Catalytic Potential of Cupin-Type Enzymes (Tm1010, Tm1459)

MASTER THESIS DEFENCE | BIOCATALYSIS

Catalytic Potential of Tm1010, Tm1459, and GtHNL in Biocatalytic Reactions

Effects of Copper on Expression and Various Reactions

Mathijs Zoon

5406889

Supervisors: U. Hanefeld, J. Berger | Faculty of Applied Sciences, TU Delft

April 2026

Faculty of Applied Sciences | Delft University of Technology

INTRODUCTION | BIOCATALYSIS

The World of Enzymes: Nature's Molecular Machines

Enzymes are biological catalysts that speed up reactions without being consumed

Cupin-type enzymes: a superfamily with a remarkable beta-barrel fold

Metal-dependent activity: manganese (Mn²⁺) and copper (Cu²⁺) as key cofactors

GtHNL-wt, Tm1010 & Tm1459: our three cupin heroes from this study

Why it matters

Enzymatic reactions occur under mild conditions — green chemistry for a sustainable future

Tm1010

Tm1459

GtHNL-wt

Faculty of Applied Sciences | Delft University of Technology

Reactions & Research Question: Cu²⁺ vs Mn²⁺

HNL Activity

Cleavage of cyanohydrins to aldehyde + HCN

Oxidative Cleavage

Alkene → carbonyl compounds (with TBHP)

Nitroaldol (Henry)

C-C bond formation, β-nitro alcohols

Michael Addition

Conjugate addition to α,β-unsaturated carbonyls

How does metal identity (Mn²⁺ vs Cu²⁺) and incorporation strategy affect the structural integrity and catalytic performance of Tm1010, Tm1459, and GtHNL-wt?

Motivated by: previous studies showing Tm1459 catalyzes oxidative cleavage (Hajnal et al. 2015) and copper-cupin systems enable non-native C-C bond forming reactions (Fujieda et al. 2020)

Methodology: How We Answer the Research Question

Each method was chosen to link enzyme structure to catalytic function

Structural Analysis

Metal Quantification: ICP-MS

Binding Thermodynamics: ITC

Catalytic Activity Assays

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Two Strategies: Mn²⁺ vs Cu²⁺ Incorporation

Introduction — How We Make and Prepare the Enzymes

Both strategies were compared — Mn²⁺ expression + Cu²⁺ saturation was primarily used for reactions due to superior activity

Faculty of Applied Sciences | Delft University of Technology

RESULTS

Results: Expression & Purification

All three enzymes (GtHNL-wt, Tm1010, Tm1459) successfully expressed in E. coli

Protein bands visible after IPTG induction; absent before induction

French Press (FP) method yielded purer samples than Heat Shock (HS) alone

FP: less protein loss in pellet fractions

Target protein bands: ~15-16 kDa for Tm1459, ~15 kDa for Tm1010 & GtHNL-wt

FP method selected for all subsequent copper expression experiments due to superior purity

Faculty of Applied Sciences | Delft University of Technology

RESULTS

Results: Dialysis & PD10 Desalting

Faculty of Applied Sciences | Delft University of Technology

RESULTS

Results: Structural Analysis — Oligomeric State

SEC-HPLC Results

Tm1010

Apparent MW: 42.2 kDa | Monomer: 14.93 kDa

→ STABLE DIMER ✓

Tm1459

Apparent MW: 24.2 kDa | Monomer: 13.1 kDa

→ STABLE DIMER ✓

GtHNL-wt

Apparent MW: 19.0 kDa | Monomer: 14.29 kDa

→ PARTIAL DISSOCIATION ⚠️ (lower than expected for tetramer)

GtHNL-wt shows apparent instability under chromatographic conditions — dilution effects or protein-column interactions may cause dissociation. This may affect active site integrity.

Native PAGE Confirmation

Native PAGE confirms oligomeric assemblies — but GtHNL-wt discrepancy with SEC suggests dynamic dissociation in dilute conditions.

Key finding: Tm1010 & Tm1459 = stable dimers | GtHNL-wt = reduced oligomeric stability → impacts catalytic performance

Faculty of Applied Sciences | Delft University of Technology

RESULTS

Metal Binding — ICP-MS & ITC

Tm1459

Most defined metal binding. Enthalpy-driven Cu²⁺, entropy-driven Mn²⁺. Clear metalloenzyme behavior.

GtHNL-wt

Moderate, largely non-specific binding for both metals. Multiple surface sites.

Tm1010

Weak, poorly defined interactions for both metals. Lacks functional metal-binding site.

Structural stability (dimer) correlates with better-defined metal binding → better catalysis

Faculty of Applied Sciences | Delft University of Technology

Slide 9

RESULTS

Results: HNL Activity & Reverse HNL (Mandelonitrile Synthesis)

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RESULTS

Results: Oxidative Cleavage (GC Analysis)

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RESULTS

Non-Native Copper Reactions: Nitroaldol & Michael Addition

Nitroaldol (Henry) Reaction

Michael Addition

Faculty of Applied Sciences | Delft University of Technology

Slide 12

Conclusions & Recommendations

CONCLUSIONS

RECOMMENDATIONS

What Did We Find?

Where to Go Next?

Structural stability:

Tm1010 & Tm1459 = stable dimers. GtHNL-wt = partial dissociation → impacts catalytic performance.

Metal binding:

All enzymes bind Cu²⁺. Tm1459 shows most defined metal coordination. Tm1010 weakest binding.

Mn²⁺ preserves native HNL activity:

Cu²⁺ expression reduces HNL activity 4-10×. Mn²⁺-expression + Cu²⁺-saturation = optimal strategy.

Oxidative cleavage:

Tm1459-Mn²⁺ = 57% conversion (benchmark). Cu²⁺ generally reduces efficiency but timing of incorporation matters.

Nitroaldol (Henry):

Cu²⁺-bound enzymes achieve 60-70% conversion. Significant Cu²⁺ background reaction (~20%). Low enantioselectivity.

Michael addition:

Low conversion (<8%) across all systems. Protein scaffold enables moderate stereocontrol (up to 66% ee). Limited catalysis overall.

Overall:

Cu²⁺ unlocks non-native reactivity. Mn²⁺ = superior for native enzymatic function. Performance governed by structure, metal identity, and incorporation.

Optimize HNL conditions:

Use acidic pH (3.5-5) for reverse HNL to minimize background reaction and improve <i>ee</i>.

Improve immobilization:

Switch from Celite R-640 to R-633 (literature standard). Explore covalent immobilization to prevent leaching and deactivation.

Enhance enantioselectivity:

Protein engineering / directed evolution of active site residues (e.g., C106L in Tm1459). Focus on substrate positioning.

ITC optimization:

Improve Tm1010 binding experiments — signal quality was poor. Consider alternative buffer conditions.

Expand metal panel:

Test other divalent metals (Co²⁺, Zn²⁺, Ni²⁺) to further explore the catalytic versatility of cupin enzymes.

Flow chemistry:

Test Henry reaction in continuous flow with immobilized enzyme for improved process control.

Structural studies:

X-ray crystallography of Cu²⁺-loaded variants to directly visualize non-specific binding sites.

Faculty of Applied Sciences | Delft University of Technology

Thank You

Any Questions?

Mathijs Zoon

Master Thesis Biocatalysis | Student No: 5406889

Special thanks to U. Hanefeld, J. Berger, L. Koekkoek, M. Stampraat, S. Eustace, R. van Oosten, N. Karakitsou, and the entire BOC department

Faculty of Applied Sciences | Biocatalysis Department | Delft University of Technology | April 2026