Effect of Glycosylation of β-Glucuronidase on its Catalytic Properties in Ionic Liquids

Licorice, the root of Glycrrhiza spp. (Fabaceae), has been used for thousands of years in China. In traditional Chinese medicine, licorice is one of the most frequently used drugs1. Glycyrrhizin (GL), an important triterpenoid saponin2, is the main active pharmacological ingredient of licorice. GL has anti-inflammatory, anti-ulcerous, anti-viral, and anti-allergic efficacy, and is also commonly used as sweetener and toner in the food industry. GL has two derivatives: glycyrrhetic acid 3-O-mono-b-D-glucuronide (GAMG) and glycyrrhetic acid (GA), which can be produced by hydrolyzing one or two glycosidic bonds from GL. GAMG is attractive due to its stronger physiological functions in comparison with GL, and its sweetness is 5-fold higher than that of GL with lower calories3. In addition, GA can be used as an additive in cosmetics due to its scavenging ability of oxygen free radicals. Therefore, GAMG and GA are expected to be better food additives as well as therapeutic agent, which has more commercial potential than GL4. In our previous work, we screened a fungal strain Penicillium purpurogenum Li-3, which used GL as a carbon source and converted it to GAMG by the secreted b-glucuronidase5. The gene was cloned (GenBank Accession No. EU095019) and overexpressed in Pichia pastoris GS115 for the high production of b-glucuronidase. Although a large amount of N-glycosylated PGUS-P wasproduced, the glycan content of PGUS-P was estimated to be 14.42 %6. Glycosylation is one of the major naturally occurring modifications of protein’s structure in eukaryotic cells, and the attached carbohydrate chains plays an integral role in the functional properties of glycoproteins7–10. Basically, there are two different types of protein glycosylation: N-glycosylation, at asparagine residues within the consensus sequence Asn-X-Ser/Thr and O-glycosylation, at hydroxyl groups of serine and threonine residues11,12. It has been enumerated that over half of the proteins present in nature are glycosylated, with more than three-quarters of these glycoproteins containing N-linked carbohydrates13. Scientists have assessed that glycosylation plays an important role in maintaining the thermostability and activity of proteins14–16. Effect of Glycosylation of β-Glucuronidase on its Catalytic Properties in Ionic Liquids