Folate analysis in complex food matrices: Use of a recombinant Arabidopsis ¨-glutamyl hydrolase for folate deglutamylation
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Natural folates have a ¿-linked polyglutamyl tail attached to their structure; this tail is removed enzymatically for quantitative analysis by ¿-glutamyl hydrolases (GGHs). Animal GGHs have been generally used for folate analysis; however, they can be inhibited by some extracts, hindering folate quantification. This study evaluated the use of two recombinant GGHs from Arabidopsis thaliana (AtGGH1, AtGGH2) to remove the tails of folylpolyglutamates from tomato fruit, black bean and peanut seeds, and alfalfa sprouts extracts. Their ability to hydrolyze was compared to the GGH present in rat plasma. AtGGH2 alone was able to completely deconjugate folates using 50-100¿g/g of sample for 1. h at 37°C in all matrices tested. Conversely, the conjugase from rat plasma recovered less than 30% monoglutamylated folates in legume seeds and 60% in tomato and alfalfa during the same incubation time. Rat plasma conjugase in addition showed differences in substrate specificity that were more evident in the presence of plant extracts. The use of a recombinant enzyme from plant origin is proposed as a better alternative to standardize folate analysis protocols. © 2013 Elsevier Ltd.