Purification and characterization of a Bacillus sp. SAM1606 thermostable ?-glucosidase with transglucosylation activity

Abstract

We purified a novel alpha-glucosidase to homogeneity from an Escherichia coli recombinant transformed with the alpha-glucosidase gene from thermophilic Bacillus sp. SAM1606. The enzyme existed as mono- and multimeric forms of a promoter protein with a relative molecular weight of 64,000 and isoelectric point of 4.6. We isolated a monomeric form of the enzyme and characterized it. The enzyme was unique among the known alpha-glucosidases in both broad substrate specificity and high thermostability. The enzyme hydrolysed a variety of O-alpha-D-glucopyranosides such as nigerose, maltose, isomaltose, sucrose, and trehalose efficiently. The molecular activity (k0) and the Michaelis constant (Km) values at 55 degrees C and pH 6.0 for sucrose were 54.6 s-1 and 5.3 mM, respectively. The optimum pH and temperature for hydrolysis were pH 5.5 and 75 degrees C, respectively. The enzyme exhibited a high transglucosylation activity: it reacted with 1.8 M sucrose at 60 degrees C for 70 h to yield oligosaccharides containing theanderose in a maximum yield of 35% (w/w). High thermostability of the enzyme (stable up to 65 degrees C at pH 7.2 for 10 min) permits the transglucosylation reaction at high temperatures, which would be beneficial for continuous production of oligosaccharides from sucrose.

Structure and expression of a gene coding for thermostable α‐glucosidase with a broad substrate specificity from Bacillus sp. SAM1606

“…previously [8]. 1 U enzyme activity was defined as the amount of enzyme that catalyses the formation of 1 pmole p-nitrophenolate min-' at 55 "C. The absorption coefficient for p-nitrophenolate under the assay conditions were 13400 cm-' M-'.…”

“…SAM1606 [8], which produced a thermostable a-glucosidase, was isolated from a hot spring in Japan. Bacillus thermoglucosidasius KP1006 (DSM2542) was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, and Bacillus cereus ATCC7064 was from American Type Culture Collection.…”

“…However, very low growth rates and enzyme productivities of the bacterium have hampered purification, further characterization, and application of the enzyme [8]. Therefore, we attempted to clone the gene of the SAM1606 a-glucosidase to overexpress it.…”

“…Characterization of the expressed a-glucosidase revealed that the enzyme is one of the a-glucosidases with the broadest substrate specificity. Particularly, it is the only a-glucosidase that can efficiently hydrolyse the a-l,l -glucosidic linkage of a,a'-trehalose, as well as the a-1,3, a-1,4 and a-1,6 linkages of nigerose, maltose and isomaltose, respectively, and 1-0-aryl a-glucosides [8]. Furthermore, the enzyme is one of the most thermostable a-glucosidases whose transglucosylation activities have been reported to date [8].…”

“…Particularly, it is the only a-glucosidase that can efficiently hydrolyse the a-l,l -glucosidic linkage of a,a'-trehalose, as well as the a-1,3, a-1,4 and a-1,6 linkages of nigerose, maltose and isomaltose, respectively, and 1-0-aryl a-glucosides [8]. Furthermore, the enzyme is one of the most thermostable a-glucosidases whose transglucosylation activities have been reported to date [8]. Thus, comparison of the primary structure of this unique a-glucosidase with those of the other a-glucosidases with different substrate specificity and thermostability should provide important information for insights into the structure-function relationship of a-glucosidases.…”