# Tuning Chemoenzymatic Pd/Laccase Conformation Toward Optimized Heterogeneous Aerobic Oxidation

Fangfang Yang, Pierre Rousselot Pailley, Rénal Backov, Elise Courvoisier-Dezord, Agnès Amouric, Thierry Tron, Yasmina Mekmouche

► To cite this version:

Fangfang Yang, Pierre Rousselot Pailley, Rénal Backov, Elise Courvoisier-Dezord, Agnès Amouric, et al. Tuning Chemoenzymatic Pd/Laccase Conformation Toward Optimized Heterogeneous Aerobic Oxidation. ChemBioChem, 2024, 25 (3), 10.1002/cbic.202300781. hal-04460072

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# Tuning Chemoenzymatic Pd/Laccase Conformation Toward Optimized Heterogeneous Aerobic Oxidation

Fangfang Yang,[a,b] Pierre Rousselot Pailley,[a] Rénal Backov,[c] Elise Courvoisier-Dezord,[a] Agnès Amouric,[a] Thierry Tron[a] and Yasmina Mekmouche*[a]

[a] Dr. F. Yang, Dr. P. Rousselot Pailley, E. Courvoisier-Dezord, Dr. A. Amouric, Dr. T. Tron, Dr. Y. Mekmouche
Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, Marseille, France
E-mail: y.mekmouche@univ-amu.fr

[b] Dr. F. Yang
College of Chemistry and Chemical Engineering, Yantai University, 264005 Yantai, China

[c] Pr. R. Backov
Université de Bordeaux, CNRS, CRPP, UMR5031, 115 Avenue Albert Schweitzer, 33600 Pessac, France
Supporting information for this article is given via a link at the end of the document.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** The chemoenzymatic catalytic system. **A.** Structure of the Pd complex (LPdCl₂= complex 1). **B.** Structural models of laccase variants bearing a unique lysine (K) aminoacid residue highlighted in red for UNIK₇₁ and blue for UNIK₁₅₇. Coppers are represented by orange bullets. T1 oxidation center and trinuclear reduction center (TNC) are highlighted. **C.** Schematic representation of a hybrid 1<uniK (either UNIK₁₅₇ or UNIK₇₁) grafted to the NH₂ activated silica foam and oxidation of veratryl alcohol into veratryl aldehyde (VA). X: chloride or water.</figcaption></figure>

**Abstract:** Heterogeneous chemoenzymatic catalysts differing in their spatial organization and relative orientation of their enzymatic laccase and Pd units confined into macrocellular silica foams were tested on veratryl alcohol oxidation. When operating under continuous flow, we show that the catalytic efficiency of hybrids is significantly enhanced when the Pd(II) complex is combined with a laccase exhibiting a surface located lysine next to the T1 oxidation site of the enzyme.

## Introduction

The development of chemoenzymatic catalysts for the synthesis of chemical goods has gained a spectacular interest in the past few years.[1] Combining enzyme and metal or organocatalysts together or generating their hybrids often overcomes limitations

encountered when operated separately. More, catalysts combination often offers better reactivity compared to mono-catalytic processes, such as higher turnover numbers (TON), synergistic behavior, selectivity and conversion yields. Besides, the rational design of advanced materials is prompting a plethora

of 3D catalytic supports that allow the tuning of pore size and/or surface functionalization.[2] Among other examples, dual enzyme-metal nanoparticles (single enzyme molecules or cross-linked enzyme aggregates (CLEAs)) combined with metal nanoparticles,[3] zeolite nanocrystals-enzyme,[4] metal-organic framework (MOF)-enzymes[5] and artificial metalloenzyme[6] are certainly remarkable combinations of inorganic-enzyme catalysts. Beyond immobilization, the overall final assembly efficiency can be seriously hampered by random orientations of the nested catalysts with respect to their support surfaces, pointing the architecture of the hybrid as a major parameter to master
regarding the whole catalytic process. Similarly, the spatial
organization of the active centers relative to each other when
they are multiple is fundamental. A critical milestone is the
design of such sophisticated catalysts and how to address their
counterparts interplay ranging from the choice of enzymes,
catalysts, host supports and their functionalization to their
relative orientation and loading.[6],[7]

We have recently shown that immobilization of a preformed
laccase-(Pd complex) hybrid catalyst within the porous scaffold
of

<figure><img src="image_3.png" /><figcaption>**Scheme 1.** Description of the immobilization methods used in this study. Method **A**: Pd nanoparticle (PdnPs) immobilization prior to laccase immobilization. Method **B**: Ligand immobilization, Pd(II) salt post-immobilization prior to laccase immobilization. Method **C**: tuning of laccase loads (UNIK<sub>71</sub> or UNIK<sub>157</sub>), various complex 1-laccase ratios, low Pd(II) complex loading (no organic solvent). Method **D**: tuning of complex 1 load (in organic solvent), various complex 1: laccase ratios, mixed laccase immobilizations. Method **E**: Hybrid (1⊂UNIK<sub>71</sub> or 1⊂UNIK<sub>157</sub>) immobilization. Tuning of hybrids loading with a fixed 1:1 complex 1: laccase ratio</figcaption></figure>

NH₂-activated Si(HIPE) monoliths (High internal Phase Emulsion) (Fig. 1) allows the efficient oxidation of veratryl alcohol, a biomass model compound, into veratryl aldehyde (VA) in mild conditions over several catalytic cycles.[8] In such a construction, the Pd-complex ensures a metal-based oxidation of the substrate, while the laccase - a typical multicopper oxidoreductase[9] acts as a sink of electrons coming from a transiently formed Pd(0) species and in turn reduces O₂ into H₂O.[10] Hybrids (the Pd complex covalently linked to the enzyme) are stacked at the surface of the host macropores rather than embedded within their walls mesoporosity. Fungal laccases are typical multicopper oxidoreductase containing a surface located T1 site responsible for substrate oxidation connected to a trinuclear center (TNC) composed of a T2 copper site and a T3 pair of antiferromagnetically coupled copper ions.[9] Location variations of the graft at the surface of the enzyme influence the overall activity, resulting from the synergy between the catalytic centers. Changing their relative orientation to each other leads to substantial variations in alcohol oxidation. Thus, the site-directed grafting of a biquinoline-Pd complex (1) at a unique surface-located lysine (either K<sub>71</sub> or K<sub>157</sub>) of fungal laccase variants (UNIK<sub>157</sub> and UNIK<sub>71</sub>) either close to (1⊂UNIK<sub>157</sub>) or opposite to (1⊂UNIK<sub>71</sub>) the enzyme's oxidation center (noted T1) led to a 1⊂UNIK<sub>157</sub> twice as active as 1⊂UNIK<sub>71</sub> after immobilization. Postulating a minimal intermolecular electron transfer (ET) occurring between hybrid units during catalysis, we explained these variations as a consequence of the localization of the Pd center relative to the T1 center in these hybrids.[8]

To assess the efficiency of a chemoenzymatic system
immobilized in the cavity of a macroporous material, it is
necessary to explore the spatial organization of its components
in relation to each other and to the support. We compared five
different methods of covalent immobilization differing in the order
of immobilization of a Pd moiety and a laccase moiety (either
UNIK<sub>157</sub> or UNIK<sub>71</sub>), assuming that whatever the method in use,
the loading and the organization of the catalyst may affect the
catalytic event (Scheme 1). Here, we compare these

constructions' efficiency while catalyzing the aerobic oxidation of
veratryl alcohol into VA as a proof-of-concept model reaction
(Scheme 2). The catalytic efficiencies of the co-immobilized
moieties are compared with those of bare Pd. The impact of the
catalyst orientation/loading on the Pd/UNIK<sub>71</sub> and Pd/UNIK<sub>157</sub>
systems' performances is discussed.

**Results and Discussion**

Two methods commonly employed to immobilize palladium
into materials - as well-defined Pd nanoparticles or with the
help of an ancillary ligand - [11] were initially compared. Following
a procedure described by Bäckwall and coworkers,[12] highly
dispersed palladium nanoparticles (PdnPs) were
heterogeneously nucleated and grown within macrocellular
Si(HIPE), producing a first functionalized support labelled
hereafter as PdnPs@NH₂@Si(HIPE) (Scheme 1, Method **A**).
Characterizations of monoliths through scanning electron
microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy
and Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
were carried out to ascertain the elemental compositions and

<figure><img src="image_4.png" /><figcaption>**Scheme 2.** Oxidation of veratryl alcohol into veratryl aldehyde (VA).</figcaption></figure>

morphology of the PdnPs@NH₂@Si(HIPE) nanocomposite
(Fig.S1). Typical hollow spherical aggregated structures of
Si(HIPE) were found to be conserved and not affected by the
presence PdnPs as controlled by SEM (Fig.S1A). C, O, Si and
Pd elements were clearly observed at different energies in the
EDX spectrum (Fig.S1B). Quantification of the Pd content by
ICP-MS accounted for final loads of 0.4-0.6 mmol of Pd per g of
foam corresponding to a weight percentage of Pd of 6.3%, a
value comparable to the 8.25% previously reported.[12]
PdnPs@NH₂@Si(HIPE) monoliths were then tested toward their
ability to oxidize veratryl alcohol into VA in various conditions,
including in the presence of enzyme (Table 1). Turnover
numbers (representing the number of moles of product/ number of moles of catalyst) expressed relative either to Pd (TON<sup>Pd</sup>) or to enzyme (TON<sup>E</sup>, when synergy occurs), witness the catalytic performances for the different organizations tested (loading in Pd or laccase may be different by one order of magnitude). The synergy between Pd and laccase can be simply assessed by examining the TON of the bimolecular system (1/UNIK or 1<sub>c</sub>UNIK) relative to that of the bare Pd complex 1 (values above 1.0 attest of the synergy). This ratio will be noted Increase Fold (IF) for all comparisons.

Using water as solvent, immobilized PdnPs were active at a reaction temperature of 90°C (11 TON<sup>Pd</sup>) (Table 1, entry 1). A

<table><caption>**Table 1.** Experimental conditions used for aerobic alcohol oxidation with immobilized Pd particles (PdnPs) Method A.[a]</caption><thead><tr><th>Entry</th><th>Conditions</th><th>T/°C</th><th>Product<br>mM</th><th>TON<sup>Pd</sup>[b]</th><th>TON<sup>E</sup>[c]</th></tr></thead><tbody><tr><td>1</td><td>Water</td><td>90</td><td>14.7 ± 3.1</td><td>11.0 ± 2.3</td><td>-</td></tr><tr><td>2</td><td>Toluene</td><td>90</td><td>40.5 ± 5.5</td><td>30.4 ± 4.1</td><td>-</td></tr><tr><td>3</td><td>Buffer<sup>[d]</sup></td><td>25</td><td>3.8 ± 1.8</td><td>2.9 ± 1.4</td><td>-</td></tr><tr><td>4</td><td>UNIK<br>laccase<sup>[e]</sup>,<br>Buffer<sup>[d]</sup></td><td>25</td><td>1.6 ± 0.6</td><td>1.2 ± 0.5</td><td>na</td></tr></tbody></table>

[a] [substrate] = 100 mM in 15 mL solvent; PdnPs@NH2@Si(HIPE), 50 mg with 0.02 mmol PdnPs (1.3 mol% Pd); under air; t=24 hours for each cycle. For UNIK/PdnPs@NH2@Si(HIPE), 50 mg with 0.02 mmol PdNPs (1.3 mol% Pd) and 0.016 µmol (27.8 mg g-1) UNIK157 or UNIK71 0.012 µmol (20.2 mg g-1) used following a protocol described by Engström et al.;[13] under air; t=24 hours. [b] TONPd is calculated as the number of moles of product divided by that of Pd. [c] TONE is calculated as the number of moles of product divided by that of laccase. [d] 100 mM Sodium Acetate buffer, pH 5.7. [e] Either UNIK157 or UNIK71. Average of duplicates. na: not applicable since no synergy occurs between Pd and UNIK.

higher conversion of alcohol into VA (41 % yield, 30 TON<sup>Pd</sup>) was achieved using toluene as solvent (Table 1, entry 2). This was expected as the Pd zero-valent nanoparticles are rather hydrophobic, while the silica host has a good hydrophobic-hydrophilic balance despite bearing silanol groups. Although apparently less efficient than the system previously reported (yield 80-99%, TON<sup>Pd</sup> ≈ 65),[12] these results further attest that PdnPs are efficient while being loaded inside silica foam macroporosity,[14] Operating veratryl alcohol oxidation in reaction conditions favorable to enzyme activity (100 mM acetate buffer pH 5.7, 25°C), PdnPs@NH2@Si(HIPE) yielded only 3.8 mM of

aldehyde (3.8 % yield, 2.9 TON<sup>Pd</sup>) (Table 1, entry 3). Having verified that the buffer composition is not much influencing the PdnPs@NH2@Si(HIPE) activity (data not shown), the operational temperature appears to be the main factor. Under these conditions, the PdnPs@NH2@Si(HIPE) was used during three consecutive cycles with no loss of activity (Fig.S2).

UNIK<sub>157</sub> and UNIK<sub>71</sub> are genetically engineered laccase enzyme variants with a unique surface-accessible lysine residue, issued from the wild-type laccase LAC3 of the fungus *Trametes* sp. C30.[15,16] From our previous studies on laccase immobilization, i) immobilization proceeds *via* stable covalent interaction,[8],[17] ii) the surface lysine is probably the only exposed primary amino group that can be modified.[8],[15],[17] Post-immobilization of UNIK variants through Schiff-base-forming reactions into a previously synthesized PdnPs@NH2@Si(HIPE) yields a significant decrease in aldehyde production from 3.8 mM to 1.6 mM (Table 1, entry 3 & 4). Almost no catalytic activity is observed after a second cycle. Under similar conditions, Moradi *et al.* have reported a conversion yield of 45% in 10 h, a yield that jumped to 99% in the presence of hydroquinone.[18] If the presence of this mediator is obviously playing an essential role in the catalytic performance of their system, reaching a tenth of their yield in the absence of a mediator was puzzling. The reason may lie in using highly purified enzymes, whereas the use of a commercial laccase preparation without further purification would indicate that the pigments present in the preparation could act as mediators. Nevertheless, we can conclude that, in our case, the co-immobilization of PdnPs and laccase does not lead to synergy. Rather, the presence of the enzyme appears deleterious for the activity.

Alternatively, an ancillary ligand (i.e. a graftable biquinoline) was post-immobilized into NH<sub>2</sub>-activated Si(HIPE), namely NH<sub>2</sub>@Si(HIPE), prior to introducing a Pd salt [Pd(OAc)<sub>2</sub>] and then the enzyme (Scheme 1, Method B). Catalysis performed with the *in situ*-formed Pd complex yields only 20% of the aldehyde obtained previously with the immobilized PdnPs (Table 1, entry 1 vs. entry 3). When laccase (0.1:1 and 1:1 LAC:Pd ratio) is immobilized in turn, whatever the use of UNIK<sub>71</sub> or UNIK<sub>157</sub>, no activity is observed anymore (Table S1, entry 2). Therefore, as for Method A, the presence of laccase appears to be deleterious for the activity. As several examples of Pd salt interacting with peptides or proteins through cysteine and histidine residues have been reported in the literature, one can speculate on such interactions occurring within the foam.[19] If exogenous substrate cannot bind to the complex due to the

<figure><img src="image_5.png" /><figcaption>**Figure 2** Evolution of the catalytic performance with Method C (Panel a,b) and Method D (Panel c, d). Reaction in 100 mM acetate buffer pH 5.7 at 25°C. [substrate]= 100 mM; t = 24 hours. TON are calculated as the number of moles of product divided by that of complex 1 (TON<sup>Pd</sup>) or that of UNIK (TON<sup>E</sup>). Loadings are as follows for Method C: 5 and 40 eq. 1 vs UNIK (1.2 and 7.9 µmol g<sup>-1</sup> of 1, respectively, and 0.01 µmol g<sup>-1</sup> of UNIK). Loadings are as following for Method D: for 15, 5 and 3 eq. 1 vs UNIK (1.8 and 2.2 µmol g<sup>-1</sup> of 1 and 0.15, 0.3 and 0.6 µmol g<sup>-1</sup> of UNIK respectively); for 1.5 eq. 1 vs UNIK (0.5 µmol g<sup>-1</sup> of 1 and 0.3 µmol g<sup>-1</sup> of UNIK) and it is annotated as red dotted. IF (Increased Fold) in green is the number that defines the synergy between 1 and UNIK. IF = TON (1/UNIK) / TON (1).</figcaption></figure>
presence of more stable protein ligands, catalysis may not occur. In that case, the observed loss of activity could be the result of the inactivation of the catalyst by the enzyme, as previously observed.[10] Beyond this, with this sequential construction, it is even harder to predict the nature and position of the Pd responsible for the activity. We then focused the study on the immobilization of the Pd(II)-biquinoline preformed catalyst complex **1** (Fig. 1A) using different combination of laccase. Three immobilization strategies were investigated (Scheme 1, Method **C**, **D** and **E**). Technically, the complex **1**/laccase ratio varies as a consequence of the grafting order (Method **C**: **1** grafted prior to enzyme immobilization; method **D**: reversed order; method **E**: **1** grafted to the enzyme surface prior to enzyme immobilization), and this impacts catalysis. As previously described, immobilization of enzymes onto NH₂-activated silica foam by Schiff-base-forming reactions requires an initial modification of their unique lysine groups to produce aldehyde functionalized enzymes from glutaraldehyde (namely UNIK<sub>71</sub>-CHO and UNIK<sub>157</sub>-CHO).[17],[20] Stable site-directed covalent bonds were assigned through this immobilization process.

<table><caption>**Table 2.** Best catalytic performances obtained for veratryl alcohol oxidation for immobilization methods **C**, **D** and **E**[a]</caption><thead><tr><th>Entry</th><th>System</th><th>Method</th><th>1/UNIK ratio</th><th>TON<sub>Pd</sub><sup>[b]</sup><br>(TON<sup>E</sup>)<sup>[c]</sup></th><th>IF<sup>[d]</sup></th></tr></thead><tbody><tr><td>1</td><td>1</td><td></td><td>-</td><td>1.4 ± 0.1<br>(n.a.)</td><td>-</td></tr><tr><td>2</td><td>1 + UNIK<sub>71</sub></td><td>C</td><td>40.0</td><td>5.9 ± 0.4<br>(236 ± 16)</td><td>4</td></tr><tr><td>3</td><td>1 + UNIK<sub>157</sub></td><td></td><td>40.0</td><td>16 ± 1<br>(636 ± 40)</td><td>11</td></tr><tr><td>4</td><td>1</td><td></td><td>-</td><td>8.9 ± 1.0<br>(n.a.)</td><td>-</td></tr><tr><td>5</td><td>UNIK<sub>71</sub> + 1</td><td>D</td><td>5.0</td><td>28 ± 4<br>(130 ± 19)</td><td>3</td></tr><tr><td>6</td><td>UNIK<sub>157</sub> + 1</td><td></td><td>5.0</td><td>116 ± 21<br>(547 ± 96)</td><td>13</td></tr><tr><td>7</td><td>1</td><td></td><td>-</td><td>27 ± 3<br>(n.a.)</td><td>-</td></tr><tr><td>8</td><td>1 &lt; UNIK<sub>71</sub></td><td>E</td><td>1.0</td><td>89 ± 12<br>(89 ± 12)</td><td>3</td></tr><tr><td>9</td><td>1 &lt; UNIK<sub>157</sub></td><td></td><td>1.0</td><td>197 ± 30<br>(197 ± 30)</td><td>7</td></tr></tbody></table>

[a] [substrate] =100 mM in 15 mL acetate buffer (100 mM, pH 5.7), flow rate = 3 mL min⁻¹, 24 hours catalysis, 50 mg silica (0.8 mm diameter). [b] TON<sup>Pd</sup> is calculated as the number of moles of product divided by that of Pd. [c] TON<sup>E</sup> is calculated as the number of moles of product divided by that of UN. [d] Increase fold (IF) is defined as TON<sup>Pd</sup> of (1/UNIK)/TON<sup>Pd</sup> of (1). For Method C: [1] = 7.9 µmol g⁻¹; [UNIK<sub>71</sub>] = [UNIK<sub>157</sub>] = 0.2 µmol g⁻¹. For Method D: [1] = 1.8 µmol g⁻¹; [UNIK<sub>71</sub>] = [UNIK<sub>157</sub>] = 0.3 µmol g⁻¹. For method E [8]: [1] = [1 &lt; UNIK<sub>71</sub>] = [1 &lt; UNIK<sub>157</sub>] = 3 µmol g⁻¹.[8] na stands for not applicable. Average of triplicates.

With Method **C** (Scheme 1), over a high load of **1** (reached in organic solvent), mixed UNIK immobilization (either grafted directly to the NH₂@Si(HIPE) and/or to the surface exposed lysine of the enzyme unit) can be achieved owing to the bifunctional nature of the biquinoline ligand. Two **1**/UNIK ratios

were tested (5 and 40 equivalents of **1**/UNIK) (Fig. 2a-b, Table 2 entries 1-3, Table S2). With both loadings, the synergy between laccase and **1** is preserved within the foam. Despite reasonable TONs (45 ± 3 and 64 ± 1 TON<sup>Pd</sup> (225 ± 15 and 320 ± 5 TON<sup>E</sup>) respectively for **1**/UNIK<sub>71</sub> and **1**/UNIK<sub>157</sub>) a **1**/UNIK ratio of 5 leads to comparable IFs for both UNIK<sub>157</sub> and UNIK<sub>71</sub> (between ca. 3-5) (Fig. 2a-b). The best IFs are obtained with 40 equivalents of **1**: IFs of ca. 4 and 11 are observed with UNIK<sub>71</sub> and UNIK<sub>157</sub> respectively. Considering the enzyme as the limiting partner, the best TON is reached with the **1**/UNIK<sub>157</sub> combination (636 ± 40 TON<sup>E</sup>, 16 ± 1 TON<sup>Pd</sup>). We have previously shown that the laccase immobilization leads to two possible environments for the substrate T1 oxidation site, either “exposed” to the solvent (for UNIK<sub>71</sub>) or “hidden” at the enzyme/material surface (for UNIK<sub>157</sub>), thus influencing its oxidation strength, UNIK<sub>157</sub> being less active than UNIK<sub>71</sub> in the oxidation of ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), a typical laccase substrate.[17] In the present configuration (grafting of **1** to NH₂@Si(HIPE) then UNIK at a molar ratio of 40 and comparable enzyme loading), UNIK<sub>157</sub> appears to be more efficient when interacting with complex **1** than UNIK<sub>71</sub>. Therefore, despite a less favorable interaction with the material, ET between a transient Pd(0) formed during catalysis and the T1 oxidation site of UNIK<sub>157</sub> is favored. Nonetheless, due to the lack of enzyme grafting control with this method (either to the material or to **1**) as a result of the chosen loading sequence (grafting of **1** then UNIK) and reaction (Schiff-base), we reversed the grafting sequence order, i.e., the enzyme then complex **1**.

With Method **D** (Scheme 1), enzyme was first immobilized within the foam macroporosity prior to complex **1**, allowing thus a control of both the orientation of the enzyme and the ratio of complex **1** vs. laccase (Fig. 2c-d, Table 2 entries 4-6, Table S3). In any case, enzyme loading is catalytic as compared to **1** (15 to 1.5 times less than **1**). Synergy is maintained with both laccase orientation and loading, with the highest IF ever obtained for the proposed orientation of the UNIK<sub>157</sub>'s grafting (13 vs. 3 with UNIK<sub>71</sub>, Fig.2c, Table 2 entries 5-6). Laccase crowding seems to affect both the synergy and the catalytic activity, as previously shown.[15] This effect appears to be more consequential for UNIK<sub>157</sub> when combined with **1**. Beyond an optimal value (0.3 µmol g⁻¹ for both UNIK), over-increasing the enzyme population may promote enzyme-enzyme interactions with the risk of altering their intrinsic accessibility and inherent efficiency (Fig. 2 c-d, Table S3). The best activity is obtained with a UNIK:1 ratio of 1:5. TON's of 28 ± 4 TON<sup>Pd</sup> and 116 ± 21 TON<sup>Pd</sup> (130 ± 19 TON<sup>E</sup> and 547 ± 96 TON<sup>E</sup>) are obtained for UNIK<sub>71</sub>/**1** and UNIK<sub>157</sub>/**1** respectively. At this enzyme loading (0.3 µmol g⁻¹), decreasing complex **1** from 5 to 1.5 equivalents vs. enzyme, affects both the catalytic performance and the synergistic effect differently for the two enzyme orientations (Fig 2c,d). While the TON<sup>Pd</sup> is moderately affected for UNIK<sub>157</sub>, it is doubled for UNIK<sub>71</sub>. On the contrary, UNIK<sub>157</sub>'s IF is the most affected, with a drop from 13 to 6, while UNIK<sub>71</sub>'s IF remains unchanged: the IF for UNIK<sub>157</sub>/**1** appeared only 1.8-fold better than the one of UNIK<sub>71</sub>/**1** (instead of 4-fold at higher complex loading) (Fig.2c, Table S1). Despite a higher TON<sup>Pd</sup> with UNIK<sub>71</sub>, synergy
between **1** and UNIK<sub>157</sub> appears to be the most affected. A decrease of TON<sup>E</sup> is observed for both orientations with a larger extent for UNIK<sub>157</sub>: TON<sup>E</sup> drops from 547 ± 96 to 161 ± 5 for UNIK<sub>157</sub> (ca. 3.4-fold) and from 130 ± 19 to 88 ± 9 for UNIK<sub>71</sub> (ca. 1.5-fold) (Fig. 2c). Therefore, as observed for Method **C**, UNIK<sub>157/1</sub> appears as the best combination, supporting that ET to the T1 of UNIK<sub>157</sub> is favored. Additionally, only 5 equivalents of **1** vs. UNIK allow reaching a higher TON<sup>D</sup> and a comparable TON<sup>E</sup> (compared to 40 equivalents with Method **C**, Table 2, entry 3), thus reflecting a better control of enzyme immobilization for Method **D**.

With Method **E**,[⁸] as previously described, **1** was primarily grafted at the laccase surface by reductive amination of a single surface exposed lysine, then followed by a Schiff-based immobilization onto NH₂@Si(HIPE) foams, therefore fixing the **1**/UNIK ratio to **1**. With both hybrids, the catalytic activity of **1** was increased (increase fold IF = 3 and 7, respectively, for 1⊂UNIK<sub>71</sub> and 1⊂UNIK<sub>157</sub>) as compared to **1** alone indicating a promiscuity between the active metallic centers of both enzymatic and chemical partner within the foam (Table 2, entries 7-9). Despite a better control of Pd-ratio and location, the TON reached with these hybrids is lower than that obtained with other configurations. This probably reflects a lower flexibility of the construction than that proposed for Method **D**, where **1** and UNIK are both directly connected to the silica wall. In this case, it makes sense to propose that several complex **1** can be electronically connected to the T1 oxidation site of the enzyme.

## Conclusion

We rationally explored different approaches in our search for the best immobilized chemoenzymatic hybrid catalysts. We found that three out of the five proposed strategies lead to hybrids active for veratryl alcohol oxidation (Methods **C**, **D** and **E**). Parameters including catalyst loads, location of the Pd complex relative to the Cu active sites (T1 or TNC), were tested by using grafting point imprinted at the surface of the enzyme. Varying catalysts sequential loads and enzyme orientations, a fine-tuning of the catalyst efficiency is demonstrated. These results highlight the combination of **1** with UNIK<sub>157</sub> (either covalently linked together or not) as the most efficient one. This may be attributed to the fact that (i) lysine 157, the grafting point, and the T1 copper site are in close proximity addressing a cooperative action mode; (ii) the ET between transient Pd(0) produced during the catalytic event and the T1 copper site is favored rather than the TNC. We have to underline that this conformational study was possible as the silica host is macroporous and that all the catalytic sites and activities are occurring at the macropore vicinity, minimizing strong molecular hindrance while optimizing accessibility. Overall, this study illustrates that it is of first importance to tune every level of the catalytic system design to optimize the catalyst's performance and sustainability. This relies on the

important, although challenging, task of site-specific immobilization, which-preserve the intrinsic functions and favors specific properties of confined catalysts, thus setting grounds on the role of the "matrix support". Extension of this work will require determining the parameters driving an efficient architecture and obtaining insights on the confined enzyme structure at the material surface. Tailor-made strategies will be facilitated by investigating alternative routes for controlling neighboring hybrid catalysts within 3D materials.

## Experimental Section

Tetraethyl Tetradecyltrimethylammonium bromide (TTAB), dodecane, tetraethyl-orthosilane (TEOS), hydrochloric acid (37 %), glutaradehyde Grade I 25% in H₂O (wt.%) and (3-Aminopropyl)triethoxysilane (APTES) from Sigma. 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and veratryl alcohol were purchased from Fisher. All buffer solutions were made with deionized water. All other chemicals and reagents used in this study are of analytical grade and are used without any further purification. Si(HIPE) monoliths (HIPE stands for High Internal Phase Emulsion) were synthesized from Tetraethylorthosilane (TEOS), tetradecyltrimethylammonium bromide (TTAB), dodecane and hydrochloric acid (37%) with a protocol adapted from Carn *et al.*[21] The synthesis and characterization of NH₂@Si(HIPE) and glutaraldehyde activated Si(HIPE) were described in a recent paper from us.[15] UNIK variants were constructed from the wild type sequence of laccase LAC3 originally from the organism *Trametes* sp C30.[15] They were heterologously expressed in *Aspergillus niger* and purified according to previously published procedures.[16] Ligand **L** and complex **1** synthesis, the chemical modification of UNIK variants to produce UNIK<sub>71</sub>-CHO or UNIK<sub>157</sub>-CHO were performed and characterized according to previously published procedures.[8]

**PdnPs@NH₂@Si(HIPE) preparation:** PdnPs@NH₂@Si(HIPE) were synthesized with a protocol adapted from Johnston *et al.*[20a] In a typical experiment, the silica monoliths NH₂@Si(HIPE) obtained from the previous steps (approximately 50 mg each) were immersed in a K₂PdCl₄ solution in 4 mL deionized water (0.01 mol L⁻¹). The mixture was placed under dynamic vacuum for 30 min and then under static vacuum overnight. The foam turned brownish. It is then re-immersed in water (15 mL), followed by slow addition of a NaBH₄ suspended in water (0.1M, 5 mL), and placed under dynamic vacuum for another 1 hour. The foam turns dark, attesting for Pd reduction. After impregnation, the monoliths are washed with distilled water for three times. The presence of Pd particles was visualized by EDX and SEM (Fig. S1) and the Pd loading was determined by ICP-MS. The immobilized palladium particles are named as PdnPs@NH₂@Si(HIPE).

**Method A: UNIK immobilization into PdnPs@NH₂@Si(HIPE)**: Prior to enzyme immobilization, PdnPs@NH₂@Si(HIPE) monoliths were first activated with glutaraldehyde. PdnPs@NH₂@Si(HIPE) monoliths were
immerged in a DPBS (Dulbecco's phosphate-buffered saline: 137 mM Sodium chloride, 2.7 mM Potassium chloride, 8.1 mM Disodium hydrogen phosphate, 1.47 mM Potassium dihydrogen phosphate), pH7.6 solution with glutaraldehyde (0.625 % (w/w)). First, a dynamic vacuum and an overnight static vacuum were applied for impregnation. After washing with distilled water, the silica foams were dried at room temperature for 24 h. The resulting materials were then introduced in a solution containing UNIK<sub>71</sub> or UNIK<sub>157</sub> at a desired concentration in DPBS-pH7.6 and placed under dynamic (30 min) and static vacuum (overnight). After impregnation, the foams were then washed three times with DPBS buffer solution (3 mL, 3x10 min) to remove unbound enzyme. Enzyme loading was calculated by subtracting the detected enzyme in residual and wash solutions from the initial impregnation solution (based on the T1 copper absorption at 610 nm) after filtration.

**Method B: Ligand L immobilization and Pd salt post-immobilization followed by UNIK post-immobilization:** A NH₂@Si(HIPE) monolith (approximately 50 mg) was introduced in 10 mL CH₂Cl₂ with 50 mg of ligand L under vacuum for 10 minutes and kept the bottle sealed overnight (without vacuum). After that, the foam with ligand was taken out of the CH₂Cl₂ and washed three times with CH₂Cl₂ (3× 3 mL). Ligand immobilization was estimated to be 40-45 % of the initial load. The foam with the ligand was dried at 80 °C overnight. Then the foam was introduced in 4 mL of CH₂Cl₂ containing 7.0 mg (0.031 mmol), Pd(OAc)₂ salt under vacuum for 10 minutes, and the bottle was sealed overnight (without vacuum). Finally, the foam was washed with CH₂Cl₂ (3×3 mL) and water (3×3 mL). The resulting materials were then introduced in a solution containing UNIK<sub>71</sub> or UNIK<sub>157</sub> at a desired concentration (1000 mg L⁻¹) in DPBS-pH 7.6 for enzyme immobilization, followed by washing and quantification as described for method A.

**Method C: Complex 1 immobilization, then UNIK post-immobilization:** (i) *Pd complex 1 immobilization:* In a typical experiment, a NH₂@Si(HIPE) monolith (approximately 50 mg) was introduced in a mixture containing 3 mL of DMSO and palladium complex at a desired concentration (higher than 100 µM). When the initial palladium concentration is lower than 100 µM, the solvent for impregnation is 90% DPBS+10% DMSO. The mixture was placed under dynamic vacuum in a desiccator for 30 mins and then under static vacuum overnight. After impregnation, the monoliths were washed with DMSO (3 mL, 3x10 min) to remove the excess palladium complex. Palladium complex loading was calculated by subtracting the detected palladium in the residual and washing solutions from the initial impregnation solution (based on the absorbance at 340 nm). The bound amount was verified by the quantification of palladium ions using ICP-MS. The presence of the complex is evaluated by FTIR (Fig. S3) The immobilized complex was named as 1@NH₂@Si(HIPE) and further used for hybrid catalyst immobilization. (ii) *Glutaraldehyde functionalization and UNIK post-immobilization:* 1@NH₂@Si(HIPE) monoliths were then immersed in DPBS containing glutaraldehyde (0.625 % (w/w)). As stated earlier, a dynamic vacuum firstly and an overnight static vacuum were applied for impregnation. After washing with distilled water, the silica foams were dried at room temperature

for 24 h. The resulting materials were then introduced in a solution containing UNIK<sub>71</sub> or UNIK<sub>157</sub> at a desired concentration (1000 mg L⁻¹) in DPBS for enzyme immobilization, then followed by washing and quantification as previously described. Enzyme loading was calculated by subtracting the detected enzyme in residual and wash solutions from the initial impregnation solution (based on the T1 copper absorption at 610 nm) after filtration. Quantification of bound copper ions was further detected by ICP/MS (Table S2). The presence of the laccase was further identified by FTIR (Fig. S4).

**Method D: Primary UNIK immobilization and complex 1 post-immobilization:** (i) *UNIK immobilization:* The NH₂@Si(HIPE) (approximately 50 mg) is introduced in a mixture containing 3 mL of DPBS, pH 7.0 and the UNIK-CHO at a desired concentration. The mixture was placed under dynamic vacuum in a desiccator for 30 mins and then under static vacuum overnight. The foams are then washed three times with DPBS buffer solution (3 mL, 3x10 min) to remove the excess enzyme. Different initial enzyme concentrations ranging from 50 mg L⁻¹ to 2000 mg L⁻¹ for UNIK<sub>157</sub> and UNIK<sub>71</sub> are used for immobilization. Enzyme loading was calculated by subtracting the detected enzyme in residual and washing solutions from the initial impregnation solution (based on the T1 copper). The residual and wash solution were filtered before detecting concentration. Quantification of bound copper ions was further detected by ICP/MS. (ii) *Complex 1 post-immobilization:* The Si(HIPE) foam with enzyme was then introduced in 100 µM or 20 µM complex 1 solution (10% DMSO+90% DPBS). Palladium complex loading was calculated by subtracting the detected palladium from the residual and wash solutions from the initial solution (based on the absorbance at 340 nm). Quantification of bound Pd ions was further detected by ICP/MS (Table S3). The morphology of the foam after immobilization is observed by SEM (Fig. S5). The presence of the laccase was further identified by FTIR (Fig. S6).

**Method E: Primary complex 1 grafting to UNIK and post-immobilization.** This immobilization method has been extensively described in a recent paper from us.[8]

**Typical Alcohol oxidation assay:** In a typical heterogeneous condition experiment,[8] the catalytic performance was performed with a recycling reactor containing 15 mL of 100 mM veratryl alcohol in 100 mM acetate buffer pH 5.7 cycling at a flow rate of 3 mL min⁻¹ at room temperature. Samples were collected for analysis after filtration through a 0.22 µm filter then loaded onto a C18 nucleosil column (300 Å, 250*4.6 mm) after quenching with one volume of an acetonitrile solution containing 2 mM benzophenone as a standard for quantification. Experimental details for elution and analysis are provided in the supporting information. Veratryl alcohol was converted into veratryl aldehyde only. No other oxidation products were observed.

## Supporting Information

All other characterization data, original spectra are provided in the Supporting Information. Additional references cited within the Supporting Information.[22-25]
Acknowledgements

The authors would like to thank the Chinese Scholarship Council, the Frenchbic and ED250 doctoral school for financial support.

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Entry for the Table of Contents

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

Different spatial organizations of a heterogeneous chemoenzymatic Pd/Laccase system were tested for synergistic veratryl alcohol oxidation. Best catalysts are obtained when the Pd complex is combined with a laccase exhibiting a surface-located lysine next to the T1 oxidation site with two different orders of immobilization (①②). This work sets the ground for controlling the immobilization of a Pd/laccase/silica foam hybrid catalyst.

Institute and/or researcher Twitter usernames: @ism2_fr and @YasminaMekmouc1