Purification, Gene Cloning, and Biochemical Characterization of a β-Glucosidase Capable of Hydrolyzing Sesaminol Triglucoside from Paenibacillus sp. KB0549

Abstract

The triglucoside of sesaminol, i.e., 2,6-O-di(β-D-glucopyranosyl)-β-D- glucopyranosylsesaminol (STG), occurs abundantly in sesame seeds and sesame oil cake and serves as an inexpensive source for the industrial production of sesaminol, an anti-oxidant that displays a number of bioactivities beneficial to human health. However, STG has been shown to be highly resistant to the action of β-glucosidases, in part due to its branched-chain glycon structure, and these circumstances hampered the efficient utilization of STG. We found that a strain (KB0549) of the genus Paenibacillus produced a novel enzyme capable of efficiently hydrolyzing STG. This enzyme, termed PSTG, was a tetrameric protein consisting of identical subunits with an approximate molecular mass of 80 kDa. The PSTG gene was cloned on the basis of the partial amino acid sequences of the purified enzyme. Sequence comparison showed that the enzyme belonged to the glycoside hydrolase family 3, with significant similarities to the Paenibacillus glucocerebrosidase (63% identity) and to Bgl3B of Thermotoga neapolitana (37% identity). The recombinant enzyme (rPSTG) was highly specific for β-glucosidic linkage, and k cat and k cat/K m values for the rPSTG-catalyzed hydrolysis of p-nitrophenyl-β-glucopyraniside at 37°C and pH 6.5 were 44 s−1 and 426 s−1 mM−1, respectively. The specificity analyses also revealed that the enzyme acted more efficiently on sophorose than on cellobiose and gentiobiose. Thus, rPSTG is the first example of a β-glucosidase with higher reactivity for β-1,2-glucosidic linkage than for β-1,4- and β-1,6-glucosidic linkages, as far as could be ascertained. This unique specificity is, at least in part, responsible for the enzyme’s ability to efficiently decompose STG.

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…β-Glucosidase varies in quaternary structure arrangement, for example, monomeric [159], dimeric [160], trimeric [147], tetrameric [151] enzymes have been reported. Native molecular weight therefore may be higher than identified by SDS-PAGE [161]. For instance, β-glucosidase reported from P. italicum had a native molecular weight of 354 kDa as determined by gel filtrations and 88.5 kDa as shown by SDS-PAGE suggesting that the native protein is a tetramer [74].…”

Lignans of Sesame (Sesamum indicum L.): A Comprehensive Review

“…To overcome the limitation of commercially available glycosylases, researchers from Kiyomoto Co. in collaboration with two universities in Japan designed a smart screening strategy to identify microbial sources of a glucosidases suitable for the hydrolysis of lignan glycosides, especially sesaminol triglucoside [ 138 ]. They collected samples of decaying sesame oil cake, extracted them with chloroform, and analyzed the extract for traces of sesaminol.…”

Sesaminol prevents Parkinson's disease by activating the Nrf2-ARE signaling pathway

“…However, most sesaminol exists as a glycoside, and sesaminol remains in the form of glycoside in the defatted residue after the extraction of sesame oil. A recent method for purifying sesaminol from sesaminol glycoside was established, and the effective use of defatted sesame, which was treated as industrial waste, is expected [17].…”