aliquots tempered at 4\u0026deg;C for 12 h at pH values of 3, 4, 5, 6, 7, 8, 9, and 10. Buffers used included 0.05 M acetate buffer (pH 2\u0026ndash;5), 0.05 M phosphate buffer (pH 6\u0026ndash;7), and 0.05 M Tris-HCl buffer (pH 8\u0026ndash;10). The enzymatic activity was evaluated at each pH value.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Determination of best temperature\u003c/h2\u003e \u003cp\u003eThe best temperature for rNcXR was found by incubating the reaction mixtures for one hour at temperatures of 25, 35, 45, 55, and 65\u0026deg;C, and the enzymatic activity (U/mL) was measured at each temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Stability of rNcXR at different pHs and temperatures\u003c/h2\u003e \u003cp\u003eAliquots of 250 \u0026micro;L of the enzyme were tempered for 12h at 4\u0026deg;C, at pH values of 3, 4, 5, 6, 7, 8, 9 and 10, using 0.05 M acetate buffer (pH 2\u0026ndash;5), 0.05 M phosphate buffer (pH 6\u0026ndash;7), and 0.05 M Tris-HCl buffer (pH 8\u0026ndash;10). The pH activity was measured by using the procedure conditions shown in section \u003cspan refid=\"Sec8\" class=\"InternalRef\"\u003e2.6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe stability of the enzyme activity concerning temperature was measured by heating aliquots of 250 \u0026micro;L of the enzyme to 10, 20, 30, 40, 50, and 60\u0026deg;C during a one-hour period. The enzymatic activity was evaluated every 10 min, as shown in section \u003cspan refid=\"Sec8\" class=\"InternalRef\"\u003e2.6\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Enzyme purification\u003c/h2\u003e \u003cp\u003ePurification of Recombinant rNcXR Purification was performed using Immobilized Metal Affinity Chromatography (IMAC). A 5 mL syringe was packed with 1 mL of Ni-NTA agarose resin. The column was equilibrated with 50 mL of 50 mM phosphate buffer to remove any contaminants.\u003c/p\u003e \u003cp\u003eSubsequently, 6 mL of the periplasmic extract was loaded onto the Ni-NTA column. The column was washed with 50 mL of buffer containing 20 mM imidazole to remove non-specifically bound proteins. Finally, the recombinant rNcXR was eluted using 100 mM imidazole. Fractions containing the purified protein were collected and stored at 4\u0026deg;C for further analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Design of the rNcXR expression construct\u003c/h2\u003e \u003cp\u003eTo evaluate rNcXR production, a synthetic construct was engineered (Fig.\u0026nbsp;1). The construct measured 1296 bp in length and comprised an expression cassette featuring a 100-bp 5\u0026prime; UTR region flanked by \u003cem\u003eBglII and NdeI\u003c/em\u003e restriction sites; this region included the T7 RNA polymerase promoter as well as the ribosome binding site. A 66-bp segment encoded the ompT signal peptide sequence for periplasmic protein secretion, while a 969-bp sequence represented the optimized synthetic xylose reductase gene from \u003cem\u003eN. crassa\u003c/em\u003e, bordered by \u003cem\u003eBamHI\u003c/em\u003e and \u003cem\u003eNotI\u003c/em\u003e restriction sites. Furthermore, the construct incorporated six CAT codons for 6XHis tag addition between \u003cem\u003eNotI\u003c/em\u003e and \u003cem\u003eHindIII\u003c/em\u003e restriction sites. Within the 3' UTR region, opal and ochre stop codons were introduced, and the final 43 bp contained the T7 RNA polymerase terminator sequence.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1\u003c/b\u003e. Schematic representation of the synthetic construct and alignment with the native sequence of the \u003cem\u003eNeurospora crassa\u003c/em\u003e XR.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eshows that the wild-type XR sequence changed according to the codon preference of \u003cem\u003eEscherichia coli\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmino acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWild gene\u003c/p\u003e \u003cp\u003eXR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSynthetic gene XR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCodon usage in E. coli\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGG, GGA, GGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutamate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAspartate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTA, GTT, GTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCA, GCT, GCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGCG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGA, CGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLysine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsparagine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsoleucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATA, ATT, ATC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThreonine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eACG, ACA, ACT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCysteine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTyrosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTAT, TAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenylalanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGT, TCG, TCA, TCT, TCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistidine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTTG, TTA, CTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCA, CCT, CCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe synthetic XR gene was introduced into the 2752-bp pUCIDT (AmpR) expression vector, designated as pITD02 (Fig.\u0026nbsp;2). Successful incorporation of the gene was confirmed by restriction enzyme analysis using \u003cem\u003eBamHI\u003c/em\u003e and \u003cem\u003eHindIII\u003c/em\u003e, as illustrated in Fig.\u0026nbsp;3.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2.\u003c/b\u003e The plasmid map of pITD02 with the optimized gene for xylose reductase production flanked by \u003cem\u003eBglII\u003c/em\u003e and \u003cem\u003eHindIII\u003c/em\u003e restriction sites, the OmpT signal peptide for periplasmic export, a 6XHis tag for purification, and an ampicillin resistance cassette.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 3\u003c/b\u003e. Restriction analysis of plasmid pITD02. \u003cb\u003eLine 1\u003c/b\u003e: DNA ladder 1 kb. \u003cb\u003eLine 2\u003c/b\u003e: Restriction digest with the \u003cem\u003eBgl\u003c/em\u003eII and \u003cem\u003eHind\u003c/em\u003eIII enzymes. A band of approximately 2.8 kb (corresponding to the 2752 bp empty plasmid backbone) was observed just below the 3000 bp marker, and the smaller band corresponds to the rNcXR expression constructs of 1200 bp.\u003c/p\u003e \u003cp\u003eSeveral factors affect recombinant protein expression. For example, codons that encode hydrophobic amino acids, such as phenylalanine (Phe), leucine (Leu), and isoleucine (Ile), are more often used than codons that encode hydrophilic amino acids, such as serine (Ser), threonine (Thr), and lysine (Lys); the translation machinery of \u003cem\u003eE. coli\u003c/em\u003e adapts better to these codons, resulting in more efficient protein expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Expression and periplasm production of rNcXR\u003c/h2\u003e \u003cp\u003eThe ability of the recombinant BL21-SI strain to regulate protein expression via the T7 promoter in the \u003cem\u003epITD02\u003c/em\u003e plasmid was examined. As shown in \u003cb\u003eFig.\u0026nbsp;4a\u003c/b\u003e, a protein band of approximately 37 kDa corresponding to the calculated molecular weight of the mature rNcXR secreted to the periplasm was present even at the beginning of the assay in the non-induced culture. However, the intensity of this protein band increased significantly over time in the assay where NaCl was added as an inducer (Fig.\u0026nbsp;4b). The recombinant enzyme (rNcXR) displayed an apparent molecular mass of approximately 37 kDa in both SDS-PAGE and Western blot analysis (Fig.\u0026nbsp;4). While the native monomeric XR from \u003cem\u003eN. crassa\u003c/em\u003e has a theoretical molecular weight of ~\u0026thinsp;34 kDa, the observed shift is attributed to the presence of the N-terminal OmpT leader sequence (~\u0026thinsp;2.5 kDa) and the C-terminal 6xHis-tag (~\u0026thinsp;0.8 kDa) used for periplasmic secretion and affinity purification, respectively.\u003c/p\u003e \u003cp\u003eThe calculated theoretical mass for the fusion protein, including the translation of the additional vector-derived amino acids, is 37.3 kDa, which is in full agreement with the experimental migration observed in our electrophoresis assays. This confirms the successful expression of the full-length recombinant fusion protein.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 4\u003c/b\u003e. Production kinetics of the rNcXR protein were conducted in LBON. a) Protein production assay without inducer addition; b) Protein production assay with 0.3 M NaCl added as an inducer.\u003c/p\u003e \u003cp\u003eTo confirm that the observed band corresponded to rNcXR and not a host \u003cem\u003eE. coli\u003c/em\u003e protein, the identity was verified using a Western blot assay (Fig.\u0026nbsp;5a). A specific protein band of approximately 37 kDa was detected. Its intensity remained constant in the absence of the inducer (Fig.\u0026nbsp;6a) and increased markedly when NaCl was used (Fig.\u0026nbsp;5b).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 5.\u003c/b\u003e Western blot analysis using the 6xHis tag antibody. \u003cb\u003eLine 1\u003c/b\u003e: Unstained protein standard. Lines 2\u0026ndash;8 (a) show the samples of the kinetic protein production over 6 h. Gels show a recombinant protein of 37 kDa marked with antibodies. Figure b shows the production kinetics of rNcXR without the supply of the inducer over 6 h.\u003c/p\u003e \u003cp\u003eThe resultant optimized gene showed 76% identity with the native gene. Codon optimization was not the only factor regulating protein expression. Elements such as the correct vector are strong inducible promoters with high affinity for polymerase enzymes, such as a T7 promoter, which can increase protein expression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The observation of protein production without the use of an inducer suggests leaky regulation of the T7 promoter, allowing for basal expression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTranscriptional regulation failures have been documented in other pET systems, in which the amount of T7 RNA polymerase (RNAP) produced after induction exceeds the inhibitory capacity of T7 lysozyme. Consequently, free T7 RNAP can engage in transcription, causing basal production of recombinant proteins [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]​. While basal expression can sometimes lead to toxicity and plasmid loss [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], no defects associated with toxicity were observed in this study. This confirms that the recombinant rNcXR is not toxic to the expression strain. Furthermore, the results support previous findings that the addition of a signal peptide (OmpT) and a 6xHis-tag can facilitate the solubility, extraction, and detection of the produced proteins [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Kinetic characterization of rNcXR\u003c/h2\u003e \u003cp\u003eThe kinetic characterization of rNcXR was performed at 25\u0026deg;C using xylose as a substrate. The specific activity of this enzyme was 13.93 U/mg, surpassing the highest activity reported for XR of \u003cem\u003eN. crassa\u003c/em\u003e, which varies between 3.54 and 9.2 U/mg [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These results underscore the influence of culture conditions, measurement techniques, and microbial strains on enzyme activity.\u003c/p\u003e \u003cp\u003eTo further elucidate the structural basis for the kinetic parameters observed in our rNcXR (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e = 4.53 mM, specific activity\u0026thinsp;=\u0026thinsp;13.93 U/mg, and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e = 51,979 min⁻\u0026sup1;), molecular docking simulations were performed (Fig.\u0026nbsp;10). These kinetic values contrast with those typically reported for the native \u003cem\u003eN. crassa\u003c/em\u003e enzyme. We hypothesize that this functional divergence stems directly from the specific sequence extensions introduced by our expression vector. The incorporation of the OmpT signal peptide and the C-terminal 6xHis-tag successfully enhanced the structural protein stability and significantly improved its solubility, extraction, and purification [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, as suggested by previous literature, the addition of a 6xHis-tag can alter secondary conformations, potentially increasing the overall stability of the recombinant protein [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our docking model supports this hypothesis by revealing a highly stabilized cofactor-binding pocket. We postulate that the mechanical tension exerted by these terminal additions induces a subtle geometric shift in the C-terminal loops, which shortens the interaction distances between the Lys279/Arg280 pair and the NADH cofactor. Consequently, this engineered genetic design not only facilitates efficient downstream processing but also yields a highly stable and active biocatalyst. The optimization of enzyme performance is crucial for both industrial and biomedical research. The kinetic parameters of xylose reductase produced by different microorganisms (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and the values obtained in this study (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e, 4.53 mM; \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e, 13.36 mM/min; and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e, 51,979 min⁻\u0026sup1;) fall within the typical range reported in the literature. However, upon comparing the models of both enzymes, differences were clear in all loops, including the rNcXR, suggesting a slight change in protein folding. This alteration significantly affected enzyme activity and stability, as shown in the present study. The catalytic activity, kinetic parameters, pH, and temperature improved enzyme stability, making rNcXR an attractive candidate for enzymatic xylitol production.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKinetic parameters of xylose reductase enzymes from different microorganisms reported in the literature and in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMicroorganism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecific activity (U/mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKm (mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKcat (min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKcat/Km (min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e/mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpathaspora passalidarum (rXR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.R.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN.R.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePichia stipitis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.R.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN.R.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeurospora crassa (rXR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e104.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeurospora crassa (rXR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51979\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e*rXR Recombinant Xilose Reductase\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDifferences in expression systems, induction strategies, and tag removal protocols likely account for the observed discrepancies in kinetic parameters between previously reported recombinant xylose reductases and the rNcXR described in this study [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. With some differences, our protein was expressed in the \u003cem\u003eE. coli\u003c/em\u003e BL21-SI strain. They used IPTG as an inducer, while we used NaCl as an inducer, and we did not remove the histidine tag to perform kinetic analysis. These expression differences may explain the significant differences in the kinetic parameters (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) between these recombinant proteins.\u003c/p\u003e \u003cp\u003eA deeper analysis showed that the V\u003csub\u003emax\u003c/sub\u003e values follow a similar trend. The same could not be said for the K\u003csub\u003em\u003c/sub\u003e values, in which an essential improvement in the affinity between NADH and rNcXR was seen in this study. This improvement could be due to the presence of phosphate ions in the buffer, which could alter the proteins\u0026rsquo; activity. It is also essential to mention that the NADH and xylose concentrations used in these experiments differed, which may explain the differences in the kinetic parameters of both proteins. However, our rNcXR production occurred in the \u003cem\u003eE. coli\u003c/em\u003e BL21-SI strain periplasm, in which the oxidizing conditions favored protein folding. Xylose reductase is usually produced in the cytoplasm of \u003cem\u003eE. coli\u003c/em\u003e (DE3 strain), where the oxidizing properties may influence protein folding. Reducing the medium can promote the formation of inclusion bodies and protein misfolding, altering the catalytic activity of the expressed enzyme [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of pH on specific enzymatic activity\u003c/h2\u003e \u003cp\u003eThe effect of pH on rNcXR stability was evaluated over time. As shown in Fig.\u0026nbsp;6, the enzyme exhibited maximum stability at pH 6.0. Interestingly, at this pH, the enzymatic activity did not decrease but rather showed a slight increase throughout the incubation period, maintaining values above 1.0 U/mL. In contrast, at pH values of 5.0 and 7.0, the enzyme retained approximately 80% of its initial activity after 60 min. Outside this range (pH\u0026thinsp;\u0026lt;\u0026thinsp;4.0 or pH\u0026thinsp;\u0026gt;\u0026thinsp;8.0), a significant loss of activity was observed, with residual activity falling below 80%. Regarding the catalytic conditions, Fig.\u0026nbsp;7 illustrates the dependence of rNcXR activity on the reaction pH. The data was fitted to a polynomial regression, revealing an optimal pH of approximately 6.5. This finding is consistent with values reported for other xylose reductases [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Notably, there is a distinct difference between the optimal catalytic pH (6.5) and the theoretical isoelectric point of the protein (pI 5.04).\u003c/p\u003e \u003cp\u003eIn our study, rNcXR exhibited a maximum activity at an optimum pH of 6.5, maintaining over 80% of its catalytic efficiency within a functional range of pH 6.0 to 7.5. This profile suggests that the enzyme is well-suited for bioprocesses where moderate acidity is required to prevent microbial contamination while maintaining high reaction rates, aligning favorably with industrial requirements.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 6.\u003c/b\u003e Stability profile of recombinant rNcXR incubated at different pH values over 60 min.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 7.\u003c/b\u003e Effect of reaction pH on the enzymatic activity of recombinant rNcXR.\u003c/p\u003e \u003cp\u003eOur findings regarding stability align with those of Woodyer et al. (2005) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], who noted that the addition of a 6xHis-tag to the N-terminus can increase enzyme activity compared to the native protein. However, kinetic parameters such as Km (4.54 mM) were lower than the reported value for \u003cem\u003eCandida tenuis\u003c/em\u003e (34 mM). pH significantly influences enzyme structure and catalytic efficiency. At the optimal pH, amino acid residues adopt the appropriate conformation for efficient substrate-cofactor binding. At acidic pH values, the enzyme may undergo aggregation due to the exposure of hydrophobic regions, while at alkaline pH, alterations in the tertiary structure can lead to deactivation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Furthermore, protein stability is also known to depend on factors such as the presence of polyols, which protect the native structure [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The negative charge at the optimal pH likely favors a more stable rNcXR conformation through electrostatic interactions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effect of Temperature on rNcXR Activity\u003c/h2\u003e \u003cp\u003eThe thermal stability of rNcXR was assessed by incubating the enzyme at different temperatures over a period of 60 min (Fig.\u0026nbsp;8). The temperature dependence profile of the purified rNcXR was evaluated. As expected from standard thermodynamic behavior, the catalytic activity at 25\u0026deg;C was approximately twofold higher than the activity observed at 15\u0026deg;C (corresponding to a temperature coefficient, Q\u003csub\u003e10\u003c/sub\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;2). This indicates a typical Arrhenius-type increase in the reaction rate before reaching the enzyme's optimal temperature. However, this was followed by a sharp decline. In terms of sustained stability, the enzyme performed best at 45\u0026deg;C and 55\u0026deg;C. Conversely, temperatures above 60\u0026deg;C proved detrimental; at 65\u0026deg;C, the activity decreased rapidly, reaching near-zero levels after 60 min. Regarding the optimal reaction temperature, Fig.\u0026nbsp;9 shows a clear profile with maximum catalytic activity at 45\u0026deg;C (0.38 U/mL). Although activity decreased at 55\u0026deg;C, the enzyme still retained approximately 85% of its maximal activity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 8.\u003c/b\u003e The thermal stability profile of recombinant rNcXR is over 60 min.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 9.\u003c/b\u003e Effect of temperature on the catalytic activity of recombinant rNcXR.\u003c/p\u003e \u003cp\u003eThe optimal temperature range observed aligns with findings for xylose reductases from various sources, which typically show optimal activity at 50\u0026deg;C [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The stability of recombinant rNcXR at temperatures up to 55\u0026deg;C suggests it can be effectively used in industrial processes without significant activity loss [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of optimal temperature and thermal stability parameters of Xylose Reductase from various microorganisms.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMicroorganism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003csub\u003eOptimal\u003c/sub\u003e (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDenaturalization half-time\u003c/p\u003e \u003cp\u003e(min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDenaturalization temperature\u003c/p\u003e \u003cp\u003e(\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBibliographic Reference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLactobacillus brevis KU15006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCandida mogii\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCandida tropicallis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeurospora crassa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the recombinant rNcXR exhibits competitive thermal properties. Protein stability at elevated temperatures is often attributed to subtle structural alterations and increased rigidity via hydrogen or ionic bonds [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, stability depends on the source microorganism; for instance, Nair and Zhao (2008) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] found that native XR from N. crassa retained\u0026thinsp;\u0026gt;\u0026thinsp;50% activity between pH 4.5 and 8.5, consistent with our results. In the case of rNcXR, hydrophobic amino acids (such as Ala and Gly) influence protein folding via hydrophobic collapse, contributing to the stability of the structure against pH and temperature changes [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Structural Analysis of XR recombinant enzyme\u003c/h2\u003e \u003cp\u003eSequence alignment between the native \u003cem\u003eN. crassa\u003c/em\u003e XR (Woodyer et al., 2005) and our recombinant rNcXR construct revealed strict conservation of the catalytic core. As observed in homologous enzymes, the catalytic residue Asp44 is crucial for xylose binding at the active site. During this process, Tyr49, a hydrophobic residue, plays a pivotal role in ensuring the efficient binding of the protein-ligand complex. In contrast, Lys78 facilitates the addition of NAD(P)H to the catalytic site through a precise interaction with the ribose phosphate, while His111 forms a catalytic duo with His27. Despite this active site conservation, specific sequence extensions were introduced by our cloning strategy. rNcXR is a fusion protein containing internal and N-terminal additions (e.g., Glu2, Thr3, Pro18, Gln19) resulting from signal peptide processing and restriction site scars. Most notably, rNcXR possesses a distinct C-terminal extension comprising a flexible linker (Ala333-Ala335) derived from the \u003cem\u003eNotI\u003c/em\u003e restriction site, followed immediately by the 6xHis purification tag (His336-His341). Previous structural studies on aldo-keto reductases demonstrate that bulky sequence extensions at the termini can exert mechanical tension on them, likely inducing a conformational shift in the adjacent flexible loops that govern cofactor binding. To further elucidate the structural basis for the enhanced specific activity and improved kinetic parameters observed in our rNcXR compared to previously reported \u003cem\u003eN. crassa\u003c/em\u003e enzymes, molecular docking simulations were performed (Fig.\u0026nbsp;10). The docking model reveals a highly stabilized cofactor-binding pocket. Specifically, the residues Lys279, Arg280, and Ser281 form a tight electrostatic interaction network with the ribose phosphate region of the cofactor. We postulate that the structural tension exerted by the before mentioned N-terminal additions and the C-terminal 6xHis-tag, combined with the oxidative folding environment of the periplasm, induces a subtle geometric shift in these C-terminal loops. This shift likely shortens the interaction distances between the Lys279/Arg280 pair and the cofactor, thereby enhancing binding affinity (\u003cem\u003eKm\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.53 mM) and catalytic rate (\u003cem\u003eVmax\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.36 mM/min). Consequently, these structural divergences provide a mechanistic explanation for how our rNcXR creates a highly active biocatalyst (13.93 U/mg) distinct from the wild-type enzyme.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 10.\u003c/b\u003e Comparative 3D structural models of the native \u003cem\u003eNeurospora crassa\u003c/em\u003e xylose reductase (Native XR) and the engineered recombinant enzyme (rNcXR).\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eThe present study confirms that modifying a plasmid with T7 regulatory sequences, an OmpT signal peptide, and a 6xHis-tag enables the highly efficient, salt-inducible production and periplasmic secretion of a recombinant xylose reductase (rNcXR) from \u003cem\u003eNeurospora crassa\u003c/em\u003e in \u003cem\u003eE. coli\u003c/em\u003e BL21-SI. The customized pITD02 expression vector successfully prevented inclusion body formation, allowing for the recovery of soluble, active protein without hindering high-yield production, despite the inherent basal expression of the T7 promoter. Biochemical characterization revealed that rNcXR possesses an optimal pH of 6.5, an optimal temperature range of 45\u0026ndash;50\u0026deg;C, and robust thermal stability, retaining significant activity for up to 60 min at 55\u0026deg;C\u0026mdash;traits that are critical for industrial operations.\u003c/p\u003e \u003cp\u003e​Beyond cost-effective expression and thermal stability, structural analysis via molecular docking highlighted the dual purpose of our genetic design. The specific sequence insertions at the termini, particularly the C-terminal flexible linker and the 6xHis-tag, not only optimize the NADH-binding pocket to enhance substrate affinity but also serve as highly reactive, accessible anchoring points for directed enzyme immobilization. Consequently, this engineered rNcXR stands out as an ideal biocatalyst for future integration onto solid matrices, such as nickel-chelated surfaces or carbon-based supports. This work not only provides a robust soluble enzyme for large-scale xylitol production but establishes the critical structural foundation for developing stable, reusable enzymatic systems tailored for continuous industrial production bioprocesses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eUse of Artificial Intelligence\u003c/h2\u003e \u003cp\u003e During the preparation of this work, the authors used Generative AI technologies (Gemini and Copilot) solely to improve the language, clarity, and readability of the manuscript. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that no specific funding was received for this study other than the Ph.D. scholarship provided to M. J. Reyes-Rosas by CONACYT.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM. J. Reyes-Rosas: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft. J. L\u0026oacute;pez-Miranda, J. A. Rojas-Contreras: Conceptualization, Resources, Writing - Review \u0026amp; Editing, Supervision, Project administration. M. Garc\u0026iacute;a-Curiel, J. C. Aguilar-Cordero, D. Reyes-Jacquez, J. B. P\u0026aacute;ez-Lerma, N. O. Soto-Cruz: Methodology, Validation, Writing - Review \u0026amp; Editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eM. J. Reyes-Rosas thanks the National Council of Science and Technology (CONACYT) for the grant (No. 775313) to conduct PhD studies.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHern\u0026aacute;ndez-P\u0026eacute;rez AF, Chaves-Villamil AC, de Arruda PV, dos Santos JC, Felipe MGA (2020) Sugarcane syrup improves xylitol bioproduction from sugarcane bagasse and straw hemicellulosic hydrolysate. 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J Sci Food Agric 102:4462\u0026ndash;4472. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/JSFA.11800\u003c/span\u003e\u003cspan address=\"10.1002/JSFA.11800\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Neurospora crassa, xylose reductase, periplasmic expression, xylitol biosynthesis, salt-inducible","lastPublishedDoi":"10.21203/rs.3.rs-9316107/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9316107/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eXylitol is a sugar alcohol widely used in the food industry due to its lower caloric value compared to sucrose. Industrial xylitol production relies primarily on chemical hydrogenation processes that require high energy input. Although biotechnological routes represent a more sustainable alternative, their industrial implementation is often limited by enzyme stability and cofactor costs.\u003c/p\u003e \u003cp\u003eThis study presents the design, expression, and biochemical analysis of recombinant xylose reductase from \u003cem\u003eNeurospora crassa\u003c/em\u003e (rNcXR). The optimized gene was cloned into a salt-inducible T7-based expression system and expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e BL21-SI. The design incorporated an OmpT signal peptide to encourage secretion into the periplasm, and a 6\u0026times;His tag at the C-terminus to simplify purification.\u003c/p\u003e \u003cp\u003eThe recombinant enzyme exhibited a specific activity of 13.93 U/mg, exceeding values previously reported for native and recombinant xylose reductases. rNcXR displayed high stability over a temperature range of 25\u0026ndash;45\u0026deg;C and retained significant activity at elevated temperatures. These results demonstrate that the engineered rNcXR represents a robust and efficient biocatalyst with strong potential for enzymatic xylitol production.\u003c/p\u003e","manuscriptTitle":"Enhancing catalytic efficiency of a salt-induced Neurospora crassa xylose reductase for xylitol bioprocesses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-17 05:14:29","doi":"10.21203/rs.3.rs-9316107/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-23T04:07:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T19:55:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-14T20:39:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292404907808409111513550385655103229924","date":"2026-04-10T11:09:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267367263605623976431543231963980842613","date":"2026-04-09T12:51:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-09T03:31:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-07T08:04:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-07T02:55:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Microbiology","date":"2026-04-03T20:37:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c7631470-0c71-4aee-8928-60e8a98da1b4","owner":[],"postedDate":"April 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T06:24:54+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-17 05:14:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9316107","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9316107","identity":"rs-9316107","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}