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

Abstract

When saccharides are produced as foodstuffs in fairly large quantities, starch is a very fascinating source. Maltooligosaccharides such as glucose, maltose and maltotetraose are produced by hydrolysis of starch. Cyclodextrins (cyclic oligosaccharides composed of 6, 7, or 8 D glucosyl residues linked by 1,4 linkages ) , 1) isomalto oligosaccharides 2) and nigero oligosaccharides 3) are also produced from starch by enzymatic transfer reactions. In additions, these saccharides can be converted into branched cyclodextrins, sorbitol and maltiotol chemically or enzymically. Starch is used as donor substrate to produce various glycosides such as glucosyl ascorbic acid, 4) and as a source for the production of many saccharide related products. The reason seems to be that not only starch is inexpensive but also various starch related enzymes are present in nature. Indeed, there are many oligosaccharides synthesized enzymically. However, nonreducing glucooligosaccharides synthesized from starch are very few in kind. Examples of such oligosaccharides are trehalose 5 11) and cyclodextrins. Cyclic tetrasaccharide (CTS; cyclo{ 6) D Glup (1 3) D Glucp (1 6) D Glucp (1 3) D Glup (1 }) is also one of the nonreducing glucooligosaccharides.12) A system of synthesizing this cyclic oligosaccharide from starch was recently found in Bacillus globisporus C11. A CTS synthesizing mechanism controlled by two enzymes and the sequence of the genes encoding them were reported in succession. 13,14) These studies indicate that there is a good possibility of industrial production of CTS and a gene cluster related to the synthesis and transport of CTS is present in microorganisms. In this paper, we describe the current study of CTS by focusing on the synthesizing system of CTS from starch.Isolation of CTS-producing bacteria and the producing system in B. globisporus C11. 13)Among some 3000 bacterial strains, strain C11 was isolated from soil from Okayama, Japan, by screening for bacteria producing nonreducing oligosaccharides. Morphological, culture, and physiological characterizations according to Bergey's Manual of Systematic Bacteriology classified strain C11 as a strain of Bacillus globisporus. A nonreducing oligosaccharide was purified from a reaction mixture containing Pinedex #100 (partial hydrolyzate of starch, 1.3% hydrolysis) and the supernatant from B. globisporus C11. This saccharide was found to be identical 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 belon...