# Catalytic Potential of Cupin-Type Enzymes (Tm1010, Tm1459)
> Exploration of metal-dependent activity in cupin enzymes for biocatalysis, focusing on Mn²⁺ vs Cu²⁺ in HNL reactions and oxidative cleavage.

Tags: biocatalysis, enzymes, cupin-proteins, green-chemistry, biotechnology, tu-delft, metalloenzymes
## Master Thesis: Catalytic Potential of Cupin-Type Enzymes

This research investigates the catalytic activity of **Tm1010**, **Tm1459**, and **GtHNL** in various biocatalytic reactions, specifically focusing on the effects of manganese (Mn²⁺) and copper (Cu²⁺) substitution.

## Key Reactions Studied
*   **HNL Activity:** Cleavage of cyanohydrins to aldehyde and HCN.
*   **Oxidative Cleavage:** Transformation of alkenes to carbonyl compounds using TBHP.
*   **Nitroaldol (Henry):** C-C bond formation to create β-nitro alcohols.
*   **Michael Addition:** Conjugate addition to α,beta-unsaturated carbonyls.

## Structural and Binding Results
*   **Oligomeric State:** Tm1010 and Tm1459 were confirmed as stable dimers (~24-42 kDa), while GtHNL-wt showed partial dissociation which negatively impacted its performance.
*   **Binding Thermodynamics:** Tm1459 showed the most defined coordination. Cu²⁺ binding was generally enthalpy-driven, while native Mn²⁺ binding was entropy-driven.

## Performance Highlights
*   **Mn²⁺** was found to better preserve native HNL activity and reached up to **57% conversion** in oxidative cleavage benchmark tests.
*   **Cu²⁺** incorporation unlocked non-native reactivity, achieving **60-70% conversion** in Nitroaldol reactions, though with low enantioselectivity.
*   Michael addition remained a challenge across all variants with conversions under 8%.

## Conclusions
Structural stability and metal identity are primary drivers of catalytic performance. The study recommends protein engineering (e.g., C106L mutation in Tm1459) and optimized immobilization strategies for future biocatalytic applications.
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