Category
Basic
Description
Peroxidases are found throughout nature and are valuable biocatalysts due to their versatility in catalyzing oxidative reactions using hydrogen peroxide as a co-substrate. The peroxidase from horseradish root is widely used in diagnostics and synthetic applications, however, plant peroxidases from other plant sources remain underexplored. Recently, a peroxidase from the skin of Cucurbita maxima (PKS) was isolated and demonstrated promising biocatalytic properties related to the decomposition of a wide variety of fluorinated aromatic compounds. Purification and characterization of this enzyme revealed a protein sequence that was similar to the well characterized HRP, with some key amino acid substitutions. A second enzyme from the skin of cucurbita moscate (butternut Squash) was also identified as having excellent potential, but it was not fully purified or characterized at the time. The primary objectives of the current study were (1) to generate a recombinant PKS enzyme via expression in E.coli and to purify active PKS enzyme from whole sale lysates, and (2) to develop a purification strategy to isolate the peroxidase from the skin of Cucurbita moscata (Butternut squash).
For objective 1: Following codon optimization, a synthetic gene for PKS was cloned into an expression vector under control of a T7 promotor, and the E.coli strain BL-21 DE3 was transformed with the vector. Expression of the PKS was demonstrated following induction with IPTG in a time dependent manner using SDS-PAGE, under initial growth conditions, active enzyme was not observed in the whole cell lysates. Subsequent analysis of the membrane fractions suggested the PKS enzyme formed inclusion bodies. Additional studies are planned to overcome this by varying growth temperature, aeration rate and inclusion of the heme precursor ALA. For Objective 2: A purification strategy for the BNS peroxidase was developed based on the protocol used previously for PKS An additional step, including the use of a final size exclusion chromatography step, produced pure enzyme. Initial characterization of the BNS enzyme with regard to ligand binding and catalysis indicated that the properties are virtually identical to the PKS enzyme. Blast analysis of the PKS protein identified 1 sequence in butternut squash with 97% homology.
Production, Isolation and Characterization of Novel Peroxidases from Cucurbita Family
Basic
Peroxidases are found throughout nature and are valuable biocatalysts due to their versatility in catalyzing oxidative reactions using hydrogen peroxide as a co-substrate. The peroxidase from horseradish root is widely used in diagnostics and synthetic applications, however, plant peroxidases from other plant sources remain underexplored. Recently, a peroxidase from the skin of Cucurbita maxima (PKS) was isolated and demonstrated promising biocatalytic properties related to the decomposition of a wide variety of fluorinated aromatic compounds. Purification and characterization of this enzyme revealed a protein sequence that was similar to the well characterized HRP, with some key amino acid substitutions. A second enzyme from the skin of cucurbita moscate (butternut Squash) was also identified as having excellent potential, but it was not fully purified or characterized at the time. The primary objectives of the current study were (1) to generate a recombinant PKS enzyme via expression in E.coli and to purify active PKS enzyme from whole sale lysates, and (2) to develop a purification strategy to isolate the peroxidase from the skin of Cucurbita moscata (Butternut squash).
For objective 1: Following codon optimization, a synthetic gene for PKS was cloned into an expression vector under control of a T7 promotor, and the E.coli strain BL-21 DE3 was transformed with the vector. Expression of the PKS was demonstrated following induction with IPTG in a time dependent manner using SDS-PAGE, under initial growth conditions, active enzyme was not observed in the whole cell lysates. Subsequent analysis of the membrane fractions suggested the PKS enzyme formed inclusion bodies. Additional studies are planned to overcome this by varying growth temperature, aeration rate and inclusion of the heme precursor ALA. For Objective 2: A purification strategy for the BNS peroxidase was developed based on the protocol used previously for PKS An additional step, including the use of a final size exclusion chromatography step, produced pure enzyme. Initial characterization of the BNS enzyme with regard to ligand binding and catalysis indicated that the properties are virtually identical to the PKS enzyme. Blast analysis of the PKS protein identified 1 sequence in butternut squash with 97% homology.
