Glycosylation of Ganoderic Acid F by Bacillus Glycosyltransferase

: Ganoderma lucidum is a medicinal fungus and has been used for improvements of health or prevention of certain diseases in Asia for thousands of years. Despite numerous kinds of triterpenoids having been identiﬁed from G. lucidum , few natural Ganoderma triterpenoids exist in the form of glycosides (saponins). To expand the diversity of Ganoderma triterpenoids and ﬁnd rare Ganoderma saponins, ganoderic acid F (GAF), a Ganoderma triterpenoid, was biotransformed by a glycosyltransferase ( Bs GT110) from Bacillus subtilis ATCC (American type culture collection) 6633. The results showed that Bs GT110 catalyzed biotransformation of GAF to produce a metabolite, which was conﬁrmed as a GAF glucoside by mass–mass spectroscopy. The GAF glucoside showed 89-fold higher aqueous solubility than that of GAF. The present study highlights the utility of Bs GT110 in the production of novel Ganoderma triterpenoid saponins, and the newly identiﬁed and highly soluble GAF glucoside can be studied for its bioactivity in the future.


Introduction
Ganoderma lucidum (in Chinese, "Lingzhi") has been used as a nutrition supplement by Chinese for more than 2000 years. This fungus is used in the prevention of some diseases, including immunomodulatory and antitumor activities [1]. Many bioactive constituents have been isolated from G. lucidum, such as polysaccharides and triterpenoids. To date, more than 300 different triterpenoids have been isolated and identified from Ganoderma spp. [2]. Although many bioactive triterpenoids have been isolated from G. lucidum, few studies have focused on the biotransformation of Ganoderma triterpenoids. Among various biotransformations, glycosylation (the attachment of a bulky sugar group to triterpenoids) can improve physical and chemical stability and aqueous solubility and reduce the cytotoxicity of natural triterpenoids. Increasing aqueous solubility and decreasing cytotoxicity could expand the applications of the triterpenoids. However, Ganoderma contains more than 374 triterpenoids, while only three triterpenoid glycosides exist [1,2].
To expand the diversity of Ganoderma triterpenoids and find new Ganoderma saponins, BsGT110 was used to glycosylate ganoderic acid F (GAF), a Ganoderma triterpenoid, in the present study. One metabolite was produced and the structure and the solubility of the product were determined.

BsGT110 Biotransforming GAF
The previous study showed that BsGT110 could glycosylate the C-26 carboxyl groups of GAA [7] and GAG [8]. Thus, to produce rare Ganoderma saponins, the enzyme was evaluated for its glycosylation activity toward another Ganoderma triterpenoid, GAF ( Figure 1). GAF contains only one functional group for glycosylation, the C-26 carboxyl group, which is identical with those of GAA and GAG. The results showed that one metabolite, compound (1), appeared in the HPLC analysis after the biotransformation ( Figure 2). The result revealed that BsGT110 could catalyze biotransformation of GAF.
BsGT110 was the first identified GT catalyzing the glycosylation of a triterpenoid at the C-26 position. The study revealed that BsGT110 might have glycosylation activity toward other Ganoderma triterpenoids.
To expand the diversity of Ganoderma triterpenoids and find new Ganoderma saponins, BsGT110 was used to glycosylate ganoderic acid F (GAF), a Ganoderma triterpenoid, in the present study. One metabolite was produced and the structure and the solubility of the product were determined.

BsGT110 Biotransforming GAF
The previous study showed that BsGT110 could glycosylate the C-26 carboxyl groups of GAA [7] and GAG [8]. Thus, to produce rare Ganoderma saponins, the enzyme was evaluated for its glycosylation activity toward another Ganoderma triterpenoid, GAF ( Figure 1). GAF contains only one functional group for glycosylation, the C-26 carboxyl group, which is identical with those of GAA and GAG. The results showed that one metabolite, compound (1), appeared in the HPLC analysis after the biotransformation ( Figure  2). The result revealed that BsGT110 could catalyze biotransformation of GAF.

GAF Glucoside Produced
To determine whether the biotransformation product was the glycoside derivative of GAF, the biotransformation was scaled up to 10 mL. About 1.7 mg of the product in the 10 mL reaction was purified with preparative HPLC. The chemical structure of the purified compound (1)

GAF Glucoside Produced
To determine whether the biotransformation product was the glycoside derivative of GAF, the biotransformation was scaled up to 10 mL. About 1.7 mg of the product in the 10 mL reaction was purified with preparative HPLC. The chemical structure of the purified compound (1) was resolved with mass and mass-mass spectral analysis. The mass analysis of the compound showed an [M-H]ion peak at m/z: 731.5 in the electrospray ionization mass (ESI-MS) spectrum corresponding to the m/z signal of molecular weight 732 of GAF-glucoside (570+180−18) at the negative mode ( Figure S1a). In addition, two significant peaks appeared from mass-mass spectral analysis at m/z 551.5 and 179.0, which represented the signals of molecular weight 552 of GAF with loss of a H 2 O (570-18) and molecular weight 180 of glucose from the breakdown of compound (1) ( Figure S1b). Accordingly, compound (1) was assigned as a GAF monoglucoside. GAF is commercially available in tiny amounts, and only 1.7 mg of the GAF monoglucoside was isolated from the biotransformation. Therefore, the chemical structure of the GAF monoglucoside was not resolved by nucleic magnetic resonance spectral analysis in advance. However, GAF contains only one functional group for glycosylation, the C-26 carboxyl group, which is identical with those of GAA and GAG. Moreover, our previous study showed that BsGT110 could glycosylate the C-26 carboxyl groups of GAA and GAG. Taken all these results together, it was concluded that BsGT110 glycosylated GAF to a new Ganoderma triterpenoid saponin, GAF glucoside, whose chemical structure is predicted as GAF-26-O-glucoside. Figure 3 illustrates the assumed biotransformation of GAF by BsGT110 to produce GAF-26-O-glucoside.

GAF Glucoside Possessing High Aqueous Solubility
Previous studies have proven that the aqueous solubility of flavonoids can be improved through glycosylation [9], which would advance the oral bioavailability of the original molecules [10,11]. Thus, the aqueous solubility of GAF and the GAF glucoside were determined with the methods we used in a previous study [6]. The results showed that the aqueous solubility of the GAF glucoside was 89.2-fold higher than that of GAF (Table 1). Due to its high aqueous solubility, the produced novel Ganoderma saponin could have broader applications in biotechnology. Thus, it is worthy to study the bioactivities of the newly identified GAF glucoside in the future.  1 The mean (n = 3) is shown, and the standard deviations are represented by error bars. 2 The fold of aqueous solubility of GAF glucoside is expressed as relative to that of GAF, normalized to 1.

Enzymes and Chemicals
Recombinant BsGT110 enzyme was prepared from the previous study [6]. UDP-G

GAF Glucoside Possessing High Aqueous Solubility
Previous studies have proven that the aqueous solubility of flavonoids can be improved through glycosylation [9], which would advance the oral bioavailability of the original molecules [10,11]. Thus, the aqueous solubility of GAF and the GAF glucoside were determined with the methods we used in a previous study [6]. The results showed that the aqueous solubility of the GAF glucoside was 89.2-fold higher than that of GAF (Table 1). Due to its high aqueous solubility, the produced novel Ganoderma saponin could have broader applications in biotechnology. Thus, it is worthy to study the bioactivities of the newly identified GAF glucoside in the future.

Enzymes and Chemicals
Recombinant BsGT110 enzyme was prepared from the previous study [6]. UDP-G was obtained from Cayman Chemical (Ann Arbor, MI, U.S.A.). A total of 20 mg of GAF was purchased from Baoji Herbest Bio-Tech (Xi-An, Shaanxi, China).

In Vitro Biotransformation Assay
The in vitro biotransformation was conducted with the purified BsGT110. The reaction (0.1 mL) containing 25 µg of the tested enzyme, 1 mg/mL of GAF, 10 mM of UDP-G, 10 mM of MgCl 2 , and 50 mM of phosphate buffer (PB) at pH 6.0 was carried out at 40 • C for 24 h. After the reaction, the mixture was stopped by adding an equal volume of methanol and analyzed with high-performance liquid chromatography (HPLC).

HPLC Analysis
HPLC was performed with the Agilent 1100 series HPLC system (Santa Clara, CA, USA) equipped with a gradient pump (Waters 600, Waters, Milford, MA, USA). The stationary phase was a C18 column (5 µm, 4.6 i.d. × 250 mm; Sharpsil H-C18, Sharpsil, Bei-jing, China), and the mobile phase was 1% acetic acid in water (A) and methanol (B). The elution condition was a linear gradient from 0 min with 50% B to 20 min with 95% B, an isocratic elution from 20 min to 25 min with 95% B, a linear gradient from 25 min with 95% B to 28 min with 50% B, and an isocratic elution from 28 min to 34 min with 50% B. All eluants were eluted at a flow rate of 1 mL/min. The sample volume was 10 µL. The detection condition was set at 254 nm.

Isolation and Identification of the Biotransformation Product
To purify compound (1), the biotransformation reaction as described in Section 3.2 was scaled up to 10 mL. After reaction, compound (1) was purified by a preparative YoungLin HPLC system. The elution corresponding to the peak of compound (1) in the HPLC analysis was collected, condensed under a vacuum, and then dehydrated by freeze drying. Finally, 1.7 mg of compound (1) was obtained. The structure of the compound was confirmed with mass spectral analyses.

Determination of Solubility
The aqueous solubility of GAF and its glucoside were examined by the previous method [6]. An excess of GAF (1 mg/200 µL) and the purified glucoside (0.5 mg/20 µL) were mixed with double-deionized water by vertexing for 10 min at 25 • C, followed by incubation for 1 h in an ultrasonic bath (Branson, Danbury, CT, USA) to maximize the solubility of the compound. The mixture was then centrifuged and the supernatant was filtrated with a nylon membrane for HPLC analysis.

Conclusions
A new Ganoderma triterpenoid saponin, GAF glucoside, was produced and identified from the biotransformation of GAF by BsGT110, which was proven to be a promiscuous biocatalyst for production of novel Ganoderma saponins. The newly identified GAF glucoside possessed 89-fold higher aqueous solubility than that of GAF and might have unique bioactivities that could benefit the medicinal industry.