Abstract Sulfur-containing aroma compounds, which are key determinants of the sensory profile and consumer acceptance of many foods, have received limited research attention regarding the liberation of these thiol aromas via exogenous enzymes. In this study, we characterized and successfully overexpressed an active cysteine-S-conjugate β-lyase (MetC) from the marine bacterium Shewanella putrefaciens, and we also established its optimal catalytic conditions. Kinetic analysis demonstrated effective activity of the enzyme toward natural substrates and aroma mercaptoketone precursors. Size-exclusion chromatography indicated a tetrameric structure for MetC. For most substrates, the apparent catalytic efficiency (<InlineEquation ID="IEq1"><EquationSource Format="TEX">${V}_{\text{m}\text{a}\text{x}}$</EquationSource><EquationSource Format="MATHML"><math><msub><mi>V</mi><mtext>max</mtext></msub></math></EquationSource></InlineEquation>/<InlineEquation ID="IEq2"><EquationSource Format="TEX">${K}_{\text{m}}$</EquationSource><EquationSource Format="MATHML"><math><msub><mi>K</mi><mtext>m</mtext></msub></math></EquationSource></InlineEquation>)app was calculated from the initial slopes of the velocity-concentration curves, which was used to evaluate catalytic efficiency due to solubility limits. Notably, only Cys-4MP-one reached saturation and conformed to sigmoidal model, indicating a unique cooperative behavior. These findings provide a quantitative basis for comparing the substrate specificity and catalytic effectiveness of MetC toward diverse precursors. The practical application of the enzyme was validated through preparative enzymatic conversion of cysteine-4-mercapto-4-methylpentan-2-one to 4-mercapto-4-methylpentan-2-one, with a 48.5% conversion achieved from its cysteine-conjugated precursor. Our findings demonstrate that S. putrefaciens MetC functions effectively as a biocatalyst for aroma thiol mercaptoketone production. Additionally, this research provides perspectives on the enzymatic production of sulfur-containing aroma compounds for applications in the fragrance and food industries. Key points • Eco-friendly enzymatic bioconversion produced aroma mercaptoketone. • Cysteine-S-conjugate β-lyase from Shewanella putrefaciens exhibited sigmoidal kinetics toward Cys-4MP-one, while other substrates followed linear kinetics. • A conversion of 48.5% was achieved for 4MMP during the preparative-scale biotransformation.

Cysteine-S-conjugate β-lyase: a green catalyst for the production of flavor-active mercaptoketones

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2026年7月3日

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Yi-Chi Huang,
Hao-Kai Li,
Yu-Lun Lin,
Hsi Ho,
Tze-Chia Lin,
Hong-Ting Victor Lin

摘要

Abstract

Sulfur-containing aroma compounds, which are key determinants of the sensory profile and consumer acceptance of many foods, have received limited research attention regarding the liberation of these thiol aromas via exogenous enzymes. In this study, we characterized and successfully overexpressed an active cysteine-S-conjugate β-lyase (MetC) from the marine bacterium Shewanella putrefaciens, and we also established its optimal catalytic conditions. Kinetic analysis demonstrated effective activity of the enzyme toward natural substrates and aroma mercaptoketone precursors. Size-exclusion chromatography indicated a tetrameric structure for MetC. For most substrates, the apparent catalytic efficiency (${V}_{\text{m}\text{a}\text{x}}$ $V_{max}$ /${K}_{\text{m}}$ $K_{m}$ )_app was calculated from the initial slopes of the velocity-concentration curves, which was used to evaluate catalytic efficiency due to solubility limits. Notably, only Cys-4MP-one reached saturation and conformed to sigmoidal model, indicating a unique cooperative behavior. These findings provide a quantitative basis for comparing the substrate specificity and catalytic effectiveness of MetC toward diverse precursors. The practical application of the enzyme was validated through preparative enzymatic conversion of cysteine-4-mercapto-4-methylpentan-2-one to 4-mercapto-4-methylpentan-2-one, with a 48.5% conversion achieved from its cysteine-conjugated precursor. Our findings demonstrate that S. putrefaciens MetC functions effectively as a biocatalyst for aroma thiol mercaptoketone production. Additionally, this research provides perspectives on the enzymatic production of sulfur-containing aroma compounds for applications in the fragrance and food industries.

Key points

• Eco-friendly enzymatic bioconversion produced aroma mercaptoketone.

• Cysteine-S-conjugate β-lyase from Shewanella putrefaciens exhibited sigmoidal kinetics toward Cys-4MP-one, while other substrates followed linear kinetics.

• A conversion of 48.5% was achieved for 4MMP during the preparative-scale biotransformation.