Enhanced catalytic stability for L-Dopa synthesis through cross-linking of Al₂O₃ nanocrystals with Bacillus subtilis tyrosine hydroxylase

Abstract

This study focuses on enhancing L-Dopa synthesis by producing tyrosine hydroxylase from Bacillus subtilis SDSC-Env-i6 and its conjugation with aluminium oxide (Al₂O₃) nanoparticles. The free enzyme exhibited moderate activity, which was significantly improved through nanoparticle conjugation. Structural characterisation confirmed successful binding, with Al₂O₃ nanoparticles showing a crystalline nature and sizes ranging from 124 to 130 nm. Compared to the free enzyme, the Al₂O₃-conjugated tyrosine hydroxylase demonstrated markedly enhanced catalytic activity, reaching up to 15.3 ± 0.05 U/mL under optimised conditions. In contrast, maximum activities for the free enzyme were 5.5 ± 0.05 U/mL and 6.3 ± 0.11 U/mL in the presence of CuCl₂ and CaCl₂, respectively. The cross-linked enzyme also showed superior stability and efficiency across varying conditions. L-Dopa production was significantly higher with the conjugated enzyme, yielding between 0.531 and 1.105 mg/mL under optimised incubation conditions. These values were notably greater than those achieved using the free enzyme across different pH levels, volumes, and incubation times. Spectroscopic and microscopic analyses confirmed the integrity and functionality of the enzyme-nanoparticle complex. The observed improvements in both enzyme activity and L-Dopa yield were statistically significant ( p ≤ 0.05), underscoring the potential of Al₂O₃ nanoparticle-conjugated tyrosine hydroxylase as an efficient and scalable biocatalyst for commercial L-Dopa production. Determination of kinetic parameters showed maximum V max /K m (1.25 × 10 6 ) was observed when immobilized tyrosinase activity was characterized with tyrosine as substrate, with significant K cat value of 1.48 × 10 18 s − 1 .

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