A Novel Glucanotransferase that Produces a Cyclomaltopentaose Cyclized by an .ALPHA.-1,6-Linkage

Abstract

Enzymatic syntheses of various nonreducing oligosaccharides from starch have been reported by many researchers. Nonreducing oligosaccharides are generally divided into two groups: linear and cyclic oligosaccharides. Trehalose (α D glucopyranosyl α D glucopyranoside) is a typical linear nonreducing oligosaccharide occurring in bacteria, yeasts, fungi, plants, and invertebrates. Mass production of trehalose from starch has been developed using two bacterial enzymes, maltooligosyltrehalose synthase (EC 5.4.99.15) and maltooligosyltrehalose trehalohydrolase (EC 3.2.1.141), 1 3) and now this saccharide is used in the fields of food, cosmetics, and pharmaceuticals. Cyclomaltohexaose (α cyclodextrin), one of the most well known cyclic oligosaccharides, is produced from linear α 1,4 glucans by the intramolecular α 1,4 transglycosylation reaction of a cyclomaltodextrin glucanotransferase (CGTase; EC 2.4.1.19).4) The cyclic oligosaccharide has a hydrophobic cavity in the center of the structure. Guest molecules of suitable size can enter the cavity, and the formation of the inclusion complex is used for stabilizing labile materials, 5) masking odors 6) and modifying viscosity. 7) Co te and co workers first reported that a cyclic tetrasaccharide consisting ofproduced from a dextran like polysaccharide, alternan, by its degrading enzyme.8 10) The cyclic oligosaccharide was designated cycloalternan (CA). Recently, we discovered two novel enzymes, 6 α glucosyltransferase and 3 α isomaltosyltransferase, in Bacillus globisporus, and succeeded in the mass production of this saccharide from starch by the joint reaction of the two enzymes.11 13) More recently, we found a new enzymatic system for synthesizing a cyclic maltosyl (1 6) 14,15) We consequently obtained two kinds of cyclic tetrasaccharides by discovering CMM. To distinguish the two kinds of cyclic tetrasaccharides, in this review we designate the cyclic nigerosyl (1 6 The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme (IgtZ) was purified. The enzyme acted on maltooligosaccharides that have a DP of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme, which has a novel amino-acid sequence, should be assigned to α-amylase. It is notable that both IGTase and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.

Cloning, Sequencing, and Expression of the Genes Encoding an Isocyclomaltooligosaccharide Glucanotransferase and an α-Amylase from aBacillus circulansStrain

“…The enzyme activity of IgtY (ICG5-forming activity) was assayed as described in a previous paper. 11) The activity of IgtZ was assayed as follows:…”

“…Our previous report showed that the novel glucanotransferase, isocyclomaltooligosaccharide glucanotransferase (IgtY), converted -1,4-glucan into ICG5 and ICG6 by a one-step reaction. 11) We now report the 8,325-bp sequence of the genomic DNA from B. circulans AM7, which contains igtY and two open reading frames (ORFs). We also report the amino-acid sequences deduced from igtY and the ORFs.…”

Production of Isocyclomaltopentaose from Starch Using Isocyclomaltooligosaccharide Glucanotransferase

“…The activities of IGTase and CGTase were assayed as reported in previous publications. 13,14) Isoamylase (EC 3.2.1.68) from Pseudomonas amylodelamosa was prepared in our laboratories. The activity of isoamylase was assayed as reported in a previous publication.…”

“…1). 13) In this study, we report on the reaction conditions y To whom correspondence should be addressed. Tel: +81-86-276-3142; Fax: +81-86-276-8670; E-mail: h-watanabe@hayashibara.co.jp required for the production of ICG5 from starch using IGTase.…”

“…The enzyme also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular -1,4-glycosyl transfer reaction (disproportionation reaction). 13) Because of the high frequency of the disproportionation reaction with increases in the substrate concentration, the cyclization reaction takes place less readily, and the yield of ICG5 decreased. Based on this result, a substrate concentration of 1.0% was considered optimal for ICG5-production.…”