Oligosaccharide oxidase for the enzymatic synthesis of glucosaminic acids

doi:10.21203/rs.3.rs-7586791/v1

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Oligosaccharide oxidase for the enzymatic synthesis of glucosaminic acids

https://doi.org/10.21203/rs.3.rs-7586791/v1

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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 C₁-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

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

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).

H₂O₂ inhibition assay

To determine the potential of H₂O₂ to inhibit LbChi7A and GOX activity, 25 mM–860 mM H₂O₂ 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 H₂O₂. 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.

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 C₂ 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 10³
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 10²	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 10³	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 C₁-OH group of GlcNAc and the N₅ atom of the FAD cofactor in GOX exceeded 4.5 Å (Fig. 1A), 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 C₂ substituted glucosamines (Fig. 1B), the active site of ChitO is much larger with the solvent accessible pocket volume of 839 Å³, compared to 279 Å ³ of LbChi7A (Fig. 1C).

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. 2A). At 500 mM Glc, however, the Glc conversion by GOX increased to 50%, underscoring the advantage of GOX at high Glc concentrations (Fig. 2D). By contrast, the conversion of GlcN and GlcNAc by LbChi7A was higher than GOX at both substrate concentrations (Fig. 2B, 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. 2C, F).

LC-MS confirmed the oxidized product of GlcN and GlcNAc as D-glucosaminic acid (2-amino-2-deoxy-D-gluconic acid) (Fig. 3A) and N-acetyl-D-glucosaminic acid (2-acetamido-2-deoxy-D-gluconic acid) (Fig. 3B), 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.

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 C₁-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.

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.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

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You are reading this latest preprint version

Oligosaccharide oxidase for the enzymatic synthesis of glucosaminic acids

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Materials and methods

Materials

LbChi7A production and purification

Activity assays and kinetic analyses

H₂O₂ inhibition assay

High Performance Anion‑exchange chromatography with Pulsed Amperometry Detection

Liquid Chromatography- Mass Spectrometry (LC-MS) analyses

Computational docking analysis

Results and Discussion

Steady-state kinetics of enzymes

Conversion yield of monosaccharides to C1- acids

Conclusions

Declarations

References

Supplementary Files

Status:

Version 1

Oligosaccharide oxidase for the enzymatic synthesis of glucosaminic acids

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Materials and methods

Materials

LbChi7A production and purification

Activity assays and kinetic analyses

H2O2 inhibition assay

High Performance Anion‑exchange chromatography with Pulsed Amperometry Detection

Liquid Chromatography- Mass Spectrometry (LC-MS) analyses

Computational docking analysis

Results and Discussion

Steady-state kinetics of enzymes

Conversion yield of monosaccharides to C1- acids

Conclusions

Declarations

References

Supplementary Files

Status:

Version 1

H₂O₂ inhibition assay