Heterologous Expression and Characterization of Cellouronate (β-1,4-Glucuronan) Lyase from a Human Intestinal Bacterium Bacteroides luhongzhouii

Abstract

Cellouronate, β-1,4-glucuronan, is synthesized from regenerated cellulose via 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical-mediated oxidation. Human intestinal bacteria were cultured in a medium containing cellouronate to evaluate its utilization. These experiments showed Bacteroides luhongzhouii to grow well in this medium. Several putative cellouronate lyases belonging to polysaccharide lyase family 38 from B. luhongzhouii were identified. Among these candidate enzymes, BlCUL1, which displayed the most similarity to authentic cellouronate lyases, was heterologously expressed and characterized. The recombinant BlCUL1 (rBlCUL1) showed the highest activity at pH 8.0 and was deactivated by treatment at pH 3.0 for 24 h or heating above 50 °C for 10 min. Moreover, the activity of rBlCUL1 was enhanced in the presence of Mg²⁺, Ca²⁺, or EDTA, but suppressed by Al³⁺ and completely inactivated by Fe³⁺. Analysis of the final reaction mixture generated from the rBlCUL1 mediated degradation of cellouronate revealed an oligomer as the main product, but the monomer was barely detectable. This study is the first to report and characterize a cellouronate lyase from human intestinal bacteria.

Abbreviations

TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl; PL, polysaccharide lyase; OD₆₆₀, optical density at 660 nm; rBlCUL1, recombinant BlCUL1; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CMC, carboxymethyl cellulose; TLC, thin-layer chromatography; NMR, nuclear magnetic resonance; rCUL-I, recombinant CUL-I

INTRODUCTION

Cellouronate, a polyuronate composed of glucuronate units linked by β-1,4-glycosidic bonds, is a semi-synthetic acidic polysaccharide derived from regenerated cellulose via 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical-mediated oxidation [1]. In a previous study, the mycorrhizal fungus Ensifer meliloti (formerly Rhizobium meliloti) was reported to produce an exopolysaccharide β-1,4-linked glucuronan with variable O-acetylation at the C2 and/or C3 positions [2]. In other examples, it is known that the mucor fungus, Mucor rouxii, and the green algae, Ulva Lactuca, possess β-1,4-linked glucuronan [3, 4]. However, in these cases only trace amounts of β-1,4-linked glucuronan are present suggesting it might be scarce in nature.

Various polyuronates were synthesized from curdlan, konjac glucomannan, chitin, and chitosan via TEMPO-mediated oxidation [5, 6, 7, 8, 9]. By contrast to the precursor polysaccharide, these polyuronates are water-soluble. Moreover, cellouronate is biodegradable [10], and we reported purification and characterization of the enzyme responsible for this degradation from the soil bacterium Brevundimonas sp. SH203 [11, 12]. This enzyme, named CUL-I, cleaves the β-1,4-linkage by β-elimination in an endo-type manner (cellouronate lyase; endo-β-1,4-glucuronan lyase, EC 4.2.2.14, Fig. 1) and was recently reported to belong to polysaccharide lyase (PL) family 38, a newly created PL family [12]. In addition to soil bacteria, cellouronate lyases have been identified in fungi, viruses, and scallops, and cellouronate lyases are known to be associated with multiple PL families [13, 14, 15, 16, 17, 18, 19]. Given that cellouronate lyases have been detected in organisms living in diverse environments, cellouronate degradation is anticipated to commonly occur in nature. However, it is not known whether human intestinal bacteria utilize cellouronate. Clarification of this issue will contribute to a better understanding of cellouronate degradation in nature and serve as a first step toward the application of cellouronate in the food sector. In this study, we evaluated the ability of human intestinal bacteria to utilize cellouronate by culturing in a medium containing cellouronate. A candidate cellouronate lyase gene from the cellouronate-utilizing bacterium Bacteroides luhongzhouii was engineered for heterologous expression, and the recombinant protein purified and characterized.

Fig. 1. Reaction scheme for cellouronate lyase.

MATERIALS AND METHODS

Bacterial strains and materials

All bacterial strains used in this study were purchased from Japan Collection of Microorganisms (Table 1). Unless stated otherwise, all reagents were purchased from either FUJIFILM Wako Pure Chemical Co., Ltd. (Osaka, Japan) or Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Cellouronate, curduronate (β-1,3-glucuronan), and amylouronate (α-1,4-glucuronan) were prepared from regenerated cellulose (BEMCOT M-3 II; Asahi Kasei Co., Ltd., Tokyo, Japan), curdlan, and soluble starch, respectively, via TEMPO-mediated oxidation as described previously [1, 5, 20].

Table 1. Strains used in this study.

Species	Strain
Akkermansia muciniphila	JCM 30893
Anaerostipes caccae	JCM 13470
Anaerotruncus colihominis	JCM 15631
Bacteroides caccae	JCM 9498
Bacteroides clarus	JCM 16067
Bacteroides luhongzhouii	JCM 33480
Bacteroides thetaiotaomicron	JCM 5827
Bifidobacterium bifidum	JCM 1209
Bifidobacterium longum subsp. infantis	JCM 1222
Faecalibacterium duncaniae	JCM 31915
Mediterraneibacter gnavus	JCM 6515
Phocaeicola vulgatus	JCM 5826
Roseburia faecis	JCM 17581

Assessment of cellouronate utilization

Bacterial cultivation was performed in an anaerobic workstation (DG250; Don Whitley Scientific Ltd., Bingley, UK) under an atmosphere of N₂, CO₂, and H₂ (8:1:1 vol/vol/vol) at 37 °C. The bacteria were pre-cultured overnight in a medium containing 10 g/L tryptone, 2.5 g/L yeast extract, 4 g/L NaHCO₃, 6 g/L glucose, 1 g/L L-cysteine hydrochloride monohydrate, 0.45 g/L K₂HPO₄, 0.45 g/L KH₂PO₄, 0.9 g/L (NH₄)₂SO₄, 0.9 g/L NaCl, 0.09 g/L MgSO₄･7H₂O, 0.09 g/L CaCl₂･2H₂O, 0.01 g/L hemin, 4.25 g/L acetic acid, 1.42 g/L propionic acid, 0.22 g/L n-valeric acid, 0.22 g/L isovaleric acid, 0.23 g/L isobutyric acid, 1 mg/L resazurin, 10 μg/L biotin, 10 μg/L cyanocobalamin, 30 μg/L p-aminobenzoic acid, 50 μg/L folic acid, 150 μg/L pyridoxine hydrochloride, 50 μg/L thiamine hydrochloride, and 50 μg/L riboflavin at pH 7.45. The pre-cultured bacteria were transferred to a glucose-containing medium, a saccharide-free medium, or a cellouronate-containing medium. The composition of the medium, except for the saccharide, corresponded to that of the previous medium. Cellouronate was sterilized by passage through a 0.2 μm filtration unit. After culturing for 24 h the optical density at 660 nm (OD₆₆₀) of the medium was monitored using a V730 Bio spectrophotometer (Jasco Co., Ltd., Tokyo, Japan). The OD₆₆₀ of the saccharide-free medium was used as control. The increase in OD₆₆₀ of the glucose- or cellouronate-containing medium relative to the saccharide-free medium was determined. Phylogenetic analysis based on 16S ribosomal RNA of the bacteria used in this study was performed using the MASCLE algorithm in MEGA11, and the phylogenetic tree was constructed by the neighbor-joining method. The 16S ribosomal RNA sequences were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/).

The NCBI database was also used to acquire the amino acid sequences of the intestinal bacteria. Amino acid sequences of putative cellouronate lyases were identified using dbCAN3 (https://bcb.unl.edu/dbCAN2/index.php) [21]. The sequences were phylogenetically analyzed by aligning them with known cellouronate lyase sequences using the MUSCLE alignment program in MEGA11.

Cloning of BlCUL1 from B. luhongzhouii

Protein sequences from B. luhongzhouii that displayed homology to the PL family 38 enzyme from Brevundimonas sp. SH203 (Accession number: GAW41138.1) were identified by carrying out an alignment. Candidate sequences that displayed significant homology were subjected to phylogenetic analysis and a BLASTp search in BioEdit (https://thalljiscience.github.io/).

Bacteroides luhongzhouii cells cultured in cellouronate-containing medium were collected by centrifugation. Total RNA was extracted using the TRIzol method and reverse-transcribed with SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific Inc., Waltham, MA, USA). DNA fragment coding BlCUL1 (Accession number: WP_143257364.1) and linearized pColdI were amplified by PCR with PrimeSTAR GXL DNA polymerase (TaKaRa Bio Inc., Shiga, Japan) and BlCUL1-specific or pColdI-specific primers, respectively (Table S1; see J. Appl. Glycosci. Web site). The amplified BlCUL1 was ligated to linearized pColdI using the In-Fusion^® Snap Assembly Master Mix (TaKaRa Bio Inc.), and the plasmid vector, named pColdI-BlCUL1, was introduced into Stellar^TM Competent Cells (TaKaRa Bio Inc.). The pColdI-BlCUL1 was extracted and purified with the FastGene^TM PlasmidMini kit (Nippon Genetics Co., Ltd., Tokyo, Japan), and sequence analysis was conducted using Genewiz from Azenta Life Sciences Inc. (Burlington, MA, USA).

Heterologous expression and purification of recombinant BlCUL1

Escherichia coli BL21-CodonPlus(DE3)-RIL (Agilent Technologies Inc., Santa Clara, CA, USA) cells were transformed with pColdI-BlCUL1. The transformation mix was plated on Plusgrow II agar supplemented with 100 μg/mL ampicillin. After selection, a single colony was picked and cultured in Plusgrow II medium supplemented with 100 μg/mL ampicillin at 37 °C for 1-2 h at 150 rpm. Heterologous expression of recombinant BlCUL1 (rBlCUL1) was then induced by addition of 50 μM isopropyl-β-D-1-thiogalactopyranoside and growth was continued at a reduced temperature of 15 °C for 24 h.

The bacterial cells were collected by centrifugation, resuspended in 20 mM sodium-phosphate (pH 7.4) with 0.5 M NaCl and 5 mM imidazole, and lysed by six 15-second rounds of ultrasonication (VibraCell VC50 Probe Sonicator; Sonics and Materials Inc., Newtown, CT, USA), at ice temperature. The resulting lysate was centrifuged and filtered through a 0.2 μm filter unit. The clarified lysate was then applied to a His GraviTrap^TM TALON column (Cytiva Inc., Marlborough, MA, USA). Bound rBlCUL1 was subsequently eluted by washing the column with 20 mM sodium-phosphate (pH 7.4) containing 0.5 M NaCl and increasing amounts of imidazole in the order of 5 mM, 20 mM, and 200 mM. The eluate containing 200 mM imidazole was concentrated by ultrafiltration (Vivaspin Turbo 15 PES; Sartorius Stedim Biotech SA, Göttingen, Germany) and the buffer replaced with 10 mM sodium-phosphate (pH 7.0). The purity of the preparation was assessed by conducting sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was determined by the Bradford method using bovine serum albumin as a standard.

Enzymatic analysis of BlCUL1

The rBlCUL1 activity was assayed by monitoring the increase in absorbance at 235 nm during the enzymatic reaction arising from the formation of a double bond between C4 and C5 at the non-reducing end of the reaction product. Unless otherwise stated, reactions were performed at room temperature with a standard reaction mixture containing 0.2 % (w/v) cellouronate, 50 mM Britton-Robinson buffer (50 mM acetate, phosphate, and borate) at pH 8.0, and rBlCUL1 solution. One unit (U) of rBlCUL1 was defined as the amount of enzyme that produces 1 μmol of unsaturated product per minute. A molar extinction coefficient of 4,837 M⁻¹ cm⁻¹ was used as reported by Konno et al. [14].

Effects of pH on the enzymatic reaction were evaluated by measuring the activity in 50 mM Britton-Robinson buffer at pH 3-10. To analyze the pH stability, rBlCUL1 was incubated in 50 mM Britton-Robinson buffer at pH 3-10 at 4 °C for 24 h. The residual activity was measured under standard conditions using the treated enzyme. The optimum temperature was determined by measuring enzymatic activity at 20-60 °C under standard conditions. Thermal stability was evaluated by measuring residual activity after treatment at 10-70 °C for 10 min in 50 mM Britton-Robinson buffer at pH 4, 7, or 10. The effect of metal ions was analyzed by measuring enzyme activity in the standard reaction mixture supplemented with LiCl, KCl, MgCl₂, CaCl₂, FeCl₃, CoCl₂, MnCl₂, AlCl₃, or EDTA at a final concentration of 2 mM. Substrate specificity was evaluated by measuring enzyme activity in the standard reaction mixture but substituting cellouronate with various polysaccharides (curduronate (β-1,3-glucuronan), amylouronate (α-1,4-glucuronate), polygalacturonate, alginate, hyaluronate, chondroitin sulfate, or carboxymethyl cellulose (CMC)). Kinetic parameters were measured using cellouronate at concentrations ranging from 0.05 to 10 mg/mL. The Michaelis constant (K_m) and maximum velocity (V_max) were determined by fitting to the Michaelis-Menten equation using a nonlinear regression model.

The degradation products of cellouronate generated by rBlCUL1 under standard conditions for 24 h were analyzed by thin-layer chromatography (TLC) with n-butanol:acetate:water (9:4:7, vol/vol/vol) as the developing solvent. The products were subsequently visualized with 5 % (v/v) sulfuric acid in ethanol. To identify the final degradation products of cellouronate, cellouronate was incubated with BlCUL1 for 48 h at 25 °C. The degradation product was purified using TOYOPEARL SuperQ-650M (Tosoh Corp., Tokyo, Japan) with 0-0.4 M NH₄HCO₃ as the eluent. The fractions containing the target product were concentrated using a rotary evaporator in a water bath set at 60 °C. The structure of the main degradation product was determined by ¹H and ¹³C nuclear magnetic resonance (NMR), as well as two-dimensional NMR techniques including ¹H-¹H COSY and HSQC, using a Bruker Avance Neo-500 NMR spectrometer (Bruker BioSpin GmbH, Ettlingen, Germany). For NMR analysis, 10 mg of the sample was dissolved in 700 μL of D₂O containing 0.05 % trimethylsilyl propanoic acid-d4, and all measurements were conducted at 25 °C.

RESULTS AND DISCUSSION

The carbohydrate utilization ability of the genus Bacteroides

To evaluate the cellouronate utilization ability of human intestinal bacteria, 13 species of human intestinal bacteria were cultured in a medium containing cellouronate (Fig. 2). Most of the bacteria failed to grow in the presence of cellouronate, especially butyric acid bacteria and Bifidobacterium. By contrast, B. clarus showed a low level of growth in this medium, and B. luhongzhouii grew to levels comparable to that of the glucose-containing medium, exhibiting an OD₆₆₀ of approximately 2.0 in the cellouronate-containing medium. Candidate genes encoding cellouronate lyase were identified using the dbCAN3 sever. Both B. clarus and B. luhongzhouii were found to possess three putative endo-cellouronate lyase genes belonging to PL family 38 (WP_195500923.1, WP_087425212.1, and WP_087426659.1 from B. clarus: WP_143259981.1, WP_143257364.1, and WP_143257365.1 from B. luhongzhouii), and B. caccae had a putative exo-cellouronate lyase gene belonging to PL family 8 (WP_005681457.1). However, no genes encoding putative cellouronate lyases were identified from the other bacteria.

Fig. 2. Phylogenetic tree based on 16S ribosomal RNA of human intestinal bacteria cultured in this study.

　Relative increase in the optical density at 660 nm (OD₆₆₀) when each strain was cultured in a medium containing cellouronate is shown on the right hand side. OD₆₆₀ values when cultured in a medium containing glucose were arbitrarily set to 100 %.

Of the human intestinal bacteria tested in this study for their ability to utilize cellouronate, only strains belonging to the genus Bacteroides showed any growth. Bacteroides is one of the most potent utilizers of polysaccharides in the human gut. We reasoned that cellouronate is likely degraded by Bacteroides in a manner resembling that of other polysaccharides. To date, the source of cellouronate in the gut has not been elucidated. On the other hand, cellouronate-like polysaccharides have been identified across diverse taxonomic groups, including bacteria, fungi, and green algae. Therefore, it is possible that cellouronate-like polysaccharides are supplied from the diet as well as from the cell walls and/or exopolysaccharides of intestinal microbes.

Cloning of the gene encoding cellouronate degrading enzymes

Given that B. luhongzhouii showed the highest level of growth on cellouronate among the strains tested, cellouronate lyases from this organism were investigated. Phylogenetic and homology analyses were performed to identify amino acid sequences from B. luhongzhouii that belong to PL family 38 and display similarity to the known cellouronate lyase from Brevundimonas sp. SH203 (GAW41138.1). The phylogenetic analysis showed that WP_143257364.1 and WP_143259981.1 formed the same clade with GAW41138.1 (Fig. 3A). Additionally, BLASTp analysis showed that WP_143257364.1 was more homologous (Score: 284 bits, Identity: 41 %, E-value: 1e-80) to GAW41138.1 than WP_143259981.1 (Score: 217 bits, Identity: 34 %, E-value: 1e-60). Consequently, WP_143257364.1 was selected for further study. WP_143257364.1 was named BlCUL1 and engineered for heterologous expression in E. coli.

Fig. 3. (A) Phylogenetic analysis of cellouronate lyases belonging to PL family 38 from B. luhongzhouii and Brevundimonas sp. SH203. The score, identity, and E-values were calculated using a BLASTp search to compare amino acid sequences of PL family 38 from B. luhongzhouii and CUL-I from Brevundimonas sp. SH203. (B) SDS-PAGE analysis of rBlCUL1 purified by immobilized metal affinity chromatography. Proteins were visualized by staining with Coomassie Brilliant Blue. Standard molecular mass markers (kDa) are shown in the left hand lane.

The rBlCUL1 was generated that included a polyhistidine tag, which facilitated its purification. SDS-PAGE analysis indicated that the molecular mass of rBlCUL1 was approximately 41 kDa (Fig. 3B), close to the predicted value based on the amino acid sequence (approximately 45 kDa).

Enzymatic characterization of BlCUL1

The effect of pH on the enzymatic activity of rBlCUL1 was investigated by measuring its activity under differing pH conditions (Fig. 4A). The optimum pH of the enzyme was between 7.0 to 8.0, with very slight or no activity being observed at pH 3, 4, and 10. The pH stability was evaluated by measuring the residual activity after exposure to various pH conditions for 24 h at 4 °C. This analysis showed that rBlCUL1 was stable from pH 4 to pH 10 (Fig. 4B). BlCUL1 exhibited the highest activity at 50 °C (Fig. 4C). The residual activity after treatment at various temperatures for 10 min was used to evaluate thermal stability. The rBlCUL1 remained stable up to 30 °C at pH 4 or 10, and 40 °C at pH 7 (Fig. 4D). An analysis of the effect of metal ions revealed that MgCl₂, CaCl₂, and EDTA activated the enzymatic reaction, while FeCl₃ and AlCl₃ markedly suppressed it (Fig. 4E).

Fig. 4. The enzymatic properties of BlCUL1.

　(A) Effects of pH on the BlCUL1 reaction. The highest activity was arbitrarily set to 100 %. (B) pH stability of BlCUL1. The enzymatic activity at each pH before treatment was arbitrarily set to 100 %. (C) Effects of temperature on the BlCUL1 reaction. The highest activity was arbitrarily set to 100 %. (D) Thermal stability of BlCUL1 at pH 4, 7, and 10. The enzymatic activity prior to thermal treatment were arbitrarily set to 100 %. Gray filled square: pH 4, black filled circle: pH 7, open triangle: pH 10. (E) Effects of metal ions on BlCUL1. The enzymatic activity without metal ions was arbitrarily set to 100 %.

In previous studies, some endo-cellouronate lyases were purified, and their biochemical properties characterized [11, 12, 13, 14, 18]. Compared with the effects of pH on CUL-I from Brevundimonas sp. SH203, rBlCUL1 exhibited activity over a broader pH range than CUL-I. Moreover, rBlCUL1 was stable at pH 4 and 5 whereas CUL-I was not [11, 12]. These observations suggest that rBlCUL1 is more tolerant to pH changes than previously characterized cellouronate lyases. However, the thermal stability of rBlCUL1 was almost the same as that of other cellouronate lyases that have been studied. The optimum temperature was higher than that of MyAly (cellouronate lyases belonging to PL family 14 from Mizuhopecten yessoensis, scallop). This difference is presumed to result from evolutionary adaptation to the distinct environmental conditions in which the respective source organisms thrive. Intriguingly, the activity of both recombinant CUL-I (rCUL-I) and TrGL (cellouronate lyases belonging to PL family 20 from Trichoderma reesei) were significantly affected by metal ions [11, 12, 14]. Specifically, rCUL-I lost its activity in the presence of CuSO₄ or FeCl₃, whereas, TrGL exhibited calcium dependence, with its activity increasing some 8-fold in the presence of calcium ions. Although rBlCUL1, like rCUL-I, was also deactivated in the presence of FeCl₃, it did not require metal ions for activity. The rBlCUL1 degraded not only cellouronate (31.1 U/mg) but also a small amount of alginate (3.7 U/mg) and did not exhibit activity toward the other substrates, indicating it to have a substrate specificity analogous to that of known cellouronate lyases (Table 2). The K_m, k_cat, and k_cat/K_m values of rBlCUL1 were 0.028 mg mL⁻¹, 24.5 s⁻¹, and 879 mg⁻¹ mL s⁻¹, respectively.

Table 2. Substrate specificity of BlCUL1

Substrate	Specific activity (U/mg)
Cellouronate	31.1
Curduronate	N.D.
Amylouronate	N.D.
Polygalacturonate	N.D.
Alginate	3.7
Hyaluronate	N.D.
Chondroitin sulfate C	N.D.
CMC	N.D.

N.D., Not detected

The enzymatic reaction products of rBlCUL1 were analyzed by TLC. The results revealed that the monomer was barely detectable. However, a lower-mobility product than the monomer was detected as the main reaction product (Fig. 5). The product was subsequently purified, and its structure was analyzed by NMR. The analysis revealed that the main reaction product was unsaturated di-glucuronate; the observed ¹³C NMR signals were consistent with those reported in a previous study (Fig. 5, Table S2, and Fig. S1; see J. Appl. Glycosci. Web site) [22]. The final reaction products of CUL-I have been reported to be unsaturated di-glucuronate and α-keto-glucuronic acid, which is formed from unsaturated glucuronate through non-enzymatic reactions [11]. Furthermore, CUL-II, an exo-cellouronate lyase from Brevundimonas sp. SH203, was reported to have a high affinity to unsaturated di-glucuronate, and cellouronate was efficiently degraded through the cooperative action of CUL-I and CUL-II [22]. Therefore, B. luhongzhouii is postulated to degrade cellouronate via the cooperative action of BlCUL1 and an enzyme with the same function as CUL-II that is capable of degrading unsaturated di-glucuronate.

Fig. 5. TLC analysis of degradation products generated from cellouronate by the BlCUL1 reaction; the structure and ¹³C NMR spectrum of the main product, unsaturated di-glucuronate.

　GlcA: glucuronate.

CONCLUSION

Although cellouronate is a semi-synthetic polysaccharide, many organisms are capable of degrading this compound and possess enzymes that display specificity for cellouronate. Here, we identified human intestinal bacterial strains that can utilize cellouronate. Bacteroides luhongzhouii showed the best growth on cellouronate and we characterized the cellouronate lyase (BlCUL1) from this organism. The properties of BlCUL1 were similar to the known cellouronate lyase from Brevundimonas sp. SH203, CUL-I, with respect to the effects of pH, temperature, and metal ions. Based on the results of this study and previous reports, we propose that BlCUL1 acts cooperatively with other enzymes to degrade cellouronate into its monomeric form. This is the first study to establish that cellouronate is degraded by human intestinal bacteria, potentially paving the way for its application in the food sector.

CONFLICTS OF INTEREST

The authors declared that they have no conflicts of interest.

ACKNOWLEDGEMENTS

This work was supported by a Grant-in-Aid for JSPS Fellows (23KJ0230) from the Japan Society for the Promotion of Science (JSPS) to Y.T., and a Grant-in-Aid for Scientific Research (23K05329) from JSPS to N.H.

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