**3. Discussion**

According to the Carbohydrate-Active Enzymes (CAZy) database, GTs can be classified into 107 families, in which GTs that catalyze the glycosylation of small molecules, such as flavonoids and triterpenoids, are classified as GT1 [18]. Although over 500 thousands of GT have been discovered, there are only six bacterial GTs reported to catalyze glycosylation of triterpenoids, including BsYjiC from *B. subtilis* 168 [11,19–22], UGT109A1 from *B. subtilis* CTCG 63501 [23,24], BsGT1 from *B. subtilis* KCTC 1022 [25], BsUGT398 and BsUGT489 from *B. subtilis* ATCC 6633 [17], and BsGT110 from *B. subtilis* ATCC 6633 [present study]. Among them, the BsYjiC group (BsYjiC, BsUGT489, UGT109A1, and BsGT1) were highly similar, sharing more than 90% identity in their amino acid sequences [17], BsGT110 and BsUGT398, however, were not grouped with the BsYjiC group, and only had 31% and 33% identity, respectively, with the BsYjiC group (Figure 6). On the other hand, some bacterial GT1 catalyzed glycosylation of flavonoids. Thus, BsGT110 was compared with the flavonoid-catalyzing GTs. The results showed that the amino acid identity between BsGT110 and other flavonoid-catalyzing bacterial GTs was also lower than 40% (Figure S5 and Table S2). Furthermore, the evolutionary tree is shown in Figure 6 also demonstrated the dissimilarity between BsGT110 and BsUGT398, indicating that BsGT110 is a unique bacterial GT with glycosylation activity toward triterpenoids.

There are two reaction mechanisms for GT, inverting and retaining reactions, depending on the outcome of the reaction [26]. There are two stereochemical outcomes for reactions that result in the formation of a new glycosidic bond: the anomeric configuration of the product is either retained or inverted with respect to the donor substrate. The mechanistic strategy for inverting GTs involves a side chain of a residue on the active-site of GT that serves as a base catalyst that deprotonates the incoming nucleophile of the acceptor, facilitating direct displacement of the activated (substituted) phosphate leaving the group of the sugar donor, UDP-glucose [26]. Up to now, all GT1s are inverting GTs and were not reported to show optimal activities in acidic conditions [26]. For example, the well-known triterpenoid-catalyzing BsGT1 [25] had optimal activity at pH 7, BsYjiC [20], BsUGT398, and BsUGT489 [17] had optimal activity at pH 8, and UGT109A1 [23] had optimal activity at pH 9–10. These triterpenoid-catalyzing GTs had a broad neutral-alkaline range in their triterpenoid glycosylation activity [17,20–25]. According to the reaction mechanism of the inverting GTs, the side-chain of a key residue in the catalytic site of the enzyme should be deprotonated to serve as a base during the reaction. Thus, it is reasonable that GT1 enzymes have optimal activities at neutral-alkaline conditions, which would favor the deprotonation of the side chain. Accordingly, we identified the glycosylation activity of the selected five BsGTs toward GAA under a standard GT reaction condition: 10 mM Mg2+, 40 ◦C, and pH 8, and found that only BsUGT398 and BsUGT489 can catalyze C-15 glycosylation of GAA [17], but other candidates, including BsGT110, were unable to catalyze glycosylation of GAA under the standard GT reaction condition. Thus, the previous study did not observe the novel acidic glycosylation activity (C-26 glycosylation of GAA) of BsGT110 toward GAA. Hence, the BsGT110 that we identified in this work was much more capable of catalyzing GAA into pure triterpenoid glucoside (GAA-26-*O*-β-glucoside) under acidic conditions (pH 5–6) (Figures 2 and 4, and Table 1). In addition, the results of the kinetics study showed that the turnover number of BsGT110 toward GAA at pH 6 was 3-fold higher than that at pH 7 (Figure 5 and Table 2). Furthermore, the catalytic efficiency (kcat/Km) of BsGT110 toward GAA at pH 6 was 1.35-fold higher than that at pH 7. Taken together, our results are unique in that they indicate that BsGT110—unlike other GT1s, which are most active at regular neutral-alkaline pH—is most active at a narrow, more acidic range of pH values (pH 5–6), specifically toward the C-26 position of GAA.


**Figure 6.** Aligned amino acid sequences and phylogenetic analysis using the Maximum Likelihood method. In total, 407 amino acids were aligned by Clustal W in MEGA X [27]. '.' denoted as identical amino acid, '-' denoted as indel(s). The phylogenetic tree was inferred using the Maximum Likelihood method and General Reversible Mitochondrial model [28]. The tree with the highest log likelihood (−3197.28) was shown. The percentage of trees in which the associated taxa clustered together was shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, the topology with highest log-likelihood value was then selected. The tree was drawn to scale, with branch lengths measured based on the number of substitutions per site. This analysis involved six amino acid sequences. All positions with less than 95% site coverage were eliminated—i.e., less than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 382 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [27].

A few reports demonstrated that triterpenoid glycosides may improve the bioactivity of the triterpenoid aglycone [5]. Liang et al. produced an unusual ginsenoside, 3β, 12β-di-*O*-Glc-protopanaxadiol (PPD), from the glucosylation of PPD by UGT109A1, and showed that the ginsenoside had anti-cancer capabilities in the Lewis lung cancer xenograft mouse model [23]. Wang et al. used BsGT1 to produce 3β-*O*-Glc-ginsenoside F1, which inhibited melanin and tyrosinase activities [25]. Dai et al. reported the enzymatic synthesis of glycyrrhetinic acid (GA) glucosides—GA-30-*O*-β-glucoside and GA-3-*O-*β-glucoside—by BsYjiC and found that the two triterpenoid glucosides had higher water solubility and higher cytotoxicity against human liver cancer cells HepG2 and breast cancer cells MCF-7 than GA aglycone [20]. Therefore, the new GAA glucoside obtained in the present study, GAA-26-*O*-β-glucoside, warrants future investigation to determine whether it also has a higher bioactivity than GAA aglycone.

In summary, even though over 300 triterpenoids have been found in *G. lucidum*, very few triterpenoid glycosides have been identified [4]. Our study was the first to reveal that a single bacterium, the *Bacillus subtilis* ATCC 6633 strain, can biotransform GAA into both GAA-26-*O*-β-glucoside by BsGT110 in specific acidic conditions and GAA-15-*O*-β-glucoside by BsGT398 and BsGT489 in neutral-alkaline conditions.
