Oxalate Oxidase: Germin

Commonly referred to as Germin, oxalate oxidase is an enzyme that degrades oxalate into carbon dioxide and hydrogen peroxide through a redox reaction. This process is catalyzed by the binding of a manganese (Mn²⁺) ion at the enzyme's active site. Naturally occurring in Triticum aestivum (wheat), Germin plays a crucial role in the plant's defense mechanism by degrading oxalate produced by pathogenic fungi, thus preventing fungal infections.

In plants, the accumulation of oxalate can be harmful as it can chelate essential minerals like calcium, so the ability to degrade oxalate is vital for maintaining homeostasis.

Synthetic Biology Applications

In synthetic biology, oxalate oxidase has been codon-optimized for expression in Escherichia coli to ensure efficient production and functionality of the enzyme. Codon optimization increases the likelihood that the host organism (E. coli) can express the protein at high levels, avoiding issues of translational inefficiency due to rare codons.

Reaction Mechanism

The enzymatic reaction is initiated when oxalate binds to the active site. In the presence of oxygen, the oxalate undergoes oxidation to produce hydrogen peroxide (H₂O₂) and carbon dioxide (CO₂). This mechanism involves the donation of electrons from oxalate to oxygen, facilitated by the manganese ion, which acts as an electron mediator in the reaction.

The production of hydrogen peroxide is particularly significant in nature as it serves as an antimicrobial agent, contributing to the host's immune response against fungal invasion.

The reaction can be summarized as follows:

Oxalate + O₂ → CO₂ + H₂O₂ + O₂

Oxalate Oxidase Variants

Several variants of oxalate oxidase exist, with different levels of activity and substrate specificity. The wild-type form, commonly found in wheat, has been studied extensively for its potential applications in both agriculture and medicine.

Industrial and Biomedical Applications

Oxalate oxidase's ability to degrade oxalate makes it a promising candidate for treating hyperoxaluria, a condition where excess oxalate in the human body leads to the formation of kidney stones. Engineered strains of E. coli expressing this enzyme could serve as a probiotic treatment for breaking down oxalate in the human gut before it gets absorbed and accumulates in the kidneys.

Plasmid Construction

We ordered a premade pBAD plasmid from VectorBuilder containing our gene of interest, which was codon-optimized for E. coli. We utilized a pBAD plasmid with an araBAD promoter system and a selectable marker for ampicillin resistance, allowing for efficient selection of transformants.

Expression and Transformation

After receiving the plasmid, we transformed it into E. coli competent cells (strain TOP10). The cells were plated on LB agar plates containing ampicillin to select for transformants. Positive control plates were prepared using the same plasmid without any insert, and negative control plates were set up with no DNA.

Results

Transformation results were successful, as evident by the growth of E. coli colonies on all positive control plates and none on the negative controls.

We observed clear colony formation on the positive plates and no growth on negative controls, indicating the specificity of the antibiotic resistance marker.

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