Single-round QuikChange PCR for engineering multiple site-directed mutations in plasmid DNA.

Mutational study is a cornerstone methodology in biochemistry and genetics, and many mutagenesis strategies have been invented to promote the efficiency of gene engineering. In this study, we developed a simple and timesaving approach to integrate simultaneous mutagenesis at discrete sites. By using plasmid as a template and compatible oligonucleotide primers per the QuikChange strategy, our method was able to introduce multiple nucleotide insertions, deletions and replacements in one round of polymerase chain reaction. The longest insertion and deletion were achieved with 28 bp and 16 bp mismatch respectively. For minor nucleotide replacements (mismatch no more than 4 bp), mutations were achieved at up to 4 discrete locations. Usually, a successful clone with all desired mutations was found by screening 5 colonies. Clones with a subset of mutations may be stocked into the library of mutants or used as templates in the next rounds of mutagenic PCR to accomplish the entire construction project. This method can be applied to build up a combinatory library of mutants through saturation mutagenesis at multiple sites. It is promising to facilitate the research of protein biochemistry, forward genetics and synthetic biology.

Disease-associated missense mutations in the pore loop of polycystin-2 alter its ion channel function in a heterologous expression system.

Polycystin-2 (PC2) is mutated in ∼15% of patients with autosomal dominant polycystic kidney disease (ADPKD). PC2 belongs to the family of transient receptor potential (TRP) channels and can function as homotetramer. We investigated whether three disease-associated mutations (F629S, C632R or R638C) localized in the channel's pore loop alter ion channel properties of human PC2 expressed in Xenopus laevis oocytes. Expression of wildtype (WT) PC2 typically resulted in small but measurable Na+ inward currents in the absence of extracellular divalent cations. These currents were no longer observed, when individual pore mutations were introduced in WT PC2. Similarly, Na+ inward currents mediated by the F604P gain-of-function (GOF) PC2 construct (PC2 F604P) were abolished by each of the three pore mutations. In contrast, when the mutations were introduced in another GOF construct, PC2 L677A N681A, only C632R had a complete loss-of-function effect, whereas significant residual Na+ inward currents were observed with F629S (∼15 %) and R638C (∼30 %). Importantly, the R638C mutation also abolished the Ca2+ permeability of PC2 L677A N681A and altered its monovalent cation selectivity. To elucidate the molecular mechanisms by which the R638C mutation affects channel function, molecular dynamics (MD) simulations were used in combination with functional experiments and site-directed mutagenesis. Our findings suggest that R638C stabilizes ionic interactions between Na+ ions and the selectivity filter residue D643. This probably explains the reduced monovalent cation conductance of the mutant channel. In summary, our data support the concept that altered ion channel properties of PC2 contribute to the pathogenesis of ADPKD.

Mechanistic Characterization of De Novo Generation of Variable Number Tandem Repeats in Circular Plasmids during Site-Directed Mutagenesis and Optimization for Coding Gene Application.

Site-directed mutagenesis for creating point mutations, sometimes, gives rise to plasmids carrying variable number tandem repeats (VNTRs) locally, which are arbitrarily regarded as polymerase chain reaction (PCR) related artifacts. Here, the alternative end-joining mechanism is reported rather than PCR artifacts accounts largely for that VNTRs formation and expansion. During generating a point mutation on GPLD1 gene, an unexpected formation of VNTRs employing the 31 bp mutagenesis primers is observed as the repeat unit in the pcDNA3.1-GPLD1 plasmid. The 31 bp VNTRs are formed in 24.75% of the resulting clones with copy number varied from 2 to 13. All repeat units are aligned with the same orientation as GPLD1 gene. 43.54% of the repeat junctions harbor nucleotide mutations while the rest don't. Their demonstrated short primers spanning the 3' part of the mutagenesis primers are essential for initial creation of the 2-copy tandem repeats (TRs) in circular plasmids. The dimerization of mutagenesis primers by the alternative end-joining in a correct orientation is required for further expansion of the 2-copy TRs. Lastly, a half-double priming strategy is established, verified the findings and offered a simple method for VNTRs creation on coding genes in circular plasmids without junction mutations.

Improving Catalytic Efficiency of L-Arabinose Isomerase from Lactobacillus plantarum CY6 towards D-Galactose by Molecular Modification.

L-Arabinose isomerase (L-AI) has been commonly used as an efficient biocatalyst to produce D-tagatose via the isomerization of D-galactose. However, it remains a significant challenge to efficiently synthesize D-tagatose using the native (wild type) L-AI at an industrial scale. Hence, it is extremely urgent to redesign L-AI to improve its catalytic efficiency towards D-galactose, and herein a structure-based molecular modification of Lactobacillus plantarum CY6 L-AI (LpAI) was performed. Among the engineered LpAI, both F118M and F279I mutants showed an increased D-galactose isomerization activity. Particularly, the specific activity of double mutant F118M/F279I towards D-galactose was increased by 210.1% compared to that of the wild type LpAI (WT). Besides the catalytic activity, the substrate preference of F118M/F279I was also largely changed from L-arabinose to D-galactose. In the enzymatic production of D-tagatose, the yield and conversion ratio of F118M/F279I were increased by 81.2% and 79.6%, respectively, compared to that of WT. Furthermore, the D-tagatose production of whole cells expressing F118M/F279I displayed about 2-fold higher than that of WT cell. These results revealed that the designed site-directed mutagenesis is useful for improving the catalytic efficiency of LpAI towards D-galactose.

Rational Design of a Yeast-derived 3',5'-bisphosphate Nucleotidase with Improved Substrate Specificity.

In recent years, a convenient phosphatase-coupled sulfotransferase assay method has been proven to be applicable to most sulfotransferases. The central principle of the method is that phosphatase specifically degrades 3'-phosphoadenosine-5'-phosphate (PAP) and leaves 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Our group previously acquired a yeast 3',5'-bisphosphate nucleotidase (YND), which showed a higher catalytic activity for PAP than PAPS and could be a potential phosphatase for the sulfotransferase assay. Here, we obtained a beneficial mutant of YND with markedly improved substrate specificity towards PAP via rational design. Of 9 chosen mutation sites in the active site pocket, the mutation G236D showed the best specificity for PAP. After optimization of the reaction conditions, the mutant YNDG236D displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YNDG236D to the assay of human SULT1A1 and SULT1A3 with their known substrate 1-naphthol, indicating that the mutant could be used to evaluate sulfotransferase activity by colorimetry. Analysis of the MD simulation results revealed that the improved substrate specificity of the mutant towards PAP may stem from a more stable protein conformation and the changed flexibility of key residues in the entrance of the substrate tunnel. This research will provide a valuable reference for the development of efficient sulfotransferase activity assays.

[Molecular modification of cyclodextrin glucosyltransferase and its application in the synthesis of α-arbutin].

α-arbutin has important applications in cosmetics and medicine. However, the extraction yield from plant tissues is relatively low, which restricts its application value. In this study, we investigated the synthesis of α-arbutin using maltodextrin as the donor and hydroquinone as the acceptor, using a cyclodextrin glucosyltransferase (CGTase) from Anaerobranca gottschalkii. We performed site-saturated and site-directed mutagenesis on AgCGTase. The activity of the variant AgCGTase-F235G-N166H was 3.48 times higher than that of the wild type. Moreover, we achieved a conversion rate of 63% by optimizing the reaction pH, temperature, and hydroquinone addition amount. Overall, this study successfully constructed a strain with improved conversion rate for the synthetic production of α-arbutin and hydroquinone. These findings have significant implications for reducing the industrial production cost of α-arbutin and enhancing the conversion rate of the product.

A high-affinity, cis-on photoswitchable beta blocker to optically control ß2-adrenergic receptors in vitro and in vivo.

This study introduces (S)-Opto-prop-2, a second-generation photoswitchable ligand designed for precise modulation of ß2-adrenoceptor (ß2AR). Synthesised by incorporating an azobenzene moiety with propranolol, (S)-Opto-prop-2 exhibited a high PSScis (photostationary state for cis isomer) percentage (∼90 %) and a favourable half-life (>10 days), facilitating diverse bioassay measurements. In vitro, the cis-isomer displayed substantially higher ß2AR binding affinity than the trans-isomer (1000-fold), making (S)-Opto-prop-2 one of the best photoswitchable GPCR (G protein-coupled receptor) ligands reported so far. Molecular docking of (S)-Opto-prop-2 in the X-ray structure of propranolol-bound ß2AR followed by site-directed mutagenesis studies, identified D1133.32, N3127.39 and F2896.51 as crucial residues that contribute to ligand-receptor interactions at the molecular level. In vivo efficacy was assessed using a rabbit ocular hypertension model, revealing that the cis isomer mimicked propranolol's effects in reducing intraocular pressure, while the trans isomer was inactive. Dynamic optical modulation of ß2AR by (S)-Opto-prop-2 was demonstrated in two different cAMP bioassays and using live-cell confocal imaging, indicating reversible and dynamic control of ß2AR activity using the new photopharmacology tool. In conclusion, (S)-Opto-prop-2 emerges as a promising photoswitchable ligand for precise and reversible ß2AR modulation with light. The new tool shows superior cis-on binding affinity, one of the largest reported differences in affinity (1000-fold) between its two configurations, in vivo efficacy, and dynamic modulation. This study contributes valuable insights into the evolving field of photopharmacology, offering a potential avenue for targeted therapy in ß2AR-associated pathologies.

Characterization of metallothionein genes from Broussonetia papyrifera: metal binding and heavy metal tolerance mechanisms.

BACKGROUND: Broussonetia papyrifera is an economically significant tree with high utilization value, yet its cultivation is often constrained by soil contamination with heavy metals (HMs). Effective scientific cultivation management, which enhances the yield and quality of B. papyrifera, necessitates an understanding of its regulatory mechanisms in response to HM stress. RESULTS: Twelve Metallothionein (MT) genes were identified in B. papyrifera. Their open reading frames ranged from 186 to 372 bp, encoding proteins of 61 to 123 amino acids with molecular weights between 15,473.77 and 29,546.96 Da, and theoretical isoelectric points from 5.24 to 5.32. Phylogenetic analysis classified these BpMTs into three subclasses: MT1, MT2, and MT3, with MT2 containing seven members and MT3 only one. The expression of most BpMT genes was inducible by Cd, Mn, Cu, Zn, and abscisic acid (ABA) treatments, particularly BpMT2e, BpMT2d, BpMT2c, and BpMT1c, which showed significant responses and warrant further study. Yeast cells expressing these BpMT genes exhibited enhanced tolerance to Cd, Mn, Cu, and Zn stresses compared to control cells. Yeasts harboring BpMT1c, BpMT2e, and BpMT2d demonstrated higher accumulation of Cd, Cu, Mn, and Zn, suggesting a chelation and binding capacity of BpMTs towards HMs. Site-directed mutagenesis of cysteine (Cys) residues indicated that mutations in the C domain of type 1 BpMT led to increased sensitivity to HMs and reduced HM accumulation in yeast cells; While in type 2 BpMTs, the contribution of N and C domain to HMs' chelation possibly corelated to the quantity of Cys residues. CONCLUSION: The BpMT genes are crucial in responding to diverse HM stresses and are involved in ABA signaling. The Cys-rich domains of BpMTs are pivotal for HM tolerance and chelation. This study offers new insights into the structure-function relationships and metal-binding capabilities of type-1 and - 2 plant MTs, enhancing our understanding of their roles in plant adaptation to HM stresses.

Green, yellow, or cyan? Introduction of color change mutations into a green thermostable fluorescent protein and characterization during an introduction to biochemistry lab course.

Green fluorescent protein has long been a favorite protein for demonstrating protein purification in the biochemistry lab course. The protein's vivid green color helps demonstrate to students the concept(s) behind affinity or ion exchange chromatography. We designed a series of introduction to biochemistry labs utilizing a thermostable green protein (TGP-E) engineered to have unusually high thermostability. This protein allows students to proceed through purification and characterization without the need to keep protein samples on ice. The 5-week lab series begins with an introduction to molecular biology techniques during weeks 1 and 2, where site-directed mutagenesis is used introduce, a single nucleotide change that shifts the fluorescent spectra of TGP-E to either cyan (CTP-E) or yellow (YTP-E). Students identify successful mutagenesis reaction by the color of a small expression sample after induction with IPTG. Next, students purify either the TGP-E (control-typically one group volunteers), YTP-E, or CTP-E protein as a 1-week lab. During the following week's lab, students run SDS-PAGE to verify protein purity, bicinchoninic acid assay to quantify protein yield, and absorbance and fluorescence spectra to characterize their protein's fluorescent character. The final lab in the series investigates the thermostability of YTP-E and CTP-E compared with TGP-E using a fluorescence plate reader. This 5-week series of experiments provide students with experience in several key biochemistry techniques and allows the students to compare properties of mutations. At the end of the course, the students will write a research report and give a short presentation over their results.

Current status and emerging frontiers in enzyme engineering: An industrial perspective.

Protein engineering mechanisms can be an efficient approach to enhance the biochemical properties of various biocatalysts. Immobilization of biocatalysts and the introduction of new-to-nature chemical reactivities are also possible through the same mechanism. Discovering new protocols that enhance the catalytic active protein that possesses novelty in terms of being stable, active, and, stereoselectivity with functions could be identified as essential areas in terms of concurrent bioorganic chemistry (synergistic relationship between organic chemistry and biochemistry in the context of enzyme engineering). However, with our current level of knowledge about protein folding and its correlation with protein conformation and activities, it is almost impossible to design proteins with specific biological and physical properties. Hence, contemporary protein engineering typically involves reprogramming existing enzymes by mutagenesis to generate new phenotypes with desired properties. These processes ensure that limitations of naturally occurring enzymes are not encountered. For example, researchers have engineered cellulases and hemicellulases to withstand harsh conditions encountered during biomass pretreatment, such as high temperatures and acidic environments. By enhancing the activity and robustness of these enzymes, biofuel production becomes more economically viable and environmentally sustainable. Recent trends in enzyme engineering have enabled the development of tailored biocatalysts for pharmaceutical applications. For instance, researchers have engineered enzymes such as cytochrome P450s and amine oxidases to catalyze challenging reactions involved in drug synthesis. In addition to conventional methods, there has been an increasing application of machine learning techniques to identify patterns in data. These patterns are then used to predict protein structures, enhance enzyme solubility, stability, and function, forecast substrate specificity, and assist in rational protein design. In this review, we discussed recent trends in enzyme engineering to optimize the biochemical properties of various biocatalysts. Using examples relevant to biotechnology in engineering enzymes, we try to expatiate the significance of enzyme engineering with how these methods could be applied to optimize the biochemical properties of a naturally occurring enzyme.

CDR grafting and site-directed mutagenesis approach for the generation and affinity maturation of Anti-CD20 nanobody.

BACKGROUND: Recently, new and advanced techniques have been adopted to design and produce nanobodies, which are used in diagnostic and immunotherapy treatments. Traditionally, nanobodies are prepared from camelid immune libraries that require animal treatments. However, such approaches require large library sizes and complicated selection procedures. The current study has employed CDR grafting and site-directed mutagenesis techniques to create genetically engineered nanobodies against the tumor marker CD20 (anti-CD20 nanobodies) used in leukemia treatment. METHODS AND RESULTS: In this study, we utilized the swapping method to graft CDRs from the VH Rituximab antibody to VHH CDRs. We aimed to enhance the binding affinity of the nanobodies by substituting the amino acids (Y101R-Y102R-Y107R) in the VHH-CDR3. To assess the binding capacity of the mutated nanobodies, we conducted an ELISA test. Moreover, through flow cytometry analysis, we compared the fluorescence intensity of the grafted CD20 and mutant nanobodies with that of the commercially available human anti-CD20 in Raji cells. The results showed a significant difference in the fluorescence intensity of the grafted nanobodies and mutant nanobodies when compared to the commercially available human anti-CD20. CONCLUSION: The approach we followed in this study makes it possible to create multiple anti-CD20 nanobodies with varying affinities without the need for extensive selection efforts. Additionally, our research has demonstrated that computational tools are highly reliable in designing functional nanobodies.

Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK's function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.

Structure, dimeric conformation, and coenzyme versatility of p-hydroxybenzoate hydroxylase from Arthrobacter sp. PAMC25564.

p-Hydroxybenzoate hydroxylase (PHBH) catalyzes the ortho-hydroxylation of 4-hydroxybenzoate (4-HB) to protocatechuate (PCA). PHBHs are commonly known as homodimers, and the prediction of pyridine nucleotide binding and specificity remains an ongoing focus in this field. Therefore, our study aimed to determine the dimerization interface in AspPHBH from Arthrobacter sp. PAMC25564 and identify the canonical pyridine nucleotide-binding residues, along with coenzyme specificity, through site-directed mutagenesis. The results confirm a functional dimeric assembly from a tetramer that appeared in the crystallographic asymmetric unit identical to that established in previous studies. Furthermore, AspPHBH exhibits coenzyme versatility, utilizing both NADH and NADPH, with a preference for NADH. Rational engineering experiments demonstrated that targeted mutations in coenzyme surrounding residues profoundly impact NADPH binding, leading to nearly abrogated enzymatic activity compared to that of NADH. R50, R273, and S166 emerged as significant residues for NAD(P)H binding, having a near-fatal impact on NADPH binding compared to NADH. Likewise, the E44 residue plays a critical role in determining coenzyme specificity. Overall, our findings contribute to the fundamental understanding of the determinants of PHBH's active dimeric conformation, coenzyme binding and specificity holding promise for biotechnological advancements.

Elucidating the Activation Mechanism of the Proton-sensing GPR68 Receptor.

GPR68 is a proton-sensing G-protein Coupled Receptor (GPCR) involved in a variety of physiological processes and disorders including neoplastic pathologies. While GPR68 and few other GPCRs have been shown to be activated by a decrease in the extracellular pH, the molecular mechanism of their activation remains largely unknown. In this work, we used a combined computational and in vitro approach to provide new insight into the activation mechanism of the receptor. Molecular Dynamics simulations of GPR68 were used to model the changes in residue interactions and motions triggered by pH. Global and local rearrangements consistent with partial activation were observed upon protonation of the inactive state. Selected extracellular histidine and transmembrane acidic residues were found to have significantly upshifted pKa values during the simulations, consistently with their previously hypothesised role in activation through changes in protonation state. Moreover, a novel pairing between histidine and acidic residues in the extracellular region was highlighted by both sequence analyses and simulation data and tested through site-directed mutagenesis. At last, we identified a previously unknown hydrophobic lock in the extracellular region that might stabilise the inactive conformation and regulate the transition to the active state.

Integrated strategies for efficient production of Streptomyces mobaraensis transglutaminase in Komagataella phaffii.

Transglutaminase (TGase) from Streptomyces mobaraensis commonly used to improve protein-based foods due to its unique enzymatic reactions, which imply considerable attention in its production. Recently, TGase exhibit broad market potential in non-food industries. However, achieving efficient synthesis of TGase remains a significant challenge. Herein, we achieved a substantial amount of a fully functional and kinetically stable TGase produced by Komagataella phaffii (Pichia pastoris) using multiple strategies including Geneticin (G418) screening, combinatorial mutations, promoter optimization, and co-expression. The active TGase expression reached a maximum of 10.1 U mL-1 in shake flask upon 96 h of induction, which was 3.8-fold of the wild type. Also, the engineered strain exhibited a 6.4-fold increase in half-life and a 2-fold increase in specific activity, reaching 172.67 min at 60 °C (t1/2(60 °C)) and 65.3 U mg-1, respectively. Moreover, the high-cell density cultivation in 5-L fermenter was also applied to test the productivity at large scale. Following optimization at a fermenter, the secretory yield of TGase reached 47.96 U mL-1 in the culture supernatant. Given the complexity inherent in protein expression and secretion, our research is of great significance and offers a comprehensive guide for improving the production of a wide range of heterologous proteins.

Combined Strategies for Improving Aflatoxin B1 Degradation Ability and Yield of a Bacillus licheniformis CotA-Laccase.

Aflatoxin B1 (AFB1) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from Bacillus licheniformis ANSB821 previously reported by our laboratory showed great potential to degrade AFB1 without redox mediators. However, the use of this CotA-laccase to remove AFB1 in animal feed is limited because of its low catalytic efficiency and low expression level. In order to make better use of this excellent enzyme to effectively degrade AFB1, twelve mutants of CotA-laccase were constructed by site-directed mutagenesis. Among these mutants, E186A and E186R showed the best degradation ability of AFB1, with degradation ratios of 82.2% and 91.8% within 12 h, which were 1.6- and 1.8-times higher than those of the wild-type CotA-laccase, respectively. The catalytic efficiencies (kcat/Km) of E186A and E186R were found to be 1.8- and 3.2-times higher, respectively, than those of the wild-type CotA-laccase. Then the expression vectors pPICZαA-N-E186A and pPICZαA-N-E186R with an optimized signal peptide were constructed and transformed into Pichia pastoris GS115. The optimized signal peptide improved the secretory expressions of E186A and E186R in P. pastoris GS115. Collectively, the current study provided ideal candidate CotA-laccase mutants for AFB1 detoxification in food and animal feed and a feasible protocol, which was desperately needed for the industrial production of CotA-laccases.

Dataset on double mutation in PGIP of Glycine max improves defense to PG of Sclerotinia sclerotiorum.

The cell wall of the Glycine max altered by the polygalacturonases (PGs) secreted by the fungus Sclerotinia sclerotiorum, causes disease and quality losses. In soybeans, a resistance protein called polygalacturonases-inhibiting proteins (PGIPs) binds to the PG to block fungal infection. The active site residues of PGIP3, VAL170 and GLN242 are mutated naturally by various amino acids in different types of PGIPs. Therefore, the mutation of VAL170 to GLY is ineffective but the GLN242 amino acid mutation by LYS significantly alters the structure and is crucial for interacting with the PG protein. Docking and Molecular Dynamics simulation provide a comprehensive evaluation of the interactions between gmPGIP and ssPG. By elucidating the structural basis of the interaction between gmPGIP and ssPG, this investigation lays a foundation for the development of targeted strategies in-order to enhance soybean resistance against Sclerotinia sclerotiorum. By leveraging this knowledge, researchers can potentially engineer soybean varieties with improved resistance to the fungus, thereby reducing disease incidence and improving crop yields.

Conjugating Hemoglobin and Albumin by Strain-Promoted Azide-Alkyne Cycloaddition.

A one-to-one conjugate of cross-linked human hemoglobin and human serum albumin results from a strain-promoted azide-alkyne cycloaddition (SPAAC) of the modified proteins. Additions of a strained alkyne-substituted maleimide to the Cys-34 thiol of human serum albumin and an azide-containing cross-link between the amino groups of each b-unit at Lys-82 of human hemoglobin provide sites for coupling by the SPAAC process. The coupled hemoglobin-albumin conjugate can be readily purified from the unreacted hemoglobin. The oxygen binding properties of this two-protein conjugate demonstrate an oxygen affinity and binding cooperativity suitable as an acellular oxygen carrier.

A new halotolerant xylanase from Aspergillus clavatus expressed in Escherichia coli with catalytic efficiency improved by site-directed mutagenesis.

Daily agro-industrial waste, primarily cellulose, lignin, and hemicellulose, poses a significant environmental challenge. Harnessing lignocellulolytic enzymes, particularly endo-1,4-ß-xylanases, for efficient saccharification is a cost-effective strategy, transforming biomass into high-value products. This study focuses on the cloning, expression, site-directed mutagenesis, purification, three-dimensional modeling, and characterization of the recombinant endo-1,4-ß-xylanase (XlnA) from Aspergillus clavatus in Escherichia coli. This work includes evaluation of the stability at varied NaCl concentrations, determining kinetic constants, and presenting the heterologous expression of XlnAΔ36 using pET22b(+). The expression led to purified enzymes with robust stability across diverse pH levels, exceptional thermostability at 50 °C, and 96-100% relative stability after 24 h in 3.0 M NaCl. Three-dimensional modeling reveals a GH11 architecture with catalytic residues Glu 132 and 22. XlnAΔ36 demonstrates outstanding kinetic parameters compared to other endo-1,4-ß-xylanases, indicating its potential for industrial enzymatic cocktails, enhancing saccharification. Moreover, its ability to yield high-value compounds, such as sugars, suggests a promising and ecologically positive alternative for the food and biotechnology industries.