Abstract
Strain GA A07 was identified as an intestinal Bacillus bacterium of zebrafish, which has high efficiency to biotransform the triterpenoid, ganoderic acid A (GAA), into GAA-15-O-β-glucoside. To date, only two known enzymes (BsUGT398 and BsUGT489) of Bacillus subtilis ATCC 6633 strain can biotransform GAA. It is thus worthwhile to identify the responsible genes of strain GA A07 by whole genome sequencing. A complete genome of strain GA A07 was successfully assembled. A phylogenomic analysis revealed the species of the GA A07 strain to be Bacillus thuringiensis. Forty glycosyltransferase (GT) family genes were identified from the complete genome, among which three genes (FQZ25_16345, FQZ25_19840, and FQZ25_19010) were closely related to BsUGT398 and BsUGT489. Two of the three candidate genes, FQZ25_16345 and FQZ25_19010, were successfully cloned and expressed in a soluble form in Escherichia coli, and the corresponding proteins, BtGT_16345 and BtGT_19010, were purified for a biotransformation activity assay. An ultra-performance liquid chromatographic analysis further confirmed that only the purified BtGT_16345 had the key biotransformation activity of catalyzing GAA into GAA-15-O-β-glucoside. The suitable conditions for this enzyme activity were pH 7.5, 10 mM of magnesium ions, and 30 °C. In addition, BtGT_16345 showed glycosylation activity toward seven flavonoids (apigenein, quercetein, naringenein, resveratrol, genistein, daidzein, and 8-hydroxydaidzein) and two triterpenoids (GAA and antcin K). A kinetic study showed that the catalytic efficiency (kcat/KM) of BtGT_16345 was not significantly different compared with either BsUGT398 or BsUGT489. In short, this study identified BtGT_16345 from B. thuringiensis GA A07 is the catalytic enzyme responsible for the 15-O-glycosylation of GAA and it was also regioselective toward triterpenoid substrates.
Highlights
Glycosyltransferase (GT, EC 2.4.x.y) exists in all living beings and is able to catalyze the glycosylation of molecules such as proteins, nucleic acids, polysaccharides, and lipids
According to a carbohydrate-activating enzyme (CAZy) database, GTs are classified into 107 families [1]
Dozens of reports showed that triterpenoid glycosides, ginseng saponins, from the medicinal plant ginseng, possess more bioactivities involved in the central nervous system, cardiovascular system, immune system, anticarcinogenic activities, and diabetes mellitus, than do ginseng triterpenoid aglycones [13]
Summary
Glycosyltransferase (GT, EC 2.4.x.y) exists in all living beings and is able to catalyze the glycosylation of molecules such as proteins, nucleic acids, polysaccharides, and lipids. GTs that use small molecules (such as flavonoids or triterpenoids) as sugar acceptors are classified into the GT1 family. Biotransformation of xenobiotics by either a microorganism’s whole cells or purified enzymes may form more-bioactive metabolites than the precursor molecules [5,6,7,8,9]. Glycosylation was shown to improve water solubility, stability, and bioactivities of flavonoids, such as anti-oxidant and anti-allergic activities [10,11,12]. Glycosylation of triterpenoids to form saponins can improve some bioactivities of the triterpenoid precursors. Dozens of reports showed that triterpenoid glycosides, ginseng saponins, from the medicinal plant ginseng, possess more bioactivities involved in the central nervous system, cardiovascular system, immune system, anticarcinogenic activities, and diabetes mellitus, than do ginseng triterpenoid aglycones [13]. Using GT to biotransform xenobiotics to new glycoside compounds is a worthy field of study
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