# Pycnoporus cinnabarinus glyoxal oxidases display differential catalytic efficiencies on 5-hydroxymethylfurfural and its oxidized derivatives

Craig Faulds, Mariane Daou, Bassem Yassine, Saowanee Wikee, Eric Record, Françoise Duprat, Emmanuel Bertrand

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Craig Faulds, Mariane Daou, Bassem Yassine, Saowanee Wikee, Eric Record, et al. Pycnoporus cinnabarinus glyoxal oxidases display differential catalytic efficiencies on 5-hydroxymethylfurfural and its oxidized derivatives. Fungal Biology and Biotechnology, 2019, 6, 10.1186/s40694-019-0067-8. [https://doi.org/10.1186/s40694-019-0067-8](https://doi.org/10.1186/s40694-019-0067-8)

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<table><tr><td>Manuscript Number:</td><td colspan="2"></td></tr><tr><td>Full Title:</td><td colspan="2">Pycnoporus cinnabarinus glyoxal oxidases display differential catalytic efficiencies on 5-hydroxymethylfurfural and its oxidized derivatives</td></tr><tr><td>Article Type:</td><td colspan="2">Research</td></tr><tr><td rowspan="4">Funding Information:</td><td>Ambassade de France en Thailande (na)</td><td>Dr Saowanee Wikee</td></tr><tr><td>H2020 European Research Council (720303)</td><td>Prof Craig Faulds<br>Dr Marianne Daou</td></tr><tr><td>FP7 International Cooperation (KBBE-2013-7-613549)</td><td>Prof Craig Faulds<br>Dr Marianne Daou<br>Dr Eric Record</td></tr><tr><td>ECCOREV (na)</td><td>Mr Bassem Yassine<br>Dr Françoise Duprat<br>Dr Emmanuel Bertrand</td></tr><tr><td>Abstract:</td><td colspan="2">Background<br>5-hydroxymethylfurfural (HMF), a major residual component of a lignocellulosic bio-refinery process, can be transformed into fundamental building blocks for green chemistry via oxidation. While chemical methods are well established, interest is also being directed into the enzymatic oxidation of HMF into the bio-plastic precursor 2,5-furandicarboxylic acid (FDCA).<br>Results<br>We demonstrate that three glyoxal oxidases (PciGLOX) isoenzymes from the Basidiomycete fungus Pycnoporus cinnabarinus were able to oxidize HMF, with PciGLOX2 and PciGLOX3 being the most efficient. The major reaction product obtained with the three isoenzymes was 5-hydroxymethyl-2-furancarboxylic (HMFCA) a precursor in polyesters and pharmaceuticals production, and very little conversion of this compound was observed. However, small concentrations of 2,5-furandicarboxylic acid, a substitute for terephthalic acid in the production of polyesters, were also obtained. The oxidation of HMF was significantly boosted in the presence of catalase for PciGLOX2 leading to 70% HMFCA yield. The highest conversion percentages were observed on 2,5-Furandicarboxaldehyde (DFF), a minor product from the reaction of PciGLOX on HMF. To bypass HMFCA accumulation and exploit the efficiency of PciGLOX in oxidizing DFF and FFCA towards FDCA production, HMF was oxidized in a cascade reaction with an aryl alcohol oxidase (UmaAAO). After 2 hours of reaction, UmaAAO completely oxidized HMF to DFF and further to FFCA, with FDCA only being detected when PciGLOX3 was added to the reaction. The maximum yield of 16% FDCA was obtained 24 hours after the addition of PciGLOX3 in the presence of catalase.<br>Conclusions<br>At least two conversion pathways for HMF oxidation can be considered for PciGLOX however the highest selectivity was seen towards the production of the valuable polyester precursor HMFCA. The three isoenzymes showed differences in their catalytic efficiencies and substrate specificities when reacted with HMF derivatives.</td></tr><tr><td>Corresponding Author:</td><td colspan="2">Craig Faulds, PhD<br>Aix-Marseille Université/INRA<br>Marseille, FRANCE</td></tr><tr><td>Corresponding Author Secondary</td><td colspan="2"></td></tr></table>
<table><tr><td>Information:</td><td></td></tr><tr><td>Corresponding Author's Institution:</td><td>Aix-Marseille Universite/INRA</td></tr><tr><td>Corresponding Author's Secondary Institution:</td><td></td></tr><tr><td>First Author:</td><td>Craig Faulds, PhD</td></tr><tr><td>First Author Secondary Information:</td><td></td></tr><tr><td>Order of Authors:</td><td>Craig Faulds, PhD<br>Marianne Daou<br>Bassem Yassine<br>Saowanee Wikee<br>Eric Record<br>Françoise Duprat<br>Emmanuel Bertrand</td></tr><tr><td>Order of Authors Secondary Information:</td><td></td></tr><tr><td>Opposed Reviewers:</td><td>Angel Martinez<br>Centro de Investigaciones Biologicas<br>Competitor in this area<br>Martin Hofrichter<br>Technische Universitat Dresden<br>Competitor in this area<br>Marco Fraaije<br>Rijksuniversiteit Groningen<br>Competitor in this area</td></tr><tr><td>Additional Information:</td><td></td></tr><tr><td>Question</td><td>Response</td></tr><tr><td>&lt;b&gt;Is this study a clinical trial?&lt;/b&gt;&lt;hr&gt;&lt;i&gt;A clinical trial is defined by the World Health Organisation as 'any research study that prospectively assigns human participants or groups of humans to one or more health-related interventions to evaluate the effects on health outcomes'.&lt;/i&gt;</td><td>No</td></tr></table>
1 **Pycnoporus cinnabarinus** glyoxal oxidases display

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2 differential catalytic efficiencies on 5-hydroxymethylfurfural

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3 and its oxidized derivatives

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5 Marianne Daou¹, Bassem Yassine¹, Saowanee Wikee¹, Eric Record¹,

6 Françoise Duprat², Emmanuel Bertrand¹, Craig B. Faulds¹ #

7 ¹Aix Marseille Université, INRA UMR1163 Biodiversité et Biotechnologie

8 Fongiques (BBF), 13009 Marseille, France

9 ²Aix-Marseille Univ, CNRS, Centrale Marseille, M2P2, Marseille, France

10 # Address correspondence to craig.faulds@univ-amu.fr

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21 **Abstract**

22 **Background:** 5-hydroxymethylfurfural (HMF), a major residual component of
23 a lignocellulosic bio-refinery process, can be transformed into fundamental
24 building blocks for green chemistry via oxidation. While chemical methods are
25 well established, interest is also being directed into the enzymatic oxidation of
26 HMF into the bio-plastic precursor 2,5-furandicarboxylic acid (FDCA).

27 **Results:** We demonstrate that three glyoxal oxidases (*PciGLOX*) isoenzymes
28 from the Basidiomycete fungus *Pycnoporus cinnabarinus* were able to oxidize
29 HMF, with *PciGLOX2* and *PciGLOX3* being the most efficient. The major
30 reaction product obtained with the three isoenzymes was 5-hydroxymethyl-2-
31 furancarboxylic (HMFCA) a precursor in polyesters and pharmaceuticals
32 production, and very little conversion of this compound was observed.
33 However, small concentrations of 2,5-furandicarboxylic acid, a substitute
34 for terephthalic acid in the production of polyesters, were also obtained. The
35 oxidation of HMF was significantly boosted in the presence of catalase for
36 *PciGLOX2* leading to 70% HMFCA yield. The highest conversion percentages
37 were observed on 2,5-Furandicarboxaldehyde (DFF), a minor product from
38 the reaction of *PciGLOX* on HMF. To bypass HMFCA accumulation and
39 exploit the efficiency of *PciGLOX* in oxidizing DFF and FFCA towards FDCA
40 production, HMF was oxidized in a cascade reaction with an aryl alcohol
41 oxidase (*UmaAAO*). After 2 hours of reaction, *UmaAAO* completely oxidized
42 HMF to DFF and further to FFCA, with FDCA only being detected when
43 *PciGLOX3* was added to the reaction. The maximum yield of 16% FDCA was
44 obtained 24 hours after the addition of *PciGLOX3* in the presence of catalase.
45 **Conclusions:** At least two conversion pathways for HMF oxidation can be considered for PciGLOX however the highest selectivity was seen towards the production of the valuable polyester precursor HMFCA. The three isoenzymes showed differences in their catalytic efficiencies and substrate specificities when reacted with HMF derivatives.

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51 **Keywords:** 5-Hydroxymethylfurfural, bio-catalysis, glyoxal oxidase, 2,5-furancarboxylic acid, furan derivatives

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66 Background

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67 With the growing concerns about the depleting supply of fossil fuel and the global problem of climate change, the demands for sustainable substitutes for petroleum-based products are increasing. Being one of the most abundant renewable natural materials on earth, plants, especially those low-cost residues from agro-industrial processing, have become a major candidate for this role. Approximately 75% of annual production of agro-industrial residues is in the form of carbohydrates, making this material highly exploitable [1].
74 Efforts are being directed for the conversion of plant carbohydrates into valuable chemicals and fuels. Among the valuable platform chemicals that can be obtained from biomass carbohydrates are 5-hydroxymethylfurfural (HMF) and furfural [2], produced by triple dehydration of hexoses [3] and acid hydrolysis of pentosans respectively [4].

79 HMF is particularly important due to its biodegradability and its versatility as a precursor for a wide selection of furan-based products, and has been recognized as top value-added molecule in biotechnology [5]. Important molecules derived from HMF include dimethylfuran, levulinic acid, 2,5-furancarboxylic acid (FDCA), 2,5-diformylfuran (DFF), 3,5-dihydroxymethylfuran, 5-hydroxy-4-keto-pentenoic acid and 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) [6]. DFF is a stable derivative of HMF. This molecule is considered important for the synthesis of pharmaceutical compounds [7], antifungal products [8], electroconductors [9] and polymeric materials [10]. Another important product from the oxidation of the aldehyde group of HMF is HMFCA. This is particularly important for the production of polyesters [11] and interleukin inhibitors [12], and to have in vivo antitumor
91 activity against Sarcoma 180 cells [13]. Furan-based derivatives also include
92 2,5-furandicarboxylic acid (FDCA), which was considered among the 12 most
93 promising sugar-based molecules for the production of bio-based material
94 [14]. FDCA is particularly important because it can replace fossil-based
95 terephthalic acid for the production of bio-based polyesters [15]. Chemically,
96 HMF derivatives are produced using metals as catalysts under extreme
97 conditions such as high temperature, high pressure and in the presence of
98 toxic solvents. The development of bio-catalytic processes is therefore
99 advantageous since enzymes work under mild conditions which can reduce
100 the energy required and the production cost.

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102 Among the enzymes that were previously tested and used for the oxidation of
103 HMF are three alcohol oxidases (EC 1.1.3.13) and one galactose oxidase
104 (GAO; EC 1.1.3.9) that have been reported to oxidize HMF leading to the
105 formation of DFF as the sole product following the reaction presented in
106 scheme 1 [16]. Synthesis of HMFCA by bio-catalysis remained limited due to
107 the need of high reaction selectivity in order to oxidize the aldehyde group and
108 leave the hydroxyl group intact [17]. Xanthine oxidase (EC 1.17.3.2) from
109 *Escherichia coli* was used for the selective oxidation of HMF to HMFCA [16].
110 Oxidative conversion of HMF to FDCA was also performed following a two-
111 steps reaction using fungal aryl-alcohol oxidase (AAO; EC 1.1.3.7) and an
112 unspecific heme peroxygenase (UPO; EC 1.11.2.1) [18]. Full HMF conversion
113 with high FDCA yields was also achieved using an FAD-dependent oxidase,
114 named HMF oxidase (HMFO; EC 1.1.3.47) [19, 20].
115 Although HMF derivatives are highly important, limited examples describing the synthesis of these compounds via enzymatic reactions were previously described and the pursuit for new enzymes that can be involved in this process is still highly interesting. Promising candidates are enzymes with versatile substrate specificity able to perform the successive oxidation steps required in the process by recognizing HMF and its derivatives as substrates.

121 Glyoxal oxidases (GLOX; EC 1.2.3.15) are metalloenzymes containing one copper metal ion, and belong to a class of enzymes called radical copper oxidases (CRO) [21]. These enzymes have been grouped within the AA5 family in the CAZymes (Carbohydrate Active Enzymes database; http://www.cazy.org/) classification system [22, 23]. GLOX catalyzes the oxidation of aldehydes to their corresponding carboxylic acids, generating simultaneously hydrogen peroxide ($H_2O_2$) [24, 25]. This enzyme was first isolated in ligninolytic cultures of the white-rot Basidiomycete *Phanerochaete chrysosporium* under restricted nitrogen supply, suggesting a role in lignin modification and peroxidase $H_2O_2$-dependent action [26]. Subsequent sequencing of the *P. chrysosporium* genome has indicated the presence of only one gene encoding GLOX in this fungus [27]. The genome of another Polyporale fungus, *Pycnoporus cinnabarinus* [28] indicated that this fungus contained 3 *glox* genes. *In vitro* characterization of these GLOX revealed their ability to act on a broad range of substrates including toxic and inhibitory aldehydes [24, 25]. In addition, GLOX were able to recognize and oxidize the alcohol group of glycerol extending further the specificity range of these enzymes [29, 25]. Although the utilization of GLOX for biotechnological applications had not previously been elaborated upon, the reactions catalyzed
140 by this enzyme made it a promising candidate, especially for the oxidation of
141 HMF and its derivatives.

142 In this work, the ability of two previously produced and characterized GLOX
143 from *P. cinnabarinus* BRFM 137 (*PciGLOX1* and *PciGLOX2*) [25] and a third
144 isoform from this fungus (*PciGLOX3*) to oxidize HMF and its derivatives was
145 investigated.

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147 RESULTS

148 PciGLOX3 Characterization

149 *PciGLOX3* shared 84% and 89% protein sequence identity with *PciGLOX1* and *PciGLOX2*, respectively, and the amino acids at the active site were highly conserved (**Fig. 1**). *PciGLOX3* showed a generally lower activity on the tested substrates compared to the other two *PciGLOX* enzymes, however, the specificity range for all three isoenzymes was similar except in the case of glycerol, which was only oxidized by *PciGLOX2* and *PciGLOX3* (**Table 1**).

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One major difference observed was that highest activity detected on formaldehyde for *PciGLOX3* while the two other enzymes were most active on glyoxylic acid. Differences in the kinetic constants were also observed for *PciGLOX3* that showed the highest specificity and catalytic efficiency on glycerol compared to the other enzymes (Table 2). In addition, *PciGLOX3* was less efficient in oxidizing glyoxylic acid compared to the two other GLOX.

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Enzyme Stability under the Conditions of Reaction

163 The standard reaction conditions for the oxidation of HMF and its derivatives
164 were chosen based on the stability and performance of the enzymes under
165 these conditions. The optimal pH was 6 and the enzymes were active in
166 dimethylsuccinate and tartrate buffers [25]. The reactions were performed in
167 tartrate buffer, as dimethylsuccinate could not be resolved from the substrates
168 and expected products of the enzymatic reaction in the HPLC analysis. The
169 stability of *PciGLOX* enzymes under these conditions and over a prolonged
170 period of time were assessed. *PciGLOX2* was the least stable of the three
171 isoenzymes and lost around 90% of its activity after 2 hours of incubation
172 (Figure 2a). On the other hand, *PciGLOX1* and *PciGLOX3* retained 70% and
173 40% of their activities after 48 hours of incubation, respectively. The stability
174 of the three enzymes was also assessed in the presence of 3 mM HMF. In the
175 presence of this substrate, no activity was detected for *PciGLOX2* after 2
176 hours of incubation (Figure 2b). On the other hand, the activities of
177 *PciGLOX1* and *PciGLOX3* were relatively stable with *PciGLOX3* showing
178 increased residual activity after 48 hours. The stability of the three *PciGLOX*
179 enzymes was further investigated in the presence of 3 mM HMFA the major
180 theoretically expected product of the reaction on HMF. In the presence of this
181 molecule, the significant loss in activity after 24 hours of incubation was
182 comparable between the three isoforms (Figure 2c). HRP was highly stable
183 under the tested conditions (Additional file 1).

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185 Stability towards H₂O₂

186 The stability of the three *PciGLOX* isoenzymes was investigated in the
187 presence of varying concentrations of H₂O₂. *PciGLOX1* was the most stable
188 enzyme in the presence of this reaction product, retaining 60% of its activity
189 when incubated with 10 mM H₂O₂ for 24 hours (Figure 3). Although more
190 stable than PciGLOX3, PciGLOX2 significantly lost activity with increasing
191 concentrations of H₂O₂ and lost more than 95% of its activity after incubation
192 in the presence of 8 mM H₂O₂. PciGLOX3 on the other hand lost 60% of its
193 activity with only 2 mM H₂O₂ and was completely inactive with 10 mM H₂O₂
194 after 24 hours of incubation.

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196 Oxidation of HMF and its derivatives by PciGLOX

197 The three PciGLOX isoenzymes were found to be active on HMF and this
198 became evident by the decrease in HMF concentration and the appearance of
199 the oxidation products over time (Figure 4, Additional file 2). Although the
200 catalytic efficiencies of the three enzymes were comparable on HMF (Table
201 3), the conversion percentage of this substrate varied between the PciGLOX
202 enzymes (14% for PciGLOX1, 34% for PciGLOX2 and 28% for PciGLOX3). In
203 addition, the three isoenzymes noticeably differed in their products' patterns.

204 The major product of the reactions of the PciGLOX isoenzymes on HMF was
205 HMFCA with PciGLOX2 and PciGLOX3 producing the highest yields (39%
206 and 41%, respectively; Figure 4b, c). By adding catalase to the reaction of
207 PciGLOX2, the amount of produced HMFCA was considerably increased to
208 76% (Figure 4b right). This increase was coupled to a significant boost in
209 HMF conversion. Interestingly, the addition of catalase seemed to shift the
210 reaction more towards the production of HMFCA and decreased the amount
211 of FFCA produced from 5% to 0.7%. This activity-enhancing effect in the
212 presence of catalase was also observed with *PciGLOX3* but not with *PciGLOX1*.

214 Interestingly, small amounts of DFF, the alternative oxidation product of HMF, were also detected. The concentration of detected DFF slightly increased over time in the reaction with *PciGLOX1* (0.017 mM at 24 hours). In the cases of *PciGLOX2* and *PciGLOX3*, smaller DFF concentrations were detected early in the reaction. The substrate specificity and catalytic efficiencies of *PciGLOX2* and *PciGLOX3* on DFF as a substrate (*Km* =0.2 mM; *Kcat/Km* = 23403 s⁻¹ M⁻¹ and *Km* =0.2 mM; *Kcat/Km* = 7267 s⁻¹ M⁻¹, respectively) were significantly better than that of *PciGLOX1* (*Km* =4.3 mM; *Kcat/Km* = 124.3 s⁻¹ M⁻¹). This was further supported in the results obtained when DFF was used as the initial substrate and 80% to 84% converted by *PciGLOX2* and *PciGLOX3*, respectively (**Figure 5a**).

225 The three *PciGLOX* were also reacted with HMFCA as the initial substrate. Although this compound was produced as the major product in HMF oxidation, the three *PciGLOX* showed very weak activity towards HMFCA, with *PciGLOX3* having the best conversion rate (2%; **Figure 5b**). The extent of HMFCA to FFCA conversion by *PciGLOX* was not sufficient to calculate catalytic constants. In addition, Higher concentrations of FFCA and FDCA were obtained when the enzymes were reacted with DFF and FFCA as substrates, respectively (**Figure 5a and c**).

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234 GLOX and AAO Cascade Reactions

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235 A cascade reaction where UmaAAO was used to oxidize HMF followed by the addition of PciGLOX was tested. PciGLOX3 was used in this experiment since this enzyme showed the highest conversion percentage on DFF (84% after 24 hours) and the highest FDCA yields (14% after 24 hours on FFCA).

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Two conditions were tested: (1) PciGLOX3 was added after 2 hours of UmaAAO reaction on HMF, and (2) PciGLOX3 was added after 24 hours of UmaAAO reaction on HMF. Under condition (1) HMF was completely oxidized by UmaAAO and the major product before the addition of PciGLOX3 was DFF whereas in condition (2) FFCA was the predominant product (Figure 6; Additional file 2).

245 After 4 hours of adding PciGLOX3, FFCA was the major product (2.6 mM) in the reaction and a little amount of DFF (0.5 mM) was detected (Table 4).
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247 Twenty hours later, DFF was almost completely consumed and the concentration of FDCA increased (0.03 mM). In the presence of catalase, DFF was completely oxidized after 4 hours of reaction and the concentration of FDCA obtained was 6 folds higher after 24 hours (Table 4). The yield of FDCA was further increased in condition (2) to reach 14% and 16% after 24 hours in the absence and presence of catalase, respectively (Table 4). The obtained yields in both tested conditions were comparable to the ones obtained when PciGLOX3 was reacted with DFF and FFCA as the initial substrates (Figure 5).

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257 Discussion

258 As mentioned in the introduction, HMF, a residue of the 2G lignocellulosic
259 biorefinery, is of particularly industrial importance due to its biodegradability
260 and its versatility as a precursor for a wide selection of furan-based products,
261 and as such has been recognized as top value-added molecule in
262 biotechnology. Currently, large chemical plants are being constructed to
263 produce HMF, but a number of biological conversions have been identified in
264 recent years, including the use of glyoxal oxidases as described in this paper.
265 Although the three GLOX from P. cinnabarinus have a high protein sequence
266 similarity and a conserved copper center, significant catalytic differences were
267 observed and it was therefore interesting to compare the activity of all three
268 enzymes on the conversion of HMF and its derivatives. The observed catalytic
differences, in addition to the fact that PciGLOX3 has not yet been found to be
270 secreted during fungal growth on different substrates [28], suggests different
physiological roles of these enzymes during fungal growth.
271 While enzymatic assays with PciGLOX were performed at 30°C, the
272 temperature at which fungi usually grows and secretes these enzymes in
273 vitro, the pH and buffer system were also previously found to be critical
274 factors for activity [25]. PciGLOX1 and PciGLOX3 were very stable under the
275 reaction conditions used, while PciGLOX2 lost significantly activity over time,
276 especially in presence of its substrate, HMF. The stability of AAO from P.
277 eryngii, which can also catalyze the oxidation of HMF to DFF, was previously
determined in the presence of 3 mM HMF, and the enzyme was found to lose
279 30% of its activity after 24 hours [18]. Similarly to HMF, the major reaction
280 product HMFA seems to alter considerably the catalytic properties of the
281 PciGLOX enzymes. However, it is difficult to determine in the presence of
282 HMF and HMFA if the observed effect is related to changes in stability or if it
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284 is due to these molecules remaining in or close to the active sites of the
enzymes, especially in the case of PciGLOX2 that was completely inactive on
GA in the presence of HMF.

287 When incubated with different concentrations of H₂O₂ over time, the three isoenzymes again showed differences in stability. The inhibition of PchGLOX by exogenous H₂O₂ has previously been reported [30]. However, the three PciGLOX isoenzymes were found to be more stable than PchGLOX, which retained only 25% of its initial activity in the presence of 2.1 mM exogenous H₂O₂ in the reaction mixture [30]. Furthermore, the detection of the reaction product of PchGLOX on glycerol was only possible when the activity was extended by adding catalase [29], which is not the case for PciGLOX [25].
295 H₂O₂ can oxidize proteins and alter the functional groups of certain amino acids leading to cleavage of the polypeptide chain and protein aggregation [31]. Therefore, one could postulate that the differences in stability observed between the three PciGLOX in the presence of H₂O₂ could be explained by structural variations between the three isoenzymes.

300 Significant difference in the kinetic parameters and products' yields for the oxidation of HMF were observed between the three enzymes, with the highest catalytic efficiency on HMF obtained with PciGLOX3 (Kcat/Km = 118.35 s⁻¹ M⁻¹). The oxidation of HMF using a phylogenetically different GLOX from *Myceliophthora thermophila* M77 (MtGLOX) has been recently published [32].
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304 Similarly to PciGLOX described in this paper, low conversion levels were observed with HMF. However MtGLOX showed a higher catalytic efficiency and specificity towards HMF (Kcat/Km = 787 s⁻¹ M⁻¹, Km = 0.02 M⁻¹)
308 compared to *PciGLOX*. Interestingly MtGLOX oxidized the alcohol group of
309 HMF leading to the formation of DFF.

310 Higher specificity and two-fold higher catalytic efficiency of AAO from *P. eryngii* were reported on HMF (*Kcat*/Km = 210 s⁻¹ M⁻¹) compared to that recorded in this study on *PciGLOX* [18]. In a more recent study using AAO for the oxidation of HMF, Karich and co-workers reported varying catalytic constants for the homologously produced AAO from *P. eryngii* on HMF with catalytic efficiency comparable to that obtained for *PciGLOX* [33]. However, *P. eryngii* AAO was less specific on HMF. Another enzyme used for the oxidation of HMF is the FAD-dependent oxidase, HMFO, which was more specific on HMF compared to *PciGLOX* and has a significantly higher catalytic efficiency (*Kcat*/Km = 7000 s⁻¹ M⁻¹) [19]. A 95% conversion yield of HMF to FDCA was achieved using HMFO, however the concentration used was 60 times higher than the one used for *PciGLOX* in this work. HMFO also requires oxygen as an electron acceptor.

323 The major product of the *PciGLOX* reaction on HMF was HMFCA. This result was expected since GLOX recognizes the aldehyde group of its substrate [25], HMF in this case (Scheme 1). The need for this specificity has limited the number of described bio-catalytic pathways for the production of HMFCA.
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326 A previously described enzyme for this purpose is the bacterial xanthine oxidase (XO), which recognizes the formyl group of HMF [16]. Using 2.2 µM of this enzyme, HMFCA was obtained with a yield of 94% after 7 hours of reaction on 26 mM HMF. The amount of *PciGLOX* used in this work was comparable to XO, but the yield of HMFCA obtained with *PciGLOX2* after 24 hours of reaction was significantly lower. However, a two fold increase in
333 HMFCA yields was observed when catalase was added to PciGLOX2 reaction. This was in agreement with the results showing that PciGLOX2 was very sensitive to accumulating H₂O₂ concentrations. Catalase converts H₂O₂ to O₂, protecting the enzymes from oxidative damage and supplying O₂. This enzyme was previously used to eliminate the accumulating H₂O₂ from the action of alcohol oxidases on HMF [16]. In addition, catalase was found to boost HMF conversion in the coupled reaction of GAO and HRP, which is similar to the effect observed in this work [16].

341 In addition to HMFCA, small amount of DFF were detected at early stages of the reaction especially with PciGLOX1. This result shows that PciGLOX are, to a smaller extent, able to act on the alcohol group of HMF. This is the second example of alcohol oxidation by these enzymes, which have been previously found to be active on glycerol [25, 29]. HMF oxidation to DFF as the only product was recently observed with MtGLOX; however this enzyme was unable to further oxidize DFF in the reaction [32]. Enzymes known to convert HMF to DFF include also AAO, alcohol oxidase, GAO, and HMFO [16, 18, 34, 19] and these enzymes showed higher yields of DFF compared to PciGLOX. However, the substrate specificity and catalytic efficiencies of PciGLOX2 and PciGLOX3 on DFF as a substrate (Km =0.2 mM; Kcat/Km = 23403 s⁻¹ M⁻¹ and Km =0.2 mM; Kcat/Km = 7267 s⁻¹ M⁻¹, respectively) were significantly better than that of AAO from P. eryngii (Km =3.3 mM; Kcat/Km = 158 s⁻¹ M⁻¹) [18] and HMFO (Km =1.7 mM; Kcat/Km = 940 s⁻¹ M⁻¹) [19]. PciGLOX2 and PciGLOX3 were also more efficient than PciGLOX1 in oxidizing DFF which explains the presence of constant concentrations of DFF with PciGLOX1. On the other hand, in the cases of
358 *PciGLOX2* and *PciGLOX3*, it is more likely that DFF was produced and consumed very fast in the reaction.

360 On the contrary, the major reaction product, HMFCA was very weakly oxidized by *PciGLOX*. In addition, although the three enzymes and especially *PciGLOX2* and *PciGLOX3* were able to oxidize HMF, higher concentrations of FFCA and FDCA were obtained when the enzymes were reacted with DFF and FFCA as substrates, respectively.

365 In the reactions of *PciGLOX* on HMF, very little DFF is produced compared to the "dead-end product" HMFCA which might be strongly altering the stability of the enzymes at early stages of the reaction preventing further oxidation steps. In addition, the obtained results suggest that similarly to AAO, HMFO and chloroperoxidase [18, 19, 34], *PciGLOX* enzymes produce FFCA mainly via the oxidation of DFF (**Scheme 1**), which is a minor product in the oxidation of HMF by *PciGLOX*. However, *PciGLOX* are more specific and catalytically efficient than these enzymes in oxidizing DFF. For these reasons a cascade reaction, where *UmaAAO* was used to oxidize HMF followed by the addition of *PciGLOX*, was tested. AAO was previously used in tandem reactions with an unspecific oxidase for the oxidation of HMF [18, 33]. Similarly to AAOs from *Pleurotus eryngii*, and *Pleurotus ostreatus* AAO, the AAO from *U. maydis* (*UmaAAO*) generates FFCA via the oxidation of HMF to DFF [18, 33]. *UmaAAO* was also found as efficient as *P. eryngii* AAO in oxidizing HMF leading to full conversion after 2 hours of reaction. In contrary to *P. eryngii* AAO, *UmaAAO* seems less efficient in converting DFF to FFCA in the reaction. However, the used concentration of *UmaAAO* in this study is significantly lower and a kinetic study is needed to determine the efficiency.
383 In the current *PciGLOX* tandem reaction, the yields of obtained FDCA were
lower compared to the ones obtained in the cascade reactions with UPO [18,
33]. A limiting step in the reaction of *PciGLOX* seems to be the conversion of
384
385
386 FFCA to FDCA and this was observed when FFCA was used as initial
substrate. This effect is most possibly due to exogenous factors affecting the
387
388 reaction since the enzymes exhibit relatively high specificity and catalytic
efficiency towards FFCA. A probable factor could be the inhibition of the
389
390 enzymes by the substrate and/or the product of the reaction. A second
possible factor affecting this reaction is the accumulation of H₂O₂ throughout
391
392 the first oxidation steps inhibiting the progress of the reaction in the FFCA
oxidation stage. The sensitivity of *PciGLOX* enzymes in the presence of H₂O₂
393
394 supports this hypothesis. It is also interesting to mention that this effect was
395 previously reported for *PostAAO* where the sensitivity of FFCA oxidation
reaction to H₂O₂ was 300-times higher than that of the initial reaction [33].
396
397

397

398 **Conclusion**

399 The potential of *PciGLOX* for the production of valuable furan derivatives from
HMF was investigated in this work. Interestingly, the three GLOX belonging to
400
the same organism and sharing high sequence similarity of 84-89%, showed
401
differences in their catalytic properties and product patterns on the same
substrates. Although known for their activity on aldehydes, these proteins
402
403
were also able to act on the alcohol group of the substrate. Previously, this
404
activity has been explained by the hypothesis that enzymes such as HMFO
405
for example recognizes the hydrated form of the aldehyde in the reaction
406
407 which explains their activity on DFF that rapidly forms hydrate in buffer pH 5-8
408 [19]. When the substrate has a carboxylic acid group in addition to the
409 aldehyde group such as in the case of FFCA, the formation of hydrate is
410 highly unfavorable and this explain the low activity of HMFO on FFCA [35,
411 19]. However, PciGLOX were active on FFCA and were found to efficiently
412 oxidize the aldehyde group of HMF. HMF was previously reported not to form
413 gem-diol under conditions similar to the ones used in this work [18]. This
414 shows that PciGLOX are able to recognize and oxidize both, aldehyde and, in
415 certain cases, alcohol groups. HMFCA remains the major product of the
416 reaction preventing the production of FDCA. However, the specificity of
417 PciGLOX towards producing HMFCA itself can be considered of high
418 biotechnological significance. The addition of catalase, to remove the
419 influence of self-inhibition through the production of H2O2, improved reaction
420 yields, while the addition of an AAO prior to the addition of PciGLOX, shifted
421 the reaction pathway through DFF and towards the production of FDCA, the
422 desired precursor for bioplastic synthesis.

423 MATERIALS AND METHODS

424 Chemicals and Enzymes

425 All chemicals were of analytical grade. HMF, DFF, HMFCA, 5-formyl-2-furan
426 carboxylic acid (FFCA), FDCA, 2,2'-azino-bis(3-ethylbenzothiazoline-6-
427 sulphonic acid (ABTS) and glyoxylic acid (GA), together with the enzymes
428 horseradish peroxidase (HRP) and catalase were purchased from Sigma
429 Aldrich (Lyon, France). PciGLOX1 and PciGLOX2 enzymes were
430 heterologously produced in Aspergillus niger as described previously [25].
431 The same protocol was used to produce the third isoform, *PciGLOX3*, and is reported here for the first time. AAO from *Ustilago maydis* was produced as previously reported in our laboratory [36]. The sequences of *PciGLOX1*, *PciGLOX2* and *PciGLOX3* are available in GenBank under accession numbers KU215437, KU215438 and MK268804, respectively.

436

437 Enzyme Activity Assay

438 The activity of the three *PciGLOX* enzymes was assayed in a coupled reaction with HRP following the protocol previously described [25]. When purified, GLOX enzymes are inactive and their oxidative activation was found to be possible in the presence of lignin peroxidase or HRP, or by the addition of strong oxidants such as molybdicyanide (*K*<sub>3</sub>Mo(CN)<sub>8</sub>), hexachloroiridate (*Na*<sub>2</sub>IrCl<sub>6</sub>), or *Mn*<sub>3+</sub> EDTA [24, 37]. HRP was used in this study since it is readily obtained commercially, highly stable under the conditions used, and does not act on any of the substrates under investigation for oxidation by GLOX. The reaction mixture consisted of 50 mM sodium tartrate buffer (pH 6) containing HRP (8 U), 0.1 mM ABTS, *PciGLOX* (1 µg) and GLOX substrate at varying concentrations depending on the reaction in 1 mL final volume. The lag period was eliminated by adding 5 µM H<sub>2</sub>O<sub>2</sub>. The reaction was initiated by the addition of the GLOX substrate and the oxidation of ABTS was followed at 436 nm for 1.5 minute. The standard assay was performed at 30 °C. All assays were performed in triplicate.

453

454 *PciGLOX3* Characterization
455 The substrate specificity of *PciGLOX3* on 10 mM of previously tested molecules (Table 1) with *PciGLOX1* and *PciGLOX2* was investigated following the activity test described above. The same standard assay was used to determine the kinetic constants for *PciGLOX3* on methylglyoxal (0.02 to 5 mM), glyoxal (0.1 to 7 mM), glyoxylic acid (0.1 to 10 mM), and glycerol (0.3 to 80 mM) as substrates. Lineweaver-Burk plots, obtained using the GraFit (version 4) program [38], were used to calculate the kinetic parameters.

462
463 The three *PciGLOX* sequences were aligned using the ClustalW program
464 within the MegAlign module (version11.0.0) of DNAStar software (Madison,
465 WI).

466
467 Enzyme Stability

468 The three *PciGLOX* were incubated at 30 °C and 800 rpm agitation for 48 hours in 50 mM sodium tartrate buffer pH6. Samples were taken after 2, 4, 24 and 48 hours and the residual activity was measured using GA as substrate and calculated as a percentage of the activity at time zero. The same experiment was also performed in the presence of 3 mM HMF or HMFCA to determine the stability of the enzymes in the presence of these molecules.
471
472
473
474 The stability of HRP was also assessed under the same conditions. All
475 measurements were performed in triplicates.

476
477 Kinetic Studies
478 The coupled reaction with HRP and ABTS under standard conditions was
479 used to determine the steady-state kinetic parameters for PciGLOX oxidation
480 of HMF (0.3-50 mM), DFF (0.01-2 mM), HMFCA (0.1-3 mM) and FFCA (0.2-5
481 mM). Kinetic parameters were obtained by fitting the data to the Michaelis-
482 Menten equation using R statistical software (R Core Team, Vienna Austria).

483

484 HPLC Analysis of Reaction Products

485 All oxidation reactions were performed at 30 °C under agitation at 800 rpm in
486 Eppendorf ThermoMixer tubes (Eppendorf, Montesson, France) over 24 hours
487 period in 100 mM tartrate buffer, pH6. To follow the reaction of the three
488 PciGLOX isoenzymes on HMF, DFF, HMFCA and FFCA over time, reaction
489 mixtures containing PciGLOX (20 µg), HRP (8 Units), ABTS (0.1 mM) and the
500 substrate (3 mM) were prepared. Samples were taken after 0, 0.5, 1, 1.5, 2, 3,
4, 5, 6, 8 and 24 hours and analyzed. Reactions containing the same
492 constituents except PciGLOX were followed for the same period of time and
493 used as controls. All measurements were performed in duplicates.

494 Reaction mixtures were separated on Aminex HPX-87H column (300 x 7.8
495 mm) (BioRad) at 45 °C, with 2.5 mM sulfuric acid as the mobile phase with a
496 flow rate of 0.6 mL/min. Eluted compounds were detected using a diode array
497 detector at 280 nm. The reactions were stopped by incubating the mixture at
498 90 °C for 10 min and centrifuging at 15000 xg for 15 minutes. The samples
499 were then filtered using 0.45µm polyvinylidene difluoride syringe filters
500 (Restek, Lisses, France) before injection in the column. Peak areas from the
501 obtained chromatograms were converted to molar concentration using

502 calibration curves of pure substrates and products standards.

503 The percentage conversion of the used substrate was determined according

504 to the following equation:

$$505 \%Conversion = \left(1 - \frac{\text{Concentration of substrate}}{\text{Initial concentration of substrate}}\right) \times 100$$

506 The percentage yield of the reaction products was determined according to

507 the following equation:

$$508 \%Yield = \left( \frac{\text{Concentration of product}}{\text{Initial concentration of substrate}} \right) \times 100$$

509

510 Effect of H₂O₂ on HMF Oxidation

511 The stability of the three PciGLOX isoenzymes in the presence of H₂O₂ was

512 assessed by measuring the residual activity of the enzymes after pre-

513 incubation with different concentrations of H₂O₂ over 24 hours. The H₂O₂ was

514 removed before adding the enzyme to the reaction mixture by washing the

515 samples with buffer in an Amicon ultrafiltration unit with a 10-kDa-molecular-

516 mass-cut-off membrane (Merck Millipore). All measurements were performed
517 in duplicate.

518 To determine the effect of hydrogen peroxide on the oxidation of HMF and its

519 derivatives by PciGLOX, the standard reaction was performed in the presence

520 of 10 µg catalase (2000 – 5000 Units/mg protein). The reaction was followed

521 over time as described above. A control reaction containing all the

522 components except PciGLOX was analyzed to determine the effect of

523 catalase on HMF oxidation.
524

525 **PciGLOX and UmaAAO Cascade Reactions**

526 The oxidation of HMF by UmaAAO in the presence and absence of 10 µg

527 catalase was followed. The reaction mixture contained UmaAAO (20 µg) and

528 HMF (3 mM) in 100 mM tartrate buffer, pH6. Samples were taken after 0, 0.5,

529 1, 1.5, 2, 3, 4, 5, 6, 8 and 24 hours and analyzed on HPLC. All measurements

530 were performed in duplicates.

531 UmaAAO was then used in a cascade reaction with PciGLOX3 for the

532 oxidation of HMF and its derivatives. The reaction was performed by reacting

533 UmaAAO with HMF for 2 or 24 hours before adding PciGLOX3. Following the

534 addition of PciGLOX3, the samples were further incubated for 4 or 24 hours

535 and then analyzed. The reactions were again performed in the presence and

536 absence of catalase.

537

538 **List of abbreviations**

539 HMF, 5-hydroxymethylfurfural; FDCA, 2, 5-Furandicarboxylic Acid; DFF,

43 diformylfuran; HMFCA, 5-hydroxymethyl-2-furancarboxylic acid; GAO,

44 galactose oxidase; AAO, aryl alcohol oxidase; UPO, unspecific peroxidase;

45 HMFO, HMF oxidase; GLOX, glyoxal oxidase; FFCA, 5-formyl-2-furan

46 carboxylic acid; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid;

48 GA, glyoxylic acid; HRP, horseradish peroxidase

49 carboxylic acid; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid;

50 GA, glyoxylic acid; HRP, horseradish peroxidase

51 carboxylic acid; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid;

52 GA, glyoxylic acid; HRP, horseradish peroxidase

53 GA, glyoxylic acid; HRP, horseradish peroxidase

54 GA, glyoxylic acid; HRP, horseradish peroxidase

55 GA, glyoxylic acid; HRP, horseradish peroxidase

545

58 59 60 61 62 63 64 65

64 65

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64 65
546 **Declarations**

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547 **Ethics approval and consent to participate**

548 Not applicable.

549 **Consent for publication**

550 Not applicable.

551 **Availability of data and materials**

552 The datasets used and/or analyzed during the current study are available

553 from the corresponding author on reasonable request.

554 **Competing interests**

555 The authors declare that they have no competing interests.

556 **Funding**

557 This project has received funding from the Bio Based Industries Joint

558 Undertaking under the European Union's Horizon 2020 research and

559 innovation program under grant agreement No. 720303 (project Zelcor), and

560 the FP7 program within the INDOX project (KBBE-2013-7-613549). We would

561 also gratefully acknowledge the research federation ECCOREV (ECosystème

562 CContinentaux et Risques EnVironnementaux) for providing a Masters grant to

563 BY, and to the French Embassy in Thailand for a Junior Research Fellowship

564 for SW.

565 **Authors' contributions**
566 MD, CBF and ER designed the experiments. MD and SW performed the production and biochemical characterization of *PciGLOX3*. MD and BY performed the reactions and HMF and the HPLC analysis. MD, EB, CBF and ER analyzed and interpreted the data and results. MD was the major contributor in writing the manuscript. CBF, EB, FD and ER provided critical feedback and helped in writing the manuscript. All authors read and approved the final manuscript.

573 Acknowledgements

574 Not applicable.

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704 [37] Whittaker MM, Kersten PJ, Nakamura N, Sanders-Loehr J, Schweizer ES, Whittaker JW. Glyoxal oxidase from Phanerochaete chrysosporium is a new radical-copper oxidase. J Biol Chem.1996;271:681-687.

707 [38] Leatherbarrow, RJ. GraFit version 4.0. Erithacus Software Ltd., Staines, 1998. United Kingdom.

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<table><caption>**Table 1.** Substrate specificity of *PciGLOX3* compared to previously characterized *PciGLOX1* and *PciGLOX2* (Daou et al. 2016).</caption><thead><tr><th rowspan="2">Substrate (10 mM)</th><th colspan="3">Activity (nkat/mg)</th></tr><tr><th>PciGLOX1</th><th>PciGLOX2</th><th>PciGLOX3</th></tr></thead><tbody><tr><td>Methyl glyoxal</td><td>814</td><td>291</td><td>90</td></tr><tr><td>Glyoxal</td><td>392</td><td>163</td><td>68</td></tr><tr><td>Glyoxylic acid</td><td>1562</td><td>384</td><td>59</td></tr><tr><td>3-phenylpropionaldehyde</td><td>10</td><td>135</td><td>51</td></tr><tr><td>Formaldehyde</td><td>50</td><td>202</td><td>133</td></tr><tr><td>DL-glyceraldehyde</td><td>11</td><td>66</td><td>26</td></tr><tr><td>Dihydroxyacetone</td><td>108</td><td>157</td><td>64</td></tr><tr><td>Glycerol</td><td>2</td><td>61</td><td>88</td></tr><tr><td>2,4-dimethoxybenzaldehyde</td><td>ND</td><td>2</td><td>ND</td></tr><tr><td>Veratraldehyde</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>4-hydroxybenzaldehyde</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>Phenyl glyoxilic acid</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>Formic acid</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>D-Glucose</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>D-Galactose</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>D-Xylose</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>Methanol</td><td>ND</td><td>ND</td><td>ND</td></tr></tbody></table>

723 ND, activity not detected under these assay conditions
724

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<table><caption>**Table 2.** Kinetic constants of *PciGLOX3* on Methylglyoxal, glyoxal, glyoxylic acid and glycerol compared to *PciGLOX1* and *PciGLOX2* (Daou et al. 2016).</caption><thead><tr><th rowspan="2">Substrate</th><th colspan="2">PciGLOX1</th><th colspan="2">PciGLOX2</th><th colspan="2">PciGLOX3</th></tr><tr><th>K<sub>m</sub> (mM)</th><th>K<sub>cat</sub>/K<sub>m</sub><br>(s<sup>-1</sup> mM)</th><th>K<sub>m</sub> (mM)</th><th>K<sub>cat</sub>/K<sub>m</sub><br>(s<sup>-1</sup> mM)</th><th>K<sub>m</sub> (mM)</th><th>K<sub>cat</sub>/K<sub>m</sub><br>(s<sup>-1</sup> mM)</th></tr></thead><tbody><tr><td>Methylglyoxal</td><td>1.3</td><td>58.4</td><td>0.2</td><td>7</td><td>0.1</td><td>75.8</td></tr><tr><td>Glyoxal</td><td>13.1</td><td>6.3</td><td>2.2</td><td>0.6</td><td>0.7</td><td>11.2</td></tr><tr><td>Glyoxylic acid</td><td>0.08</td><td>2136.3</td><td>0.1</td><td>17</td><td>0.5</td><td>15.3</td></tr><tr><td>Glycerol</td><td>660.5</td><td>0.04</td><td>9.4</td><td>0.06</td><td>5.5</td><td>1.4</td></tr></tbody></table>

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<table><caption>Table 3. Kinetic parameters for the oxidation of HMF, DFF, and FFCA by the three PciGLOX isoenzymes.</caption><thead><tr><th>Substrate</th><th></th><th>PciGLOX1</th><th>PciGLOX2</th><th>PciGLOX3</th></tr></thead><tbody><tr><td rowspan="3">HMF</td><td>K<sub>m</sub> (mM)</td><td>15.66</td><td>5.872</td><td>6.353</td></tr><tr><td>K<sub>cat</sub> (s<sup>-1</sup>)</td><td>2.59</td><td>0.56</td><td>0.75</td></tr><tr><td>K<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup> M<sup>-1</sup>)</td><td>101.66</td><td>96.04</td><td>118.35</td></tr><tr><td rowspan="3">DFF</td><td>K<sub>m</sub> (mM)</td><td>4.38</td><td>0.205</td><td>0.176</td></tr><tr><td>K<sub>cat</sub> (s<sup>-1</sup>)</td><td>0.54</td><td>4.80</td><td>1.28</td></tr><tr><td>K<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup> M<sup>-1</sup>)</td><td>124.39</td><td>23403.66</td><td>7267.72</td></tr><tr><td rowspan="3">FFCA</td><td>K<sub>m</sub> (mM)</td><td>0.85</td><td>1.404</td><td>0.608</td></tr><tr><td>K<sub>cat</sub> (s<sup>-1</sup>)</td><td>0.03</td><td>2.02</td><td>0.04</td></tr><tr><td>K<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup> M<sup>-1</sup>)</td><td>38.55</td><td>1435.45</td><td>72.03</td></tr></tbody></table>

<caption>Table 3. Kinetic parameters for the oxidation of HMF, DFF, and FFCA by the three PciGLOX isoenzymes.</caption>
**Table 4.** Percentage content of HMF, DFF, FFCA and FDCA in the cascade reactions of UmaAAO and PciGLOX3 following condition (1) that consisted of reacting UmaAAO with HMF for 2 hours before adding PciGLOX3 and condition (2) in which PciGLOX3 was added after 24 hours of the reaction of UmaAAO with HMF. Reactions were performed in the presence (C) and absence of catalase (NC) for both conditions. PciGLOX3 was reacted for 2 or 24 hours before analyzing the reaction products.

750

<table><thead><tr><th rowspan="3"></th><th colspan="4">Condition (1)</th><th colspan="4">Condition (2)</th></tr><tr><th colspan="2">After 2 hours</th><th colspan="2">After 24 hours</th><th colspan="2">After 2 hours</th><th colspan="2">After 24 hours</th></tr><tr><th>NC</th><th>C</th><th>NC</th><th>C</th><th>NC</th><th>C</th><th>NC</th><th>C</th></tr></thead><tbody><tr><th>HMF</th><td>ND</td><td>ND*</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><th>DFF</th><td>17%</td><td>ND</td><td>1%</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><th>FFCA</th><td>83%</td><td>96%</td><td>98%</td><td>95%</td><td>91%</td><td>88%</td><td>87%</td><td>84%</td></tr><tr><th>FDCA</th><td>ND</td><td>4%</td><td>1%</td><td>5%</td><td>9%</td><td>12%</td><td>13%</td><td>16%</td></tr></tbody><tfoot><tr><td colspan="9">*ND, values below 1 %</td></tr></tfoot></table>

751
752 **List of Schemes**

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753 **Scheme 1.** The oxidation reaction pathways of HMF to FDCA (Carro et al.

754 2015)

755 **List of Figures**

756 **Figure 1.** Custal W alignment of *P. cinnarbarinus* GLOX proteins. Identical

757 residues are highlighted in red. Arrows indicate residues essential for

758 catalysis

759 **Figure 2.** Residual activity of the three *PciGLOX* after incubation at 30°C and

760 800 rpm shaking in 50 mM tartrate buffer pH6 in the absence (a) and

761 presence (b) of 3 mM HMF, and in the presence of 3 mM HMFCA (c). The

762 residual activity was calculated as a percentage of the activity before

763 incubation.

764 **Figure 3.** Stability of the three *PciGLOX* in increasing concentrations of

765 hydrogen peroxide after an incubation period of 24 hours.

766 **Figure 4.** HMF oxidation reaction follow-up over time in the absence (left) and

767 presence (right) of catalase by (a) *PciGLOX1*, (b) *PciGLOX2* and (c)

768 *PciGLOX3*.

769 **Figure 5.** Follow-up of the reactions of *PciGLOX1*, *PciGLOX2* and *PciGLOX3*

770 on (a) DFF, (b) HMFCA and (c) FFCA as initial substrates.
771 **Figure 6.** Oxidation of HMF overtime by UmaAAO in the presence (A) and

772 absence (B) of catalase.

773

774 **Additional files**

775

776 **Additional file 1.** Residual activity of HRP on ABTS after incubation for

777 different time periods in tartrate buffer pH 6 at 30°C and 800 rpm in the

778 presence and absence of HMF or HMFCA.

779 **Additional file 2.** HPLC chromatogram of the reactions of (A) the three

780 *PciGLOX* enzymes on HMF after 24 hours of incubation and of (B) the

781 reaction of *UmaAAO* on HMF overtime.
PciGLOX1 -APSAHGRD LAERGIVALES VSPTRALWDS-SDD QENHAGALNET 58
PciGLOX2 APSTPTGRNLAERGIVALES VSPTRALWDS-TNDQENHAGALNET 59
PciGLOX3 -APSAHGRD LAERGIVALES VSPTRALWDS-SDD QENHAGALNET 58

<figure><img src="image_3.png" /></figure>

PciGLOX1 SVTRFNLNACRGALLERSKAVCGTRFPNPNINCNCAILTEPARTD 118
PciGLOX2 SVTRFNLNACRGALLERSKAVCGTRFPNPNINCNCAILTEPARTD 119
PciGLOX3 SVTRFNLNACRGALLERSKAVCGTRFPNPNINCNCAILTEPARTD 118

<figure><img src="image_4.png" /></figure>

PciGLOX1 CCTVFDQLLLEDRYPSIIIFDCGSLVQQTWNENYLHANFSEFFPKSS 178
PciGLOX2 CCTVFDQLLLEDRYPSIIIFDCGSLVQQTWNENYLHANFSEFFPKSS 179
PciGLOX3 CCTVFDQLLLEDRYPSIIIFDCGSLVQQTWNENYLHANFSEFFPKSS 178

PciGLOX1 PWSARLESPANLTFALDPKVMVANQSIYDIHANERLPTIINNVRYNI 238
PciGLOX2 PWSARLESPANLTFALDPKVMVANQSIYDIHANERLPTIINNVRYNI 239
PciGLOX3 PWSARLESPANLTFALDPKVMVANQSIYDIHANERLPTIINNVRYNI 238

PciGLOX1 ICGSAILLETIFVYIYVRCGTTSTPLD-SLASSQTATTCCSRKDEGAKGW 297
PciGLOX2 ICGSAILLETIFVYIYVRCGTTSTPLD-SLASSQTATTCCSRKDEGAKGW 298
PciGLOX3 ICGSAILLETIFVYIYVRCGTTSTPLD-SLASSQTATTCCSRKDEGAKGW 297

PciGLOX1 CVENLEPTKPHVLPGNIANGARGFANIKQDILNRSFVAPVTPK 357
PciGLOX2 CVENLEPDKPHVLPGNIANGARGFANIASDVGKNAHVLPVTPK 358
PciGLOX3 CVENLEPDKPHVLPGNIANGARGFANIASDVGKNAHVLPVTPK 357

Tyr377

<figure><img src="image_5.png" /></figure>

His378

PciGLOX1 AWLOCRISAAKSSGARVHSLIPGNEILARSNNGNTT--VGFVTPSSFW 415
PciGLOX2 AWLOCRISAAKSSGARVHSLIPGNEILARSNNGNTT--VGFVTPSSFW 416
PciGLOX3 AWLOCRISAAKSSGARVHSLIPGNEILARSNNGNTT--VGFVTPSSFW 415

His471

<figure><img src="image_6.png" /></figure>

PciGLOX1 CQLDTTFVFREKINTERANLKTFVYISVTTASPGAVVSMDLGTSAHAF 475
PciGLOX2 CQLDTTFVFREKINTERANLKTFVYISVTTASPGAVVSMDLGTSAHAF 476
PciGLOX3 CQLDTTFVSVREKINTERANLKTFVYISVTTASPGAVVSMDLGTSAHAF 475

PciGLOX1 SSARLVFMDATERRRLFTTPNGRYEPSPRTVFLTTGYTREAWXNGSGSP 535
PciGLOX2 SSARLVFNAKSGKBLRTFTTPNGRYEPSPRTVFLTTGYTREAWXNGSGSP 536
PciGLOX3 SSARLVFNGKSLRTFTTPNGRYEPSPRTVFLTTGYTREAWXNGSGSP 535

PciGLOX1 SSARLVFMDATERRRLFTTPNGRYEPSPRTVFLTTGYTREAWXNGSGSP 538
PciGLOX2 SSARLVFNAKSGKBLRTFTTPNGRYEPSPRTVFLTTGYTREAWXNGSGSP 539
PciGLOX3 SSARLVFNGKSLRTFTTPNGRYEPSPRTVFLTTGYTREAWXNGSGSP 538
<figure><img src="image_7.png" /><figcaption>a.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>b.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>c.</figcaption></figure>

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /></figure>
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>
<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>
<figure><img src="image_20.png" /><figcaption>A.</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_22.png" /></figure>
<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>
Click here to access/download

**Supplementary Material**

Daou et al COVER_LETTER.pdf
Click here to access/download
**Supplementary Material**
Additional file 1.pdf
Click here to access/download
**Supplementary Material**
Additional file 2.pdf