Integration of transcriptomic and proteomic data from Phlebodium aureum identifies a functional hydroxynitrile lyase

Area of Science:

• Biochemistry
• Enzymology
• Plant Science

Background:

• Hydroxynitrile lyases (HNLs) are crucial biocatalysts for synthesizing cyanohydrins stereoselectively.
• The first fern HNL was isolated from Phlebodium aureum, but its genetic sequence remained unknown, hindering its application.
• Elucidating the HNL coding sequence from P. aureum is essential for its biotechnological use.

Purpose of the Study:

• To identify and characterize the coding sequence of a hydroxynitrile lyase (HNL) from Phlebodium aureum (PhaHNL).
• To recombinantly express and functionally verify the enzymatic activity of PhaHNL.
• To determine the optimal conditions and kinetic properties of the newly identified fern HNL.

Main Methods:

• Transcriptome sequencing of Phlebodium aureum and integration with proteomic data to identify the HNL coding sequence.
• Recombinant expression of the identified PhaHNL isoform 1 in E. coli.
• Enzymatic assays to confirm HNL activity, determine optimal pH and temperature, assess stability, and analyze kinetics.

Main Results:

• The coding sequence for PhaHNL isoform 1 was successfully identified and characterized.
• Recombinantly expressed PhaHNL demonstrated hydroxynitrile lyase activity, converting mandelonitrile.
• Optimal activity was observed at pH 5.2 and 30 °C, with notable stability and positive cooperativity for mandelonitrile.

Conclusions:

• This study provides the first elucidated coding sequence for a hydroxynitrile lyase from Phlebodium aureum (PhaHNL).
• PhaHNL exhibits functional biocatalytic activity and favorable kinetic properties, making it a promising candidate for cyanohydrin synthesis.
• The findings significantly advance the understanding of HNLs in ferns and their potential biotechnological applications.