Improved biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate by adding l-glutamine plus glycine instead of NAD+ in β-cyclodextrin–water system

• l-Glutamine and glycine were used for enhancing the reductase activity.
• Effective biotransformation was obtained in β-cyclodextrin–water media.
• Ethyl 4-chloro-3-oxobutanoate (3000 mM) was effectively biotransformed.
• Ethyl (S)-4-chloro-3-hydroxybutanoate (>99% ee) could be obtained at 98% yield.
• A possible biocatalytic mechanism in β-cyclodextrin–water system was proposed.

To reduce dependence on the expensive cofactor and effectively biotransform ethyl 4-chloro-3-oxobutanoate, l-glutamine and glycine were found to enhance the content of intracellular NADH and the reductase activity. Adding the mixture of 200 mM of l-glutamine and 500 mM of glycine to the reaction media, a 1.67-fold of reductase activity was increased over the control without the addition of the two compounds. Moreover, β-cyclodextrin (0.4 mol β-cyclodextrin/mol ethyl 4-chloro-3-oxobutanoate) was also added into this reaction media, and the biocatalytic activity of the whole-cell biocatalyst of Escherichiacoli CCZU-K14 was increased by 1.34-fold than that without β-cyclodextrin. In this β-cyclodextrin–water media containing l-glutamine (200 mM) plus glycine (500 mM), ethyl (S)-4-chloro-3-hydroxybutanoate (>99% ee) could be obtained from 3000 mM ethyl 4-chloro-3-oxobutanoate in the yield of 98.0% after 8 h. All the positive features demonstrate the potential applicability of the bioprocess for the large-scale production of ethyl (S)-4-chloro-3-hydroxybutanoate.

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