Production of Cyclic Tetrasaccharide with 6-.ALPHA.-Glucosyltransferase and .ALPHA.-Isomaltosyltransferase

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Cyclic tetrasaccharide (CTS; cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}) is a cyclic oligosaccharide four D-glucosyl residues linked in an alternating α-1,3- and α-1,6-fashion. CTS has been reported for enzymatic synthesis from alternan, an α-(1→3)-α-(1→6)-D-glucan synthesized from sucrose by Leuconostoc mesenteroides NRRL B-1355 alternansucrase, by endo-glucanase (alternanase) from Bacillus sp. NRRL B-21195. While searching for nonreducing oligosaccharides that can be produced from α-1,4-glucan as the substrate, we screened bacteria isolated from soil in our own way, and obtained Bacillus globisporus C11, which produces CTS from starch. Two kinds of glycosyltransferase, 6-α-glucosyltransferase (6GT) and α-isomaltosyltransferase (IMT), were purified from the culture supernatant of this strain. It was found that CTS is produced by the sequential action of both enzymes. The genes for IMT (CtsY) and 6GT (CtsZ) were linked together on the chromosome, forming ctsYZ. Both of the gene products showed similarities to α-glucosidases belonging to glycoside hydrolase family 31 and conserved two aspartic acids corresponding to the putative catalytic residues of these enzymes. CtsYZ and four open reading frames upstream of ctsYZ form a gene cluster, ctsUVWXYZ. The reaction conditions for CTS synthesis were examined using 6GT and IMT from B. globisporus C11. The optimum reaction conditions to obtain CTS from Pinedex #100 (partial hydrolyzate of starch, 1.3%-hydrolysis) were the following: substrate concentration, 3%; pH, 6-7; temperature, 30°C; enzyme dosages, 1 U/g-dry solid 6GT, 10 U/g-dry solid IMT. In these optimum conditions the CTS yields reached 62% at the reaction time of 48 h. A mass-production method of highly purified CTS crystals at a reasonable cost was established, and the functions and characteristics of CTS were studied.