BIOSYNTHESIS OF RIBOFLAVIN GLYCOSIDES

BY PLANT GRAINS*

Yukio SUZUKI and Kei UCHIDA

In a communicationり， we pointed out that 5'-D-riboflavin-α-D-glucopyranoside (B2-5'・ーα-gl)could be synthesized by enzyme preparations from plant grains. We have now shown that not only B2-5'-α-gl but also a number of different riboflavin glycosid回， e.g.， 5'-D-riboflavin-β-D-glucopyranoside (B2-5'-s-gI)， 5'-D-riboflavin-αーD-galactopyranoside(~-5'-α-ga) and 5'-D-riboflavin-β-D-galactopyranoside (B2-5'-β-ga) are easily synthesized by the action of enzym田 preparedfrom glutinous millet grains. These riboflavin glycosid田 havebeen isolated in crystal-line forms and characterized.

EXPERIMENT AL

1. Plant grains. The seeds of glutinous millet obtained from Sakata Nu四百yCo.， Yokohama， Kanagawa-ken， ]apan， were sown on April 14th in 1966 in the experimental field of the Institute and harvested on August 8th. Millet grains harvested on August 8th were used in the experiments.

2. Chemicals and enzyme preparations for analysis. Riboflavin (Bz) was obtained from E. Merck， Darmstadt， Germany. Melibiose was the product of Difco Laboratories， Detroit， Michigan， U. S. A. All other chemical reagents used were of analyt~cal grade and were obtained from Ishizu Pharmaceutical Co.， Ltd.， Osaka， ]apan. Crystalline ~-5'ーα-glwas isolated in our laboratory from a sucrose-Bz culture of Leuconostoc mesenteroides2>. α-Glucosidase from Leuc. mesenteroides NRRL B-1299 (=IAM 1151) was prepared as reported previouslyの.β-Gluc侶 idasefrom almond (emulsin) was purcha田 dfrom Sigma Chemicals Co.， St. Louis， Missouri， U. S. A. This β-glucosidase preparation manifested hydrolytic activity toward β-D-glucosides，βーD田 galactosidesand α-D-galactosides， but was inert toward α由 glucosides.αーGalactosidaseu from Mortierella vinacea (a lyophilized preparation purified by DEAE-Sephadex chromatography) was kindly supplied by Dr. H. Suzuki， Fermentation Research Institut怠， Agency of Industrial Science and Technology， Chiba， Japan. Crystalline β-galactosidase from Escherichia coli was obtained from Boehringer Mannheim GmbH， Mannheim， Germany.

3. Assays of riboflavin glycosides. The procedures and methods u舘 dfor the a岱 ayof riboflavin glycosid田 weresimilar to th偲 ereported

本 Theout1ine of this work was presented at the meeting of Kansai Division of the Agricultural Chemical S∞iety of ]apan. Kyoto. Aprll 27. 1968.

188 y. Suzuki & K. Uchida

previously5). After two developments with n-butanol-pyridine-wa飽r(6: 4: 3， by volume) (solvent A)， the location of B2由 glycosideon paper chromatogram was examined by ultraviolet light and the fl.uorescent area was cut out. The excised stri伊 ofpaper were then eluted with deionized water by the descending method， and each of the eluate was filtered. B2-Glycoside in each filtrate was estimated by light absorption at 450 mμin a Shimadzu spectrophotometer model QR 50. The quantity of B2-g1ycoside was computed from a standard recoverable curve of each crystalline B2-g1ycoside which was app1ied to paper， developed and eluted as described above.

4. Preparation of crude enzyme solution. The grains of glutinous mi11et were soaked in 0.596 Na2S204 solution for 20 minutes， washed with tap water， ground in a mortar， suspended in 0.1 M acetate bu旺er(pH 5.3) at 40C， strained through two layers of cloth， and centrifuged. (NHc)2S04 was added to the supernatant solution to 0.95 saturation and the preci-pitate was collected， dissolved in acetate bu百'er，and centrifuged. The supernatant solution was used as the crude enzyme solution. Nine hundred ml of crude enzyme solution was prepared from 23 kg of grain powders of glutinous mi11et.

5. Isolation of 5'-D-ribofl.avin-βートglucopyranoside，5'-D-ri加fl.avin-α-D-galactopyranoside and 5'-D-ri加fl.avin-βーD-galactopyranoside.

Reaction mixtures for three di百'erentB2-g1ycoside formation were as follows : (a) Biosynthesis of B2-5ιβ-gl; a reaction mixture containing 120 g of cellobiose， 1200 mg of B2， 1200 ml of 0.1 M acetate bu百'er(pH 5.3) and 1200 ml of curde enzyme solution was incubated in the dark at 250C for 6 hours. Two batches were incubated in this way. (b) Biosynthesis of B2-5仁α-ga;a reaction mixture containing 90 g of meli-biose， 900 mg of B2， 900 ml of 0.1 M acetate bu百'er(pH 5.3) and 900 ml of crude enzyme solution was incubated in the dark at 250C for 6 hours. Two batches were incubated in this way. (c) Biosynthesis of B2-5'-β-ga; a rl伺 ctionmixture containing 98 g of lactose， 980 mg of B2， 980 ml of 0.1 M acetate bu町er(pH 5.3) and 980 ml of crude enzyme solution was incubated in the dark at 250C for 6 hours. Seven batches were incubated in this way. After incubation， each reaction mixture was treated with equal volume of ethanol， kept overnight at OOC， and filtered to remove the precipitate. The filtrate was concentrated under reduced pressure， followed by the addition of (NHc)zSOc to 0.66 saturation， and the resulting precipitate was removed by centrifugation. The yellow solution was shaken vigorously with phenol in a separatory funnel， and the phenol layers combined. A sample of this pheno1ic solution was analyzed by paper chromatography (Fig. 1)， and each yield of B2-5'-β-gl， B2-5'ーα-gaand B2-5'-β-ga was estimated by light absorption at 450 mμto be 132，

Riぷ??]1.

2.

3.

Biosynthesis of Ri凶flavinGlycosides

I

2

3

4

Left

Cellobiose

B2

Bz-5'ーβ-gl

1

2

'3

4

5

(Bz-oligosaccharide -like)

Bz

1

2

3

4

Middle

Lactose

(unknown)

Bz-5'ーβ-ga

189

Right

Melibiose

B2

(unknown)

B2-5ια-ga

4. |(B2-oligosaccharide-like)

(B2-oligolsikaecc) haride- (B2-olig田likaeCC)haride-

5. (Broligolsikaecc) haride-

Fig. 1. Paper chromatograms of reaction products from ri凶 flavinand cellobim泥 (melibioseor lactωe) by enzyme preparation from gluti-nous millet grains- Papers were developed twice by the ascコendtng司

method With solvent A.

190 Y. Suzuki & K. Uchida

74.4 and 473 mg， respectively. The phenolic solution was shaken with ether and water in order to transfer B2 compounds into the water layer. The water layer was concentrated and centrifuged. All of the super-natant solution were applied as the band (2一mlaliquots of the super-natant solution per a Toyo No. 2 filter paper sheet， 30 x 60 cm) along a starting line previously drawn in parallel to the bottom of the sheet. At the sametime， a trace amount of B2 solution was spotted as the control on the same starting line. The paper sheets were developed twice by the ascending method with solvent A (1st paper chromatography). After two developments， the band of B2-g1ycoside-like compound on the chromatogram was cut out. The excised strips of paper were then eluted chromatographically by the descending method with deionized water. The eluate， after concentration， was re-applied to paper and developed twice with solvent A (2nd paper chromatography). The zone containing B2-g1ycoside-like compound on chromatograms was eluted; the eluate was filtered and concentrated in vσ'Cuo. The concen tra te was subjected to paper chromatography， using water saturated with isoamyl-alcohol (solvent B) as a developing solvent in order to remove sugar impurities present in the concentrate (3rd paper chromatography). After appropriate sectioning， elution and concentration， the concentrate was rechromatographed with solvent B (4th paper chromatography). The band of B2-g1ycoside-like com戸 undwas eluted and the eluate was concentrated. The ∞ncentrate was applied to paper， followed by two developments with solvent C [n-butanol-formic acid-water (4: 1: 1， by volume)J (5th paper chromatography). The eluate containing B2-g1ycoside-like compound was reconcentrated and rechromatographed twice on paper using solvent C (6th paper chromatography). Several repet.itions of the chromatographic isolation with three different solvents yielded chromato-graphically pure B2-g1ycoside-like compound. The band of B2-g1ycoside-like compound on paper was cut out and eluted with deionized water. The eluate was evaporated to dryness at 350C under reduced pressure， and dissolved in a small volume of 80 % ethanol. The insoluble impuri-ties were filtered 0百， and the filtrate was evaporated in vacuo leaving yellow residue. The residue was recrystallized several times from 80 % ethanol， yielding whitish yellow crystals of each Bcglycoside-like com-pound: 36 mg (Bc5'-β-gl)， 14.4 mg (B2-5'-α-ga) and 125 mg (B2-5にβ-ga)(Fig. 2).

6. Characterization of 5'-D-ribofiavin-β-D-glucopyranoside， 5'-D-ribofiavin-α-D-galactopyranoside and 5'-D-ribofiavin-β-D-galactopyrano・side. As .shown in Table 1， this purified B2-β-gl-like compound (Com-pound I) was different from crystalline B2-5'-α-gl obtained with rat liver， Leuc. mesenteroides and plant grains in characteristics like Rf values，

Biosynthesis of Ri凶fIavinGlyc田 ides 191

CrystaIs of 5'ーo-ribofIavin-β-0-gI ucopyrano!"ide.

Crystals of 5'-ローri凶flavin-a-o-galactopyranoside.

Crystals of 5'-o-ribofIavin-β-D-galactopyranoside.

Fig. 2. CrystaIs of ribofIavin glycosides.

192 Y. Suzuki & K. Uchida

mp， and the behavior in hydrolysis by glucosidases. Hydrolysis of Com-pound 1 by 1 N HCl at 1000C for 2.5 hours gave glucose and B2 in a molar ratio of 1: 1 as determined by both colorimetric6

) and manometrid) methods. Glucose was identified as a single reducing product of hydro-lysis with ani1ine hydrogen phthalate on paper chromatograms developed with four di旺erentsolvent systems: solvents A， C， n-butanol-acetic acid-water (4: 1 : 1， by volume)， and phenol-water (4: 1， by volume). Isolation and purification of B2 from the hydrolyzate were carried out by multiple paper chromatography， followed by recrystallization from ethanol. B2 thus obtained was identical with an authentic specimen in mp， absorption spectrum and Rf values on paper chromatograms developed wi th three di旺'erentsolvents A， B and C. Oxidation of purified crystals with sodium metaperiodate by the method described in the previous paperめ resultedin consumption of 4.0 mole of oxidant per mole of Compound I. Sweet almond emulsin caused a breakdown of Compound 1 into B2' whi1e a preparation of α-glucosidase from Leuc. mesenteroides3

)

had no e旺'ectupon Compound I. Compound 1 was not converted to higher members of Bcglucosy101ig凶 a∞harid白 8)in the 何回en白 ofsucrose by dextransucrase from Leuc. mesenteroides9

). The ph凶 phatetest by the method of Youngburg cmd Youngburg1G

) was negative in仕leacid hydro-lyzate of Compound I. Thus， the crystalline Compound 1 was identified as Bl-5'-β-g1.

Pure crystals of Bl-α-ga-like (or Bl-β-ga-1ike) compound were shown to be Br5'-α-ga (or B2-5'-β-ga) by comparison of the properties of the crystals， viz.， Rf values， absorbance at 220 to 500 mμ， detection of galactose and Bl in the acid hydrolyzate， and hydrolytic behavior with glycosidas白， with those of both crysta11ine Bz-5'-α-gl and crysta1line Bz-5'-β-gl synthesized by the enzyme preparations from glutinous millet grains (Table I).

From these experiments， it has been shown that glycosyl transfer reactions to ribofiavin (Disaccharide + ribofiavin一一~ ribofiavin glycoside + monosaccharide) are also distributed in plants. The relationship of the transferases utilizing ribofiavin as acceptor to the non-specific glycosidases (such asα-glucosidase，β-glucosidase，α-galactosidase and β-galactosidase) is now under investigation.

Acknowlegements. We thank Miss A. Mino and Mrs. N. Kawakami for their technical assistance.

T ABLE 1.

Properties of Crystalline Riboflavin G

lyc伺

ides

Sour

，田of

e田yme

Leuconostoc

Glutinous niil1et(

1)

Glutinous m

il1et

Glutinous m

il1et

Glutinous m

i11et

mesenteroidesω

5'-CDTrytsttmaflllainve in-

5'-CD-rdysbtoaflllainve in-

5'-CDr-yris回ta目llainvein-

5'ーCD-rydBMtanllainvein-

βーD5-'gーCaDlTrayctsttmtoapnlylairnvaeinno-side

α-D-glucopyran国

ide

α-D-glucopyranoside

β-D-glucopyranoside

α-D-galactopyran偶

ide

RI valu回

ホ

B. A.W.

0.20

0.20

0.17

0.18

0.16

P.W.

0.40

0.40

0.36

0.41

0.37

W. i田

A.

0.50

o.回

0.43

0.50

0.48

B.F.W.

0.05

0.05

0.04

0.03

0.03

Melting point (OC)

247-249

247-249

269-270

247-248

258-259

(d回ompc:渇

ed)

Ratios omf μo/p4t5ic0al mdμ .ensity

2到o

mp/450 m

p

2. 25

2.26

2.25

2.21

2.23

275 mμ/450 m

p

2.03

2.04

2.05

2.05

2.05

375 mμ/450 mμ

0.85

0.85

0.87

0.84

0.85

Position of maxtma in 2a 日bs，o3r7p3t，ion

spectrum (

mp)

“5

2回.373.“5

2 日.3百，

445

袋路.373.445

2 日.373，445

PahRydr rcohlyrozamte atogmm glouf eaαc 払idrtboflavin

gluco艶

.ri加

f1avin

glu∞

se，ri加f1avin

galacto 記

.ribof1avin

galactose，

ri加f1avin

Molar ratio of dboflavin:{ogr 1uα

斑e

galact

仮調e)

colorimetric

1:0.95

1:0.98

1:1.13

1:0.99

1:1.02

manωnetric

1:0.97

1:0.83

1:0.

邸

Periαiate uptake

4.04/1

3.89/1

3.98/1

3.71/1

3.田/1

(mole/mole of crystalline Bs

-glyc蝿

ide)

Hydrolysis b

y

α-gluc国

idase

+

+

β-gluα

lSidase

紳+

+

+

a-galact

田idase

+

β-galactosidase

一+

Presence of phosphate料

*

ホ:B. A. W

.: n-butanol-acetic acid-water (4:1:5，

by volume)

P. W.:

Na2HPO..12HzO-water (5:1∞

)

W. i釦

A.:

water，

saturated with i

田amylal∞hol

B. F. W.: n-butanol-formic acid-water (7れ

10:13.

by volume)

榊:

Com

mercialβ

ーgluc

伺idasefrom almond (emulsin) revealed the abilities to hydrolyze cellobi

個e.

melibiose and lactose. but not maltωe.

紳ホ

:After acid hydrolysis with 1

N HCl at 1∞。

Cfor 2.5 hours. ph国

phatein the hydrolyzate was examined by the method of Youngburg

and Youngburg.lO )

思 S U B S g Z R E Z同 - s z E『 n S E g ]「∞ ω

194 Y. Suzuki & K. Uchida

REFERENCES

1. Suzuki. Y. and Uchida. K. 1969. Arch. Bi∞hem. Biophys.. 150.回3.2. Suzuki. Y. and Uchida. K. 1967. Nippω1 Nogeikagaku Kaishi. 41. 125 3. Uchida. K. and Suzuki. y. 1後氾. Nippon Nogeikagaku Kaishi. 42. 502. 4. Suzuki. H.. Li. S. C. and Li. Y. T. 1970. J. Blol. Chem.. 245. 781. 5 Suzuki. Y. and Katagirl. H. 1963. J. Vitamino1.. 9. 285. 6. Ui. O. 1958. “Seikagaku Ryoiki ni okeru Kodenhishokuho，" (in Japanese. ed. by

Sekine. T.. Sasagawa. T.. Morita. 8.. Kimura. T. and Kuratomi. K. (Methods in photo' electric colorimetry for analysis of biolog1cal materlals). Vo1. 2. p.27. Nankodo Press. Tokyo.

7. Kaplan. N. O. 1957. “Methods ln Enzymology，" ed. Colowick. S. P. and Kaplan. N. 0.. Vol. 3. p. 107. Academic Pぉss.New York.

8. Suzuki. Y. and Uchida. K. 1967. Vltamins (Kyoto). 35， 31. 9. Suzuki. Y. and Uchida. K. 1967. Vltamins (Kyoto). 35. 27. 10. Youngburg. G. E. and Youngburg. M. V. 1930. J. Lab. C1in. Med.. 16. 158.