Recombinant Arabidopsis y-glutamyl Hydrolase for Superior Deglutamylation

In order to test for natural folates in food products, scientists use pteroylpoly-γ-glutamyl-hydrolases (GGHs) to cleave the polyglutamyl tail so that the folates present as monoglutamylated (MG) species for accurate quantification. Unfortunately, most GGHs are sourced from animals and require complicated preparations to accommodate inhibitory food matrix effects in plant extracts. This can result in cumbersome protocols and inaccurate results.

Recently, Ramos-Parra et al.¹ proposed an alternative method for folate quantification that relies upon recombinant plant GGHs obtained from Arabidopsis thaliana (AtGGH1 and AtGGH2). Since these are plant-derived, they may escape inhibition by plant food extracts as seen with those sourced from animals. The team compiled data on AtGGH1/2 deglutamylation of red ripe tomato fruit (Lycopersicon esculentum), black bean seeds (Phaseolus vulgaris), and alfalfa sprouts (Medicago sativa) and compared the outcomes to rat plasma conjugase.

To do this, the researchers transformed Escherichia coli plasmids with expression vectors containing AtGGH1 and AtGGH2. The yield for the recombinant AtGGHs was 3 to 6 mg. SDS-PAGE revealed 80-85% purity. Then, they homogenized 1 g of tomato fruit or alfalfa sprout tissue, added folate extraction buffer (10 mL) and used a boiling water bath for enzyme deactivation and the release of protein-bound folates. They centrifuged the samples, separated and flushed the supernatants, and resuspended the pellet in extraction buffer (5 mL) before re-boiling and re-centrifuging. After combining the supernatants, the team flushed with N₂ and incubated with GGH treatments. Enzyme concentrations per assay varied with a range of 5 to 100 μg for AtGGHs and 29 to 58 mg for rat plasma. The researchers adjusted the pH to 6.0 for samples treated with AtGGHs and maintained it at pH 7.9 for samples treated with rat plasma.

The black bean seeds required slight modifications to this protocol due to the complexity of the matrix. They were pulverized in a mortar rather than homogenized. After boiling, the researchers added protease (47 U/g) by incubation followed by boiling for enzyme deactivation. They also added  α-amylase (3000 U/g) during deglutamylation. The team used an equilibrated solid-phase extraction cartridge and folate-binding column before separation by HPLC and analysis via a four-channel electrochemical detector (Thermo Scientific).

Ramos-Parra et al. found that total folate values were 14% higher than USDA reported values for alfalfa sprouts, on level with previously reported values for black beans, and variable for tomato fruits. The value for the tomato samples was on level with previously reported values for cherry tomatoes in general but twice as high as those reported for the specific variety. Discrepancies are likely results of not only the quantification method but also differences in growing seasons, storage, and sampling times.

Overall, this study offers a compelling alternative for the deglutamylation of folates in the biological matrices of plant extracts. AtGGH2 in particular outperformed the conjugase derived from rat plasma under these experimental conditions. Using 25 μg/g samples of black bean seeds and 50 μg/g samples of tomato fruits and alfalfa sprout extracts, AtGGH2 completely hydrolyzed the folates to MG forms within a one-hour incubation period. In comparison, the rat plasma conjugase only hydrolyzed 28, 55, and 57% of the these samples, respectively, during the same incubation period. The researchers suggest further investigation into whether this finding can be generalized to other animal conjugases, as well.

Reference:

1 Ramos-Parra, P.A. et al. (2013) ‘Folate analysis in complex food matrices: Use of a recombinant Arabidopsis γ-glutamyl hydrolase for folate deglutamylation.’ Food Research International, 54, 177–185.