Vol.. 1 8, No. 1 (1 966) 23
ENZYMATIC HYDROLYSIS OF SOYBEAN POLYSACCHARIDES
INTERMEDIATE PRODUCTS IN THE HYDROLYSIS
OF THE HEMICELLULOSE Bi OF SOYBEAN
COTYLEDONS BY TAKA-DIASTASE
(Laboratory of Agricultural Products Technology)
In the first paper of this series(') hydrolysis of the hemicellulose B1 of soybean cotyledons by Taka-diastase was reported. Taka -diastase liberated xylose, alabinose, galactose, and galacturonic acid in the hydrolyzate and gave an unhydrolyzable polysaccharide residue rich in r hamnose, f ucose, xylose, glucose, and galactur onic acid. Further, five intermediate hydrolysis products were formed i n an early stage of the incubation and two of them remained unchanged in the hydr olyzate after 48 hours' incubation. These results seemed to indicate that some informations on the components and the structures of the hemicellulose B1 would be obtained by examination of the intermediate hydrolysis products.
The present investigation was undertaken to see if examination of the intermediate hydrolysis products could qive any informations for the components and the structures of the hemicellulose B1.
1. Materials and Methods
The hemicellulose BI and the enzyme solution were prepared according t~ the procedures reported in the pr ecedinp; paper
Enzymatic hydrolysis was performed under the standard conditions described in t h e pre- ceding paper.
2. Separation of the Intermediate Hydrolysis Products
To 100 ml each of the reaction mixtures at 4 hr and 48 hr incubation 200 ml of absolute ethanol was added to stop the reaction and to precipitate the residual polysaccharides. After centrifugation, The supernatant clear solutions were concentrated in vacuo below 40" C to about 0.5 ml. The obtained sugar solutions were used for the separation of the inter- mediate hydrolysis products by ascending paper chromatography on Toyo Roshi No.50 filter paper sheets of 40 X40 cm. T h e solvent mixtures were n-butanol-acetic acid-water (4: 1 :2, double development) and n-butanol-pyridine- water (6:2: 3, single development)
.T h e sepa- rated areas were detected by consulting the guid strips colored by
P-anisidhe-HC1. The zones containing each intermediate hydrolysis products were cut and extracted with 10 ml of water. The obtained sugar solutions were hydrolyzed with 2 N Hz SO4 (final concentration) in boiling water for 4 h r . The hydrolyzates were neutralized with BaCOs and the formed
24 Tech.. Bull. Fac. Agr.. Kagawa Univ..
precipitate was removed by centrifugation and filtration through Toyo Roshi No. 5-C filter paper. The component sugars of these hydrolyzates were determined by paper chromato- graphy.
3 . The Intermediate Products at 4 Hoursr Incubation
Paper chromatograms of the acid hydrolyzates of the intermediate hydrolysis products are g i v e n in Fig. 1
BuOH-C5HsN Hz0 (single development)
Fig ? Component sugars of the oligcsaccha~ides in
4 hours' enzymatic hydrolyzate
This enzymatic hydrolyzate contained 5 intermediate products, A, B , C, I),and E, as r epor ted in the preceding paper
The spot A gave only sylose by hydrolysis with H2S04. T h e spot B consisted of galactose and trace amounts of xylose and arabinose. The spot E gave arabinose and galac- tose in the ratio of 1 : 2.
4 . The Intermediate Products at
The reaction mixture incubated for 48 hr contained only 2 intermediate products, C and
D, as shown in Fig 2.
Vol 18, No.. 1 (1966) 25
(double debelopment) (sinfile development) lactose and hydrolyzed to the spot Fig 2
.Component sugars of the oligosaccha~~des
in 48 hours' enzymatic hydrolyzate C, which appeared to be ar abinose- galactose and resistant t o Taka- diastase, because t h e spot E disappeared after
8hr incubation while t h e spot C increased with the progress of incubation and was detected from the reaction mixture incubated
.The behaviors of the spots B, C, and E seemed to confirm the presence of an arabogalactan in the hemicellulose B1. The spot D gradually increased with the progress of incubation as t h e spot C and was detectable in the reaction mixture incubated for
h r . This spot may be a xylosyl-galacturonic acid resistant to t h e activity of Taka-diastase. The increase and decrease of t h e spots A a n d D during t h e incubation seemed to establish t h e presence of a galacturonic acid-containing xylan in the hemicellulose B.1. I t is well known t h a t plant xylans contain galacturonic acid. The above results seemed to show that no essential difference exists between wood xylans and a xylan of soybean cotyledons
BUOH-ACOH -HZO BUOH -CSHSN-HZO spot E seemed to be arabinose-ga-
The author is grateful to prof. Sin'itiro KAWAMURA, Laboratory of Biological Chemistry. this University, for his interest in this work. Valuable suggestions of prof. Akira KAJI, Laboratory of Fermentation Chemistry, this University, are greatefully acknowledged.
A part of expenditure was defrayed by the research fund donated by the Ministxy of Education to T . NARASAKI (Enzymatic hydrolysis of soybean hemicelluloses, 1962) and to
The spot C consisted of arabinose and galactose in the ratio of 1
: I .
The spot D gave xylose and gal- acturonic acid in the ratio of 1 : 1.
The spot A indicates t h e presence of xylose-xylose linkage and the linkage can be hydrolyzed by Take-diastase, since the spot disap- peared after 12 hr incubation as has been shown in Fig
.6in the pre- ceding report. The spot B shows the presence of galactose -galactose linkage and this linkage can also be hydrolyzed by Taka-diastase, be- cause this spot appeared in an early stage of the reaction and was not detected after
8hr incubation. The
ai?3 Rha E U C Xyl 1 , " c lu G ~ I t c Ga?A
-@ - Rh4 1. uc X,I A ~ J Gal Gal A D . 4
26 Tech. Bull. Fac. Agr
A . KAJI (Improvement of the utilization of agricultural and horticultural products by t h e ase of enzymes, 1962-1963).
(1) NARASAKI, T. : Kagawa Daigaku Nogakubu Gakuz,yutu Hokoku, 18,16(1966).
EP EJ;JBK$sL\-CPjsP7zP--k?vcd: 6 - $ C l v e -2 B ~ D f i n ; k f i % d : , -.
:C l v e - X B I D # & & % ~f a D t z g % w n E % % k k a s & + E g ~ k .
$ $ E ? B @ % ~ ~ R K ~ : Q T & F ~ # B E Q ~ ~ % ~ E % & ~ E L ,? h b D @ @ & & i B ~ 6 c k K d : ~-Cj?D@&%%fiiB L k . X ~ $ E K % L ~ # ~ ~ + @ @ D W G E & # D E @ ~ ? D @ X @ & i k ; 5 ~ b ~
<C P m -2 @@,$@%R#KBY
5 ~ j 5 ' 5 P ~ k j 5 ' 5 9 7 ~ ~ @ & ~ ~ + i . ~ ~ d ~ ~ & f 6 t k % @ % L k . X P j f P ; I ; P X P - + ? k 2 7 4 Y l - X : j5'
E--%$sd:U\'+Ve - 2 : j 5 ' 5 9 Y o ~ ~ D ~ ~ % 5 3 ~ 3 - a Z G r 5 j T $ ? k L > C k f i ~ ; h d a 3 k .
$ ~ % - t r f i ~ j ~ ;5 ~ ; b j k
~ ~ P ~ B ~ B B ~ ~ ; ~ ~ ~ ) I I H ~ S - I B ~ ~ * ~ : U \ ' ~ ~ ~ ~ B K R <& % L a + .
DWF%DBBD-ZBfiBsS:%PPRPWf%R(tiBRi37+E&thlWfRR, U@J*, Y 4 z'- ?
#$ ; tiBa37, 38+ERMWF%B7 B % K 1 6BZ!SBYflD?%E%UH K M f 6