Oligosaccharide oxidase for the enzymatic synthesis of glucosaminic acids

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Vuong, Nadia Davoudvandi, Emma R. Master This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7586791/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2026 Read the published version in Biotechnology Letters → Version 1 posted 4 You are reading this latest preprint version Abstract Background D-Glucosaminic acid is a valuable amino acid useful in food and medical applications. It is a highly sought after enantiopure molecule important in synthesis of drugs and glycopeptides. Current enzymatic synthesis pathways to D-glucosaminic acid carry disadvantages such as low product yield, long reaction times, and high cost due to increase in enzyme usage. Results Herein, the Auxiliary Activity 7 chito-oligosaccharide oxidase from Lentinus brumalis , LbChi7A, was shown as a potent biocatalyst capable of efficiently converting D-glucosamine (GlcN) and N -acetyl-D-glucosamine (GlcNAc) to their respective C 1 -acids. Due to a unique substrate specificity towards GlcN and GlcNAc, LbChi7A converts at least 90% GlcN to D-glucosaminic acid within 60 min and 100% GlcNAc to N -acetyl-D-glucosaminic acid within the same time frame. Furthermore, LbChi7A inhibition by the hydrogen peroxide co-product was not detected, even at 860 mM. This single enzymatic conversion offers an efficient process for the production of glucosaminic acids including D-glucosaminic acid or N -acetyl-D-glucosaminic acid. Conclusions The biotechnological potential of LbChi7A is demonstrated, particularly in the production of rare sugars and pharmaceutical intermediates. The ability of the enzyme to perform selective oxidation without the need for hazardous chemicals presents a cleaner and efficient alternative to traditional chemical methods. LbChi7A D-glucosaminic acid Glucose oxidase D-glucosamine biocatalysis green chemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Chitin is a major polysaccharide that is found in the exoskeleton of arthropods and fungal cell walls. The estimated annual synthesis of chitin is close to 1 trillion tonnes [ 1 ]. It is made up of 𝛽-(1∀4)-linked N -acetyl-D-glucosamine (GlcNAc) units. When processing chitin to remove protein and for demineralization, N -acetyl groups can be lost, creating a polymer with a mixture of GlcNAc and D-glucosamine (GlcN) units, known as chitosan [ 2 ]. D-Glucosaminic acid is a valuable sugar acid that can be obtained from the oxidation of GlcN. It is an enantiopure building block that has been used to synthesize various compounds such as enzyme (glucosidase) inhibitors [ 3 ], chemotherapeutic drugs [ 4 – 6 ], drug delivery agents [ 7 ], ligands for cation exchange chromatography [ 8 ], enantioselective catalysts [ 9 ], and biopolymers [ 10 ]. The diverse applications of GlcNA, including biopolymer synthesis [ 7 ], have prompted investigations into its sustainable and efficient production. Several pathways, chemical and enzymatic, can be used to produce glucosaminic acid [ 10 – 17 ]. Historically, this acid was synthesized by oxidizing GlcN with yellow mercuric oxide and hydrogen sulfide [ 17 ]. In addition to toxicity and safety concerns, this process generates many by-products that reduce product yields to less than 50% [ 17 ]. Alternatively, glucosaminic acid can be produced using activated Charcoal-Supported Palladium‐Bismuth Catalysts (Pd-Bi/C) where 70% yield of glucosaminic acid is reported [ 13 ]; however, production of the Pd-Bi/C catalyst is energy and water intensive, and requires harsh chemicals like nitric acid and formaldehyde [ 13 ]. Microbial routes to glucosaminic acid include oxidative fermentation of GlcN by Pseudomonas putida GNA5 [ 14 ] where a 95% yield is reported; however, product recovery steps introduce economic barriers to scale-up [ 18 , 19 ]. Finally, cell-free biological routes to glucosaminic acid production oxidize GlcN using the glucose oxidase (GOX) from Aspergillus niger . Despite the selectivity and high atom economy of this approach, reported product yields are below 5% [ 20 ]. Recently, LbChi7A, also known as PbChi7A, was characterized as a chito-oligosaccharide oxidase from Lentinus brumalis (formerly Polyporus brumalis) , belonging to auxiliary activity family 7 (AA7) of the Carbohydrate-Active EnZYmes Database ( http://www.cazy.org ) [ 21 ]. LbChi7A was found to oxidize GlcN and GlcNAc with catalytic efficiencies more than three orders of magnitude higher than that on glucose (Glc). LbChi7A also produced glucosaminic acid at rates similar to those achieved for oxidized chito-oligosaccharides. To further assess the biotechnological potential of LbChi7A, particularly in alternative routes to aminosugar oxidation, this study directly compared the efficiencies of LbChi7A and GOX in the oxidation of Glc, GlcN, and GlcNAc. Materials and methods Materials Glc, GlcN, GlcNAc, N -valeryl-D-glucosamine (GlcVal), N -hexanoyl-D-glucosamine (GlcHex), D-glucosaminic acid, catalase, and A. niger glucose oxidase (UniProt ID: Q9HFQ1) were purchased from Sigma, USA. Bovine serum albumin (BSA) was purchased from Thermo Fisher Scientific, USA. LbChi7A production and purification LbChi7A was produced as described previously [ 21 ]. In brief, plasmids encoding the LbChi7A gene (Genbank ID: RDX44700.1; UniProt ID: A0A371CWQ2_9APHY) were transformed into Komagataella phaffii (formerly Pichia pastoris ) strain X33 by electroporation, and positive transformants were grown in 4-L baffled flasks containing 2 L of BMGY (Buffered Glycerol-complex Medium). The cells were grown over 48 h at 30°C, harvested by centrifugation, suspended in 400 mL of BMMY (Buffered Methanol-complex Medium) in 1-L shake flask, and then incubated for 3 days at 20°C for protein production. Methanol (3%, v/v) was added to the cultivation every 24 h. Secreted proteins were separated from the cells by centrifugation and filtered using a 0.22 µm PES filter. The filtered supernatant was exchanged to 50 mM sodium phosphate pH 7.5 and purified using a Fast Protein Liquid Chromatography (FPLC) system with a 5-mL His Trap HP column (GE Healthcare, Uppsala, Sweden). The column was equilibrated at a flow rate of 2.5 mL/min with 50 mM sodium phosphate pH 7.5, 300 mM NaCl, and 20 mM imidazole. Filtered protein supernatant solution was applied to the column and eluted at a flow rate of 2.5 mL/min with 50 mM sodium phosphate pH 7.5, 300 mM NaCl, and 20–250 mM imidazole gradient over 20 column volumes. Fractions with LbChi7A were concentrated and exchanged into 50 mM sodium phosphate pH 7.5 buffer. SDS-PAGE analysis was used to assess protein purity, and protein concentration was quantified by gel densitometry using a standard curve generated with BSA. Activity assays and kinetic analyses Enzyme reactions (0.25 mL) containing 100 mM and 500 mM substrate, enzyme (0.05–5 µM LbChi7A or GOX), and 1.0 M sodium phosphate buffer (pH 8.0) were incubated at 30°C for 15, 30, and 60 min in an orbital shaking thermomixer at 700 rpm. The reactions were then filtered using a 0.2 µm PES filter, and substrate depletion and product formation were quantified using high performance anion‑exchange chromatography with pulsed amperometry detection (HPAEC-PAD) as described below. Steady-state kinetics of LbChi7A and GOX was determined by coupling the enzymatic release of hydrogen peroxide to the activity of horseradish peroxidase [ 21 ]. The kinetics on Glc (1–1,320 mM), GlcN (0.01–110 mM), and GlcNAc (0.01–10 mM) in 50 mM sodium phosphate pH 8.0 were determined by fitting the Michaelis-Menten equation using GraphPad Prism5 software (GraphPad Software, USA). H 2 O 2 inhibition assay To determine the potential of H 2 O 2 to inhibit LbChi7A and GOX activity, 25 mM–860 mM H 2 O 2 were added to the enzyme assay reactions, which have 1 mM GlcNAc for LbChi7A and 1 mM Glc for GOX, 15 nM enzyme, and 50 mM sodium phosphate buffer pH 8.0. The reactions were incubated at 30°C in a covered orbital thermomixer at 700 rpm for 6 h. Catalase (200 µg/mL) was then added to the reactions for 30 min to remove any remaining H 2 O 2 . The reactions were filtered using a 0.2 µm filter, and subsequent substrate depletion and products formation were quantified using HPAEC-PAD as described below. High Performance Anion‑exchange chromatography with Pulsed Amperometry Detection The amount of residual substrate and products formed was quantified by HPAEC-PAD equipped with a CarboPac PA1 (2 × 250 mm) analytical column and corresponding guard column (2 × 50 mm) (Dionex, Sunnyvale, CA, USA). Briefly, 12.5 µL of filtered and appropriately diluted samples were injected onto the column and eluted at flow rate of 0.25 mL/min using a gradient elution of sodium acetate in 100 mM sodium hydroxide, specifically 0–0.1 M sodium acetate over 35 min, followed by 0.1–0.2 M sodium acetate over 10 min, then 0.2–0.5 M sodium acetate over 5 min, and finally 0.5–0 M sodium acetate over 10 min to re-equilibrate the column. Data were analyzed using Chromeleon software (version 7.1.2.1478; Dionex). Liquid Chromatography- Mass Spectrometry (LC-MS) analyses Samples were prepared similarly as for HPAEC-PAD and analyzed using an Ultimate 3000 LC system with a Q-Exactive orbitrap mass spectrometer (Thermo Scientific, USA) equipped with a Hypersil GOLD column (50 × 2.1 mm) (Thermo Scientific, USA). Chromatograms were analyzed using Qual Browser in Thermo Xcalibur (v2.2) software (Thermo Scientific, USA). Computational docking analysis The 3D structural model of LbChi7A with the FAD cofactor was built using the AlphaFold3 server ( https://alphafoldserver.com/ ) while the x-ray structures of GOX (PDB ID: 3qvp) and ChitO (PDB ID: 6y0r) were downloaded from the RCSB Protein Data Bank ( https://www.rcsb.org ). The 3D structures of Glc, GlcN, GlcNAG, GlcVal and GlcHex were retrieved from PubChem with the CIDs of 5793, 441477, 439174, 22896149 and 12086638, correspondingly. All structures were energy-minimized using AMBER ff14SB force field, and docking simulation was carried out using Autodock Vina v1.1.2 ( http://vina.scripps.edu ). Pocket areas and volumes were calculated using the CASTpFold server ( https://cfold.bme.uic.edu/castpfold/ ). Figures were generated by PyMoL v3.1.0. Results and Discussion Steady-state kinetics of enzymes We previously characterized LbChi7A as an Auxiliary Activity family 7 (AA7) chito-oligosaccharide oxidase [ 21 ], with a preference for chito-oligomers and GlcNAc. In our broad screening of LbChi7A, it was discovered to have an unusually low K m for targeted monosaccharides, leading to catalytic efficiencies on those monosaccharides that were significantly higher than other previously characterized AA7s [ 21 ]. In the present study, we screened LbChi7A against Glc, GlcN, and GlcNAc. LbChi7A performance on these substrates was also compared to glucose oxidase (GOX) from Aspergillus niger , which serves as an industrially relevant benchmark. Whereas the catalytic efficiency of GOX on Glc was three orders of magnitude higher than that of LbChi7A, the catalytic efficiency of LbChi7A on GlcN was two orders of magnitude higher than that of GOX (Table 1 and Suppl. Figure S1). The catalytic efficiency of LbChi7A was even higher on GlcNAc indicating preference for substituted glucosamines; by contrast, GOX activity towards GlcNAc was not detected. Even though the presence of the C 2 acetyl group of GlcNAc increased the catalytic efficiency of LbChi7A (Table 1 ), the bulkier substitutions presented by GlcVal and GlcHex reduced enzyme efficiency. This is in contrast to ChitO from Fusarium graminearum , which displays lower K m and higher catalytic efficiency towards GlcVal and GlcHex compared to GlcNAc [ 22 ]. Recently, another flavin-dependent enzyme, N -acetyl-glucosamine oxidase (NagOx) from the bacterium Ralstonia solanacearum was reported [ 23 ]. Compared to NagOx, LbChi7A exhibits a lower K m for GlcNAc (30 nM vs 220 nM), but also a lower k cat value (2.8 s − 1 vs 140 s − 1 ). Therefore, while LbChi7A has higher substrate affinity and NagOx has greater turnover, both enzymes are capable of efficiently oxidizing GlcNAc under appropriate substrate loadings. Table 1 Kinetic parameters of LbChi7A and GOX oxidation of monosaccharides Parameters LbChi7A Glucose oxidase (GOX) Glc k cat (min −1 ) 120 ± 2 16,530 ± 170 K m (mM) 157 ± 11 7.6 ± 0.3 k cat / K m (mM − 1 min − 1 ) 0.8 2 x 10 3 GlcN k cat (min −1 ) 140 ± 1.7 50 ± 3 K m (mM) 0.24 ± 0.01 10.0 ± 1.8 k cat / K m (mM − 1 min − 1 ) 6 x 10 2 5 GlcNAc k cat (min −1 ) 165 ± 1.2 N.D. K m (mM) 0.03 ± 0.01 N.D. k cat / K m (mM − 1 min − 1 ) 5 x 10 3 N.D. N.D. = not detected; n = 3 and errors indicate standard deviation Docking analyses indicated that while all three ligands, Glc, GlcN and GlcNAc could access the active sites of LbChi7A and GOX, the distance between the C 1 -OH group of GlcNAc and the N 5 atom of the FAD cofactor in GOX exceeded 4.5 Å (Fig. 1 A), which is not favorable for either electron or proton transfer during enzymatic catalysis. Structural and docking analyses also showed that while both LbChi7A and ChitO have a secondary pocket in the active site that can accommodate C 2 substituted glucosamines (Fig. 1 B), the active site of ChitO is much larger with the solvent accessible pocket volume of 839 Å 3 , compared to 279 Å 3 of LbChi7A (Fig. 1 C). Conversion yield of monosaccharides to C1- acids We performed a time-course study to track the conversion of Glc, GlcN and GlcNAc by LbChi7A and GOX to corresponding oxidized products at different substrate and enzyme loadings (Fig. 2 and Suppl. Figure S2). Despite the higher catalytic efficiency of GOX on Glc, both LbChi7A and GOX achieved approximately 30% conversion of 100 mM Glc after 1 h (Fig. 2 A). At 500 mM Glc, however, the Glc conversion by GOX increased to 50%, underscoring the advantage of GOX at high Glc concentrations (Fig. 2 D). By contrast, the conversion of GlcN and GlcNAc by LbChi7A was higher than GOX at both substrate concentrations (Fig. 2 B, C, E, F). Whereas the conversion of 100 mM and 500 mM GlcN by LbChi7A was 90% and 70%, respectively, in less than 1 hour, corresponding conversions by GOX were less than 20%. In an earlier study using GOX from A. niger , Pezzotti et al. (2005) report 76% conversion of 558 mM GlcN, however, only after 72 h and with over an order of magnitude higher enzyme loading [ 16 ]. LbChi7A performance was even higher on GlcNAc, where an over 90% conversion was observed within 1 h (Fig. 2 C, F). LC-MS confirmed the oxidized product of GlcN and GlcNAc as D-glucosaminic acid (2-amino-2-deoxy-D-gluconic acid) (Fig. 3 A) and N -acetyl-D-glucosaminic acid (2-acetamido-2-deoxy-D-gluconic acid) (Fig. 3 B), respectively. The LC-MS analyses also confirmed the high purity of reaction products. Since hydrogen peroxide is the co-product of the GOX and LbChi7A reactions, potential inhibition of the enzymes by hydrogen peroxide was investigated. Less than 5% reduction in total activity was observed for both GOX or LbChi7A (Fig. 4 ) in reactions containing 860 mM hydrogen peroxide, indicating reactions can be performed using molar quantities of substrate without having to remove accumulating hydrogen peroxide. Conclusions In summary, the Auxiliary Activity 7 chito-oligosaccharide oxidase LbChi7A from Lentinus brumalis demonstrates significant potential as an efficient and selective biocatalyst for the oxidation of GlcN and GlcNAc to their corresponding C 1 -acids. At 100 mM substrate concentrations, the enzyme converted 90% of GlcN to D-glucosaminic acid and 100% of GlcNAc to N -acetyl-D-glucosaminic acid within 1h, without evidence of hydrogen peroxide inhibition. In comparison, the conversion of these substrates by the commercial GOX remained below 20%. These results reveal the biotechnological potential of LbChi7A in the sustainable synthesis of rare sugars and key pharmaceutical intermediates. Declarations Acknowledgments We would like to thank Dr. Jean-Guy Berrin (INRAE, Aix Marseille Univ, Biodiversité et Biotechnologie Fongiques (BBF), Marseille, France) for kindly providing the LbChi7A clone. Funding This project received funding from the NSERC CREATE for BioZone project (grant no. 528163), the NSERC Alliance “BioMax” project (grant no. ALLRP 570676-2021) and European Union’s Horizon 2020 research and innovation programme under grant agreement No 964764. 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S., Santema, L. L., Rozeboom, H. J., Xiang, R., Guallar, V., Mattevi, A. & Fraaije, M. W. (2023) Structural elucidation and engineering of a bacterial carbohydrate oxidase, Biochemistry. 62 , 429-436. Supplementary Files LbChi7Amanuscriptsupplmaterial.docx Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2026 Read the published version in Biotechnology Letters → Version 1 posted Reviewers agreed at journal 23 Dec, 2025 Reviewers invited by journal 02 Oct, 2025 Editor assigned by journal 13 Sep, 2025 First submitted to journal 11 Sep, 2025 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. 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16:33:17","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":64196,"visible":true,"origin":"","legend":"","description":"","filename":"BILED25005151structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7586791/v1/63f2f4fdfe4da244e6888434.xml"},{"id":93613704,"identity":"f2ee35c9-8aa8-4437-a5c5-293ce643bb55","added_by":"auto","created_at":"2025-10-15 16:33:18","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":70367,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7586791/v1/5a4c4648df5bd9f6953ffab8.html"},{"id":93613689,"identity":"2e49997c-f4dd-4e3e-a65e-0924a1bfdb13","added_by":"auto","created_at":"2025-10-15 16:33:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":335867,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComputational docking and structural analyses of LbChi7A, GOX and ChitO. \u003c/strong\u003e(A) Docking scores of Glc, GlcN and GlcNAc for LbChi7A and GOX; the distance in Å between the C\u003csub\u003e1\u003c/sub\u003e-OH group of ligands and the N\u003csub\u003e5\u003c/sub\u003e atom of the FAD cofactor was shown. (B) Docking of GlcNAc to LbChi7A (left) and GlcVal to ChitO (right). Active site surfaces are shown in gold, with carbon atoms of FAD in green and those of the ligands in aqua. The width of the secondary pocket is indicated by a dashed red line. (C) The 3D structural model of LbChi7A (left) and the x-ray structure of ChitO (right) with solvent-accessible, active-site pockets in red.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7586791/v1/c6da324b7afac7aabb5978d9.png"},{"id":93616277,"identity":"f2a00f94-86ec-4baa-a095-e50d04a2ac07","added_by":"auto","created_at":"2025-10-15 16:57:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":462982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTime course conversion of LbChi7A and GOX at different substrate and enzyme loadings. \u003c/strong\u003eGOX (blue) or LbChi7A (red) at 0.5 µM were incubated with 100 mM substrates (A, B, and C), or these enzymes at 5 µM were incubated with 500 mM substrates (D, E, and F) at 30 °C. Samples were aliquoted at 15 min, 30 min, and 60 min. For each time point, residual substrate was quantified by HPAEC-PAD against a standard curve. Every point is the average of three independent assays measured individually, with error bars indicating the standard error of the mean.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7586791/v1/e21adc9b6ccf3e67dcc5b72b.png"},{"id":93613691,"identity":"d10cebbb-f6bf-4bf9-9641-f98e127072ac","added_by":"auto","created_at":"2025-10-15 16:33:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":351586,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLC–MS of LbChi7A conversion of GlcN(A) and GlcNAc (B). \u003c/strong\u003eFor the reaction, 1 mM GlcN or GlcNAc, 15 nM LbChi7A, 50 mM sodium phosphate buffer pH 8.0 were incubated at 30 °C for 24 h. D-Glucosaminic acid (196.0816 m/z) and N-acetyl-D-glucosaminic acid (238.0919 m/z) were both detected in the positive mode; the relative abundance is shown in %.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7586791/v1/7aa12f11a78fb1513eacbce4.png"},{"id":93613695,"identity":"674899a0-d076-4bd4-bc6a-375d63721037","added_by":"auto","created_at":"2025-10-15 16:33:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":233209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpacts of hydrogen peroxide on enzymatic activity of LbChi7A and GOX. \u003c/strong\u003eFor each reaction, 1 mM GlcNAc for LbChi7A (grey bar) and 1 mM Glc for GOX (white bar), 15 nM enzyme, 50 mM sodium phosphate buffer pH 8.0 were incubated at 30 °C for 6 h with various concentrations of hydrogen peroxide, up to 0.860 M. Excess catalase was added at endpoint to remove any residual hydrogen peroxide. Residual substrate was quantified by HPAEC-PAD against a standard curve. Every point is the average of three independent assays measured individually, with error bars indicating the standard error of the mean.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7586791/v1/d481965be9272038852ebfc2.png"},{"id":105224854,"identity":"fa447dcf-edad-4b8b-8787-848c7fcb6d78","added_by":"auto","created_at":"2026-03-23 16:16:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2149446,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7586791/v1/430b0f94-765a-428b-82b5-dda21bb88219.pdf"},{"id":93615534,"identity":"99c3d857-3f35-418d-a9d1-2e9ad46cb714","added_by":"auto","created_at":"2025-10-15 16:49:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":266994,"visible":true,"origin":"","legend":"","description":"","filename":"LbChi7Amanuscriptsupplmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7586791/v1/017c51d055a84a1b510f6eef.docx"}],"financialInterests":"","formattedTitle":"Oligosaccharide oxidase for the enzymatic synthesis of glucosaminic acids","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChitin is a major polysaccharide that is found in the exoskeleton of arthropods and fungal cell walls. The estimated annual synthesis of chitin is close to 1 trillion tonnes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is made up of \u0026#120573;-(1\u0026forall;4)-linked \u003cem\u003eN\u003c/em\u003e-acetyl-D-glucosamine (GlcNAc) units. When processing chitin to remove protein and for demineralization, \u003cem\u003eN\u003c/em\u003e-acetyl groups can be lost, creating a polymer with a mixture of GlcNAc and D-glucosamine (GlcN) units, known as chitosan [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eD-Glucosaminic acid is a valuable sugar acid that can be obtained from the oxidation of GlcN. It is an enantiopure building block that has been used to synthesize various compounds such as enzyme (glucosidase) inhibitors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], chemotherapeutic drugs [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], drug delivery agents [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], ligands for cation exchange chromatography [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], enantioselective catalysts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and biopolymers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The diverse applications of GlcNA, including biopolymer synthesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], have prompted investigations into its sustainable and efficient production.\u003c/p\u003e\u003cp\u003eSeveral pathways, chemical and enzymatic, can be used to produce glucosaminic acid [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Historically, this acid was synthesized by oxidizing GlcN with yellow mercuric oxide and hydrogen sulfide [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition to toxicity and safety concerns, this process generates many by-products that reduce product yields to less than 50% [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Alternatively, glucosaminic acid can be produced using activated Charcoal-Supported Palladium‐Bismuth Catalysts (Pd-Bi/C) where 70% yield of glucosaminic acid is reported [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; however, production of the Pd-Bi/C catalyst is energy and water intensive, and requires harsh chemicals like nitric acid and formaldehyde [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Microbial routes to glucosaminic acid include oxidative fermentation of GlcN by \u003cem\u003ePseudomonas putida\u003c/em\u003e GNA5 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] where a 95% yield is reported; however, product recovery steps introduce economic barriers to scale-up [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Finally, cell-free biological routes to glucosaminic acid production oxidize GlcN using the glucose oxidase (GOX) from \u003cem\u003eAspergillus niger\u003c/em\u003e. Despite the selectivity and high atom economy of this approach, reported product yields are below 5% [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecently, LbChi7A, also known as PbChi7A, was characterized as a chito-oligosaccharide oxidase from \u003cem\u003eLentinus brumalis\u003c/em\u003e (formerly \u003cem\u003ePolyporus brumalis)\u003c/em\u003e, belonging to auxiliary activity family 7 (AA7) of the Carbohydrate-Active EnZYmes Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cazy.org\u003c/span\u003e\u003cspan address=\"http://www.cazy.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. LbChi7A was found to oxidize GlcN and GlcNAc with catalytic efficiencies more than three orders of magnitude higher than that on glucose (Glc). LbChi7A also produced glucosaminic acid at rates similar to those achieved for oxidized chito-oligosaccharides. To further assess the biotechnological potential of LbChi7A, particularly in alternative routes to aminosugar oxidation, this study directly compared the efficiencies of LbChi7A and GOX in the oxidation of Glc, GlcN, and GlcNAc.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eGlc, GlcN, GlcNAc, \u003cem\u003eN\u003c/em\u003e-valeryl-D-glucosamine (GlcVal), \u003cem\u003eN\u003c/em\u003e-hexanoyl-D-glucosamine (GlcHex), D-glucosaminic acid, catalase, and \u003cem\u003eA. niger\u003c/em\u003e glucose oxidase (UniProt ID: Q9HFQ1) were purchased from Sigma, USA. Bovine serum albumin (BSA) was purchased from Thermo Fisher Scientific, USA.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLbChi7A production and purification\u003c/h3\u003e\n\u003cp\u003eLbChi7A was produced as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In brief, plasmids encoding the LbChi7A gene (Genbank ID: RDX44700.1; UniProt ID: A0A371CWQ2_9APHY) were transformed into \u003cem\u003eKomagataella phaffii\u003c/em\u003e (formerly \u003cem\u003ePichia pastoris\u003c/em\u003e) strain X33 by electroporation, and positive transformants were grown in 4-L baffled flasks containing 2 L of BMGY (Buffered Glycerol-complex Medium). The cells were grown over 48 h at 30\u0026deg;C, harvested by centrifugation, suspended in 400 mL of BMMY (Buffered Methanol-complex Medium) in 1-L shake flask, and then incubated for 3 days at 20\u0026deg;C for protein production. Methanol (3%, v/v) was added to the cultivation every 24 h. Secreted proteins were separated from the cells by centrifugation and filtered using a 0.22 \u0026micro;m PES filter. The filtered supernatant was exchanged to 50 mM sodium phosphate pH 7.5 and purified using a Fast Protein Liquid Chromatography (FPLC) system with a 5-mL His Trap HP column (GE Healthcare, Uppsala, Sweden). The column was equilibrated at a flow rate of 2.5 mL/min with 50 mM sodium phosphate pH 7.5, 300 mM NaCl, and 20 mM imidazole. Filtered protein supernatant solution was applied to the column and eluted at a flow rate of 2.5 mL/min with 50 mM sodium phosphate pH 7.5, 300 mM NaCl, and 20\u0026ndash;250 mM imidazole gradient over 20 column volumes. Fractions with LbChi7A were concentrated and exchanged into 50 mM sodium phosphate pH 7.5 buffer. SDS-PAGE analysis was used to assess protein purity, and protein concentration was quantified by gel densitometry using a standard curve generated with BSA.\u003c/p\u003e\n\u003ch3\u003eActivity assays and kinetic analyses\u003c/h3\u003e\n\u003cp\u003eEnzyme reactions (0.25 mL) containing 100 mM and 500 mM substrate, enzyme (0.05\u0026ndash;5 \u0026micro;M LbChi7A or GOX), and 1.0 M sodium phosphate buffer (pH 8.0) were incubated at 30\u0026deg;C for 15, 30, and 60 min in an orbital shaking thermomixer at 700 rpm. The reactions were then filtered using a 0.2 \u0026micro;m PES filter, and substrate depletion and product formation were quantified using high performance anion‑exchange chromatography with pulsed amperometry detection (HPAEC-PAD) as described below. Steady-state kinetics of LbChi7A and GOX was determined by coupling the enzymatic release of hydrogen peroxide to the activity of horseradish peroxidase [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The kinetics on Glc (1\u0026ndash;1,320 mM), GlcN (0.01\u0026ndash;110 mM), and GlcNAc (0.01\u0026ndash;10 mM) in 50 mM sodium phosphate pH 8.0 were determined by fitting the Michaelis-Menten equation using GraphPad Prism5 software (GraphPad Software, USA).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e inhibition assay\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTo determine the potential of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to inhibit LbChi7A and GOX activity, 25 mM\u0026ndash;860 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were added to the enzyme assay reactions, which have 1 mM GlcNAc for LbChi7A and 1 mM Glc for GOX, 15 nM enzyme, and 50 mM sodium phosphate buffer pH 8.0. The reactions were incubated at 30\u0026deg;C in a covered orbital thermomixer at 700 rpm for 6 h. Catalase (200 \u0026micro;g/mL) was then added to the reactions for 30 min to remove any remaining H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The reactions were filtered using a 0.2 \u0026micro;m filter, and subsequent substrate depletion and products formation were quantified using HPAEC-PAD as described below.\u003c/p\u003e\n\u003ch3\u003eHigh Performance Anion‑exchange chromatography with Pulsed Amperometry Detection\u003c/h3\u003e\n\u003cp\u003eThe amount of residual substrate and products formed was quantified by HPAEC-PAD equipped with a CarboPac PA1 (2 \u0026times; 250 mm) analytical column and corresponding guard column (2 \u0026times; 50 mm) (Dionex, Sunnyvale, CA, USA). Briefly, 12.5 \u0026micro;L of filtered and appropriately diluted samples were injected onto the column and eluted at flow rate of 0.25 mL/min using a gradient elution of sodium acetate in 100 mM sodium hydroxide, specifically 0\u0026ndash;0.1 M sodium acetate over 35 min, followed by 0.1\u0026ndash;0.2 M sodium acetate over 10 min, then 0.2\u0026ndash;0.5 M sodium acetate over 5 min, and finally 0.5\u0026ndash;0 M sodium acetate over 10 min to re-equilibrate the column. Data were analyzed using Chromeleon software (version 7.1.2.1478; Dionex).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eLiquid Chromatography- Mass Spectrometry (LC-MS) analyses\u003c/h2\u003e\u003cp\u003eSamples were prepared similarly as for HPAEC-PAD and analyzed using an Ultimate 3000 LC system with a Q-Exactive orbitrap mass spectrometer (Thermo Scientific, USA) equipped with a Hypersil GOLD column (50 \u0026times; 2.1 mm) (Thermo Scientific, USA). Chromatograms were analyzed using Qual Browser in Thermo Xcalibur (v2.2) software (Thermo Scientific, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eComputational docking analysis\u003c/h3\u003e\n\u003cp\u003eThe 3D structural model of LbChi7A with the FAD cofactor was built using the AlphaFold3 server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafoldserver.com/\u003c/span\u003e\u003cspan address=\"https://alphafoldserver.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) while the x-ray structures of GOX (PDB ID: 3qvp) and ChitO (PDB ID: 6y0r) were downloaded from the RCSB Protein Data Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The 3D structures of Glc, GlcN, GlcNAG, GlcVal and GlcHex were retrieved from PubChem with the CIDs of 5793, 441477, 439174, 22896149 and 12086638, correspondingly. All structures were energy-minimized using AMBER ff14SB force field, and docking simulation was carried out using Autodock Vina v1.1.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://vina.scripps.edu\u003c/span\u003e\u003cspan address=\"http://vina.scripps.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Pocket areas and volumes were calculated using the CASTpFold server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cfold.bme.uic.edu/castpfold/\u003c/span\u003e\u003cspan address=\"https://cfold.bme.uic.edu/castpfold/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Figures were generated by PyMoL v3.1.0.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSteady-state kinetics of enzymes\u003c/h2\u003e\u003cp\u003eWe previously characterized LbChi7A as an Auxiliary Activity family 7 (AA7) chito-oligosaccharide oxidase [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], with a preference for chito-oligomers and GlcNAc. In our broad screening of LbChi7A, it was discovered to have an unusually low \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e for targeted monosaccharides, leading to catalytic efficiencies on those monosaccharides that were significantly higher than other previously characterized AA7s [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the present study, we screened LbChi7A against Glc, GlcN, and GlcNAc. LbChi7A performance on these substrates was also compared to glucose oxidase (GOX) from \u003cem\u003eAspergillus niger\u003c/em\u003e, which serves as an industrially relevant benchmark.\u003c/p\u003e\u003cp\u003eWhereas the catalytic efficiency of GOX on Glc was three orders of magnitude higher than that of LbChi7A, the catalytic efficiency of LbChi7A on GlcN was two orders of magnitude higher than that of GOX (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Suppl. Figure S1). The catalytic efficiency of LbChi7A was even higher on GlcNAc indicating preference for substituted glucosamines; by contrast, GOX activity towards GlcNAc was not detected. Even though the presence of the C\u003csub\u003e2\u003c/sub\u003e acetyl group of GlcNAc increased the catalytic efficiency of LbChi7A (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the bulkier substitutions presented by GlcVal and GlcHex reduced enzyme efficiency. This is in contrast to ChitO from \u003cem\u003eFusarium graminearum\u003c/em\u003e, which displays lower \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and higher catalytic efficiency towards GlcVal and GlcHex compared to GlcNAc [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Recently, another flavin-dependent enzyme, \u003cem\u003eN\u003c/em\u003e-acetyl-glucosamine oxidase (NagOx) from the bacterium \u003cem\u003eRalstonia solanacearum\u003c/em\u003e was reported [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Compared to NagOx, LbChi7A exhibits a lower \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e for GlcNAc (30 nM vs 220 nM), but also a lower \u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e value (2.8 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e vs 140 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Therefore, while LbChi7A has higher substrate affinity and NagOx has greater turnover, both enzymes are capable of efficiently oxidizing GlcNAc under appropriate substrate loadings.\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\u003eKinetic parameters of LbChi7A and GOX oxidation of monosaccharides\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLbChi7A\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGlucose oxidase (GOX)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eGlc\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e (min\u0026nbsp;\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e120\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,530\u0026thinsp;\u0026plusmn;\u0026thinsp;170\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (mM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e157\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 x 10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eGlcN\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e (min\u0026nbsp;\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e140\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (mM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6 x 10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eGlcNAc\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e (min\u0026nbsp;\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e165\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (mM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5 x 10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eN.D. = not detected; n\u0026thinsp;=\u0026thinsp;3 and errors indicate standard deviation\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eDocking analyses indicated that while all three ligands, Glc, GlcN and GlcNAc could access the active sites of LbChi7A and GOX, the distance between the C\u003csub\u003e1\u003c/sub\u003e-OH group of GlcNAc and the N\u003csub\u003e5\u003c/sub\u003e atom of the FAD cofactor in GOX exceeded 4.5 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which is not favorable for either electron or proton transfer during enzymatic catalysis. Structural and docking analyses also showed that while both LbChi7A and ChitO have a secondary pocket in the active site that can accommodate C\u003csub\u003e2\u003c/sub\u003e substituted glucosamines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), the active site of ChitO is much larger with the solvent accessible pocket volume of 839 \u0026Aring;\u003csup\u003e3\u003c/sup\u003e, compared to 279 \u0026Aring; \u003csup\u003e3\u003c/sup\u003e of LbChi7A (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eConversion yield of monosaccharides to C1- acids\u003c/h2\u003e\u003cp\u003eWe performed a time-course study to track the conversion of Glc, GlcN and GlcNAc by LbChi7A and GOX to corresponding oxidized products at different substrate and enzyme loadings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Suppl. Figure S2). Despite the higher catalytic efficiency of GOX on Glc, both LbChi7A and GOX achieved approximately 30% conversion of 100 mM Glc after 1 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). At 500 mM Glc, however, the Glc conversion by GOX increased to 50%, underscoring the advantage of GOX at high Glc concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). By contrast, the conversion of GlcN and GlcNAc by LbChi7A was higher than GOX at both substrate concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C, E, F). Whereas the conversion of 100 mM and 500 mM GlcN by LbChi7A was 90% and 70%, respectively, in less than 1 hour, corresponding conversions by GOX were less than 20%. In an earlier study using GOX from \u003cem\u003eA. niger\u003c/em\u003e, Pezzotti et al. (2005) report 76% conversion of 558 mM GlcN, however, only after 72 h and with over an order of magnitude higher enzyme loading [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. LbChi7A performance was even higher on GlcNAc, where an over 90% conversion was observed within 1 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLC-MS confirmed the oxidized product of GlcN and GlcNAc as D-glucosaminic acid (2-amino-2-deoxy-D-gluconic acid) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and \u003cem\u003eN\u003c/em\u003e-acetyl-D-glucosaminic acid (2-acetamido-2-deoxy-D-gluconic acid) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), respectively. The LC-MS analyses also confirmed the high purity of reaction products.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSince hydrogen peroxide is the co-product of the GOX and LbChi7A reactions, potential inhibition of the enzymes by hydrogen peroxide was investigated. Less than 5% reduction in total activity was observed for both GOX or LbChi7A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) in reactions containing 860 mM hydrogen peroxide, indicating reactions can be performed using molar quantities of substrate without having to remove accumulating hydrogen peroxide.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, the Auxiliary Activity 7 chito-oligosaccharide oxidase LbChi7A from \u003cem\u003eLentinus brumalis\u003c/em\u003e demonstrates significant potential as an efficient and selective biocatalyst for the oxidation of GlcN and GlcNAc to their corresponding C\u003csub\u003e1\u003c/sub\u003e-acids. At 100 mM substrate concentrations, the enzyme converted 90% of GlcN to D-glucosaminic acid and 100% of GlcNAc to \u003cem\u003eN\u003c/em\u003e-acetyl-D-glucosaminic acid within 1h, without evidence of hydrogen peroxide inhibition. In comparison, the conversion of these substrates by the commercial GOX remained below 20%. These results reveal the biotechnological potential of LbChi7A in the sustainable synthesis of rare sugars and key pharmaceutical intermediates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. Jean-Guy Berrin (INRAE, Aix Marseille Univ, Biodiversit\u0026eacute; et Biotechnologie Fongiques (BBF), Marseille, France) for kindly providing the LbChi7A clone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project received funding from the NSERC CREATE for BioZone project (grant no. 528163), the NSERC Alliance \u0026ldquo;BioMax\u0026rdquo; project (grant no. ALLRP 570676-2021) and European Union\u0026rsquo;s Horizon 2020 research and innovation programme under grant agreement No 964764.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRevathi, M., Saravanan, R. \u0026amp; Shanmugam, A. (2012) Production and characterization of chitinase from Vibrio species, a head waste of shrimp \u003cem\u003eMetapenaeus dobsonii\u003c/em\u003e (Miers, 1878) and chitin of \u003cem\u003eSepiella inermis\u003c/em\u003e Orbigny, 1848, \u003cem\u003eAdv Biosci Biotechnol. \u003c/em\u003e\u003cstrong\u003e03\u003c/strong\u003e, 392-397.\u003c/li\u003e\n\u003cli\u003eKumar, M. N. V. R. 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(2023) Structural elucidation and engineering of a bacterial carbohydrate oxidase, \u003cem\u003eBiochemistry. \u003c/em\u003e\u003cstrong\u003e62\u003c/strong\u003e, 429-436.\u003c/li\u003e\n\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"LbChi7A, D-glucosaminic acid, Glucose oxidase, D-glucosamine, biocatalysis, green chemistry","lastPublishedDoi":"10.21203/rs.3.rs-7586791/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7586791/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eD-Glucosaminic acid is a valuable amino acid useful in food and medical applications. It is a highly sought after enantiopure molecule important in synthesis of drugs and glycopeptides. Current enzymatic synthesis pathways to D-glucosaminic acid carry disadvantages such as low product yield, long reaction times, and high cost due to increase in enzyme usage.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eHerein, the Auxiliary Activity 7 chito-oligosaccharide oxidase from \u003cem\u003eLentinus brumalis\u003c/em\u003e, LbChi7A, was shown as a potent biocatalyst capable of efficiently converting D-glucosamine (GlcN) and \u003cem\u003eN\u003c/em\u003e-acetyl-D-glucosamine (GlcNAc) to their respective C\u003csub\u003e1\u003c/sub\u003e-acids. Due to a unique substrate specificity towards GlcN and GlcNAc, LbChi7A converts at least 90% GlcN to D-glucosaminic acid within 60 min and 100% GlcNAc to \u003cem\u003eN\u003c/em\u003e-acetyl-D-glucosaminic acid within the same time frame. Furthermore, LbChi7A inhibition by the hydrogen peroxide co-product was not detected, even at 860 mM. This single enzymatic conversion offers an efficient process for the production of glucosaminic acids including D-glucosaminic acid or \u003cem\u003eN\u003c/em\u003e-acetyl-D-glucosaminic acid.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe biotechnological potential of LbChi7A is demonstrated, particularly in the production of rare sugars and pharmaceutical intermediates. The ability of the enzyme to perform selective oxidation without the need for hazardous chemicals presents a cleaner and efficient alternative to traditional chemical methods.\u003c/p\u003e","manuscriptTitle":"Oligosaccharide oxidase for the enzymatic synthesis of glucosaminic acids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 16:33:13","doi":"10.21203/rs.3.rs-7586791/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-12-23T12:05:48+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-02T19:14:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-13T07:46:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology Letters","date":"2025-09-11T16:08:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f8a9003b-3d69-4911-81a5-5636af514549","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:15:06+00:00","versionOfRecord":{"articleIdentity":"rs-7586791","link":"https://doi.org/10.1007/s10529-026-03721-9","journal":{"identity":"biotechnology-letters","isVorOnly":false,"title":"Biotechnology Letters"},"publishedOn":"2026-03-17 15:58:48","publishedOnDateReadable":"March 17th, 2026"},"versionCreatedAt":"2025-10-15 16:33:13","video":"","vorDoi":"10.1007/s10529-026-03721-9","vorDoiUrl":"https://doi.org/10.1007/s10529-026-03721-9","workflowStages":[]},"version":"v1","identity":"rs-7586791","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7586791","identity":"rs-7586791","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}