Purification and Characterization of a Feruloyl Esterase from the Intestinal BacteriumLactobacillus acidophilus

Abstract

Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37°C, respectively). Ferulic acid (FA) is a common component in plant cell walls and shows strong antioxidant potential by its radical-scavenging ability (16). In recent years, the results of many in vitro and in vivo studies have indicated that FA prevents low-density lipoprotein from oxidation (2, 27, 29, 31), exhibits inhibitory effects on tumor promotion (1, 17), and protects against certain chronic diseases such as coronary heart disease and some cancers (6,20,21,37).In cereals, FA is mainly ester linked with arabinose or galactose residues (which constitute side chains of cell wall polysaccharides) (15,18,19,28,34,38). Wende et al. showed that FA was released from 2-O-␤-D-xylopyranosyl-(5-O-feruloyl)-Larabinose by rat gut microorganisms quite quickly and presumed that feruloyl esterase (FAE) would be produced by microorganisms, with high-level activity which is closely related to the release (40). Subsequently, other in vitro observations confirmed the existence of FAE in animal and human intestines (3, 23). These results suggest that FAE plays an important role in releasing free FA from different feruloylated forms in foodstuff in the gut.Although FAEs from some eukaryotic cells (4,7,11,12,14,22,26,39) and from only one prokaryotic cell (13) have been purified and characterized to date, there is no information about FAE from intestinal bacteria. It is reported that FAEs from various sources show different properties with respect to such characteristics as optimal temperature and optimal pH. Therefore, it is necessary to purify the FAE from human gut bacteria to further study the mechanism of release of FA from complex food matrices in vitro. Nishizawa et al. studied the FAE activities of typical intestinal bacteria and found that among the intestinal bacteria tested, Lactobacillus acidophilus exhibited the highest level of activity with respect to feruloylated arabinose ester (30).Some investigations indicate that the presence of xylanase enhances FAE activity. In most of these studies, however, destarched wheat bran was used as a substrate; these experiments could not supply sufficiently detailed information to explain the releasing mechanism of dietary FA and the interaction between xylanase and FAE to ...

Biochemical Properties of Two Cinnamoyl Esterases Purified from a Lactobacillus johnsonii Strain Isolated from Stool Samples of Diabetes-Resistant Rats

“…Lj1228 enzymatic activity was arrested with 1 mM of Zn 2ϩ , Fe 3ϩ , or Cu 2ϩ . Lj1228 was five times more sensitive to Fe 3ϩ than the enzyme purified from L. acidophilus (38). The addition of EDTA to the reaction mixtures did not affect the enzymatic activity of these enzymes.…”

A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters

“…These results confirmed that Est_1092 is an esterase able to hydrolyze hydroxycinnamic esters. Apart from the feruloyl esterase Lp_0796 described previously in L. plantarum strains (18), among lactic acid bacteria, only feruloyl esterases from L. johnsonii (12) and L. acidophilus (37) had been previously identified.…”

“…The buffers (100 mM) used were acetic acid-sodium acetate (pH 3 to 5), sodium phosphate (pH 6 to 7), Tris-HCl (pH 8), and glycine-NaOH (pH 9). The optimal temperature was assayed by incubating purified Est_1092 esterase in 50 mM McIlvaine buffer (pH 5.0) at different temperatures (5,20,30,37,40,45,55, and 65°C). For temperature stability measurements, the recombinant esterase was incubated in 50 mM McIlvaine buffer, pH 5.0, at 20, 30, 37, 45, 55, and 65°C for 5, 15, and 30 min and 1, 2, 4, 6, and 20 h. Aliquots were withdrawn at these incubation times to test the remaining activity under standard conditions.…”

Biochemical Analysis of a β- d -Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

“…The ethyl acetate layer was then transferred to a new tube and evaporated under N 2 gas, and the extracted ferulic acid was dissolved in 1 ml of 50% methanol. The concentration of ferulic acid was then determined by HPLC as described by Wang et al (67).…”