Production of Hesperetin Using a Covalently Multipoint Immobilized Diglycosidase from <b><i>Acremonium</i></b> sp. DSM24697

Abstract

The diglycosidase α-rhamnosyl-β-glucosidase (EC 3.2.1.168) from the fungus Acremonium sp. DSM24697 was immobilized on several agarose-based supports. Covalent multipoint immobilization onto glyoxyl-activated agarose was selected as the more stable preparation at high concentration of dimethyl sulfoxide (DMSO) and high temperature. The optimal conditions for the immobilization process involved an incubation of the enzyme with agarose beads containing 220 μmol of glyoxyl groups per gram at pH 10 and 25°C for 24 h. The hydrolysis of hesperidin carried out in 10% v/v DMSO at 60°C for 2 h reached 64.6% substrate conversion and a specific productivity of 2.40 mmol h^-1 g^-1. Under these conditions, the process was performed reutilizing the catalyst for up to 18 cycles, maintaining >80% of the initial activity and a constant productivity 2.96 ± 0.42 µmol^-1 h^-1 g^-1. To the best of our knowledge, such productivity is the highest achieved for hesperetin production through an enzymatic approach.

Effect of Hesperidin on Cardiovascular Disease Risk Factors: The Role of Intestinal Microbiota on Hesperidin Bioavailability

“…Citrus flavanones are resistant to stomach and small intestine enzymes and thus reach the colon intact. There, intestinal microbiota activity breaks down the hesperidin molecule, releasing the aglycone form, named hesperetin [28,29] (Figure 1). Once inside the intestinal epithelium, hesperetin is released into the bloodstream in form of glucuronide and sulfatate conjugates [30].…”

“…Some of them are related with the food matrix and the physical form in which they are ingested (e.g., juice, soluble extract or capsules, among others), processing methods and storage techniques, as well as the structure of the compound and the host intrinsic characteristics, including intestinal microbiota composition [18]. All these factors affect the solubility of flavanones and their uptake by the gastrointestinal tract [28,29].…”

Flavonoids as Aglycones in Retaining Glycosidase-Catalyzed Reactions: Prospects for Green Chemistry

“…αRβG I was covalently immobilized on glyoxyl-activated agarose, resulting in a catalyst that was repeatedly used in 2-h conversions in the presence of 0.52 mM hesperidin and 10% (v/v) dimethyl sulfoxide at 60 °C. The catalyst could be reused for 15 cycles without significant loss of activity; a substrate conversion rate of 65% and a productivity of 3 μmol (g immobilized catalyst · h) −1 per cycle was obtained corresponding to 2 mmol (g enzyme · h) −1 . Complete hydrolysis of mostly undissolved rutin at 185 g L –1 (0.3 M) in the absence of cosolvents was reported for two purified recombinant β-rutinosidases with productivities of 357 and 149 mmol (g enzyme · h) −1 calculated for rutin conversions of 20% and 70%, respectively .…”

“Sweet Flavonoids”: Glycosidase-Catalyzed Modifications

“…The hesperidin-specific rutinosidase from Acremonium sp. was immobilized on several chitosan and agarose-based supports to stabilize the enzyme against elevated temperature and high concentration of dimethyl sulfoxide, enabling to reutilize the enzyme for up to 18 cycles of hesperetin production [ 101 , 102 ]. This enzyme was also employed in the development of the enzymatic-spectrophotometric method for quantification of hesperidin in raw orange juices by the hydrolysis of hesperidin to hesperetin and measuring the product’s absorbance in the UV range at 323 nm without the need of flavonoid extraction [ 103 ].…”