US007223570B2

(12) United States Patent (10) Patent No.: US 7,223,570 B2 Aga et al. (45) Date of Patent: May 29, 2007

(54) BRANCHED CYCLIC TETRASACCHARIDE, 5,763,598 A 6/1998 Hamayasu et al. PROCESS FOR PRODUCING THE SAME, 5,786, 196 A * 7/1998 Cote et al...... 435,208 AND USE FOREIGN PATENT DOCUMENTS (75) Inventors: Hajime Aga, Okayama (JP); Takanobu f Higashiyama, Okayama (JP); Hikaru E. 1 . A1 is: Watanabe, Okayama (JP); Tomohiko JP 647s) 1, 1994 Sonoda, Okayama (JP); Michio JP 6-16705 1, 1994 Kubota, Okayama (JP) JP 6-298806 10, 1994 JP 10-25305 1, 1998 (73) Assignee: Kabushiki Kaisha Hayashibara JP 10-304882 11, 1998 Seibutsu Kagaku Kenkyujo, Okayama WO WO 01/90338 A1 11, 2001 (JP) WO WO O2/10361 A1 2/2002 WO WO O2.40659 A1 5, 2002 (*) Notice: Subject to any disclaimer, the term of this WO WO O2/O55708 A1 T 2002 patent is extended or adjusted under 35 U.S.C. 154(b) by 324 days. OTHER PUBLICATIONS (21) Appl. No.: 10/471.377 Biely, P. “Enzymic alpha-galactosylation . . . " Carbohyd. Res. (2001) vol. 332, pp. 299-303.* (22) PCT Filed: Mar. 8, 2002 Cote, Gregory and Biely, Peter, Enzymically produced cyclic C-1,3- linked and O-1,6-linked oligosaccharides of D-glucose, European (86). PCT No.: PCT/UPO2/O2213 Journal of Biochemistry, vol. 226, (1994), p. 641-648. Sambrook, Joseph and Russell, David, Molecular Cloning. A Labo S 371 (c)(1), ratory Manuel. 3" Edition, (Cold Spring Harbor Laboratory, New (2), (4) Date: Sep. 9, 2003 York), 2001, Chapters 15-17. (87) PCT Pub. No.: WO02/072594 k cited. by examiner Primary Examiner Leigh C. Maier PCT Pub. Date: Sep. 19, 2002 (74) Attorney, Agent, or Firm Browdy and Neimark, PLLC (65) Prior Publication Data US 2004/0236097 A1 Nov. 25, 2004 (57) ABSTRACT (30) Foreign Application Priority Data The object of the present invention is to provide a novel Mar. 9, 2001 (JP) 2001-067282 glycosyl derivative of cyclotetrasaccharide represented by • - s 1- w u way - F · · · · · · · · · · · · · · · · · · · · · · · · · · · · · cyclo->6)-O-D-glucopyranosyl-(1->3)-C-D-glucopyrano (51) Int. Cl. syl-(1->6)-C-D-glucopyranosyl-(1->3)-C-D-glucopyrano C7H 3/06 (2006.01) syl-(1->}, and it is solved by providing a branched cyclotet CI2P 19/18 (2006.01) rasaccharide, wherein one or more hydrogen atoms in the (52) U.S. Cl...... 435/97.536/12312 hydroxyl groups of cyclotetrasaccharide are replaced with (58) Field of Classification Search 536/123.12. an optionally Substituted glycosyl group, with the proviso ------43 sjo that, when only one hydrogen atom in the C-6 hydroxyl See application file for complete search histo group among the above hydrogen atoms is substituted with pp p ry. an optionally-substituted glycosyl group, the Substituted (56) References Cited glycosyl group is one selected from those excluding D-glu cosyl group. U.S. PATENT DOCUMENTS 5,366,879 A 11/1994 Kitahata et al. 29 Claims, 20 Drawing Sheets U.S. Patent May 29, 2007 Sheet 1 of 20 US 7,223,570 B2

FIG. 1

Retention time (min) U.S. Patent May 29, 2007 Sheet 2 of 20 US 7,223,570 B2

IG. 2

i

Shift value (PPM) U.S. Patent May 29, 2007 Sheet 3 of 20 US 7,223,570 B2

FIG. 3

Stift Wallue (PPM) FG. 4.

: maen:No.,……!!!!!!!!!!!) Pyle's re's pare starts

Shift value (PPV) U.S. Patent May 29, 2007 Sheet 4 of 20 US 7,223,570 B2

FIG. 5

> | -- (i) ; O) - r C

tre -- (t. || | gold 'r p- "rrentirjatikshire for - intries-y-

Wu- PP, r- T --- 100 90 8O 70 6C Shift value (PPM)

FIG. 6

------mm.

> : rrn

rr- i () : I

?9 ; | :

alJkr- || swell-appaikish-fly-fristed| seriest, Wei- eys:

- PPA, - | OO 9 O 8) - 60 Shift value (PPM) U.S. Patent May 29, 2007 Sheet 5 of 20 US 7,223,570 B2

FIG 7

> - -- 2 (1) : i -

>

?. . its W. Viview vs. PPM. ------50 OO 90 80 70 Shift value (PPM) U.S. Patent May 29, 2007 Sheet 6 of 20 US 7,223,570 B2 FIG. 8a (a)

O C

O O6

C OS

O C - r to log ar < O - - g C. co Ci ?t

-- in0 inc

H - 0 <

!

O An Estient II e A Fate L93 U.S. Patent May 29, 2007 Sheet 7 of 20 US 7,223,570 B2

FIG. 8b

O

O O

O OB

8

C. C. -- E 9) (Cg

s C rr i

i Op. \ ---

C O&

C ,

(D ------2x -

An Suent I e An-else U.S. Patent May 29, 2007 Sheet 8 of 20 US 7,223,570 B2

FLG. 9 |------

shift value (PPM) FIG. 1 O

Shift value (PPV)

U.S. Patent May 29, 2007 Sheet 10 of 20 US 7,223,570 B2

F.G. 13

Shift value (PPM)

Shift value (PPM) U.S. Patent May 29, 2007 Sheet 11 of 20 US 7,223,570 B2 U.S. Patent May 29, 2007 Sheet 12 of 20 US 7,223,570 B2

FIG. 16

t

n

c ----

g: s - \ () - O

O ar. -- ) C2 (t r 4as --- t" -

--

s

--- U.S. Patent May 29, 2007 Sheet 13 of 20 US 7,223,570 B2

FIG. 17

...------r-|-~~~~~~~~--- ?noe,JJT(I=T6-LIÐTIO()gz GZ------~--~{-~~~()------.-----···----|—l-………------1

--~~~~);o–

(sdo) An LSU33. UI U.S. Patent May 29, 2007 Sheet 14 of 20 US 7,223,570 B2

FIG. 18 –*~~~~-----,------—1–---~~~~––

||||||||| IJJTCInoetro??treaT()z)(

}

{}?!------+()()------— U.S. Patent May 29, 2007 Sheet 15 of 20 US 7,223,570 B2

FG 19

Û°C)?I

||||||||| ()~º

| {}~

| eT6ueUropaoeaJFGI |

()*[]{ U.S. Patent May 29, 2007 Sheet 16 of 20 US 7,223,570 B2

O c. O ? | || | O - c. | C ! s

-1 C --- n t - G) - C - is

O) C. d E

C.) C) C N

i

l k- -- rism c { C C C) {} - O C) O C c s i. 9. r

( : ) ofureu o nu fei, U.S. Patent May 29, 2007 Sheet 17 of 20 US 7,223,570 B2

FIG. 21

------!----;------± (O)a.L.eure?ul?Ina

---

) e fuguo nube M U.S. Patent May 29, 2007 Sheet 18 of 20 US 7,223,570 B2

FIG. 22

-

C) | - s o f s | i f A - -- : - K.) --- - t --- --1 - C. f c s

: se -- H (1) O.

c Cl \ i

| |t f k f - c. A e } / | ! |f l --- c o c C c c frn V3: t C -

U.S. Patent May 29, 2007 Sheet 20 of 20 US 7,223,570 B2

FIG. 24

f 5

-

c - ) f ? r A f | | t

-- -- i l C WW i.

-- 3. -. s

(g abue up auf!e M. US 7,223,570 B2 1. 2 BRANCHED CYCLIC TETRASACCHARIDE, ride has been expected to be used in different fields similarly PROCESS FOR PRODUCING THE SAME, as in or much more useful than conventional cyclodextrins. AND USE The method of the report, however, may not suitable for an industrial-scale production of cyclotetrasaccharide, because TECHNICAL FIELD alternan used as a starting material is not easily obtainable The present invention relates to a novel branched cyclic and the yield of cyclotetrasaccharide from the material is tetrasaccharide, more particularly, a glycosyl derivative of a insufficient in view of industrial-scale production. cyclic tetrasaccharide represented by the chemical formula The same applicant as the present invention disclosed an of cyclo{->6)-O-D-glucopyranosyl-(1->3)-O-D-glucopyra O-isomaltosyl-transferring enzyme, a novel enzyme which nosyl-(1->6)-O-D-glucopyranosyl-(1->3)-O-D-glucopyra 10 forms cyclotetrasaccharide when acts on a saccharide having nosyl-(1->}, a process for producing the same, and uses a glucose polymerization degree of at least three and having thereof. both an isomaltosyl group at the non-reducing end and the O-1,4-glucosyl bond as a linkage other than the linkage at the BACKGROUND ART non-reducing end (abbreviated as "O-isomaltosylglucosac O- B-, and Y-Cyclodextrins, which consist of 6, 7, and 8 charide” hereinafter) as disclosed in Japanese Patent Appli glucose molecules that are linked each other via the C-14 15 cation No. 149,484/2000, and Japanese Patent Application glucosyl linkage, respectively, have been known as cyclic No 229,557/2000 (International Publication No. WO 01/90, saccharides composed of glucose units. These cyclodextrins 338 A1) applied for based on the above Japanese Patent have been used in a variety of fields due to their advanta Application; and also disclosed an O-isomaltosylglucosac geous inherent properties of non-reducibility, tasteless, charide-forming enzyme, a novel enzyme which forms enclosing hydrophobic materials, etc. There have been being O-isomaltosylglucosaccharide when acts on a maltooli pursued remarkable researches directed to improve proper gosaccharide having a glucose polymerization degree of at ties of cyclodextrins and impart additional new functions least three, as disclosed in Japanese Patent Application No. thereupon. For example, Japanese Patent Kokai Nos. 9,708/ 233,364/2000 and Japanese Patent Application No. 234.937/ 94, 14,789/94, 16,705/94, 298,806/94, and 25.305/98 pro 2000 (International Publication No. WO 02/10.361 A1). posed branched cyclodextrins with different branching 25 applied for based on Japanese Patent Application No.233, structures, which are prepared by coupling a glycosyl group 364/2000. Further, the above applicant proposed a method to such as a glucosyl, galactosyl, mannosyl, glucosaminyl, or produce cyclotetrasaccharide as a main product from starch, N-acetylglucosaminyl group to cyclodextrins; processes for a widely, commonly used material for producing foods by producing the same; and uses thereof. using the C-isomaltosylglucosaccharide-forming enzyme As an example of cyclic saccharide reported recently, 30 and the O.-isomaltosyl-transferring enzyme in combination, there is a cyclic tetrasaccharide, reported by Gregory L. Cote as disclosed in Japanese Patent Application Nos. 233,364/ et al. in European Journal of Biochemistry, Vol. 226, pp. 2000 and 234.937/2000 (International Publication No. WO 641-648 (1994), composed of glucose molecules linked each 02/10.361 A1). This proposal was a breakthrough for an other via the alternating C-13 and C-1.6 bonds and having industrial scale production of cyclotetrasaccharide. the structures represented by Chemical Formulae A and Bas Thus, the study on cyclotetrasaccharide has just merely bonding fashions between atoms and between glucosyl 35 been started, and further studies for elucidating unknown groups, respectively. In addition to Chemical Formulae A functions and developing new uses of cyclotetrasaccharide and B, cyclo->6)-O-D-glucopyranosyl-(1->3)-O-D-glu are now being greatly expected. Even though cyclotetrasac copyranosyl-(1->6)-O-D-glucopyranosyl-(1-3)-C-D-glu charide is a known compound, there is found no study to copyranosyl-(1->} belongs to the above cyclic tetrasaccha produce derivatives thereof as a main object, because ride. Throughout the specification, the term 40 cyclotetrasaccharide has not yet been easily obtained. So far “cyclotetrasaccharide” means the above-identified cyclic found is merely the above report by Cote et al. that reported tetrasaccharide. only a 6-O-glucopyranosyl derivative of cyclotetrasaccha Chemical Formula A: ride, represented by Chemical Formula C, isolated and OH identified as a by-product in a negligible yield through the HO 45 action of alternanase on alternan. Chemical Formula D O represents the 6-O-glucopyranosyl derivative in terms of OH O bonding fashions between glucosyl groups. HO Chemical Formula C: HO O OH O OH 50

O HO O OH OH O OH HO OH OH O O O OH HO 55 O O HO OH HO O OH O OH

HO O Chemical Formula B: 60 O OH OH O HO The above report by Cote et al. shows that cyclotetrasac O charide is formed by allowing alternan, a type of polysac OH charide composed of glucose molecules linked via the 65 OH alternating C-13 and C-1,6 bonds to act on alternanase, a type of hydrolyzing enzyme. Thereafter, cyclotetrasaccha US 7,223,570 B2

Chemical Formula D:

6

1 C-D-Glcp

10 Similarly as in cyclodextrins, Supplying of glycosyl wherein in Formula 1, R to R each independently repre derivatives of cyclotetrasaccharide would provide a useful sents an optionally substituted glycosyl group or hydrogen knowledge for developing uses of cyclotetrasaccharide atom, with the proviso that all of R to R are not hydrogen through analyses on their properties, and also it remarkably atom at the same time and that, when either R or Rio is an influences on the development of uses of novel saccharides, 15 optionally Substituted glycosyl group, the glycosyl group R. obtained by improving or modifying the properties and or Rio is a glycosyl group other than D-glucopyranosyl functions of cyclotetrasaccharide. group. The present invention solves the second object by pro DISCLOSURE OF INVENTION viding a process for producing the branched cyclotetrasac In view of the above backgrounds, the first object of the charides of the present invention, which uses an enzyme present invention is to provide a novel glycosyl derivative of capable of transferring a glycosyl group from a monosac cyclotetrasaccharide, the second object is to provide a pro charide, oligosaccharide, or polysaccharide to cyclotetrasac cess for producing the same, and the third object is to charide; and comprises a step of forming the branched provide uses thereof. cyclotetrasaccharides by reacting the above enzyme with a To solve the above objects, the present inventors firstly 25 found that cyclotetrasaccharide-related-saccharides were mixture of cyclotetrasaccharide and the above monosaccha formed as by-products in the reaction system accomplished ride, oligosaccharide, or polysaccharide, and collecting the by the present inventors, where cyclotetrasaccharide was formed branched cyclotetrasaccharides. formed by Subjecting a partial starch hydrolyzate to the Further, the present invention solves the third object by action of C-isomaltosyl-transferring enzyme and C-isomal providing a composition in the form of a food product, tosylglucosaccharide-forming enzyme, and then tried to 30 cosmetic, or pharmaceutical, which comprises the branched isolate and identify the by-products. As a result, they con cyclotetrasaccharide(s) of the present invention. firmed that all the by-products were novel glycosyl deriva tives of cyclotetrasaccharide. Further, they reacted cyclotet BRIEF DESCRIPTION OF DRAWINGS rasaccharide, prepared by the above two-types of enzymes, with these enzymes together with well known saccharide 35 FIG. 1 is a chromatogram of cyclotetrasaccharide on relatedenzymes in the presence of different glycosyl donors. high-performance liquid chromatography (abbreviated as As a result, they found that various glycosyl derivatives “HPLC hereinafter). were obtained through the action of the above enzymes, FIG. 2 is a 'H-NMR spectrum of cyclotetrasaccharide. cyclomaltodextrin glucanotransferase, B-galactosidase, FIG. 3 is a 'C-NMR spectrum of cyclotetrasaccharide. O-galactosidase, lysozyme, and other saccharide-related 40 enzymes Such as glycosyltransferase, glycosylhydrolase, FIG. 4 is a 'C-NMR spectrum of the branched cyclotet and glycosylphosphatase. They isolated the formed glycosyl rasaccharide of the present invention, represented by Chemi derivatives of cyclotetrasaccharide and examined their prop cal Formula 1. erties, and confirmed that the glycosyl derivatives can be FIG. 5 is a 'C-NMR spectrum of the branched cyclotet advantageously used in the fields of food products, cosmet 45 rasaccharide of the present invention, represented by Chemi ics, pharmaceuticals, etc. The present invention was made cal Formula 3. based on the above self-findings by the present inventors. FIG. 6 is a 'C-NMR spectrum of the branched cyclotet The present invention solves the first object by providing rasaccharide of the present invention, represented by Chemi branched cyclotetrasaccharides, i.e., glycosyl derivatives of cal Formula 4. cyclotetrasaccharide represented by Formula 1. 50 FIG. 7 is a 'C-NMR spectrum of the branched cyclotet Formula 1: rasaccharide of the present invention, represented by Chemi OR cal Formula 5. RO FIGS. 8a and 8b are respectively a chromatogram (a) on O HPLC for a reaction mixture obtained by reacting CGTase 55 with a mixture of cyclotetrasaccharide and C-cyclodextrin, R1O and a chromatogram (b) on HPLC for a reaction mixture RO O 2 obtained by contacting glucoamylase with the above mixture O OR after reacted with CGTase. FIG. 9 is a 'C-NMR spectrum of the branched cyclotet RSO O 60 rasaccharide of the present invention, represented by Chemi O OR) cal Formula 2. OR6 O FIG. 10 is a 'C-NMR spectrum of the branched cyclotet RO rasaccharide of the present invention, represented by Chemi O cal Formula 6. OR8 OR 65 FIG. 11 is a 'C-NMR spectrum of the branched cyclotet rasaccharide of the present invention, represented by Chemi cal Formula 8.- US 7,223,570 B2 5 6 FIG. 12 is a 'C-NMR spectrum of the branched cyclotet wherein in Formula 1, R to R2 each independently repre rasaccharide of the present invention, represented by Chemi sents an optionally substituted glycosyl group or hydrogen cal Formula 7. atom, with the proviso that all of R to R are not hydrogen FIG. 13 is a 'C-NMR spectrum of the branched cyclotet atom at the same time and that when either R or Rio is an rasaccharide of the present invention, represented by Chemi optionally substituted glycosyl group, the glycosyl group R. cal Formula 9. FIG. 14 is a 'C-NMR spectrum of the branched cyclotet or Rio is a glycosyl group other than D-glucopyranosyl rasaccharide of the present invention, represented by Chemi group. The term 'glycosyl group’ as referred to as in the cal Formula 10. present invention means an atomic group represented by a FIG. 15 is an X-ray diffraction spectrum for a crystal of structure where an anomeric hydroxyl group is removed the branched cyclotetrasaccharide of the present invention, 10 from the molecular structure of a saccharide. The term represented by Chemical Formula 1. “saccharides' as referred to as in the present invention FIG. 16 is an X-ray diffraction spectrum for a crystal of means a general term for compounds including polyalcohols the branched cyclotetrasaccharide of the present invention, and their aldehydes, ketons and acids; amino Sugars and represented by Chemical Formula 2. their derivatives; and oligosaccharides, polysaccharides, and FIG. 17 is an X-ray diffraction spectrum for a crystal of 15 the branched cyclotetrasaccharide of the present invention, their condensed compounds. The term "substituents in gly represented by Chemical Formula 3. cosyl groups with an optional group’ as referred to as in the FIG. 18 is an X-ray diffraction spectrum for a crystal of present invention means Substituent groups which can Sub the branched cyclotetrasaccharide of the present invention, stitute hydrogen(s) of one or more non-anomeric hydroxyl represented by Chemical Formula 6. groups in a saccharide molecule, or of one or more non FIG. 19 is an X-ray diffraction spectrum for a crystal of anomeric hydroxyl groups and amino groups when the the branched cyclotetrasaccharide of the present invention, saccharide molecule is an amino Sugar. Examples of Such represented by Chemical Formula 7. Substituent groups are alkyl, acyl, acetyl, phosphoric acid, FIG. 20 shows a thermal property of the branched and Sulfuric acid groups. cyclotetrasaccharide of the present invention, represented by 25 Chemical Formula 1 on thermogravimetric analysis. Examples of the glycosyl groups that are positioned at the FIG. 21 shows a thermal property of the branched branched parts of the branched cyclotetrasaccharides of the cyclotetrasaccharide of the present invention, represented by present invention, i.e., one or more glycosyl groups selected Chemical Formula 2 on thermogravimetric analysis. from R to R in Formula 1 are optionally substituted FIG. 22 shows a thermal property of the branched {O-D-glucopyranosyl-(1->4)- C-D-glucopyranosyl cyclotetrasaccharide of the present invention, represented by 30 groups, with the proviso that “n” represents an integer of 0 Chemical Formula 3 on thermogravimetric analysis. or over and, when at least two of R to R are the above FIG. 23 shows a thermal property of the branched glycosyl groups, the integers of “n” in each glycosyl groups cyclotetrasaccharide of the present invention, represented by are independent each other, optionally substituted C-D- Chemical Formula 6 on thermogravimetric analysis. glucopyranosyl-(1->6)-C-D-glucopyranosyl-(1-3)-C-D- FIG. 24 shows a thermal property of the branched 35 glucopyranosyl-(1->6)- C-D-glucopyranosyl group, with cyclotetrasaccharide of the present invention, represented by the proviso that “n” represents an integer of 0 or over and, Chemical Formula 7 on thermogravimetric analysis. when at least two of R to R are the above glycosyl groups, the integers of “n” in each glycosyl groups are independent BEST MODE FOR CARRYING OUT THE each other, optionally substituted {?-D-galactopyranosyl INVENTION 40 (1->6)-3-D-galactopyranosyl groups, with the proviso that “n” represents an integer of 0 or over and, when at least Preferred embodiments of the present invention are two of R to R are glycosyl groups, the integers of “n” in described below in more detail: each glycosyl groups are independent each other, optionally 1. Branched Cyclotetrasaccharides Substituted C-D-galacropyranosyl groups; and optionally Novel branched cyclotetrasaccharides, which the present 45 substituted B-D-chitosaminyl groups. The branched invention provides, have the structure represented by For cyclotetrasaccharides of the present invention may have one mula 1. or more of the above-mentioned groups intramolecularly. The first more concrete example of the branched cyclotet 50 rasaccharides are those wherein R and/or R, in Formula 1 Formula 1: are optionally substituted C-D-glucopyranosyl-(1-4)- C-D-glucopyranosyl groups, with the proviso that 'n' rep resents an integer of 0 or over and, when both R and R, are Such glycosyl groups, the integers of “n” in each groups are 55 independent each other. Examples of the structural formulae thereof are Chemical Formulae 1 and 2 as shown in Experi ments 3-4 and 4-3. The second more concrete example of the branched cyclotetrasaccharides are those wherein R and/or Rs in 60 Formula 1 are optionally substituted C-D-glucopyranosyl (1->6)-O-D-glucopyranosyl-(1->3)-C-D-glucopyranosyl (1->6)-a C-D-glucopyranosyl groups, with the proviso that 'n' represents an integer of 0 or over and, when both R and Rs are such glycosyl groups, the integers of “n” in each 65 groups are independent each other. Examples of the struc tural formulae thereof are Chemical Formulae 3 and 4 as shown in Experiment 3-4. US 7,223,570 B2 7 8 The third more concrete example of the branched These hydrous crystals can be converted into anhydrous cyclotetrasaccharides are those wherein R and/or Rs in ones by heating at normal pressure or reduced pressure and Formula 1 are optionally substituted {?-D-galactopyrano at ambient temperature. Crystals of these branched cyclotet syl-(1->6)- (3-D-galactopyranosyl groups, with the pro rasaccharides can be identified by conventional X-ray pow viso that “n” represents an integer of 0 or over and, when 5 der diffraction analysis. For-example, upon the analysis, the both R- and Rs are such glycosyl groups, the integers of 'n' branched cyclotetrasaccharides represented by Chemical in each groups are independent each other. Example of the Formulae 1, 2, 3, 6, and 7 have main diffraction angles (20) structural formula thereof is Chemical Formula 6 as shown of (1) 8.1°, 12.2°, 14.2°, and 15.4°; (2)5.6°, 8.8°, 16.9°, and in Experiment 4-4. 21.9°; (3)7.9°, 12.19, 17.9°, and 20.29; (4) 11.0°, 12.3°, The forth more concrete example of the branched 10 12.8°, and 24.9°; and (5) 8.7°, 13.0°, 21.7°, and 26.1°, cyclotetrasaccharides are those wherein R and/or Rio in respectively. Each branched cyclotetrasaccharide can be Formula 1 are optionally substituted {?-D-galactopyrano provided in the form of a saccharide composition compris syl-(1->6)--D-galactopyranosyl groups, with the proviso ing the same as a main ingredient. The saccharide compo that And represents an integer of 0 or over and, when both sition, which contains one or more of the branched cyclotet Ra and Ro are such glycosyl groups, the integers of “n” in 15 rasaccharides in an amount, usually, of at least 50%, each groups are independent each other. Examples of the preferably, at least 60%, more preferably, at least 70%, and structural formulae thereof are Chemical Formulae 7 and 8 more preferably, at least 80% against the total amount of as shown in Experiment 4-5. Sugar components, on a dry solid basis (d.S.b.), is provided The fifth more concrete example of the branched cyclotet in the form of a solution, syrup, block, granule, crystalline rasaccharides are those wherein Ra and/or Rio in Formula 1 powder containing hydrous and/or anhydrous crystal, amor are optionally Substituted C-D-galactopyranosyl groups. phous crystalline powder, or molasses of the branched Example of the structural formula thereof is Chemical cyclotetrasaccharide(s). Formula 9 as shown in Experiment 4-6. 2. Process for Producing Branched Cyclotetrasaccharides The sixth more concrete example of the branched The process for producing the branched cyclotetrasaccha cyclotetrasaccharides are those wherein R and/or Rs in 25 rides of the present invention employs the action of an Formula 1 are optionally substituted f-D-chitosaminyl enzyme that transfers a glycosyl group from a monosaccha groups (“a chitosaminyl group' is also generally called “a ride, oligosaccharide, or polysaccharide to cyclotetrasaccha glucosaminyl group'). Example of the structural formula ride, and it is characterized in that it comprises the steps of thereof is Chemical Formula 10 as shown in Experiment 4-7. allowing the enzyme to act on a mixture of cyclotetrasac Though the above examples of the branched cyclotet 30 charide and any of the above monosaccharide, oligosaccha rasaccharides of the present invention are respectively clas ride, and polysaccharide to form the desired branched sified and exemplified based on their constituent saccharides cyclotetrasaccharides, and collecting the produced branched positioned at their branched parts, the branched cyclotet cyclotetrasaccharides. rasaccharides may be those which have either one of these branched parts or two or more of them in an appropriate 35 2.1. Preparation of Cyclotetrasaccharides combination. For example, a branched cyclotetrasaccharide The process for producing cyclotetrasaccharide is not which combinationally has both the structure of the specifically restricted. Examples of Such are (1) a process for branched part represented by the above first example, and producing cyclotetrasaccharide by contacting alternanase, the structure of the branched part represented by any of the i.e., a hydrolyzing enzyme, with alternan, i.e., a polysac above second to sixth examples. An example of Such struc 40 charide reported by Gregory L. Cote et al. in European tures is Chemical Formula 5 in Experiment 3-4. Journal of Biochemistry, Vol. 226, pp. 641-648 (1994); (2) As long as the branched cyclotetrasaccharides of the a process for producing cyclotetrasaccharide by contacting present invention have any of the above-mentioned struc C-isomaltosyl-transferring enzyme with O-isomaltosylglu tures, they should not be restricted to those which are cosaccharide; and (3) a process for producing cyclotetrasac produced by specific methods such as organic syntheses and 45 charide by contacting both C.-isomaltosylglucosaccharide include those which are produced by enzymatic reactions. forming enzyme and C-isomaltosyl-transferring enzyme However, since the branched cyclotetrasaccharides are effi with a saccharide, having a glucose polymerization degree ciently produced by the process according to the present of at least two and the C-1,4-glucosyl bond as a linkage at invention described in detail below, those which are pro its reducing end, such as a starch hydrolyzate. The terms duced by the above process are advantageously used in a 50 "O-isomaltosylglucosaccharide-forming enzyme’ and variety of fields. The branched cyclotetrasaccharides of the "O-isomaltosyl-transferring enzyme as referred to as in the present invention are provided in the form of a product present invention mean enzymes having the following enzy consisting essentially of the branched cyclotetrasaccharides matic activities (A) and (B), respectively, independently of as a saccharide component, usually, in a purified form with their other enzymatic activities, not specified in (A) and (B), a purity of at least 90%, preferably, at least 95%, and more 55 Such as physicochemical properties and origins. preferably, at least 97%; in the form of a solution, amor (A) Acting on a saccharide, which has a glucose poly phous powder, or molasses; or in the form of an isolated merization degree of “n” (“n” represents an integer of at crystal. The branched cyclotetrasaccharides in a crystal form least two) and the C-1.4 glucosyl bond as a linkage at its can be isolated by crystallizing in water, an organic solvent non-reducing end, to form a saccharide having a glucose Such as lower alcohols and dimethylformamide, or a solvent 60 polymerization degree of “n+1 and having both the C-1.6 in a mixture form of two or more of the above solvents glucosyl bond as a linkage at its non-reducing end and the selected appropriately; and further separating the resulting C.-1.4 glucosyl bond as a linkage other than the above crystals in a conventional manner. Crystals of branched linkage at its non-reducing end, without Substantially cyclotetrasaccharides in a hydrous or anhydrous form can be increasing the reducing power of the saccharide. obtained by crystallization in water. Examples of such 65 (B) Acting on a saccharide, which has a glucose poly hydrous crystal are those of the branched cyclotetrasaccha merization degree of at least three, the C-1,6 glucosyl bond rides represented by Chemical Formulae 1, 2, 3, 6 and 7. as a linkage at its non-reducing end, and the C-1,4-glucosyl US 7,223,570 B2 10 bond as a linkage other than the above linkage at its citric acid and Succinic acid. The concentration of these non-reducing end, to form cyclotetrasaccharide represented carbon Sources in nutrient culture media is appropriately by cyclo-->6)-O-D-glucopyranosyl-(1->3)-C-D-glucopyra changed depending on their types. The nitrogen Sources nosyl-(1->6)-O-D-glucopyranosyl-(1->3)-O-D-glucopyra usable in the present invention are, for example, inorganic nosyl-(1->}. nitrogen-containing compounds such as ammonium salts The formation mechanism of cyclotetrasaccharide by the and nitrates; and organic nitrogen-containing compounds above process (3) is roughly estimated as follows: Such as urea, corn steep liquor, casein, yeast extract, and beef (I) O-isomaltosylglucosaccharide-forming enzyme acts extract. The inorganic ingredients usable in the present on a glucose residue positioning at the reducing end of an invention are, for example, salts of calcium, magnesium, C.-1.4 glucan chain Such as in glycogen or starch hydrolyZ 10 potassium, Sodium, phosphoric acid., manganese, Zinc, iron, ates, and intermolecularly transfers the glucose residue to copper, molybdenum, and cobalt. If necessary, amino acids the C-6 hydroxyl group of a glucose group positioning at the and vitamins can be appropriately used in combination. non-reducing end of another intact C.-1.4 glucan chain, and Explaining the culture conditions, microorganisms are to form an O-14 glucan chain having an O-isomaltosyl preferably cultured, usually, under aerobic conditions at group at its non-reducing end. 15 temperatures, usually, of 4 to 40°C., preferably, 20 to 37°C., (II) C.-Isomaltosyl-transferring enzyme acts on the result at pHs, usually, of 4 to 10, preferably, 5 to 9, for 10 to 150 ing C-1.4 glucan chain having an isomaltosyl group at its hours. The concentration of dissolved oxygen (DO) can be non-reducing end, and intermolecularly transfers the isoma controlled during the culturing, and it can be kept within the ltosyl group to the C-3 hydroxyl group of a glucose group range of 0.5-20 ppm, for example, by means of controlling positioning at the non-reducing end of another intact C.-1.4 the aeration rate and the stirring speed, increasing or glucan chain having an isomaltosyl group at its non-reduc decreasing the oxygen concentration in gas used for aera ing end to form an O-1,4-glucan chain having an isomalto tion, and increasing or decreasing the inner pressure of Syl-1,3-isomaltosyl group at its non-reducing end. fermenters. The cultivation is freely carried out batchwise, (III) Subsequently, C.-isomaltosyl-transferring enzyme in a continuous manner, or in a semi-continuous manner, as acts on the resulting C-14 glucan chain, having an isoma 25 long as it affords the conditions in which microorganisms ltosyl-1,3-isomaltosyl group at its non-reducing end, to can grow and produce C-isomaltosyl-transferring enzyme. release the isomaltosyl-1,3-isomaltosyl group from the The cultures of Strain C9, FERM BP-7143, and Strain C-1,4-glucan chain via the intramolecular transferring action C11. FERM BP-7144, usually contain C-isomaltosyl-trans and to circularize the released group for forming cyclotet ferring enzyme and C-isomaltosylglucosaccharide-forming rasaccharide. 30 enzyme. Therefor, in the process for producing cyclotet (IV) The resulting C-14 glucan chain is sequentially rasaccharide, the above cultures can be used intact as an received the above enzymatic reactions (I) to (III) to increase enzyme agent or used after purified into an enzyme agent the yield of cyclotetrasaccharides. which contains either or both of the above enzymes. For To produce cyclotetrasaccharide on an industrial scale, example, a partially purified enzyme agent, containing both among the above processes, the processes (2) and (3) are 35 the enzymes, can be obtained by removing cells from the relatively advantageous in terms of its production cost and cultures by means of a conventional liquid-solid separation labors, particularly, the process (3) is more preferable. With method, collecting the resulting Supernatant, and optionally reference to the above process (3) mainly, the process for Subjecting the Supernatant to conventional methods for producing cyclotetrasaccharide is explained below: concentrating proteins such as Salting out using ammonium 40 Sulfate, sedimentation using acetone or alcohol, concentra 2.1.1. C.-Isomaltosyl-transferring Enzyme and C-isomalto tion in vacuo, and concentration using membranes. If nec sylglucosaccharide-forming Enzyme essary, the enzyme agent thus obtain can be further treated C-Isomaltosyl-transferring enzyme and O-isomaltosylglu by appropriately combining with conventional methods for cosaccharide-forming enzyme can be obtained, for example, purifying enzymes Such as gel-filtration chromatography, by culturing a microorganism which produces either or both 45 ion-exchange chromatography, affinity chromatography, and of the enzymes, and applying conventional methods for hydrophobic chromatography. From the separated fractions, preparing enzymes to the resulting culture. Bacillus glo those with the desired enzymatic activities are separately bisporus C9 strain was deposited on Apr. 25, 2000, with collected into enzyme agents having either of the enzymes International Patent Organism Depositary National Institute purified to the desired level. of Advanced Industrial Science and Technology Tsukuba 50 The enzymatic activity of C-isomaltosyl-transferring Central 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, enzyme can be assayed as follows: Dissolve panose in 100 Japan, and has been maintained therein under the accession mm acetate buffer (pH 6.0) to give a concentration of 2% number of FERM BP-7143; and Bacillus globisporus C11 (w/v) for a substrate solution, add 0.5 ml of an enzyme strain was deposited on Apr. 25, 2000, with the same solution to 0.5 ml of the substrate solution, and keep the depositary as above and has been maintained therein under 55 mixture at 35° C. for 30 min to proceed the formation of the accession number of FERM BP-7144, are particularly cyclotetrasaccharide from panose. In the reaction system, useful as Sources of the above enzymes because they pro glucose is formed together with cyclotetrasaccharide from duce both the enzymes (the above microorganisms may be panose. After the reaction, boil the reaction mixture for 10 called “Strain C9” and “Strain C11, hereinafter). min to Suspend the reaction. Subject the resulting reaction The nutrient culture media and culture conditions used for 60 mixture to the glucose oxidase method to quantify the culturing Strain C9, FERM BP-7143, and Strain C11, FERM glucose formed in the reaction mixture. In the present BP-7144, are as follows: Examples of the carbon sources invention, one unit activity of C-isomaltosyl-transferring usable in the present invention are starches and phytogly enzyme is defined as the enzyme amount that forms one cogens from plants; glycogens and pullulans from animals micromole of glucose per minute under the above enzymatic and microorganisms; partial hydrolyzates thereof saccha 65 reaction conditions. rides such as D-glucose, D-fructose, lactose, Sucrose, man The C-isomaltosylglucosaccharide-forming enzyme nitol, L-Sorbitol, and molasses; and organic acids Such as activity can be assayed as follows: Add 0.5 ml of an US 7,223,570 B2 11 12 enzymatic solution to 0.5 ml of a substrate solution obtained sequence listing of the present specification, SEQID NO:1 by dissolving maltotriose in 100 mM acetate buffer (pH 6.0) represents a nucleotide sequence of a coding region corre to give a concentration of 2% (w/v) is added, incubate the sponding to a precursor of C-isomaltosyl-transferring mixture at 35° C. for 60 min to proceed the formation enzyme from Strain C11 disclosed in Japanese Patent Appli reaction of isomaltosylmaltose from maltotriose. In the 5 cation No. 350,142/2000, and SEQ ID NO:2 represents a reaction system, maltose is formed together with isomalto nucleotide sequence of a coding region corresponding to a Sylmaltose from maltotriose. Thereafter, boil the reaction precursor of C-isomaltosylglucosaccharide-forming enzyme mixture for 10 min to suspend the enzymatic reaction. Subject the resulting mixture to-conventional HPLC for from Strain C11 disclosed in Japanese Patent Application detecting and quantifying maltose formed in the reaction 10 No. 5,441/2001. With reference to these nucleotide mixture to quantify the maltose. In the present invention, sequences, both of the above enzymes can be prepared by one unit activity of the C-isomaltosylglucosaccharide-form using conventional recombinant DNA technologies Such as ing enzyme is defined as the enzyme amount that forms one a DNA cloning method, site-directed mutagenesis, transfor micromole of maltose per minute under the above enzymatic mation of microorganisms, and artificial expression method reaction conditions. 15 of DNAs. Conventional recombinant DNA technologies are, Table 1 shows the physicochemical properties of both the for example, described in detail in “Molecular cloning, A enzymes obtained from Strain C9, FERM BP-7143; and LABORATORY MANUAL THIRD EDITION by J. Sam Strain C11, FERM BP-7144, which are confirmed-by the brook, published by Cold Spring Harbor Laboratory Press, index in the above defined enzymatic activities. 2001.

TABLE 1. C-Isomaltosyl-transferring C-Isomaltosylglucosaccharide enzyme forming enzyme Molecular weight About 82,000 to about 132,000 daltons About 117,000 to about 160,000 altons (Analysis method) (SDS-PAGE) (SDS-PAGE) Isoelectric point pI of about 5.0 to about 6.0 pI of about 4.7 to about 5.7 (Analysis method) (Isoelectrophoresis using ampholine) (Isoelectrophoresis using mpholine) Optimum temperature About 45° C. to about 50° C. About 40° C. to about 45° C. (Analysis condition) (Reaction at pH 6.0 for 30 min) (Reaction at pH 6.0 for 60 min) About 45° C. to about 50° C. (Reaction under the same condition as above in the presence of 1 mM Ca2:) Optimum pH pH of about 5.5 to about 6.0 pH of about 6.0 to about 6.5 (Analysis condition) (Reaction at 35° C. for 30 min) (Reaction at 35° C. for 60 min) Thermal stability About 40° C. or lower About 35° C. to about 40° C. or lower (Analysis condition) (Reaction at pH 6.0 for 60 min) (Reaction at pH 6.0 for 60 min) About 40° C. to about 45° C. or lower (Reaction under the same condition as above in the presence of 1 mM Ca2+) pH Stability pH of about 4.0 to about 9.0 pH of about 4.5 to about 10.0 (Analysis condition) (Incubated at 4° C. for 24 hours) (Incubated at 4° C. for 24 hours)

When an enzyme agent is a composition of C-isomaltosyl 2.1.2. Preparation of Cyclotetrasaccharide Using C-isomal transferring enzyme and C-isomaltosylglucosaccharide tosyl-transferring Enzyme and O-isomaltosylglucosaccha forming enzyme, it has an activity of forming cyclotetrasac- 45 ride-forming Enzyme charide from partial starch hydrolyzates. The activity of In preparing cyclotetrasaccharide from a Substrate such as forming cyclotetrasaccharide from partial starch hydrolyZ partial starch hydroly Zates by using C-isomaltosyl-transfer ates (the term “a cyclotetrasaccharide forming activity” ring enzyme and C-isomaltosylglucosaccharide-forming means the above activity, hereinafter) can be assayed as enzyme, the former enzyme is allowed to act on a Substrate, follows: Add 0.5 ml of an enzyme solution to 0.5 ml of a so usually, in a aqueous solution form, after the action of or substrate solution obtained by dissolving “PINE-DEX under the coaction of the latter enzyme. The coaction of the #100TM, a partial starch hydrolyzate commercialized by above enzymes is relatively preferable in view of the pro Matsutani Chemical Ind., Tokyo, Japan, in 50 mM acetate duction efficiency of cyclotetrasaccharide. Examples of the buffer (pH 6.0) to give a concentration of 2% (w/v), incubate Substrates used in this method are saccharides having a the mixture at 35° C. for 60 min to proceed the formation 55 glucose polymerization degree of at least two and the C-14 reaction of isomaltosylglucosaccharide-forming enzyme can glucosyl bond as a linkage at their non-reducing ends; be also obtained by the recombinant DNA technology. The maltooligosaccharides, maltodextrins, amylodextrins, amy same applicant as the present invention disclosed the nucle loses, amylopectins, Soluble starches, liquefied starches, otide sequences of DNAs which encode the O.-isomaltosyl gelatinized starches, and glycogens. Considering the pro transferring enzyme and C-isomaltosylglucosaccharide- 60 duction cost, terrestoreal starches such as corn, wheat, and forming enzyme from Strain C11, FERM BP-7144, in rice; and Subterranean starches such as potato, Sweet potato, Japanese Patent Application Nos. 350,142/2000 and 5,441/ and tapioca are preferably used as starting materials. Both of 2001. As disclosed in these specifications, each of the above the enzymes are preferably allowed to act on liquefied nucleotide sequences includes a nucleotide sequence of a starches prepared by allowing liquefying-type amylases to coding region, which corresponds to a precursor of each of 65 act on Suspensions of the above starches or heating the the enzymes having a signal peptide at the N-terminus, and suspensions under acid conditions. The lower the “DE” those in the 5’- and 3'-non-translational regions. In the (dextrose equivalent) of liquefied starch, the higher the yield US 7,223,570 B2 13 14 of cyclotetrasaccharide becomes. The DE is usually 20 or block, powder, granule, crystal, etc., by applying thereunto lower, preferably, 12 or lower, and more preferably, five or an appropriate combination of treatments such as concen lower. Prior to or in parallel with the action of C.-isomalto tration, crystallization, drying, pulverization, and dissolu Syl-transferring enzyme and C-isomaltosylglucosaccharide tion. forming enzyme on liquefied starch, debranching enzymes The above outlines the process for producing cyclotet Such as pullulanase or isoamylase can be advantageously rasaccharide, which uses C-isomaltosyl-transferring enzyme used because they may increase the yield of cyclotetrasac and O-isomaltosylglucosaccharide-forming enzyme in com charide. bination. In the method of using only C-isomaltosyl-trans The concentration of Substrates is not specifically ferring enzyme (the above-mentioned process for producing restricted as long as cyclotetrasaccharide is formed. The 10 cyclotetrasaccharide (2)). Such an enzyme is prepared simi higher the concentration, the higher the yield of cyclotet larly as above and then allowed to act on O-isomaltosylglu rasaccharide per batch becomes; usually, it is 0.1%(w/w) or cosaccharide such as panose commercialized or prepared in higher, preferably, one percent (w/w) or higher, d.s.b. a usual manner, and optionally the formed cyclotetrasaccha Though substrates can be used in a solution form with a ride can be further purified similarly as above. concentration over their water solubility, the concentration is 15 preferably set to 40% (w/w) or lower, preferably, 35% (w/w) 2.2. Enzyme Which Transfers Glycosyl Group to Cyclotet or lower, d.s.b., to ease handlings. rasaccharide The reaction condition is not specifically restricted as Any enzymes can be used in the process of the present along as cyclotetrasaccharide is formed. For example, any invention independently of their unrequisite actions and temperatures of from ambient temperature to 50° C., pref origins other than the ability of forming the above-identified erably, 30° C. to 45° C. can be suitably used; and any pHs branched cyclotetrasaccharides, represented by Formula 1, of from 4.5 to 8, preferably, from 5.5 to 7 can be preferably by transferring a glycosyl group from monosaccharides, used. Metal ions such as Ca" and Mg", which stabilize any oligosaccharides, or polysaccharides (hereinafter, these sac of the enzymes used, can be advantageously coexisted in a charides are called 'donors of glycosyl group'). Examples reaction mixture. The reaction time can be arbitrarily set in 25 of Such enzymes are cyclomaltodextrin glucanotransferase, view of the reaction progress, depending on the amount of C-isomaltosyl-transferring enzyme, C.-isomaltosylglucosac the enzymes used. charide-forming enzyme, B-galactosidase, C.-galactosidase, If necessary, other saccharide transferring enzymes can be and lysozyme. In terms of the transferring of a glycosyl advantageously used when C-isomaltosyl-transferring group to cyclotetrasaccharide (the expression of “transfer of enzyme and C-isomaltosylglucosaccharide-forming enzyme 30 glycosyl group' may be abbreviated as 'glycosyl transfer”. are allowed to act on a substrate. For example, the combi hereinafter), the properties of each enzymes are briefly nation use of cyclomaltodextrin glucanotransferase may explained in the below: increase the production yield of cyclotetrasaccharide as Cyclomaltodextrin glucanotransferase (EC 2.4.1.19) usu compared with the case without the combination use. ally transfers a glycosyl group from, as a donor of glycosyl As an enzyme agent used as C-isomaltosyl-transferring 35 group, a saccharide having the C-1.4 glucosyl bond as a enzyme and C-isomaltosylglucosaccharide-forming enzyme linkage and having a glucose polymerization degree of at to be allowed to act on a substrate, any microorganism which least two, such as maltooligosaccharide, maltodextrin, amy produces both the enzymes can be used. To use microor lodextrin, amylose, amylopectin, soluble starch, liquefied ganisms as Such an enzyme agent, for example, Strain C9, starch, gelatinized Starch, or glycogen; and usually forms the FERM BP-7143, and Strain C11, FERM BP-7144, which 40 branched cyclotetrasaccharides as in the first example of the are capable of forming both the enzymes, are cultured and aforesaid paragraph “1. Branched cyclotetrasaccharides'. proliferated up to reach the desired cell density under their C.-Isomaltosyl-transferring enzyme transfers a glycosyl proliferation conditions. As the culture conditions for the group from, as a donor of glycosyl group, C.-isomaltosyl above microorganisms, the above ones for forming enzymes glucosaccharides Such as panose to cyclotetrasaccharide and can be arbitrarily used. The resulting cultures can be allowed 45 to act on Substrates similarly as in the above enzyme agent. usually forms the branched cyclotetrasaccharides as in the The reaction mixtures thus obtained contain cyclotet second example of the aforesaid paragraph “1. Branched rasaccharide and they can be used intact as cyclotetrasac cyclotetrasaccharides', particularly, those represented by charide solutions or used after purification. To purify Chemical formulae 3 and 4. cyclotetrasaccharide, conventional purification methods for 50 C.-Isomaltosylglucosaccharide-forming enzyme usually saccharides can be appropriately employed. Examples of forms saccharides, which have the C-1,4-glucosyl bond as a Such are decoloration with activated charcoal; desalting with linkage and a glucose polymerization degree of at least three ion-exchange resins in H- and OH-forms; fractionation by Such as maltooligosaccharide, maltodextrin, amylodextrin, column chromatography using an ion-exchange resin, acti amylose, amylopectin, Soluble starch, liquefied starch, gela vated charcoal, and silica gel (usually called "chromatogra 55 tinized starch, or glycogen; and usually forms the branched phy'); separation sedimentation using organic solvents such cyclotetrasaccharides represented by Formula 1. as alcohol and acetone; separation using membranes with B-Galactosidase (EC 3.2.1.23) usually transfers a glucosyl appropriate separability; and decomposition and treatment group from lactose as a donor of glycosyl group to cyclotet to remove coexisting or remaining other saccharides, for rasaccharide and usually forms the branched cyclotetrasac example, enzymatic treatment with amylases such as 60 charides as in the third and forth examples of the above C.-amylase, B-amylase, and glucoamylase, and C-glucosi paragraph “1. Branched cyclotetrasaccharides'. Depending dase, fermentation treatment with yeasts, and alkaline treat on the origin of B-galactosidase used, the type or the ment. An appropriate combination use of the above purifi composition of the formed branched cyclotetrasaccharides cation methods advantageously increase the purity of may differ. For example, the enzymes from the microorgan cyclotetrasaccharide. The resulting purified cyclotetrasac 65 isms of the species Bacillus circulans relatively efficiently charide and Saccharide compositions containing the same form the branched cyclotetrasaccharides represented by can be prepared into the desired form of a solution, syrup, Chemical Formula 6, while those from the microorganisms US 7,223,570 B2 15 16 of the species Aspergillus niger form the branched cyclotet ing on the properties of the enzymes used. The cyclotet rasaccharides represented by Chemical Formulae 6 to 8. rasaccharide used in the present invention is prepared C-Galactosidase (EC 3.2.1.22) usually transfers a glyco according to any of the methods described in the above Syl group from melibiose as a donor of glycosyl group to paragraph 2.1. cycloterasaccharide and usually forms branched cyclotet Using a glycosyl-transferring enzyme and any of the rasaccharides, particularly, those represented by Chemical above-mentioned Substrates, i.e., cyclotetrasaccharide and a Formula 9 as in the fifth example of the above paragraph “1. donor of glycosyl group, a mixture of Substrates usually in Branched cyclotetrasaccharides”. the form of an aqueous solution is provided and then Lysozyme (EC 3.2.1.17) usually transfers to cyclotet admixed with the enzyme to effect enzymatic reaction for rasaccharide a glycosyl group from oligosaccharides or 10 forming the desired branched cyclotetrasaccharides in the polysaccharides, as a donor of a glycosyl group, such as resulting reaction mixture. The reaction conditions for the N-acetylchitooligosaccharide and chitin, which are com glycosyl-transferring enzyme are not specifically restricted posed of N-acetylchitosamine (also known as N-acetylglu as long as the desired branched cyclotetrasaccharides are cosamine) as a constituent saccharide and the B-1.4 glycosyl formed. In practicing the enzymatic reaction in an aqueous bond; and usually forms the branched cyclotetrasaccharides, 15 system, though it varies depending on the water Solubilities particularly, those represented by Chemical Formula 10 as in of Substrates used at their reaction temperatures, the con the sixth example of the above paragraph “1. Branched centrations of Substrates, i.e., the one of cyclotetrasaccharide cyclotetrasaccharides'. is usually set to 1 to 40% (w/w), preferably, 5 to 35% (w/w); The above describes the transferring reactions that are and the one of the donor of glycosyl group is set to the mainly catalyzed by the above enzymes when they each highest possible level but within the range that the donor independently act on cyclotetrasaccharide, however, com dissolves therein, usually, in an amount of at least a halftime bination use of two or more of them can form other branched of preferably, at least the same as, and more preferably, at cyclotetrasaccharides. For example, when cyclomaltodex least two times of that of cyclotetrasaccharide. The reaction trin glucanotransferase and C-isomaltosyl-transferring temperatures and pHs are arbitrarily selected in view of the enzyme are combinationally allowed to act on both cyclotet 25 enzymological properties of the glycosyl-transferring rasaccharide and an appropriate Saccharide as a donor of enzymes used as long as they do not completely inactivate glycosyl group, the branched cyclotetrasaccharides repre the enzymes during their enzymatic reactions. The amount sented by Chemical Formula 5 will be formed. Another of enzymes is arbitrarily selected to yield the desired product combination of the above enzymes may form different types at the end of enzymatic reaction depending on the Substrate of branched cyclotetrasaccharides. In addition to the above 30 concentration and the reaction time used. exemplified enzymes, saccharide-related enzymes Such as Though the resulting reaction mixture with branched kojibiose phosphorylase, disclosed in Japanese Patent Kokai cyclotetrasaccharides can be used intact as a saccharide No. 304.882/98 applied for by the same applicant as the composition containing the same, it is usually purified-from present applicant, glycogen phosphorylase (EC 2.4.1.1), the mixture before use. The branched cyclotetrasaccharides maltose phosphorylase (EC 2.4.1.8), C-glucosidase (EC 35 are purified according to conventional methods: Decolora 3.2.1.20), oligo-1,6-glucosidase (EC 3.2.1.10), and B-glu tion with an activated charcoal and desalting with ion cosidase (EC 3.2.1.21) can be arbitrarily used in the process exchange resins in H- and OH-forms can be arbitrarily used. of the present invention as long as they transfer a glycosyl If necessary, the degree of purification of the desired group from its donor to cyclotetrasaccharide and form the branched cyclotetrasaccharides can be advantageously branched cyclotetrasaccharides represented by the above 40 increased by an appropriate combination of fractionation by mentioned Formula 1. Particularly, the above kojibiose column chromatography using ion-exchange resins, acti phosphorylase can be advantageously used to meet its vated cachols, and silica gels; fractional precipitation using purpose because, depending on reaction conditions, it can organic solvents such as alcohol and acetone; separation transfer a glycosyl group from either glucose-1-phosphate, using membranes with an adequate separability; and other as a donor Saccharide, or a glycosyl group to cyclotetrasac 45 treatments of decomposing and removing the coexisting or charide and form the branched cyclotetrasaccharides repre remaining other saccharides, for example, enzymatic treat sented by Formula 1, with the proviso that one or more of ments with amylases such O-amylase, 3-amylase, and glu R. R. Ro, and R2 are oligoglucosyl groups having C-1,2- coamylase, and C-glucosidase, fermentations with yeasts, glucosyl bonds such as a glycosyl group and a kojibiosyl and alkaline treatments. The resulting purified branched group. 50 cyclotetrasaccharide(s) and Saccharide compositions con All the above-exemplified enzymes are well known in the taining the same can be treated with an appropriate combi art and any of commercialized preparations thereof or those nation of treatments such as concentration, crystallization, prepared by conventional reports on their preparations can drying, pulverization, and dissolution, and optionally mixed be used in practicing the present invention. with an appropriate Saccharide other than the branched 55 cyclotetrasaccharides of the present invention into the 2.3. Preparation of Branched Cyclotetrasaccharides from desired products in the form of a solution, syrup, block, Cyclotetrasaccharide crystalline powder containing hydrous- and/or anhydrous To produce the branched cyclotetrasaccharides of the crystals, amorphous powder, granule, isolated crystal, or present invention, an enzyme (hereinafter abbreviated as “a molasses. glycosyl-transferring enzyme in this paragraph 2.3.) is 60 In the reaction to form cyclotetrasaccharide via the action firstly selected depending on the structure of the aimed of C-isomaltosyl-transferring enzyme and O-isomaltosylglu branched cyclotetrasaccharides, and is obtained by purchas cosaccharide-forming enzyme, or the action of C-isomalto ing a commercialized enzyme preparation of Such an Syl-transferring enzyme on C.-isomaltosylglucosaccharide, enzyme or by preparing the enzyme in a conventional as shown in the above paragraph 2.1.2., the branched manner. A Suitable Saccharide as a donor of glycosyl group 65 cyclotetrasaccharides of the present invention are formed as is obtained by purchasing a commercialized product thereof by-products in different yields depending on the reaction or by preparing the donor in a conventional manner, depend conditions used. Among the branched cyclotetrasaccharides, US 7,223,570 B2 17 18 main components are those represented by Chemical For Such as pullulan, hydroxyethyl starch, or poly(Vinylpyrroli mula 1, and those in the second example of the above done). The branched cyclotetrasaccharides of the present paragraph “1. Branched cyclotetrasaccharides', and those of invention have properties such as osmotic pressure-control Chemical Formulae 3, 4, and 5. Therefore, to meet the ling ability, filling ability, gloss-imparting ability, moisture object, the branched cyclotetrasaccharides of the present 5 retaining ability, Viscosity-imparting ability, crystallization invention can be obtained by separating from cyclotetrasac preventing ability of other saccharides, and insubstantial charide in their reaction mixture obtained after the action of fermentability. Therefore, the branched cyclotetrasaccha the aforesaid both enzymes or the sole use of C.-isomaltosyl rides and the Saccharide compositions comprising the same transferring enzyme. can be advantageously used in compositions such as food 10 products, articles of taste including tobaccos and cigarettes, 3. Use of Branched Cyclotetrasaccharides feeds, baits, cosmetics, and pharmaceuticals as saccharide Since having a common basic structure, the branched Seasonings, taste-improving agents, quality-improving cyclotetrasaccharides of the present invention usually have agents, stabilizers, color-deterioration preventing agents, Substantially the same properties and functions as of and fillers. cyclotetrasaccharide. Therefore, the branched cyclotetrasac 15 The branched cyclotetrasaccharides and the saccharide charides can be used for purposes similarly as in cyclotet compositions comprising the same can be used intact as rasaccharide. The following is a brief description of the uses seasonings to give a refined taste, and if necessary, they can of the branched cyclotetrasaccharides of the present inven be used in combination with other Sweeteners such as a tion which can be used in accordance with those of the powdered starch hydrogenate, glucose, isomerized Sugar, cyclotetrasaccharide disclosed in Japanese Patent Applica Sugar, maltose, trehalose, honey, maple Sugar, Sorbitol, tion No. 234.937/2000 (International Publication No. WO maltitol, dihydrochalcones, Stevioside, C-glycosylstevio 02/10,361 A1). side, extract from Momordica grosvenori, glycyrrhizin, The branched cyclotetrasaccharides of the present inven thaumatin, L-aspartylphenylalanine methylester, Saccharin, tion are usually stable, non-reducing saccharides, which acesulfam K. Sucralose, glycine, and alanine; or fillers such have a white powder form and a relatively low- or non 25 as dextrins, starches, and lactose. Particularly, the branched sweetness, refined taste. When mixed and processed with cyclotetrasaccharide and the saccharide compositions com other materials, particularly, amino acids or materials con prising the same can be preferably used as low-caloric or taining amino acids such as oligopeptides and proteins, the diet Sweeteners in combination with one or more Sweeteners branched cyclotetrasaccharides hardly induce the browning such as meso-erythritol, xylitol, or maltitol; Sweeteners with reaction, hardly-cause undesirable Smell, and hardly spoil 30 high Sweetness Such as C-glycosylstevioside, thaumatin, the other materials mixed. Thus, the branched cyclotetrasac L-aspartylphenylalanine methylester, saccharin, acesulfam charides of the present invention can be incorporated and K, and sucralose. used as materials or bases in many fields such as food The branched cyclotetrasaccharides and the saccharide products, cosmetics, and pharmaceuticals. compositions comprising the same can be arbitrarily used Since the branched cyclotetrasaccharides of the present 35 intact or optionally mixed with fillers, excipients, and bind invention have an inclusion ability, they effectively inhibit ers; and then shaped into appropriate forms such as a the volatilization and the deterioration of flavoring ingredi granule, sphere, short stick/rod, sheet, cube, and tablet. ents and effective ingredients, and quite satisfactorily, stably The refined taste of the branched cyclotetrasaccharides keep these ingredients. In this case, if necessary, the above and the saccharide compositions comprising the same well stabilization by inclusion can be enhanced by the combina 40 harmonize with other materials having Sour-, acid-, salty tion use of other cyclotetrasaccharides such as cyclodex delicious-, astringent-, and bitter-tastes; and have a satisfac trins, branched cyclodextrins, cyclodextrans, and cyclofrac torily-high acid- and heat-tolerance. Thus, they can be tans. The cyclotetrasaccharides such as cyclodextrins should favorably used as Sweeteners, taste-improving agents, qual not be limited to those with the highest purity, and include ity-improving agents, etc., in seasonings, for example, a Soy those with a lower purity, for example, partial starch hydro 45 sauce, powdered soy sauce, miso, “funmatsu-miso' (a pow lyZates rich in maltodextrins along with cyclodextrins can be dered miso), “morom-i” (a refined sake), “hishio' (a refined arbitrarily used. soy sauce), “furikake' (a seasoned fish meal), mayonnaise, Since cyclotetrasaccharide is not hydrolyzed by amylase dressing, vinegar, 'sanbai-Zu' (a sauce of Sugar, soy sauce or C-glucosidase, it is not assimilated and absorbed by living and vinegar), “funmatsu-Sushi-Su” (powdered vinegar for bodies when orally taken, hardly fermented by intestinal 50 Sushi), 'chuka-no-moto' (an instant mix for Chinese dish), bacteria, and utilized as an aqueous dietary fiber with quite “tentsuyu' (a sauce for Japanese deep-fat fried food), “ment low calories. Also, since cyclotetrasaccharide is hardly Suyu' (a sauce for Japanese vermicelli), sauce, catsup, assimilated by dental-caries-inducing bacteria, it can be used "yakiniku-no-tare' (a sauce for Japanese grilled meat), curry as a Sweetener which does not substantially cause dental roux, instant stew mix, instant Soup mix, "dashi-no-moto' caries. Further, cyclotetrasaccharide also has a function of 55 (an instant stock mix), mixed seasoning, “mirin' (a Sweet preventing the adhesion and solidification of Solid materials sake), “shin-mirin' (a synthetic mirin), table Sugar, and within the oral cavity. The branched cyclotetrasaccharides of coffee Sugar. Also, the cyclotetrasaccharide and the saccha the present invention have the same basic structure as ride compositions comprising the same can be arbitrarily cyclotetrasaccharide and have a high utility as Saccharides used to Sweeten and improve the taste and quality of with a relatively low calorie and cariogenicity compared 60 “wagashi' (Japanese cakes) such as 'senbei' (a rice with conventional saccharides susceptible to fermentation cracker), “arare' (a rice cake cube), “okoshi' (a millet-and by dental-caries inducing bacteria. The branched cyclotet rice cake), ''gyuhi' (a starch paste), "mochi' (a rice paste) rasaccharides of the present invention are non-poisonous, and the like, “manju' (a bun with a bean-jam), “uiro” (a harmless saccharides, free from side effect, and useful as sweet rice jelly), “an' (a bean jam) and the like, “yokan' (a stable materials or bases, and when they are in the form of 65 sweet jelly of beans), “mizu-yokan' (a soft adzuki-bean a crystalline product, they can be arbitrarily processed into jelly), "kingyoku' (a kind of yokan), jelly, pao de Castella, tablets or sugar coated tablets in combination with binders and “amedama” (a Japanese toffee); Western confectioneries US 7,223,570 B2 19 20 Such as a bun, biscuit, cracker, cookie, pie, pudding, butter penicillin, erythromycin, chloramphenicol, tetracycline, cream, custard cream, cream puff waffle, sponge cake, streptomycin, and kanamycin Sulfate; liquid preparations doughnut, chocolate, chewing gum, caramel, nougat, and containing vitamins such as thiamine, riboflavin, L-ascorbic candy; frozen desserts such as an ice cream and sherbet: acid, cod liver oil, carotenoid, ergosterol, and tocopherol; processed foods of fruit and vegetables such as a jam, liquid preparation containing highly unsaturated fatty acids marmalade, and “toka' (conserves); pickles and pickled products Such as a “fukujin-Zuke' (red colored radish pick and ester derivatives thereof such as EPA, DHA, and arachi les), (ettara-lue (a kind of whole fresh radish pickles), “sen donic acid, or containing enzymes such as lipase, elastase, mai-Zuke' (a kind of sliced fresh radish pickles), and “rak urokinase, protease, B-amylase, isoamylase, glucanase, and kyo-Zuke' (pickled shallots); premixes for pickles and 10 lactase; extracts such as ginseng extract, Snapping turtle pickled products such as a “takuan-Zuke-no-moto' (a premix extract, chlorella extract, aloe extract, and propolis extract; for pickled radish), and “hakusai-Zuke-no-moto' (a premix biologically active Substances such royal jelly; and pastes for fresh white rape pickles); meat products such as a ham with alive viruses, lactic acid bacteria or yeasts. By applying and sausage; fish meat products such as a fish ham, fish to the above biologically active substances, the branched sausage, “kamaboko' (a steamed fish paste), 'chikuv wa” (a 15 cyclotetrasaccharides and the saccharide compositions con kind of fish paste), and “tenpura' (a Japanese deep-fat fried taining the same to the above biologically active substances fish paste); “chinm-i' (relish) such as a “uni-no-shiokara' (salted guts of sea urchin), “ika-no-shiokara' (Salted guts of facilitate the production of stabilized, high-quality health squid), “su-konbu’ (processed tangle), “saki-Surume' (dried foods and pharmaceuticals in the form of a liquid, paste, or squid strips), “-fugu-no-mirin-boshi' (a dried mirin-sea solid with less fear of losing or inactivating the effective soned swellfish), seasoned fish flour such as of Pacific cod, ingredients and activities of the Substances. sea bream, shrimp, etc; “tsukudani' (foods boiled down in The methods for incorporating the branched cyclotet soy sauce) Such as those of layers, edible wild plants, dried rasaccharides or the saccharide compositions containing the squids, Small fishes, and shellfishes; daily dishes such as a same into the aforesaid compositions are those which can “nimame' (cooked beans), potato Salad, and “kombu-maki” 25 incorporate them into a variety of compositions before (a tangle roll); milk products; canned and bottled products Such as those of meat, fish meat, fruit, and vegetable; completion of their processings. For Such purposes, the alcoholic beverages such as a synthetic sake, fermented following conventional methods such as mixing, kneading, liquor, sake, fruit wine, sparkling alcoholic beverage, beer; dissolving, melting, soaking, penetrating, dispersing, apply Soft drinks such as a coffee, cocoa, juice, carbonated bev 30 ing, coating, spraying, injecting, crystallizing, and solidify erage, lactic acid beverage, and beverage with lactic acid ing can be appropriately selected. The percentage of the bacteria; instant food products such as instant pudding mix, branched cyclotetrasaccharides or the saccharide composi instant hot cake mix, instant juice or soft drink, instant tions containing the same to the final compositions is usually coffee, “Sokuseki-Shiuko' (an instant mix of adzuki-bean at least 0.1%, desirably, at least 1%, d.s.b. in addition to the Soup with rice cake), and instant Soup mix; and other foods 35 above uses similar to those for cyclotetrasaccharide, the and beverages such as a food for babies, food for therapy, branched cyclotetrasaccharides of the present invention may health/tonic drink, peptide food, and frozen food. The branched cyclotetrasaccharides and the saccharide compo exhibit an activity of growing bifid bacteria and it can be sitions comprising the same can be arbitrarily used to used as a factor for Such purpose when incorporated into improve the taste preference and property of feeds and pet 40 foods, beverages, health foods, health food Supplements, and foods for animals and pets such as domestic animals, poul pharmaceuticals. In this case, other saccharides, having an try, honey bees, silk warms, and fishes; and also they can be activity of growing bifid bacteria, Such as lactosucrose arbitrary used as a taste preference-improving agent, taste (4-B-D-galactosylsucrose), N-acetyl-D-chitosamine improving agent, flavor-imparting agent, quality-improving (N-acetyl-D-glucosamine), and lactulose can be advanta agent, and stabilizer in other tastable products, cosmetics, 45 geously used together. Since the branched cyclotetrasaccha and pharmaceuticals in a solid, paste, or liquid form Such as rides of the present invention has a function of preventing a tobacco, cigarette, tooth paste, lipstick, rouge, lipcream, crystallization of cyclotetrasaccharide in an aqueous solu internal liquidmedicine, tablet, troche, cod liver oil drop, cachou, oral refreshment, and gargle. When used as a tion, it can be used as a crystallization-preventing agent for quality-improving agent or stabilizer, the branched cyclotet 50 Such purpose. For example, when the branched cyclotet rasaccharides and the Saccharide compositions comprising rasaccharides or the saccharide compositions containing the the same can be arbitrarily used in biologically active same of the present invention are added to an aqueous Substances susceptible to lose their active ingredients, as Solution, containing cyclotetrasaccharide alone or in com well as health foods and pharmaceuticals containing Such bination with another reducing saccharide(s) such as glu biologically active Substances. Examples of Such biologi 55 cose, maltose and panose, obtained by any of the above cally active Substances are liquid preparations containing methods, the resulting Solution can be concentrated into a lymphokines such as C.-, 3- and Y-interferons, tumor necrosis supersaturated solution of cyclotetrasaccharide. Further, the factor-O. (TNF-C.), tumor necrosis factor-B (TNF-B), mac rophage migration inhibitory factor, colony-stimulating fac above solution can be used after hydrogenation by reducing tor, transfer factor, and interleukin 2; liquid preparations 60 saccharides contained therein into Sugar alcohols, if neces containing hormones such as insulin, growth hormone, sary. The high cyclotetrasaccharide content solutions thus prolactin, erythropoietin, and follicle-stimulating hormone; obtained can be more efficiently stored in tanks and trans liquid preparations containing biologically active Substaces ported by pumps and tank lorries compared with Solutions Such as BCG vaccine, Japanese encephalitis vaccine, free of the branched cyclotetrasaccharides of the present measles vaccine, live polio vaccine, Smallpox vaccine, teta 65 invention. Thus, the branched cyclotetrasaccharides of the nus toxoid, Trimeresurus antitoxin, and human immunoglo present invention are remarkably useful in industrial han bulin; liquid preparations containing antibiotics such as dlings of cyclotetrasaccharide. US 7,223,570 B2 21 22 The following experiments explain the present invention conventional gas chromatography. As a result, only D-glu in more detail: cose was detected, revealing that the saccharide was cyclotetrasaccharide composed of four moles of D-glucose EXPERIMENT 1 in view of the above mass number and non-reducibility. Nuclear magnetic resonance analysis (called “NMR) of Isolation and Identification of Cyclotetrasaccharides the non-reducing saccharide gave a 'H-NMR spectrum in from Culture of Microorganism FIG. 2 and a 'C-NMR spectrum in FIG. 3. These spectra were compared with those of known saccharides and A liquid culture medium, consisting of 5% (w/v) of revealed that they coincided with those of cyclotetrasaccha panose, 1.5% (w/v) of “ASAHIMEASTTM”, a yeast extract 10 ride-as disclosed by Gregory L. Cote et al. in European commercialized by Asahi Breweries, Ltd., Tokyo, Japan, Journal of Biochemistry, Vol. 226, pp. 641-648 (1994). 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of Based on these results, the non-reducing saccharide iso sodium dihydrogen phosphate dodecahydrate, 0.05% (w/v) lated in the above method was identified with cyclotetrasac magnesium sulfate heptahydrate, and water was placed in a charide, i.e., cyclo-->6)-C-D-glucopyranosyl-(1->3)-C-D- 500-ml Erlenmeyer flask in an amount of 100 ml, was 15 glucopyranosyl-(1->6)-O-D-glucopyranosyl-(1->3)-C-D- sterilized by autoclaving at 121° C. for 20 min, cooled, and glucopyranosyl-(1->}. then seeded with a seed culture of Bacillus globisporus C9 strain, FERM BP-7143, followed by culturing under rotary EXPERIMENT 2 shaking conditions at 27°C. and 230 rpm for 48 hours and centrifuging the resulting culture to remove cells to obtain a C.-Isomaltosyl-transferring Enzyme and supernatant. The supernatant was autoclaved at 120° C. for C-isomaltosylglucosaccharide-forming enzyme 15 min and then cooled, and the resulting insoluble sub stances were removed by centrifugation to obtain a Super EXPERIMENT 2-1 natant. A non-reducing saccharide, which was positive on the Sulfuric acid-methanol method and negative on the 25 Enzyme Preparation Containing diphenylamine-aniline method, was observed in this Super C-isomaltosyl-transferring Enzyme and natant. C-isomaltosylglucosaccharide-forming Enzyme About 90 ml of the supernatant after the autoclaving was adjusted to pH 5.0 and 45° C. and then incubated for 24 A liquid culture medium, consisting of 4.0% (w/v) of hours after the addition of 1,500 units per gram of solids of 30 “PINE-DEX #4TM”, a partial starch hydrolyzate commer “TRANSGLUCOSIDASE L AMANOTM, an O-glucosi cialized by Matsutani Chemical Ind., Tokyo, Japan, 1.8% dase commercialized by Amano Pharmaceutical Co., Ltd., (w/v) of “ASAHIMEASTTM”, a yeast extract commercial Aichi, Japan, and 75 units per gram of solids of “GLU ized by Asahi Breweries, Ltd., Tokyo, Japan, 0.1% (w/v) of COTEAMTM”, a glucoamylase commercialized by Nagase dipotassium phosphate, 0.06% (w/v) of sodium dihydrogen Biochemicals, Ltd., Kyoto, Japan. Thereafter, the resulting 35 phosphate dodecahydrate, 0.05% (w/v) magnesium sulfate mixture was adjusted to pH 12-by the addition of sodium heptahydrate, and water, was placed in 500-ml Erlenmeyer hydroxide and boiled after two hours to decompose the flasks in respective volumes of 100 ml, sterilized by auto reducing Sugars in the Supernatant. After removing insoluble claving at 121° C. for 20 min, cooled, and then seeded with substances by filtration, the filtrate was decolored and a stock culture of Bacillus globisporus C9 strain, FERM deSalted with “DIAION PK218TM’ and “DIAION 40 BP-7143, followed by culturing under rotary-shaking con WA30T.M. ion-exchange resins commercialized by Mitsub ditions at 27°C. and 230 rpm for 48 hours for a seed culture. ishi Chemical Industries, Ltd., Tokyo, Japan, and further About 20 L of a fresh preparation of the same liquid desalted with “DIAION SK-1BTM’, a cation exchange resin culture medium as used in the above seed culture was placed commercialized by Mitsubishi Chemical Industries, Ltd., in a 30-L fermenter, sterilized by heating, and then cooled to Tokyo, Japan, and “AMBERLITE IRA411TM, an anion 45 27° C. and inoculated with 1% (v/v) of the seed culture, exchange resin commercialized by Japan Organo-Co., Ltd., followed by culturing at 27 c. and a pH of 6.0-8.0 for 48 Tokyo, Japan, followed by filtrating with a membrane, hours under aeration-agitation conditions. The resulting cul concentrated by an evaporator, and lyophilized in vacuo to ture was centrifuged at 10,000 rpm for 30 min to obtain obtain about 0.5 g solid of a non-reducing saccharide about 18L of a Supernatant. The obtained Supernatant had an powder. 50 activity of about 1.5 units/ml of C-isomaltosyl-transferring The purity of the non-reducing saccharide powder was enzyme, an activity of about 0.45 unit/ml of O-isomaltosyl analyzed on high-performance liquid chromatography (ab glucosaccharide-forming enzyme, and an activity of about breviated as “HPLC hereinafter). HPLC was carried out 0.95 unit/ml of cyclotetrasaccharide-forming enzyme. using “SHOWDEX KS-801 columnTM, Showa Denko The Supernatant was concentrated to give a Volume of two K.K., Tokyo, Japan, at a column temperature of 60° C. and 55 liters by using “API-2013TM”, a membrane for ultrafiltration a flow rate of 0.5 ml/min of water, and using “RI-8012TM, commercialized by Asahi Kasei Corporation, Tokyo, Japan, a differential refractometer commercialized by Tosoh Cor to obtain an enzyme preparation containing O-isomaltosyl poration, Tokyo, Japan. As shown in FIG. 1, a single peak glucosaccharide-forming enzyme and C-isomaltosyl-trans was detected at an elution time of 10.84 min, revealing that ferring enzyme. The enzyme preparation had about 8.5 the saccharide had a high purity of at least 99.9%. 60 units/ml of cyclotetrasaccharide-forming activity. Mass analysis by fast atom bombardment mass spectrom etry (called "FAB-MS) of the non-reducing saccharide, EXPERIMENT 2-2 obtained by the aforesaid method, significantly detected a proton-addition-molecular ion with a mass number of 649, Purified O-isomaltosyl-transferring Enzyme revealing that the Saccharide had a mass number of 648. 65 The above non-reducing saccharide was hydrolyzed with About 18 L of the culture supernatant of Bacillus glo Sulfuric acid and then analyzed for Sugar composition by bisporus C9 strain in Experiment 2-1, having 26.900 units of US 7,223,570 B2 23 24 O-isomaltosyl-transferring enzyme, was salted out with 80% The fractions purified on the ion-exchange resin were saturated ammonium sulfate at 4° C. for 24 hours. The pooled and dialyzed against 10 mM phosphate buffer (pH formed sediments were collected by centrifugation at 10,000 7.0) with 1 Mammonium sulfate. The dialyzed inner solu rpm for 30 min, dissolved in 10 mM phosphate buffer (pH tion was centrifuged to remove insoluble Substances, and fed 7.5), and dialyzed against a fresh preparation of the same to affinity column chromatography using 500 ml of buffer to collect a dialyzed inner solution. “SEPHACRYL HRS-200, a gel commercialized by Amer sham Corp., Div., Amersham International, Arlington The dialyzed inner solution was fed to a column packed Heights, Ill., USA. The elution of the enzyme was carried with 1,000 ml of “SEPABEADS FP-DA13TM gel, an ion out using a linear gradient decreasing from 1 M to O M of exchange resin commercialized by Mitsubishi Chemical ammonium sulfate and Subsequently a linear gradient Industries, Ltd., Tokyo, Japan, followed by collecting non 10 increasing from 0 mM to 100 mM of maltotetraose, followed adsorbed fractions. by collecting fractions eluted at around 30 mM maltotet The non-adsorbed fractions were pooled and dialyzed raose with an O-isomaltosylglucosaccharide-forming against 10 mM-phosphate buffer (pH 7.0) with 1 Mammo enzyme activity. nium Sulfate, and the dialyzed inner Solution was collected The fractions, purified on the above affinity column and centrifuged to remove insoluble Substances, and then 15 chromatography, were pooled and dialyzed against 10 mM fed to affinity column chromatography using 500 ml of phosphate buffer (pH 7.0) containing 1 Mammonium sul “SEPHACRYL HR S-200TM”, a gel commercialized by fate. The dialyzed inner solution was centrifuged to remove Amersham Corp., Div. Amersham International, Arlington insoluble Substances, and the resulting Supernatant was fed Heights, Ill., USA, followed by feeding thereunto a linear to hydrophobic column chromatography using 350 ml of gradient decreasing from 1 M to 0 M of ammonium sulfate “BUTYL-TOYOPEARL 650 M', a gel commercialized by and collecting fractions, having an O-isomaltosyl-transfer Tosoh Corporation, Tokyo, Japan, followed by feeding ring enzyme activity, eluted at around 0 M ammonium thereunto a linear gradient decreasing from 1 M to 0 M of ammonium Sulfate and collecting fractions with an O-iso sulfate. maltosylglucosaccharide-forming enzyme activity eluted at The factions purified on the above affinity column chro 25 around 0.3 Mammonium sulfate. matography were pooled and dialyzed against 10 mM phos The factions, purified on the above hydrophobic chroma phate buffer (pH 7.0) containing 1 Mammonium sulfate. tography, were poled and dialyzed against 10 mM phosphate The dialyzed inner solution was centrifuged to remove buffer (pH 7.0) containing 1 Mammonium sulfate. The insoluble Substances, and the resulting Supernatant was fed dialyzed inner Solution was centrifuged to remove insoluble to hydrophobic chromatography using 350 ml of “BUTYL Substances., and the resulting Supernatant was fed to affinity TOYOPEARL 650 M”, a gel commercialized by Tosoh 30 chromatography again under the same conditions as in the Corporation, Tokyo, Japan, followed by feeding thereunto a above, followed by collecting a fraction with the enzyme linear gradient decreasing from 1 M to 0 M of ammonium activity. Sulfate and collecting fractions, having an O-isomaltosyl The above faction was subjected to SDS-PAGE using a transferring enzyme activity, eluted at around 0.3 Mammo 7.5% (w/v) of a polyacrylamide gel and detected as a single nium sulfate. 35 protein band with a molecular weight of about 140,000+20, The factions purified in the above hydrophobic chroma 000 daltons. The specific activity of the enzyme in the fraction was calculated based on its enzyme activity and tography were pooled and dialyzed against 10 mM phos protein quantitative analysis, revealing that it had a specific phate buffer (pH 7.0) containing 1 Mammonium sulfate. activity of about 13.6 units/mg protein. Thus, a purified The dialyzed inner solution was centrifuged to remove 40 specimen of C-isomaltosylglucosaccharide-forming enzyme insoluble Substances, and the resulting Supernatant was fed was obtained. to affinity column chromatography again under the same Though the above Experiments 2-1 to 2-3 show the data conditions as above, followed by collecting a fraction with on preparations of enzymes from Bacillus globisporus C9 the enzyme activity. strain, such enzymes can be also obtained from Bacillus The faction, purified twice on the affinity column chro 45 globisporus C11 strain, FERM BP-7144, in accordance with matography, was subjected to SDS-PAGE using a 7.5% the above methods. The following are the properties of (w/v) of polyacrylamide gel, resulting in a single protein enzymes purified from Bacillus globisporus C11 strain in band at a position corresponding to a molecular weight of accordance with Experiments 2-1 to 2-3: about 112,000+20,000 daltons. The specific activity of the (A) O-isomaltosyl-transferring enzyme Molecular weight enzyme in the fraction was calculated based on its enzyme 50 on SDS-PAGE: 102,000+20,000 Daltons Specific activity and protein quantitative analysis, revealing that it activity: About 26.9 units/mg protein had a specific activity of about 26.9 units/mg protein. Thus, (B) O-isomaltosylglucosaccharide-forming enzyme a purified C-isomaltosyl-transferring enzyme specimen was Molecular weight on SDS-PAGE: 137,000+20,000 obtained. Daltons Specific activity: About 13.4 units/mg protein 55 EXPERIMENT 2-3 EXPERIMENT 3 Purified C-isomaltosylglucosaccharide-forming Production of Cyclotetrasaccharide by Enzymatic Enzyme Action on Partial Starch Hydrolyzate and Branched 60 Cyclotetrasaccharide as a By-product About 18 L of a culture supernatant of Bacillus glo bisporus C9 strain obtained by the method in Experiment EXPERIMENT 3-1 2-1, having 8,110 units of an O-isomaltosylglucosaccharide forming enzyme, was subjected to salting-out with an 80% Enzymatic Reaction saturation solution of ammonium sulfate, dialyzed, and 65 purified on ion-exchange resin according to method in 3.7 kg of “PINE-DEX #100TM”, a partial starch hydro Experiment 2-2. lyzate commercialized by Matsutani Chemical Ind., Tokyo, US 7,223,570 B2 25 26 Japan, was dissolved in 35 L of 10 mM sodium acetate (pH of 160 cm and which were cascaded in series and packed 6.0). To the resulting solution was added about 17,500 units with about 225 L of “AMBERLITE CR-1310 (Na-form)TM, of cyclotetrasaccharide forming activity of an enzyme preparation, obtained by the method in Experiment 2-1, and an ion-exchange resin commercialized by Japan Organo Co., enzymatically reacted at 30° C. for two days, followed by 5 Ltd., Tokyo, Japan; and chromatographed at a flow rate of boiling for 20 minto inactivate the remaining enzyme. The about 45 L/h of water and a column temperature of 60° C. resulting mixture was cooled to 45° C. and admixed with 11 The resulting elute was fractionated and analyzed for sac g (137,500 units) of “NEO-SPITASE PK2TM”, ano-amylase charide composition on HPLC as described in Experiment specimen commercialized by Nagase Biochemicals, Ltd., 3-1 in Such a manner of pooling fractions relatively rich in Kyoto, Japan, and 44 g (140,800 units) of “GLUCO 10 ZYMETM”, a glucoamylase specimen commercialized by cyclotetrasaccharide into a saccharide Solution with a solid Nagase Biochemicals, Ltd., Kyoto, Japan, and then adjusted content of 1,530 g. HPLC analysis of the saccharide solution to pH 6.0, followed by enzymatically reacting at 45° C. for conducted under the same conditions as above and the data one day. The reaction mixture was boiled for 20 minutes to calculated based on the results on the peak areas of HPLC inactivate the remaining enzymes, and then cooled and 15 revealed that the pooled fraction, i.e., a high cyclotetrasac filtered in a usual manner to obtain a filtrate. The filtrate was concentrated to give a solid concentration of about 16% charide content fraction, contained 79.8% cyclotetrasaccha (w/w) by using a reverse osmosis membrane. The resulting ride of 79.8% and 6.1% isomaltose to the total sugar concentrate was Subjected in a usual manner to decoloration, COntentS. desalting, filtration, and concentration to obtain about 6.1 kg The high cyclotetrasaccharide content fraction with a of a saccharide solution with a solid content of about 3.5 kg. solid content of 1,310 g was adjusted to pH 5.0 and 50° C., The saccharide solution was analyzed by the following and then incubated for 20 hours after admixed with 1,000 HPLC by using both cyclotetrasaccharide, isolated by the units/g solids of “TRANSGLUCOSIDASE LAMANOTM, method in Experiment 1, and known saccharides as a 25 an O-glucosidase commercialized by Amano Pharmaceuti standard. HPLC was run using “CCPM’, a chromatograph cal Co., Ltd., Aichi, Japan, and 60 units/g solids of “GLU commercialized by ToSoh Corporation, Tokyo, Japan, “AQ COTEAMTM”, a glucoamylase commercialized by Nagase 303 ODS', and a column with a diameter of 4.6 mm and a Biochemicals, Ltd., Kyoto, Japan. After removing insoluble length of 25 cm commercialized by YMC Co., Ltd., Tokyo, substances by filtration, the above reaction mixture was Japan, at a column temperature of 40° C. and a flow rate of 30 0.5 ml/min of water; and using “RI-8012TM”, a differential desalted with “DIAION PK218TM”, a cation exchange resin refractometer commercialized by Tosoh Corporation, commercialized by Mitsubishi Chemical Industries, Ltd., Tokyo, Japan. Among the ingredients detected on this analy Tokyo, Japan, and “AMBERLITE IRA411TM, an anion sis, the data for those with a respectively-wide peak area exchange resin commercialized by Japan Organo Co., Ltd., were shown in Table 2 including their retention times, 35 Tokyo, Japan, followed by concentrating. The concentrate names, and relative values of peak areas. was fractionated according to the conditions used in the above chromatography to obtain a fraction with a cyclotet TABLE 2 rasaccharide purity of at least 97%. The fractions were pooled and in a conventional manner decolored, desalted, Retention time 40 on HPLC (min) Name Peak area filtered, and concentrated into a saccharide solution with a solid content of about 1.260 g. After adjusted to give a solid 1O.S Cyclotetrasaccharide 43.8% 19.7 Secondary Product 1 10.6% concentration of about 50% (w/w), the saccharide solution 24.7 Secondary Product 2 3.3% was placed in a cylindrical plastic vessel and cooled from 49.9 Secondary Product 3 O.S9/o 45 58.2 Secondary Product 4 O.3% 65° C. to 20° C. over about 20 hours under gentle stirring conditions to effect crystallization. The formed crystals were * All the retention times are approximate values. **Each value is a relative value when the sum of the detected peak areas collected by separation and dried at 60° C. for three hours is regarded as 100. under normal pressure to obtain 544 g of a crystalline 50 powder. The crystalline powder was of a crystalline cyclotet Upon the above HPLC, as a whole, there exists a tendency that the higher the molecular weight of a compound tested, rasaccharide with a purity of 99.9% and a moisture content the longer the retention time of the compound becomes. of 12.7%. Thus, the results in Table 2 show that the enzymatic reaction EXPERIMENT 3-3 of this experiment usually forms saccharides with a higher 55 molecular weight than cyclotetrasaccharide, i.e., those Isolation of by-products which are composed of a larger number of saccharides than cyclotetrasaccharide, together with cyclotetrasaccharide. 6.1 kg of a saccharide solution obtained by the method in 60 EXPERIMENT 3-2 Experiment 3-1 was fractionated according to the chroma tography as described in Experiment 3-2, and the resulting Preparation of Cyclotetrasaccharides fractions were analyzed for saccharide composition on HPLC as described in Experiment 3-1. Fractions relatively 6.1 kg of a saccharide solution obtained by the method in 65 rich in the by-products 1 and 2 were pooled for Fraction 1, Experiment 3-1 was fed to columns, consisting of ten while fractions relatively rich in the by-products 3 and 4 columns, which each had a diameter of 13.5 cm and a length were pooled for Fraction 2. Fractions 1 and 2 had solid US 7,223,570 B2 27 28 contents of 320 g and 150 g, respectively. Based on the peak gyi-Nelson method, (3) Saccharide composition was deter areas of Fractions 1 and 2 on HPLC, their saccharide mined by conventional gas chromatography to analyze con compositions were determined, revealing that Fraction 1 stituent saccharides after hydrolysis with sulfuric acid, (4) contained 47.9% of the by-product 1 and 14.9% of the biological analysis was conducted by the HPLC analysis by-product 2 to the total Sugars and Fraction 2 contained described in Experiment 3-1 after treatment with TRANS components, which contained the by-products 3 and 4 had a GLUCOSIDASE LAMANOTM, an O-glucosidase commer retention time longer than that of by-product 2 when mea cialized by Amano Pharmaceutical Co., Ltd., Aichi, Japan, (5) binding fashion was determined by a conventional sured on the above HPLC, in an amount of at least 25% to methylation analysis, (6) Specific rotation was measured in the total Sugars. 10 a conventional manner, and (7) C-NMR was done in a Fraction 1 was kept at a pH of 11 or higher by the addition conventional manner. The results are as follows: of sodium hydroxide and heated at 95°C. or higher for one The by-product 1 was a Substantially non-reducing sac hour to decompose reducing saccharides, followed by decol charide with a mass number of 810. Considering the mass oring, desalting, filtering, and concentrating in a conven number and the fact that the constituent saccharides were tional manner. An adequate amount of the concentrate equal 15 only D-glucose molecules, the by-product 1 was estimated to a solid content of 50 g. by weight was subjected to to be composed of five D-glucose molecules. When treated preparative liquid chromatography using “YMN-PACK with C-glucosidase, cyclotetrasaccharide and an equimolar ODS-A R355-15 S-15 120A', a preparative column com of glucose were formed. Upon methylation analysis, 2.3- dimethylated compound, 2,3,4-trimethylated compound, mercialized by YMC Co., Ltd., Tokyo, Japan, and a purified, 2,4,6-trimethylated compound, and 2.3.4.6-tetramethylated deionized water as a moving phase. Based on the HPLC compound were detected in a molar ratio of 0.83:1.02:1.69: analysis described in Experiment 3-1, the above chromatog 1, meaning a composition ratio of 1:1:2:1. The by-product 1 raphy yielded a fraction with a solid content of 20 g had a specific rotation of C.I’d+24.6° and a 'C-NMR containing the by-product 1 with a purity of at least 97%, spectrum in FIG. 4. The data on the signal assignment of the and another fraction with a solid content of five grams 25 by-product 1 is in Table 4 below together with those of containing the by-product 2 with a purity of at least 96%. cyclotetrasaccharide and other saccharides in Experiments Similarly as in Fraction 1, the above Fraction 2 was 3-4 and 4-3. Subjected to decomposition of reducing saccharides, fol Based on these results, the by-product 1 was identified lowed by decoloring, desalting, filtrating, and concentrating. with a blanched cyclotetrasaccharide having a structure An adequate amount of the concentrate equal to a solid represented by Chemical Formula 1.

Chemical Formula 1:

4

1 C-D-Glcp

content of 10 g by weight was subjected to preparative liquid 50 The by-product 2 was a Substantially non-reducing sac chromatography by using preparative chromatography simi charide with a mass number of 972. Considering the mass larly as in Fraction 1. Based on the HPLC analysis described number and the fact that the constituent saccharides were in Experiment 3-1, the above chromatography yielded a D-glucose molecules, the by-product 2-was estimated to be fraction with a solid content of 77 mg containing the composed of six D-glucose molecules. Upon methylation by-product 3 with a purity of at least 97%, and another 55 analysis, 2,4-dimethylated compound, 0.2.3,4-trimethylated fraction with a solid content of 77 mg containing the compound, 2,4,6-trimethylated compound, and 2.3.4.6-tet by-product 4 with a purity of at least 97%. ramethylated compound were detected in a molar ratio of EXPERIMENT 3-4 0.94:2.01:1.72:1, meaning a composition ratio of 1:2:2:1. 60 By-product 2 had a specific rotation of old-246° and a Identification of By-products 'C-NMR spectrum in FIG. 5. The data on the signal assignment of the by-product 2 is in Table 4 below together Fractions rich in the by-products 1 to 4 obtained in with those of cyclotetrasaccharide and other saccharides in Experiment 3-3 were subjected to wholly or partly to the Experiments 3-4 and 4-3. following analysis: (1) Mass number was determined by 65 Based on these results, the by-product 2 was identified mass spectroscopy using the high-speed atom shocking with a blanched cyclotetrasaccharide having a structure method, (2) reducing power was determined by the Somo represented by Chemical Formula 3. US 7,223,570 B2 29 30

Chemical Formula 3:

3

1

The by product 3 was a Substantially non-reducing sac following characteristics. The blanched cyclotetrasaccha charide with a mass number of 1.296. Considering the mass rides have a basic structure represented by Formula 1, where number and the fact that the constituent saccharides were one or more of R to R are optionally substituted C-D- D-glucose molecules, the by-product 3 was estimated to be glucopyranosyl-(1->6)-C-D-glucopyranosyl-(1->3)-O-D- composed of eight D-glucose molecules and had a C 15 glucopyranosyl-(1->6)-C-D-glucopyranosyl groups, with NMR spectrum in FIG. 6. The data on the signal assignment the proviso that “n” is an integer of at least 0 and, when at of the by-product 3 is in Table 4 below together with those least two of the groups of R to R are the above groups, the of cyclotetrasaccharide and other saccharides in Experi number of each “n” is independent in each group; and in ments 3-4 and 4-3. relatively many cases, R. and/or Rs are/is optionally Substi Based on these results, the by-product 3 was identified tuted C-D-glucopyranosyl-(1->6)-O-D-glucopyranosyl with a blanched cyclotetrasaccharide having a structure (1->3)-C-D-glucopyranosyl-(1->6)- C-D-glucopyranosyl represented by Chemical Formula 4. groups, with the proviso that “n” is an integer of at least 0

Chemical Formula 4:

3

1

The by-product 4 was a Substantially non-reducing sac and, when both R- and Rs are the above groups, the number charide with a mass number of 1, 134. Considering the mass 35 of each “n” is independent in each group. number and the fact that the constituent saccharides were D-glucose molecules, the by-product 4 was estimated to be EXPERIMENT 4 composed of seven D-glucose molecules. Upon methylation analysis, 2,3-dimethylated compound, 2,4-dimethylated Glycosyl Transfer to Cyclotetrasaccharide by compound, 2,3,4-trimethylated compound, 2,4,6-trimethy 40 Isolated Enzyme lated compound, and 2.3.4.6-tetramethylated compound were detected in molar ratio of 0.78: 0.78:1.47:1.60:2, mean EXPERIMENT 4-1 ing a composition ratio of 1:1:1:2:2. The by-product 4 gave a 'C-NMR spectrum in FIG. 7. The data on the signal Glycosyl Transfer by assignment of the by-product 3 is in Table 4 below together 45 C-isomaltosylglucosaccharide-forming Enzyme with those of cyclotetrasaccharide and other saccharides in Experiments 3-4 and 4-3. A 10 mM sodium acetate buffer (pH6.0) containing 20% Based on these results, the by-product 4 was identified (w/w) of cyclotetrasaccharide, obtained by the method in with a blanched cyclotetrasaccharide having a structure Experiment 3-2, and 10% (w/w) of maltopentaose, produced represented by Chemical Formula 5. by Hayashibara Biochemical Laboratories Inc., Okayama,

Chemical Formula 5:

Judging totally the constituent saccharides and the bind Japan, was incubated at 30° C. for 24 hours after admixed ing fashions of blanched parts of blanched cyclotetrasac with 3 units/g maltopentaose of a purified C-isomaltosyl charides identified in Experiment 3-4, the blanched cyclotet 65 glucosaccharide-forming enzyme obtained by the method in rasaccharides as by-products, which were formed in the Experiment 2-3. The reaction mixture was then boiled for 20 enzymatic reaction in Experiment 3-1, would have the minutes to inactivate the remaining enzyme. US 7,223,570 B2 31 32 The resulting mixture was adjusted to pH 5.0 and incu after admixed with 10 units/g C-cyclodextrin of CGTase bated at 50° C. for one hour after admixed with 500 units/g from Bacillus Stearothermophilus, commercialized by solids of “GLUCOTEAMTM, a glucoamylase commercial Hayashibara Biochemical Laboratories Inc., Okayama, ized by Nagase Biochemicals, Ltd., Kyoto, Japan. Thereaf Japan. The reaction mixture was boiled for 20 minutes to ter, the reaction mixture was boiled for 10 minutes to inactivate the remaining enzyme. inactivate the remaining enzyme. To the reaction mixture was added 350 g of 50 mM After glucoamylase treatment, the resulting reaction mix sodium acetate buffer (pH 4.5) and then incubated at 40°C. ture was filtered in a conventional manner with a membrane, for four hours after admixed with 2,000 units of “GLU desalted, and analyzed on HPLC as in Experiment 3-1. 10 COTEAMTM”, a glucoamylase commercialized by Nagase Comparison of retention times on HPLC for the blanched Biochemicals, Ltd., Kyoto, Japan. Thereafter, the resulting cyclotetrasaccharides, which had been isolated and identi culture was boiled for 20 minutes to inactivate the remaining fied in Experiments 3-3 and 3-4, revealed that the reaction enzyme. mixture contained a branched cyclotetrasaccharide, repre The above reaction mixtures, obtained after the CGTase sented by Chemical Formula 1, in an amount of 17.3% to the 15 treatment and the combination treatment with CGTase and total Sugars, when calculated based on a relative ratio of its glucoamylase, were subjected to HPLC as in Experiment peak area on HPLC. This result indicates that the branched 3-1. While, under the same conditions as above, cyclotet cyclotetrasaccharide of the present invention can be effi rasaccharide and glucose were analyzed. Based on these, the ciently produced by contacting O-isomaltosylglucosaccha components in the above reaction mixtures were identified. ride-forming enzyme with cyclotetrasaccharide. The results on the reaction mixtures after the CGTase treatment and the combination treatment with CGTase and EXPERIMENT 4-2 glucoamylase are respectively shown in FIG. 8a and FIG. 8b. Glycosyl Transfer by C-isomaltosyl-transferring 25 A peak X with a retention time of about 10 min, com Enzyme monly observed in FIGS. 8a and 8b, is a peak for cyclotet rasaccharide, and a peak G with a retention time of about six A 10 mM sodium acetate buffer (pH6.0) containing 20% minutes, specifically observed in FIG. 8b, is a peak for (w/w) cyclotetrasaccharide obtained by the method in 30 glucose. As shown in FIGS. 8a and 8b, the reaction mixture Experiment 3-2 and 10% (w/w) of panose produced by with the CGTase treatment contained newly formed com Hayashibara Biochemical Laboratories Inc., Okayama, ponents with longer retention times than that of cyclotet Japan, was incubated at 30° C. for 24 hours after admixed rasaccharide (FIG. 8a), while that with the combination with 30 units/g panose of a purified C-isomaltosyl-transfer treatment of CGTase and glucoamylase has almost lost these ring enzyme obtained by the method in Experiment 2-2. The 35 components except for two components with peaks 1 and 2 reaction mixture was boiled for 20 minutes to inactivate the (FIG. 8b). These results indicate that the peaks, observed in remaining enzyme. FIG. 8a, except for that for cyclotetrasaccharide were for The reaction mixture was in a conventional manner glycosyl derivatives of cyclotetrasaccharide to which one filtered with a membrane, desalted, and analyzed on HPLC or -more glycosyl groups were bound. In FIG. 8b, the two as shown in Experiment 3-1. Comparison of retention times 40 peaks, i.e., peaks 1 and 2, with retention times longer than on HPLC of the blanched cyclotetrasaccharides, which had that of cyclotetrasaccharide are glucosyl derivatives of been isolated and identified in Experiments 3-3 and 3-4, cyclotetrasaccharide where a glycosyl group is bound to a revealed that the reaction mixture contained a branched specific position of cyclotetrasaccharide. The retention times cyclotetrasaccharide, represented by Chemical Formula 3, in 45 of these two components in the reaction mixture, received an amount of 4.9% to the total Sugars, when calculated based with the combination treatment of CGTase and glucoamy on a relative ratio of its peak area on HPLC. This result lase, are shown in Table 3 together with respective names revealed that the branched cyclotetrasaccharide of the and relative values of peak areas. present invention can be efficiently produced by contacting C-isomaltosyl-transferring enzyme with cyclotetrasaccha 50 TABLE 3 ride. Retention time EXPERIMENT 4-3 on HPLC (min) Name Peak area 18.7 CGTase product 1 About 35% Glycosyl Transfer by Cyclomaltodextrin 55 38.7 CGTase product 2 About 22% glucanotransferase (CGTase) *Each time determined is a rough value. *Each value is a relative value when the sum of detected peak areas is EXPERIMENT 4-3(a) regarded as 100. 60 Enzymatic Reaction EXPERIMENT 4-3(b) Tengrams of cyclotetrasaccharide obtained by the method Isolation and Identification of Reaction Product in Experiment 3-2 and 10 g of C.-cyclodextrin, produced by Hayashibara Biochemical Laboratories Inc., Okayama, 65 A reaction mixture, obtained after the combination treat Japan, were dissolved in 30 g of 50 mM sodium acetate ment of CGTase and glucoamylase in the method in Experi buffer (pH 5.5), and then incubated at 50° C. for 24 hours ment 4-3(a), was filtered with a membrane, and the filtrate US 7,223,570 B2 33 34 was desalted with “DIAION PK218TM”, a cation exchange resin commercialized by Mitsubishi Chemical Industries, TABLE 4-continued Ltd., Tokyo, Japan; and “AMBERLITE IRA411TM”, an anion exchange resin commercialized by Japan Organo Co., Chemical shift (ppm 5 Ltd., Tokyo, Japan, followed by concentrating. The concen Carbon number A. B C D E F trate was fractionated according to the chromatographic 2b 72.64 72.71 72.64 72.63 72.58 72.91 conditions in Experiment 3-2, and the resulting each fraction 3b 77.31 77.58 77.23 77.13 77.26 77.93 was analyzed on HPLC in Experiment 3-1 to obtain a 4b 73.62 73.58 73.62 73.65 73.52 73.76 Sb 74.23 74.22 74.25 74.28 74.2O 74.42 fraction containing product 1 or 2 produced by CGTase 10 6b 62.88 62.87 62.88 62.87 62.84 63.08 (called “CGTase product 1 or 2) with a purity of 97%. 1c 99.35 99.32 99.28 99.18 The fraction rich in the CGTase product 1 was in a 2c 74.22 74.25 74.28 75.47 conventional manner subjected to 'C-NMR, and the 3c 7549 75.45 75.43 75.77 obtained spectrum coincided with that of the by-product 1 of 4c 73.34 73.36 73.37 81:14 15 Sc 72.71 72.78 72.75 71.09 Chemical Formula 1 (FIG. 4) in Experiment 3-4. Based on 6c 70.15 70.08 7O.OO 70.2O the result, the CGTase product 1 was identified with a 1d 101.18 101.17 101.08 101.13 2d 72.65 72.64 72.63 72.66 branched cyclotetrasaccharide represented by Chemical for 3d 77.37 77.23 77.04 77.51 mula 1. 4d 73.58 73.57 73.58 73.52 The fraction rich in the CGTase product 2 was analyzed Sd 74.22 74.25 74.28 74.2O 6d 62.87 62.88 62.87 62.84 in accordance with the 7th analytical item in Experiment 1e 102.14 100.59 10OSO 100.54 102.34 3-4. The CGTase product 2 was a substantially non-reducing 2e 74.40 74.25 74.28 74.2O 74.60 saccharide with a mass number of 972. Considering the mass 3e 75.62 75.82 7585 75.77 75.81 number and the fact that the constituent saccharides were 4e 72.23 72.16 72.O2 72.12 72.43 25 Se 74.10 73.10 73.08 73.05 74.33 D-glucose molecules, the CGTase product 2 was judged to 6e 63.38 68.18 67.85 68.14 63.58 be composed of six molecules of D-glucose. When treated 1f 101.84 102.17 101.79 with C-glucosidase, the CGTase product 2 was hydrolyzed 2f 74.25 72.63 74.2O 3f 75.82 82.98 75.77 into cyclotetrasaccharide and glucose in a molecule ratio of 4f 72.25 72.87 72.20 1:2. Upon methylation analysis, 2,3-dimethylated com 30 5f 74SO 74.28 74.45 pound, 2,4,6-trimethylated compound, and 2.3.4.6-tetram 6f 63.18 62.87 63.13 1g 10OSO 102.11 ethylated compound were observed in a molar ratio of 2g 74.28 74.36 0.89:1:1.24, meaning a composition ratio of 1:1:1. The 3g 7585 75.57 CGTase product 2 had a specific rotation of C.I’d+24.1° and 4g 72.24 72.20 gave a 'C-NMR spectrum in FIG. 9. The assignment of 35 5g 72.99 74.06 6g 67.85 63.33 signals of the spectrum are in Table 4 below together with 1h 102.17 the those of cyclotetrasaccharide and other saccharides 2h 74.28 described in Experiments 3-4. 3h 75.92 4h 72.24 40 Sh 74.51 TABLE 4 6h 63.16 Chemical shift (ppm A: Cyclotetrasaccharide, B: By-product 1*, Carbon number A. B C D E F C: By-product 2*, 45 1a. 99.34 99.24 99.46 99.40 99.44 99.49 D: By-product 3*, 2a 74.28 75.49 72.84 72.87 72.8O 75.72 E: By-product 4*, 3a 75.45 75.75 82.47 82.98 82.45 75.98 F: CGTase 2 4a 73.35 81.18 73.81 73.72 73.76 81.38 Sa 72.78 71.14 72.45 72.46 72.38 71.34 6a 70.22 70.27 70.17 70.10 69.98 70.47 Based on these results, the CGTase product 2 was iden 1b 101.20 101.18 101.17 101.08 101.13 101.43 50 tified with a branched cyclotetrasaccharide having a struc ture represented by Chemical formula 2.

Chemical Formula 2:

4 4

1 1 US 7,223,570 B2 35 36 Totally judging from the constituent saccharides and the EXPERIMENT 4-4(b) binding fashions of the branched part of the branched cyclotetrasaccharide identified in Experiment 4-3(b) and Isolation and Identification of Reaction Product from the results of HPLC analysis on the branched cyclotet rasaccharide after the enzymatic reaction in Experiment 4-3(a), it is considered that the branched cyclotetrasaccha ride formed by the enymatic reaction in Experiment 4-3(a) To a reaction mixture obtained by the method in Experi has the following characteristics: The blanched cyclotet ment 4-4(a) was added 4.8g of Sodium hydroxide and kept rasaccharide has a basic structure represented by Formula 1, at 100° C. for one hour to decompose reducing saccharides. wherein in Formula 1 one or more of R to R2, particularly, 10 The resulting mixture was decolored, desalted, filtered, and R and/or R, in relatively many cases are/is an optionally substituted C-D-glucopyranosyl-(1->4)-}, C-D-glucopyra concentrated in a conventional manner, followed by Sub nosyl group(s), with the proviso that “n” is an integer of 0 jecting the concentrate to a preparative liquid chromatogra or more, and when at least two of R to R are the above phy using “YMN-PACK ODS-AQR355-15AQ, S-10/20 groups, “n” is each independent in each group. 15 um, 120ATM, a preparative column commercialized by EXPERIMENT 4-4 YMC Co., Ltd., Tokyo, Japan, and using a purified, deion ized water as a moving bed. The eluate was analyzed on Glycosyl Transfer by B-galactosidase from Bacillus HPLC as described in Experiment 3-1, resulting in obtaining circulans a fraction containing a product produced by B-galactosidase (or 3-galactosidase product 1) with a purity of at least 97%. EXPERIMENT 4-4(a) Enzymatic Reaction The above fraction was analyzed in accordance with 25 seven analytical items in Experiment 3-4. The B-galactosi Twenty grams of cyclotetrasaccharide obtained by the dase product 1 was a Substantially non-reducing saccharide method in Experiment 3-2 and 20 g of a special grade with a mass number of 810. The constituent saccharides of lactose, commercialized by Wako Pure Chemical Industries, the B-galactosidase product 1 were D-glucose and D-galac Ltd., Tokyo, Japan, were dissolved in 93.3 g of 20 mM tose which were present in a composition ratio of 4:1. sodium acetate buffer (pH 6.0). The solution was subjected 30 to an enzymatic reaction at 40°C. for 24 hours after admixed Judging from its mass number, the 3-galactosidase product with 3 units/g lactose of “BIOLACTAN 5TM, a B-galac 1 was considered to be composed of four molecules of tosidase from a microorganism of the species Bacillus D-glucose and one molecule of D-galactose. The B-galac circulans commercialized by Daiwa Kasei K.K., Osaka, tosidase product 1 had a specific rotation of C.I’d+200° and Japan, followed by boiling the resulting mixture for 20 35 a 'C-NMR spectrum of FIG. 10. Table 7 shows the results minutes to inactivate the remaining enzyme. of assignment of signals of the spectrum together with those The reaction mixture thus obtained and an aqueous solu of cyclotetrasaccharide and other results in Experiments 4-5 tion of cyclotetrasaccharide were analyzed on HPLC in to 4-7. Experiment 3-1. As a result, the HPLC analysis revealed the Based on these results, the B-galactosidase product 1 was formation of at least three kinds of new components, which 40 had different retention times from that of cyclotetrasaccha identified with a branched cyclotetrasaccharide represented ride (about 10 minutes), in the reaction mixture. Table 5 by Chemical Formula 6.

Chemical Formula 6:

3

1 B-D-Galp shows the retention times of the three components together Experiment 4-5 with their names and relative values of peak areas. 55 Glycosyl Transfer by B-galactosidase from TABLE 5 Asperiment niger

Retention time on HPLC (min) Name Peak area EXPERIMENT 4-5(a) 60 14.0 B-Galactosidase product 1 12.2% Enzymatic Reaction 19.7 B-Galactosidase product 2 2.9% 20.3 B-Galactosidase product 3 1.1% Twenty grams of cyclotetrasaccharide obtained by the *Each time determined is a rough value. method in Experiment 3-2 and 20 g of a special grade **Each value is a relative value when the sum of detected peak areas is 65 lactose, commercialized by Wako Pure Chemical Industries, regarded as 100. Ltd., Tokyo, Japan, were dissolved in 93.3 g of 20 mM sodium acetate buffer (pH 4.5), and then incubated at 40°C. US 7,223,570 B2 37 38 for 24 hours after admixed with 10 units/g lactose of another fraction containing a product formed by B-galac “LACTASE YA-OTM, a B-galactosidase from a microor tosidase (or B-galactosidase product 5) with a purity of at ganism of the species Aspergillus niger, commercialized by least 99%. Yakult Pharmaceutical Inc. Co., Ltd., Tokyo, Japan. There after, the resulting reaction mixture was boiled for 20 minto The above fraction of 3-galactosidase product 4 was inactivate the remaining enzyme. analyzed in accordance with the seven analytical items in The reaction mixture thus obtained and an aqueous solu Experiment 3-4. The B-galactosidase product 4 was a Sub tion of cyclotetrasaccharide were analyzed on HPLC stantially non-reducing saccharide with a mass number of described in Experiment 3-1. As a result, the HPLC analysis 973. The constituent saccharides were D-glucose and D-ga revealed the formation of at least three kinds of new com 10 ponents, which had different retention times from that of lactose, which were present in a molar ratio of 2:1. Judging cyclotetrasaccharide (about 10 minutes), in the reaction from its mass number, the B-galactosidase product 4 was mixture. Table 7 shows the retention times of the three considered to be composed of four molecules of D-glucose components together with their names and relative values of and two molecules of D-galactose. Upon methylation analy peak areas. 15 sis, 2,4-dimethylated glucose, 2,3,4-trimethylated glucose, 2,4,6-trimethylated glucose, 2,3,4-trimethylated galactose, TABLE 6 and 2.3.4.6-tetramethylated galactose were detected in a Retention time molar ratio of 1:1.86:0.96:1.34:1.12, meaning a composition on HPLC (min) Name Peak area ratio of 1:2:1:1:1. Upon 'C-NMR, the f-galactosidase 14.1 Chemical Formula 6*** 3.3% product 4 gave a spectrum of FIG. 11. Table 7 shows the 15.1 B-Galactosidase product 4 O.7% 19.1 B-Galactosidase product 5 7.1% assignment of signals of the spectrum together with those of cyclotetrasaccharide and other results in Experiments 4-5 to *Each value determined is a rough value. **Each value is a relative value when the sum of detected peak areas is 25 4-7. regarded as 100. *** Based on its retention time, it was identified with a branched cyclotet Based on these results, the B-galactosidase product 4 was rasaccharide, represented by Chemical Formula 6 in Experiment 4-4(b). identified with a branched cyclotetrasaccharide represented by Chemical Formula 8.

Chemical Formula 8:

6

1

EXPERIMENT 4-5(b) The above fraction rich in the B-galactosidase product 5 was analyzed in accordance with the seven analytical items Isolation and Identification of Reaction Product 50 in Experiment 3-4. The B-galactosidase product 5 was a Substantially non-reducing saccharide with a mass number of 810. The constituent saccharides were D-glucose and To the reaction mixture, obtained by the method in D-galactose present in a composition ratio of 4:1. Judging Experiment 4-5(a), was added 4.8 g of sodium hydroxide from its mass number, the B-galactosidase product 4 was 55 considered to be composed of four molecules of D-glucose and kept at 100° C. for one hour to decompose reducing and one molecule of D-galactose. Upon methylation analy saccharides. The resulting mixture was decolored, desalted, sis, 2,4-dimethylated glucose, 2,3,4-trimethylated glucose, filtered, and concentrated in a conventional manner. The 2,4,6-trimethylated glucose, and 2.3.4.6-tetramethylated concentrate was Subjected to preparative liquid chromatog galactose were detected in a molar ratio of 1:2.02:1.00:1.04. 60 meaning a composition ratio of 1:2:1:1. Upon 'C-NMR, the raphy using “YMN-PACK ODS-AQR355-15AQ, S-10/20 B-galactosidase product 5 gave a spectrum of FIG. 12. Table um, 120A', a preparative column commercialized by YMC 7 shows the assignment of signals of the spectrum together Co., Ltd., Tokyo, Japan, and using a purified, deionized with those of cyclotetrasaccharide and other results in water as a moving bed. The eluate was analyzed on HPLC Experiments 4-5 to 4-7. described in Experiment 3-1, resulting in obtaining a frac 65 Based on these results, the B-galactosidase product 5 was tion containing a product formed by 3-galactosidase (or identified with a branched cyclotetrasaccharide represented B-galactosidase product 4) with a purity of at least 94%, and by Chemical Formula 7. US 7,223,570 B2 39 40

Chemical Formula 7:

6

1 B-D-Galp

10 Judging totally from the constituent saccharide is and the filtered, and concentrated, the resulting concentrate was binding fashion of the branched part of the branched Subjected to preparative liquid chromatography described in cyclotetrasaccharide identified in Experiment 4-5(b), the Experiment 4-5(b) and to HPLC analysis described in branched cyclotetrasaccharide formed by the enzymatic Experiment 3-1, followed by collecting a fraction containing reaction in Experiment 4-5(a) has the following character 15 a product, formed with O-galactosidase (or C-galactosidase istics in general. The blanched cyclotetrasaccharide has a product), with a purity of at least 98%. basic structure represented by Formula 1, wherein in For The above fraction rich in C-galactosidase product was mula 1 one or more of R to R, particularly in many cases, analyzed according to seven analytical items in Experiment Re and/or R2 are/is an optionally substituted {?-D-galacto 3-4. The O-galactosidase product was a Substantially non pyranosyl-(1->6)- C-D-galactopyranosyl group(s), with reducing Saccharide with a mass number of 810. The C-ga the proviso that “n” is an integer of at least 0 and it is lactosidase product had constituent saccharides of D-glu independent when at least two of R to R have the above cose and D-galactose in a composition ratio of 4:1. Judging groups. from its mass number, the C-galactosidase product was considered to be composed of four molecules of D-glucose EXPERIMENT 4-6 25 and one molecule of D-galactose. Upon methylation analy sis, 2,4-dimethylated glucose, 2,3,4-trimethylated glucose, Glycosyl Transfer by C-Galactosidase 2,4,6-trimethylated glucose, and 2.3.4.6-tetramethylated galactose were observed in a molar ratio of 1:2.09:1.02:1.02, EXPERIMENT 4-6(a) meaning a composition ratio of 1:2:1:1. The C-galactosidase 30 product gave a 'C-NMR spectrum in FIG. 13. Table 7 Enzymatic Reaction shows the assignment of signals of the spectrum together with those of cyclotetrasaccharide and other results in Five grams of cyclotetrasaccharide obtained by the Experiments 4-5 to 4-7. method in Experiment 3-2 and five grams of a special grade Based on these results, the C-galactosidase product was melibiose, commercialized by Wako Pure Chemical Indus 35 identified with a branched cyclotetrasaccharide represented tries, Ltd., Tokyo, Japan, were dissolved in 15 g of 50 mM by Chemical Formula 9.

Chemical Formula 9:

6

1 C-D-Galp sodium acetate buffer (pH 5.0), and then incubated at 40°C. EXPERIMENT 4-7 for 30 hours after admixed with 100 units/g melibiose of “MELIBIASETM”, an O-galactosidase from a microorgan 50 Glycosyl Transfer by Lysozyme ism of the genus Mortierella, commercialized by Hokkaido Sugar Co., Ltd., Tokyo, Japan, and the reaction mixture was boiled for 20 minto inactivate the remaining enzyme. EXPERIMENT 4-7(a) The reaction mixture and an aqueous solution of cyclotet rasaccharide were subjected to HPLC described in Experi 55 Enzymatic Reaction ment 3-1. As a result, the HPLC analysis revealed the formation of at least one newly formed component with a Twenty grams of cyclotetrasaccharide obtained by the retention time of 13.3 min, which differed from cyclotet rasaccharide with a retention time of about 10 min. Upon the method in Experiment 3-2 and 10 g of “NA-COS-YTM, a HPLC analysis, the newly formed component had a peak 60 chichin oligosaccharide commercialized by Yaizu Suisanka area of about 1.0% to the total peak areas. gaku Industry Co., Ltd., Shizuoka, Japan, containing about EXPERIMENT 4-6(b) 55%, d.s.b., of a chitin oligosaccharide with a polymeriza Isolation and Identification of Reactive Product tion degree of two to six, were dissolved in 45 g of 50 mM 65 sodium acetate buffer (pH 4.5), and then incubated at 60° C. After a reaction mixture obtained by the method in for nine days after admixed with 2.8g of albumin lysozyme, Experiment 4-6(a) was in a conventional manner desalted, commercialized by Seikagaku Corporation, Tokyo, Japan. US 7,223,570 B2 41 42 Thereafter, the reaction mixture was boiled for 20 min to signals of the product together with that of cyclotetrasac inactivate the remaining enzyme. charide and other results described in Experiments 4-4 to 4-6. As a treatment before HPLC, the resulting reaction mix ture was centrifuged, then the Supernatant was filtered by TABLE 7 using “SEP-0013TM”, an ultrafiltration membrane commer cialized by Asahi Kasei Corporation, Tokyo, Japan, to Chemical shift (ppm) remove proteins and desalted in a conventional manner. The Carbon number A. B-Gal1* B-GalA* B-Gal5* C-Gal’ Lys pretreated Solution and an aqueous Solution of cyclotetrasac 10 1a. 99.34 98.96 99.39 99.36 99.2S 99.47 charide were subjected to HPLC described in Experiment 2a 74.28 73.67 74.27 74.24 74.28 72.48 3-1. As a result, the HPLC analysis revealed the formation 3a 75.45 83.59 75.4S 75.42 75.44 84.53 of at least one newly formed component with a retention 4a 73.35 73.11 73.28 73.24 73.42 74.03 time of 36.6 min, which differed from cyclotetrasaccharide Sa 72.78 72.44. 72.76 72.76 72.60 72.57 15 6a 70.22 70.06 70.38 70.3S 70.1O 70.18 with a retention time of about 10 min. Upon the HPLC 1b 101.20 101.04 101.22 101.27 101.07 101.13 analysis, the newly-formed component had a peak area of 2b 72.64 72.60 72.6S 72.62 72.60 72.69 about 7.3% to the total peak areas. 3b 77.31 77.29 77.37 77.36 77.35 77.22 4b 73.62 73.62 73.34 73.31 73.61 73.65 Sb 74.23 74.21 73.46 73.45 72.79 74.23 6b 62.88 62.86 70.66 70.59 67.94 62.84 1c 99.21 99.39 99.36 99.2S 99.31 2c 74.26 74.27 74.24 74.28 74.28 3c 7S.41 75.45 75.42 75.44 75.44 EXPERIMENT 4-7(b) 4c 73.34 73.22 73.24 73.35 73.35 25 Sc 72.70 72.76 72.73 72.60 72.77 Isolation and Identification of Reactive Product 6c 70.06 70.22 70.2O 70.1O 70.18 1d 100.95 101.22 10121 100.96 101.13 A reaction mixture, obtained by the method in Experiment 2d 72.54. 72.57 72.54. 72.60 72.63 4-7(a), was similarly treated as the pretreatment of HPLC in 3d 77.03 77.24 77.21 77.18 77.22 Experiment 4-7(a), and then Subjected to preparative liquid 30 4d 73.53 73.34 73.31 73.61 73.60 Sd 74.21 74.27 74.24 74.22 74.23 chromatography according to the method in Experiment 6d 62.86 62.88 6286 62.87 62.84 4-5(b), and to HPLC analysis described in Experiment 3-1, 1e 107.57 105.93 106.01 100.SS followed by collecting a fraction containing a product 2e 74.08 73.42 73.58 71.2O formed by lysozyme (or a lysozyme product), with a purity 35 3e 75.18 75.26 75.42 72.29 4e 71.46 71.37 71.34 71.97 of at least 99%, after analysis and confirmation on the HPLC Se 78.02 76.57 77.83 73.75 analysis in Experiment 3-1. 6e 64.18 71.6O 63.7O 63.87 1f 106.02 104.66 The above fraction rich in the lysozyme product was 40 2f 73.62 58.43 3f 75.45 76.38 analyzed according to the seven analytical items in Experi 4f 71.31 71.86 ment 3-4. The lysozyme product was a Substantially non 5f 77.87 78.52 reducing saccharide with a mass number of 851. The 6f 63.67 63.38 CO 177.58 lysozyme product had D-glucose and N-acetyl-D-glu 45 cosamine (N-acetyl-D-chitosamine) as constituent saccha CH 24.92 rides in a composition ratio of 4:1. Judging from its mass A: Cyclotetrasaccharide number, the lysozyme product was considered to be com *B-Gall means f-galactosidase product 1 isolated in Experiment 4-4(b), posed of four molecules of D-glucose and one molecule of B-Gala and B-GaliS mean f-galactosidase products 4 and 5 isolated in 50 Experiment 4-5(b), N-acetyl-D-glucosamine. Upon methylation analysis, 2,4- C-Gal means C-galactosidase product isolated in Experiment 4-6(b), and dimethylated glucose, 2,3,4-trimethylated glucose, and 2.4. Lys means lysozyme product isolated in Experiment 4–7(b). 6-trimethylated glucose were observed in a molar ratio of 1.02:1:1.67, meaning a composition ratio of 1:1:2. The Based on these results, the lysozyme product obtained in product had a specific rotation of C.I’d+246° and a C this Experiment was identified with a branched cyclotet NMR spectrum of FIG. 14. Table 7 shows the assignment of rasaccharide represented by Chemical Formula 10.

Chemical Formula 10:

3

1 B-D-GlcNAcp US 7,223,570 B2 43 44 EXPERIMENT 5 300° C. or over. These results indicate that the compound of Chemical Formula 1 in the form of a penta- or hexa-hydrous Crystal of Branched Cyclotetrasaccharide crystal is converted into a mono- or di-hydrous crystal when heated to 150° C. under normal pressure. Fraction rich in any one of the by-product 1, obtained by the method in Experiment 3-3 (a branched cyclotetrasac As shown in FIG. 21, the compound of Chemical Formula charide represented by Chemical Formula 1, and hereinafter, 2 gave a weight reduction corresponding to that of six to the branched cyclotetrasaccharides of the present invention seven moles of water as the temperature increased to about are respectively shown by their Chemical Formula num 150° C., and also gave a weight reduction due to its bers.), the by-product 2 (Chemical Formula 3), the CGTase 10 decomposition at a temperature of around 300° C. or over. product 2 obtained by the method in Experiment 4-3(b) These results indicate that the compound of Chemical For (Chemical Formula 2), the B-galactosidase product 1 mula 2 in the form of a undeca- or dodeca-hydrous crystal obtained by the method in Experiment 4-4(b) (Chemical is converted into a tetra- or penta-hydrous crystal when Formula 6), and the B-galactosidase product 5 obtained by heated to 150° C. under normal pressure. the method in Experiment 4-5(b) (Chemical Formula 7), was 15 As shown in FIG. 22, the compound of Chemical Formula concentrated, resulting in an observation of crystal. After 3 gave a weight reduction corresponding to that of six to collecting each crystal, they were dried at ambient tempera ture into five kinds of crystals of the above branched seven moles of water as the temperature increased to about cyclotetrasaccharides. The HPLC analysis described in 110° C., and also gave a weight reduction due to its Experiment 3-1 revealed that the crystals of Chemical decomposition at a temperature of around 300° C. or over. Formulae 1, 2, 6, and 7 had purities of at least 99%, and the These results indicated that the compound of Chemical one of Chemical Formula 3 had a purity of at least 98%. Formula 3 in the form of a deca- or undeca-hydrous crystal The crystalline powders were respectively analyzed on is converted into a tri- or tetra-hydrous crystal when heated the following whole or part of the items of crystalline form, to 110° C. under normal pressure. X-ray diffraction, color, moisture content, melting point, and 25 As shown in FIG. 23, the compound of Chemical Formula thermal property. The crystalline form was microscopically 6 gave a weight reduction corresponding to that of seven to observed. The X-ray analysis was examined by a conven eight moles of water as the temperature increased to about tional powdery X-ray diffraction analysis. The moisture 120° C., and also gave a weight reduction due to its content was measured by the Karl Fischer method, and the decomposition at a temperature of around 300° C. or over. melting point was measured by “MODEL MP-21TM”, a 30 These results indicate that the compound of Chemical For melting point measurement device commercialized by mula 6 in the form of a nona- or deca-hydrous crystal is Yamato Scientific Co., Ltd., Tokyo, Japan. The thermal property was analyzed based on thermogravimetric analysis converted into a mono- or di-hydrous crystal when heated to using “TG/DTA220 typeTM, a digital thermoanalyzer com 120° C. under normal pressure. mercialized by Seiko Instruments Inc., Chiba, Japan. 35 As shown in FIG. 24, the compound of Chemical Formula The microscopic observation revealed that the com 7 gave a weight reduction corresponding to that of five to six pounds of Chemical Formulae 1, 2, and 3 had a pillar moles of water as the temperature increased to about 130° needle-, and pillar-form, respectively. C., and also gave a weight reduction due to its decomposi The data on X-ray diffraction analysis of all the above tion at a temperature of around 300° C. or over. These results crystals are in FIGS. 15 to 19. Predominant diffraction 40 indicate that the compound of Chemical Formula 7 in the angles (20) observed in the analysis are tabulated in Table 8 form of a penta- or hexa-hydrous crystal is converted into an along with the results on color, moisture content, and anhydrous or monohydrous crystal when heated to 130° C. melting point. under normal pressure.

TABLE 8

Number of Main diffraction Moisture crystal of Compound angles (20) Color content Water Melting point Chemical Formula 1 8.1, 12.2 White 11.1% 5 to 6 Not measurable, as it was decomposed 14.2°, 15.4° at around 270° C. Chemical Formula 2 5.6, 8.8 White 17.5% 11 to 12 Not measurable, as it was decomposed 16.9°, 21.9° at around 280° C. Chemical Formula 3 7.9, 12.1 White 15.8% 10 to 11 Not measurable, as it was decomposed 17.9, 20.20 at around 275° C. Chemical Formula 6 11.0°, 12.3 White 17.1% 9 to 10 93° C. 12.8°, 24.9° Chemical Formula 7 8.7°, 13.0° White 11.0% 5 to 6 Not measurable, as it was decomposed 21.7°, 26.1° at around 245° C. *It was determined based on the water content measured.

The results of thermal properties of all the above crystals EXAMPLE A-1 are respectively shown in FIG. 20 to 24. As shown in FIG. Syrup Containing Branched Cyclotetrasaccharide 20, the compound of Chemical Formula 1 gave a weight Represented by Chemical Formulae 1 and 2 reduction corresponding to that of four moles of water as the 65 temperature increased to 150° C., and gave a weight reduc One part by weight of cyclotetrasaccharide obtained by tion due to its decomposition at a temperature of around the method in Experiment 3-2 and one part by weight of US 7,223,570 B2 45 46 C-cyclodextrin, commercialized by Hayashibara Biochemi and pharmaceuticals without any treatment or after concen cal Laboratories Inc., Okayama, Japan, were dissolved in trated, dried, crystallized, or pulverized into a product in the three parts by weight of 50 mM sodium acetate buffer (pH form of an amorphous powder, molasses, block, etc. 5.5). The resulting solution was incubated at 50° C. for 24 hours after admixed with 10 units/g C-cyclodextrin of 5 EXAMPLE A-3 CGTase from a microorganism of the species Bacillus Stearothermophilus, commercialized by Hayashibara Bio Syrup Containing Branched Cyclotetrasaccharide chemical Laboratories Inc., Okayama, Japan, and the reac Represented by Chemical Formula 6 tion mixture was boiled for 20 minutes to inactivate the Two parts by weight of cyclotetrasaccharide obtained by remaining enzyme. 10 the method in Experiment 3-2 and two parts by weight of To the reaction mixture was added 350 g of 50 mM lactose were mixed and dissolved in nine parts by weight of sodium acetate buffer (pH 4.5) and then incubated at 40°C. 20 mM sodium acetate buffer (pH 6.0). The resulting solu for four hours after admixed with 200 units of “GLU tion was incubated at 40°C. for 24 hours after admixed with COTEAMTM”, a glucoamylase specimen commercialized by three units/glactose of “BIOLACTANTM, a B-galactosidase Nagase Biochemicals, Ltd., Kyoto, Japan, per gram of the 15 specimen from a microorganism of the species Bacillus initially added C.-cyclodextrin. Thereafter, the resulting mix circulans commercialized by Daiwa Kasei K. K., Osaka, ture was boiled for 20 min to inactivate the remaining Japan, and the reaction mixture was boiled for 20 minutes to enzyme. inactivate the remaining enzyme. The reaction mixture thus obtained was membrane fil To the reaction mixture thus obtained was-added 0.5 part tered, desalted with “DIAION PK218TM”, a cation exchange 20 by weight of sodium hydroxide and kept at 100° C. for one resin commercialized by Mitsubishi Chemical Industries, hour to decompose reducing saccharides. The obtained Ltd., Tokyo, Japan, and “AMBERLITE IRA411TM, an reaction mixture was in a conventional manner desalted, anion exchange resin commercialized by Japan Organo Co., filtered, and concentrated. The concentrate was Subjected to Ltd., Tokyo, Japan, and concentrated. The concentrate was preparative liquid chromatography using “YMC-PACK fractionated by chromatography according to the conditions 25 ODS-AQR355-15AQ S-10/20 um, 120ATM”, a preparative used in Experiment 3-2, and the resulting each fraction was column commercialized by YMC Co., Ltd., Tokyo, Japan, analyzed on the HPLC in Experiment 3-1 to collect a where a purified, deionized water was used as a moving fraction containing a branched cyclotetrasaccharide, repre phase. The eluate was analyzed on the HPLC described in sented by Chemical Formulae 1 and 2, with a purity of 85% Experiment 3-1 to collect a fraction containing a branched or higher each. The fraction was concentrated into a syrup 30 cyclotetrasaccharide, represented by Chemical Formula 6, with a solid concentration of about 50% (w/w). with a purity of at least 85%. The fraction was concentrated The syrup can be advantageously used as a material for to obtain a syrup with a solid concentration of 40% (w/w). products in a variety fields of foods, beverages, cosmetics, The syrup can be advantageously used as a material for and pharmaceuticals without any treatment or after concen products in variety fields of foods, beverages, cosmetics, and trated, dried, crystallized, or pulverized into a product in the 35 pharmaceuticals without any treatment or after concentrated, form of an amorphous powder, molasses, block, etc. dried, crystallized, or pulverized into a product in the form EXAMPLE A-2 of an amorphous powder, molasses, block, etc. EXAMPLE A-4 Syrup Containing Branched Cyclotetrasaccharide 40 Represented by Chemical Formula 3 Syrup Containing Branched Cyclotetrasaccharide Represented by Chemical Formula 7 Two parts by weight of cyclotetrasaccharide obtained by the method in Experiment 3-2 and one part by weight of Two parts by weight of cyclotetrasaccharide by the panose commercialized by Hayashibara Biochemical Labo- 45 method in Experiment 3-2 and two parts by weight of lactose ratories Inc., Okayama, Japan, were dissolved in seven parts were dissolved in nine parts by weight of 20 mM sodium by weight of 10 mM sodium acetate buffer (pH 6.0). The acetate buffer (pH 4.5). The resulting solution was incubated resulting solution was incubated at 30° C. for 24 hours after at 40°C. for 24 hours after admixed with 10 units/g lactose admixed with 30 units/g panose of a purified C-isomaltosyl of “LACTASEYA-OTM, a B-galactosidase specimen from transferring enzyme obtained by the method in Experiment 50 a microorganism of the species Aspergillus niger commer 2-2, and the reaction mixture was boiled for 20 min to cialized by Yakult Pharmaceutical Inc. Co., Ltd., Tokyo, inactivate the remaining enzyme. Japan, and the reaction mixture was boiled for 20 min to The reaction mixture thus obtained was membrane fil inactivate the remaining enzyme. tered, desalted with “DIAION PK218TM”, a cation exchange To the reaction mixture thus obtained was added 0.5 part resin commercialized by Mitsubishi Chemical Industries, 55 by weight of sodium hydroxide and kept at 100° C. for one Ltd., Tokyo, Japan, and “AMBERLITE IRA411TM, an hour to decompose reducing saccharides. The resulting anion exchange resin commercialized by Japan Organo Co., reaction mixture was in a conventional manner desalted, Ltd., Tokyo, Japan, and concentrated. The concentrate was filtrated, and concentrated. The concentrate was subjected to fractionated by chromatography according to the conditions preparative liquid chromatography using “YMC-PACK in Experiment 3-2, and the resulting each fraction was 60 ODS-AQR355-15AQ, S-10/20 um 120ATM”, a preparative analyzed on the HPLC in Experiment 3-1 to collect a column commercialized by YMC Co., Ltd., Tokyo, Japan, fraction with a branched cyclotetrasaccharide, represented where a purified, deionized water was used as a moving by Chemical Formula 3, with a purity of 80% or higher. The phase. The eluate was analyzed on the HPLC described in fraction was concentrated into a syrup with a solid concen Experiment 3-1 to collect a fraction containing a branched tration of about 40% (w/w). 65 cyclotetrasaccharide, represented by Chemical Formula 7. The syrup can be advantageously used as a material for with a purity of at least 85%. The fraction was concentrated products in a variety fields of foods, beverages, cosmetics, into a syrup with a solid concentration of 45% (w/w). US 7,223,570 B2 47 48 The syrup can be advantageously used as a material for enzyme and the inactivation of enzyme, was Subjected to products in variety fields of foods, beverages, cosmetics, and chromatographic separation similarly as in Example A-3. pharmaceuticals without any treatment or after concentrated, The eluate was analyzed on the HPLC described in Experi dried, crystallized, or pulverized into a product in the form ment 3-1 to collect a fraction containing branched cyclotet of an amorphous powder, molasses, block, etc. rasaccharides, represented by Chemical Formula 6, with a purity of at least 96%. The fraction was concentrated and EXAMPLE A-5 then admixed with the crystal of the compound of Chemical Formula 6 obtained in Experiment 5 as a seed crystal to Crystal of Branched Cyclotetrasaccharide effect sufficient crystallization. The crystal was centrifuged Represented by Chemical Formula 1 or 2 10 and collected in a conventional manner, and the collected According to Example A-1, a reaction mixture, obtained crystal was dried at ambient temperature to obtain a crystal through the sequential treatments of CGTase and glucoamy of a branched cyclotetrasaccharide, represented by Chemical lase and the inactivation of enzyme, was Subjected to Formula 6, with a purity of at least 99%. Measurement of chromatographic separation similarly as in Example A-1. 15 moisture content described in Experiment 5 revealed that the The eluate was analyzed on the HPLC in Experiment 3-1 to crystal was a nona- or deca-hydrous crystal. collect fractions containing branched cyclotetrasaccharides, The crystal can be arbitrarily used as a material for represented by Chemical Formulae 1 and 2, with a purity of products in variety fields of foods, beverages, cosmetics, and 97% or higher each. The fractions were respectively con pharmaceuticals. centrated and then admixed with a corresponding crystal of Chemical Formula 1 or 2 obtained in Experiment 5 as a seed EXAMPLEA-8 crystal to effect sufficient crystallization. The crystals were centrifuged and collected in a conventional manner, and the Crystal of Branched Cyclotetrasaccharide collected crystals were dried at ambient temperature to Represented by Chemical Formula 7 obtain respective crystals of branched cyclotetrasaccharides, 25 represented by Chemical Formulae 1 and 2, with a purity of According to Example A-4, a reaction mixture, obtained at least 99% each. Measurement of moisture content through the treatments of B-galactosidase and the inactiva described in Experiment 5 revealed that the compound of tion of enzyme, was subjected to chromatographic separa Chemical Formula 1 was a penta- or hexa-hydrous crystal, tion similarly as in Example A-4. The eluate was analyzed while the compound of Chemical Formula 2 was an undeca 30 or dodeca-hydrous crystal. on the HPLC described in Experiment 3-1 to collect a The crystals can be arbitrarily used as materials for fraction containing branched cyclotetrasaccharides, repre products in variety fields of foods, beverages, cosmetics, and sented by Chemical Formula 7, with a purity of at least 97%. pharmaceuticals. The fraction was concentrated and admixed with the crystal 35 of the compound of Chemical Formula 7 obtained in Experi EXAMPLEA-6 ment 5 as a seed crystal to effect sufficient crystallization. The crystal was centrifuged and collected in a conventional Crystal of Branched Cyclotetrasaccharide manner, and the collected crystal was dried at ambient Represented by Chemical Formula 3 temperature to obtain a crystal of a branched cyclotetrasac 40 charide, represented by Chemical Formula 7, with a purity According to Example A-2, a reaction mixture, obtained of at least 99%. Measurement of moisture described in through the treatments of B-galactosidase and the inactiva Experiment 5 revealed that the crystal was a penta- or tion of enzyme, was subjected to chromatographic separa hexa-hydrous crystal. tion similarly as in Example A-2. The eluate was analyzed The crystal can be arbitrarily used as a material for on the HPLC described in Experiment 3-1 to collect a 45 products in variety fields of foods, beverages, cosmetics, and fraction containing branched cyclotetrasaccharides, repre pharmaceuticals. sented by Chemical Formula 3, with a purity of at least 97%. The fraction was concentrated and then admixed with the EXAMPLEA-9 crystal of the compound of Chemical Formula 3 obtained in Experiment 5 as a seed crystal to effect sufficient crystalli 50 Syrup Containing Branched Cyclotetrasaccharides Zation. The formed crystal was centrifuged and collected in a conventional manner, and the collected crystal was dried 3.7 kg of “PINE-DEX #100TM”, a partial starch hydro at ambient temperature to obtain a crystal of a branched lyzate commercialized by Matsutani Chemical Ind., Tokyo, cyclotetrasaccharide, represented by Chemical Formula 3, Japan, was dissolved in 35 L of 10 mM sodium acetate with a purity of at least 99%. Measurement of moisture 55 buffer (pH 6.0), and the solution was incubated at 30°C. for content described in Experiment 5 revealed that the crystal two days after admixed with 17,500 units of a cyclotetrasac was a deca- or undeca-hydrous crystal. charide-forming activity of an enzyme preparation obtained The crystal can be arbitrarily used as a material for by the method in Experiment 2-1, and the reaction mixture products in variety fields of foods, beverages, cosmetics, and was boiled for 20 minutes to inactivate the remaining pharmaceuticals. 60 enzyme. The reaction mixture was cooled to 45° C. and admixed with 11 g (137,500 units) of “NEO-SPITASETM, EXAMPLEA-7 an O-amylase specimen commercialized by Nagase Bio Crystal of Branched Cyclotetrasaccharide chemicals, Ltd., Kyoto, Japan, and 44 grams (140,800 units) Represented by Chemical Formula 6 of “GLUCOZYMETM”, a glucoamylase specimen commer 65 cialized by Nagase Biochemicals, Ltd., Kyoto, Japan, and According to Example A-3, a reaction mixture, obtained then adjusted to pH 6.0, followed by enzymatically reacting through the treatments of C-isomaltosyl-transferring at 45° C. for one day. The reaction mixture thus obtained US 7,223,570 B2 49 50 was boiled for 20 minutes to inactivate remaining enzymes, Since the syrup is substantially free of crystallization and and then cooled and filtered in a conventional manner to easily produced on an industrial scale and at a lesser cost, it obtain a filtrate. The filtrate was concentrated to give a solid can be arbitrarily used as a material for products in a variety concentration of about 16% (w/w) by using a reverse fields of foods, beverages, cosmetics, and pharmaceuticals. osmosis membrane. The concentrate was Subjected in a conventional manner to decoloration, desalting, filtration, EXAMPLE A-11 and concentration to obtain about 6.1 kg of a syrup con taining 3.5 kg of solids consisting of 12% of branched Syrup Containing Branched Cyclotetrasaccharides cyclotetrasaccharides, 44% of cyclotetrasaccharide, 25% of glucose, and 19% of oligosaccharides. 10 To 400 g of a syrup containing branched cyclotetrasac Since the syrup is substantially free of crystallization and charides obtained in Example A-9 was added 0.1 g/g solids easily produced on an industrial scale and at a lesser cost, it of “N154TM commercialized by Nikki Chemical Co., Ltd., can be arbitrarily used as a material for products in a variety Kanagawa, Japan, an activated Raney nickel catalyst with an fields of foods, beverages, cosmetics, and pharmaceuticals. alkali. The mixture was placed in an autoclave and hydro 15 genated by reacting at 100° C. for four hours and further at 120° C. for two hours while stirring and keeping at a EXAMPLE A-10 hydrogen pressure of 100 kg/cm. After cooled, the resulting hydrogenated mixture was taken out from the autoclave and Syrup Containing Branched Cyclotetrasaccharides filtered by passing through an activated carbon layer with about one centimeter in thickness to remove the Raney 6.1 kg of a saccharide solution, obtained by the method in nickel catalyst. The filtrate was in a conventional manner Experiment 3-1, was fed to ten columns, having an inner decolored with an activated charcoal, desalted with ion diameter of 13.5 cm and a length of 160 cm each, which had exchange resins in H- and OH-forms, purified, and concen been packed with about 225 L of “AMBERLITE CR-1310 trated to give a concentration of about 40% and to obtain a (Na-form)TM, an ion-exchange resin commercialized by 25 syrup which was substantially free of crystallization and Japan Organo Co., Ltd., Tokyo, Japan, and cascaded in composed of 12% of branched cyclotetrasaccharides, 44% series, and chromatographed at a flow rate of about 45 L/h of cyclotetrasaccharide, 25% of sorbitol, and 19% of other of water and at a column temperature of 60° C. The eluate saccharides. from the columns was fractionated and determined for Since the syrup is substantially free of crystallization and saccharide composition by the HPLC described in Experi 30 easily produced on an industrial scale and at a lesser cost, it ment 3-1. Fractions, which were relatively rich in cyclotet can be arbitrarily used as a material for products in a variety rasaccharide, were collected and pooled to obtain a saccha fields of foods, beverages, cosmetics, and pharmaceuticals. ride solution with a solid content of about 1,530 g. The saccharide solution, i.e., a high cyclotetrasaccharide content EXAMPLE A-12 fraction, was analyzed on the HPLC under the same condi 35 tions as above, and based on the peak areas determined on Syrup of Branched Cyclotetrasaccharides the HPLC analysis, it was composed of 79.8% of cyclotet rasaccharide and 6.1% of isomaltose to the total Sugars. A Substantially non-reducing and non-crystallizing syrup, The saccharide solution in an amount equal to a solid which had a solid concentration of about 55% and consisted content of 1,310 g was adjusted to pH 5.0 and 50° C., and 40 of 4% of branched cyclotetrasaccharides, 94% of cyclotet then incubated for 20 hours after admixed with 1,000 units/g rasaccharide, 1% of sorbitol, and 1% of other saccharides, solids of “TRANSGLUCOSIDASE L AMANOTM, an was obtained similarly as in Example A-11 except for C-glucosidase specimen commercialized by Amano Phar replacing 400 g of the syrup containing branched cyclotet maceutical Co., Ltd., Aichi, Japan, and 60 units/g solids of rasaccharides obtained in Example A-9 with 400 g of the GLUCOZYMETM, a glucoamylase specimen commercial 45 syrup containing branched cyclotetrasaccharides obtained in ized by Nagase Biochemicals, Ltd., Kyoto, Japan. After Example A-10. removing insoluble substances by filtration, the above enzy Since the syrup is substantially free of crystallization and matic reaction mixture was desalted with "DIAION easily produced on an industrial scale and at a lesser cost, it PK218TM”, a cation exchange resin commercialized by can be arbitrarily used as a material for products in a variety Mitsubishi Chemical Industries, Ltd., Tokyo, Japan, and 50 fields of foods, beverages, cosmetics, and pharmaceuticals. “AMBERLITE IRA411TM, an anion exchange resin com mercialized by Japan Organo Co., Ltd., Tokyo, Japan, and EXAMPLE B-1 then concentrated. The concentrate was fractionated accord ing to the conditions as used in the above chromatographic Sweetener separation to collect a fraction of cyclotetrasaccharide with 55 a purity of 97%. The fraction was in a conventional manner To 0.8 part by weight of a branched cyclotetrasaccharide decolored, desalted, filtered, and concentrated to obtain a crystal, penta- or hexa-hydrate, represented by Chemical saccharide solution with a solid content of 1,260 g. After Formula 1, obtained by the method in Example A-5, were adjusted to a solid concentration of about 50% (w/w), the homogeneously added 0.2 part by weight of “TREHATM, a saccharide Solution was placed in a cylindrical plastic vessel 60 crystalline trehalose hydrate commercialized by Hayash and cooled from 65° C. to 20° C. over about 20 hours under ibara Shoji Inc., Okayama, Japan, 0.01 part by weight of “a gentle stirring conditions. The resulting mixture was Sub G SWEETTM, an O-glycosylstevioside product commer jected to separation, and the obtained mother liquor was cialized by Toyo Sugar Refining Co., Tokyo, Japan, and 0.01 purified and concentrated into a syrup with a solid concen part by weight of “ASPALTAMETM, a product of L-aspar tration of about 45%, consisting of 4% of branched cyclotet 65 tyl-L-phenylalanine methyl ester, followed by feeding the rasaccharides, 94% of cyclotetrasaccharide, 1% of glucose, resulting mixture to a granulator to obtain a granular Sweet and 1% of other saccharides. ener. The product has a satisfactory Sweetness and an about US 7,223,570 B2 51 52 two-fold higher Sweetening power of Sucrose. Since the EXAMPLE B-5 branched cyclotetrasaccharide is hardly digestible and ferf mentable and is substantially free from calorie the product Bath Salt has only about /10 calorie of that of sucrose with respect to Sweetening power. In addition, the product is Substantially 5 One part by weight of a peel juice of "yuzu' (a Chinese free of deterioration and stable even when stored at room lemon) was admixed with 10 parts by weight of a branched temperature. Thus, the product is preferable as a high cyclotetrasaccharide crystal, undeca- or dodeca-hydrate, quality, low caloric, less cariogenic Sweetener. represented by Chemical Formula 2, obtained by the method in Example A-5, followed by drying and pulverizing the 10 mixture into a powder containing a yuzu peel extract. A bath EXAMPLE B-2 salt was prepared by mixing five parts by weight of the above powder with 90 parts by weight of grilled salt, two Hard Candy parts by weight of hydrous crystalline trehalose, one part by weight of silicic anhydride, and 0.5 part by weight of "O.G One hundred parts by weight of a 55% (w/w) sucrose 15 HESPERIDIN', an O-glucosyl hesperidin product commer solution were mixed while heating with 50 parts by weight cialized by Hayashibara Shoji, Inc., Okayama, Japan. The of a syrup containing a branched cyclotetrasaccharide rep product is a bath salt with an elegant, gentle flavor and a resented by Chemical Formula 6, obtained by the method in Superior skin moisturizing effect. Example A-3. The mixture was then concentrated by heating under a reduced pressure to give a moisture content of less EXAMPLE B-6 than 2%. The concentrate was mixed with 0.6 part by weight of citric acid and-an adequate amount of a lemon flavor, Cosmetic Cream followed by forming the resultant into the desired product in a conventional manner. The product is a stable, high quality Two parts by weight of polyoxyethylene glycol hard candy which has a satisfactory mouth feel, taste, and 25 monostearate, five parts by weight of glyceryl monostearate, flavor, less adsorbs moisture; and does neither induce crys self-emulsifying, two parts by weight of a branched tallization of Sucrose nor cause melting. cyclotetrasaccharide crystal, deca- or undeca-hydrate, rep resented by Chemical Formula 3, obtained by the method in EXAMPLE B-3 Example A-6, one part by weight of “CG RUTIN', an 30 C-glucosyl rutin product commercialized by Hayashibara Shoji, Inc., Okayama, Japan, one part by weight of liquid Beverage with Lactic Acid Bacteria petrolatum, 10 parts by weight of glyceryl tri-2-ethylhex anoate, and an adequate amount of an antiseptic were Fifty parts by weight of a syrup containing a branched dissolved by heating in a usual manner. The resulting cyclotetrasaccharide represented by Chemical Formula 7. 35 solution was admixed with two parts by weight of L-lactic obtained by the method in Example A-4, and 175 parts by acid, five parts by weight of 1,3-butylene glycol, and 66 weight of a skim milk powder, and 50 parts by weight of parts by weight of refined water. The resultant mixture was “NYUKAOLIGOTM, a high lactosucrose content powder emulsified by a homogenizer and admixed with an adequate commercialized by Hayashibara Shoji Inc., Okayama, amount of a flavor while stirring to obtain a cosmetic cream. Japan, were dissolved in 1,150 parts by weight of water. The 40 The product exhibits an antioxidant activity and has a resulting solution was sterilized at 65° C. for 30 min, then relatively high stability, and these render it advantageously cooled to 40°C., inoculated in a usual manner with 30 parts useful as a high quality Sunscreen, skin-refining agent, and by weight of lactic acid bacteria as a starter, and incubated skin-whitening agent. at 37°C. for eight hours to obtain a beverage with lactic acid bacteria. The product can be suitably used as a lactic acid 45 EXAMPLE B-7 beverage which has a satisfactory flavor and taste, contains oligosaccharides and cyclotetrasaccharide, stably retains the Tablet lactic acid bacteria, and has actions of promoting the growth of the bacteria and controlling the intestinal conditions. Fourteen parts by weight of a branched cyclotetrasaccha 50 ride crystal, nona- or deca-hydrate, represented by Chemical EXAMPLE B-4 Formula 6, obtained by the method in Example A-7, were sufficiently mixed with 50 parts by weight of aspirin and Toothpaste four parts by weight of corn starch. The mixture was then in a conventional manner tabletted by a tabulating machine A syrup containing a branched cyclotetrasaccharide rep 55 into a tablet, 680 mg, 5.25 mm in thickness each. The tablet, resented by Chemical Formula 1, obtained by the method in processed with the filler-imparting ability of the branched Example A-1, was adjusted to a solid concentration of about cyclotetrasaccharide, has a quite low hygroscopicity, Sufi 30% (w/w). A toothpaste was prepared by mixing 13 parts cient physical strength, and Superior degradability in water. by weight of water with 15 parts by weight of the above syrup, 45 parts by weight of calcium secondary phosphate, 60 INDUSTRIAL APPLICABILITY 1.5 parts by weight of sodium lauryl sulfate, 25 parts by weight of glycerol, 0.5 part by weight of polyoxyethylene As described above, the present invention was made sorbitan laurate, 0.02 part by weight of saccharine, 0.05 part based on completely novel findings by the present inventors by weight of an antiseptic, and 13 parts by weight of water. that glycosyl derivatives of cyclotetrasaccharide are formed The product has an improved after taste and a satisfactory 65 as by-products of cyclotetrasaccharide when the novel feeling after use without reducing the detergent power of the enzymes, i.e., C.-isomaltosyl-transferring enzyme and O-iso Surfactant. maltosylglucosaccharide-forming enzyme, which the US 7,223,570 B2 53 54 present inventors had previously found, are allowed to act on in a various fields of foods and beverages, cosmetics, and starch hydroly Zates; and that a variety of glycosyl deriva pharmaceuticals similarly as cyclotetrasaccharide. tives are obtained by allowing saccharide-related enzymes Advanced analysis of physical and chemical properties and Such as the above-identified enzymes, cyclomaltodextrin functions of the branched cyclotetrasaccharides of the glucanotransferase, B-galactosidase, O-galactosidase, and present invention will give an important finding that leads to lysozyme to act on cyclotetrasaccharide Since the glycosyl development of novel uses of cyclotetrasaccharide and derivatives provided by the present invention, i.e., branched improvement of the properties and functions thereof. cyclotetrasaccharides, have the intrinsic properties of The present invention with these outstanding functions cyclotetrasaccharide Such as an inclusion ability and Sub and effects is a significant and important invention that stantially non-digestibility, they can be advantageously used greatly contributes to this art.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 2

<21 Oc SEQ ID NO 1 <211 LENGTH 3282 <212> TYPE DNA <213> ORGANISM: Bacillus globisporus FEATURE NAME/KEY: CDS LOCATION: (1) . . (3282) FEATURE NAME/KEY: sig peptide LOCATION: (1) . . (87) <400 SEQUENCE: 1

atg tat gta agg aat cita aca ggit toa titc. cga. titt tot citc. tct titt 48 Met Tyr Wall Arg Asn Telu Thr Gly Ser Phe Arg Phe Ser Leu Ser Phe 1 10 15

ttg ctic tgt titc. tgt ctic titc. gto: tot att gCC att gat ggit 96 Teu Teu Cys Phe Cys Telu Phe Wall Pro Ser Ile Ala Ile Asp Gly 2O 25 30

gtt tat cat gCg cca tac gga atc gat gat citg gag att cag gog 144 Wall His Ala Pro Gly Ile Asp Asp Telu Glu Ile Glin Ala 35 40 45

acg gag Cgg agt cca aga gat cco gtt gCa ggC gat act gtg tat atc 192 Thr Glu Arg Ser Pro Arg Asp Pro Wall Ala Gly Asp Thr Val Tyr Ile 5 O 55 60

aag alta a Ca acg tgg coc att gaa toa gga Cala acg gct tgg gtg acc 240 Lys Ile Thr Thr Trp Pro Ile Glu Ser Gly Glin Thr Ala Trp Val Thr 65 70 75

acg a.a.a. aac ggit gtc aat Cala gct gct gtc gga gCa gca ttcaaa 288 Trp Thr Asn Gly Wall Asn Glin Ala Ala Wall Gly Ala Ala Phe Lys 85 90 95

tac aac agC ggC aac aac act tac tgg gaa gCg aac citt ggc act titt 336 Asn Ser Gly Asn Asn Thr Trp Glu Ala Asn Telu Gly Thr Phe 100 105 110

gCa a.a.a. ggg gac gtg atc tat acc gtt cat ggC aac aag gat ggC 384 Ala Gly Asp Wall Ile Ser Tyr Thr Wall His Gly Asn Lys Asp Gly 115 120 125

gcg aat gag aag gtt atc ggit cost titt act titt acc gta acg gga tagg 432 Ala Asn Glu Wall Ile Gly Pro Phe Thr Phe Thr Wall Thr Gly Trp 130 135 14 O

gaa too gtt agc atc agC tot att acg gat aat acg aac cqt gtt 480 Glu Ser Wall Ser Ser Ile Ser Ser Ile Thr Asp Asn Thr Asn Arg Val 145 15 O 155 160

gtg citg aat gCg gtg cc.g aat a Ca ggC a Ca aag cca aag atc aac 528 Wall Teu Asn Ala Wall Pro Asn Thr Gly Thr Telu Pro Lys Ile Asn 1.65 17 O 175

citt too titt acg gCg gat gat gto citc. cgc gta Cag gtt tot coa acc 576 Teu Ser Phe Thr Ala Asp Asp Wall Teu Arg Wall Glin Wall Ser Pro Thr 18O 185 190

US 7,223,570 B2 70

-continued

220 1225 1230 gcc att tat gcg aac ata agt aac agc ggit gtt tgg aaa tat atc. 3744 Ala le Tyr Ala Asn. Ile Ser Asn. Ser Gly Wall Trp Lys Tyr Ile 235 1240 1245 agC gta agt gat att at g g to acc aat ggit Cag ata gat gtt gga 3789 Ser Val Ser Asp Ile Met Val Thr Asn Gly Glin Ile Asp Val Gly 25 O 1255 1260

tac gtg gat to a cott ggit gga act acg citt cac att gat gat 3834 Tyr Val Asp Ser Pro Gly Gly Thr Thr Teu His Ile Asp Asp 265 1270 1275

cgc gta acc aaa caa taa 3855 Arg Val Thr Lys Glin 280

What is claimed is: 2. The branched cyclotetrasaccharide of claim 1, wherein 1. A branched cyclotetrasaccharide which is a glycosyl one or more glycosyl groups positioning at one or more derivative of cyclotetrasaccharide represented by cyclo positions of R to R2 in Formula 1 each independently {->6)-C-D-glucopyranosyl-(1->3)-O-D-glucopyranosyl represent any one of the glycosyl groups selected from those (1->6)-O-D-glucopyranosyl-(1->3)-O-D-glucopyranosyl represented by the following (1) to (5): (1->} and which has a structure represented by Formula 1: 25 (1) optionally substituted C-D-glucopyranosyl-(1-> 4)-O-D-glucopyranosyl groups, with the proviso that each “n” in the above groups means an integer of 0 or Formula 1: more, and when at least two of R to R are the above groups, each “n” is independent; OR 30 RO (2) optionally substituted C-D-glucopyrancoyl-(1->6)- O {O-D-glucopyranosyl-(1->3)-C-D-glucopyranosyl OR O O (1->6)-O-D-glucopyranosyl groups, with the proviso RO that each “n” in the above groups means an integer of RO O 2 35 0 or more, and when at least two of R to R are the O OR 11 {?-D-galactopyranosyl-(1->6)-3-D-galactopyrano syl groups, with the proviso that each “n” in the above RSO O groups means an integer of 0 or more, and when at least O OR) two of R to R2 are the above groups, each “n” is OR6 independent; O 40 RO (4) optionally Substituted C-D-galactopyranosyl groups; O and ORs OR (5) optionally Substituted B-D-chitosaminyl groups. 3. The branched cyclotetrasaccharide of claim 1, wherein 45 R and/or R, in Formula 1 are independently optionally wherein in Formula 1, R to R each independently repre substituted C-D-glucopyranosyl-(1->4)-O-D-glucopyra sents an optionally substituted glycosyl group or hydrogen, with the proviso that all of R to R2 are not hydrogen atom, nosyl groups, with the proviso that each “n” in the above and when either R or Rio is an optionally Substituted groups means an integer of 0 or more, and when both R and glycosyl group, R or Rio as the glycosyl group is a member so R, are the above groups, each “n” is independent. selected from glycosyl groups other than D-glucopyranosyl 4. The branched cyclotetrasaccharide of claim 3, which is group. the one represented by Chemical Formula 1 or 2:

Chemical Formula 1:

4

1 US 7,223,570 B2 71 72

Chemical Formula 2:

4 4

1 1 C-D-Glcp C-D-Glcp

10 5. The branched cyclotetrasaccharide of claim 1, wherein 9. The branched cyclotetrasaccharide of claim 1, wherein R and/or Rs in Formula 1 are optionally substituted C-D- Ra and/or Rio in Formula 1 are optionally substituted B-D- glucopyranosyl-(1->6)-C-D-glucopyranosyl-(1->3)-O-D- galactopyranosyl-(1->6)-?3-D-galactopyranosyl groups, glucopyranosyl-(1->6)-O-D-glucopyranosyl groups, with with the proviso that each “n” in the above groups means an the proviso that each “n” in the above groups means an 15 integer of 0 or more, and when both R and Rs are the above integer of 0 or more, and when both RandR are the above groups, each “n” is independent. groups, each “n” is independent. 6. The branched cyclotetrasaccharide of claim 5, which is 10. The branched cyclotetrasaccharide of claim 7, which the one represented by Chemical Formula 3, 4, or 5: is the one represented by Chemical Formula 7 or 8;

Chemical Formula 3:

3

1

Chemical Formula 4:

3

1

Chemical Formula 5:

4 3

1 1

7. The branched cyclotetrasaccharide of claim 1, wherein R and/or Rs in Formula 1 are optionally substituted B-D- galactopyranosyl-(1->6)-?3-D-galactopyranosyl groups, with the proviso that each “n” in the above groups means an integer of 0 or more, and when both R- and Rs are the above groups, each “n” is independent. 8. The branched cyclotetrasaccharide of claim 7, which is the one represented by Chemical Formula 6;

Chemical Formula 6:

3

1 B-D-Galp US 7,223,570 B2 73 74

Chemical Formula 7:

6

1 B-D-Galp Chemical Formula 8:

6

1

11. The branched cyclotetrasaccharide of claim 1, wherein Ra and/or Rio in Formula 1 are optionally substituted C-D- galactopyranosyl groups. 12. The branched cyclotetrasaccharide of claim 11, which is the one represented by Chemical Formula 9;

Chemical Formula 9:

6

1 C-D-Galp

13. The branched cyclotetrasaccharide of claim 1, wherein R and/or Rs in Formula 1 are optionally substituted B-D-chitosaminyl groups. 35 14. The branched cyclotetrasaccharide of claim 13, which is the one represented by Chemical Formula 10;

Chemical Formula 10:

3

1 B-D-GlcNAcp

15. The branched cyclotetrasaccharide of claim 1, which is in the form a solution, amorphous powder, or molasses. 50 -continued 16. An isolated crystal of the branched cyclotetrasaccha ride of claim 1. (3) 7.9°, 12.1°, 17.9°, and 20.2°: 17. An isolated crystal of the branched cyclotetrasaccha (4) 11.0°, 12.3, 12.8, and 24.9; and ride of claim 4, 6, 8 or 10, which is a crystal of a cyclotet (5) 8.7°, 13.0°, 21.7°, and 26.1°. rasaccharide, represented by Chemical Formula 1, 2, 3, 6, or ss 7. 20. A saccharide composition comprising the branched 18. The isolated crystal of the branched cyclotetrasaccha cyclotetrasaccharide of claim 1 and other saccharide(s) ride of claim 16, which is in the form of a hydrous- or anhydrous-crystal. except for the branched cyclotetrasaccharides. 21. The saccharide composition of claim 20, which is in 19. The isolated crystal of the branched cyclotetrasaccha 60 ride of claim 16, which has main diffraction angles (2) of any the form of a solution, amorphous powder, molasses, or one of (1) to (5) on X-ray powder diffraction analysis: crystalline powder. 22. A process for producing a branched cyclotetrasaccha ride which uses the action of an enzyme capable of trans (1) 8.1°, 12.2°, 14.2°, and 15.4°: 65 ferring a glycosyl group from a monosaccharide, oligosac (2) 5.6°, 8.8, 16.9°, and 21.9°: charide, or polysaccharide to cyclotetrasaccharide represented cyclo6)-C-D-glucopyranosyl-(1->3)-O-D-glu US 7,223,570 B2 75 76 copyranosyl-(1->6)-O-D-glucopyranosyl-(1->3)-O-D-glu Enzymatic activity (A): Acting on a saccharide, having copyranosyl-(1->}, and which is characterized in that it both a glucose polymerization degree of “n” (“n” is an comprises the following two steps of integer of two or more) and the C-1,4-glucosyl bond as (1) forming the branched cyclotetrasaccharide of claim 1 a linkage at the non-reducing end, and forming a by allowing the enzyme to act on a mixture of the Saccharide, having a glucose polymerization degree of cyclotetrasaccharide along with the monosaccharide, “n1 and having both the C-1,6 glucosyl bond as a the oligosaccharide, or the polysaccharide, and linkage at the non-reducing end and the C-1,4-glucosyl (2) collecting the formed branched cyclotetrasaccharide bond as a linkage other than the non-reducing end, in the step (1). without Substantially increasing the reducing power, 23. The process of claim 22, which further contains the 10 and following step for producing the cyclotetrasaccharide prior Enzymatic activity (B): Acting on a saccharide, having a to the step (1): glucose polymerization degree of at least three and forming the cyclotetrasaccharide by allowing an O-iso having both the C-1,6 glucosyl bond as a linkage at the maltosylglucosaccharide-forming enzyme having the non-reducing end and the C-14 glucosyl bond as a following enzymatic activity (A) and an O-isomaltosyl 15 linkage other than the non-reducing end, and forming a transferring enzyme having the following enzymatic cyclotetrasaccharide represented cyclo->6)-C-D-glu activity (B), to act on a saccharide having both a copyranosy-(1->3)-O-D-glucopyranosyl -(1->6)-C-D- glucose polymerization degree of at least two and the glucopyranosyl-(1->3)-O-D-glucopyranosyl-(1}. C.-1.4 glucosyl bond as a linkage at the non-reducing 25. The process of claim 22, which uses, as the monosac end, and collecting the formed cyclotetrasaccharide; charide, oligosaccharide or polysaccharide, one or more Enzymatic activity (A): Acting on a saccharide, having saccharides selected from the group consisting of glucose both a glucose polymerization degree of “n” (“n” is an 1-phospate, maltooligosaccharide, circular dextrin, panose, integer of two or more) and the C-1,4-glucosyl bond as isomaltosylglucosaccharide, lactose, melibiose, N-acetyl a linkage at the non-reducing end, and forming a chitooligosaccharide, dextrin, glycogen, liquefied Starch, Saccharide having a glucose polymerization degree of 25 and chitin as a monosaccharide, oligosaccharide, and “n+1 and having both the C-1,6 glucosyl bond as a polysaccharide. linkage at the non-reducing end and the C-1,4-glucosyl 26. The process of claim 22, wherein the formed branched bond as a linkage other than the non-reducing end, cyclotetrasaccharide is collected by a step comprising one or without Substantially increasing the reducing power; more purification methods selected from the group consist and 30 ing of decoloration, desalting, column chromatography, and Enzymatic activity (B): Acting on a saccharide, having a crystallization. glucose polymerization degree of at least three and 27. A method for transferring a glycosyl group using an having both the C-1,6 glucosyl bond as a linkage at the enzyme capable of transferring a glycosyl group from a non-reducing end and the C-14 glucosyl bond as a monosaccharide, oligosaccharide, or polysaccharide to a linkage other than the non-reducing end, and forming 35 cyclotetrasaccharide represented cyclo->6)-O-D-glucopy cyclotetrasaccharide represented cyclo->6)-C-D-glu ranosyl-(1->3)-C-D-glucopyranosyl-(1->6)-O-D-glucopy copyranosyl-(1->3)-O-D-glucopyranosyl-(1->6)-C-D- ranosyl-(1->3)-C-D-glucopyranosyl-(1->}; said method glucopyranosyl-(1->3)-O-D-glucopyranosyl-(1->}. comprising a step of allowing the enzyme to act on a mixture 24. The process for producing a branched cyclotetrasac of cyclotetrasaccharide and the monosaccharide, oligosac charide of claim 22, which uses one or more enzymes 40 selected from the group consisting of cyclomaltodextrin charide, or polysaccharide to form the branched cyclotet glucanotransferase, 3-galactosidase, C.-galactosidase, rasaccharide of claim 1. lysozyme, an O-isomaltosylglucosaccharide-forming 28. A composition comprising the branched cyclotetrasac enzyme having the following enzymatic activity (A), and an charide of claim 1. C-isomaltosyl-transferring enzyme having the following 45 29. The composition of claim 28, which is in the form of enzymatic activity (B), which are enzymes capable of trans a food, cosmetic, or pharmaceutical. ferring a glycosyl group from a monosaccharide, oligosac charide, or polysaccharide to cyclotetrasaccharide;