Substrate specificity and transglucosylation catalyzed by cycad β-glucosidase

BiochimicaL et Biophysics

ELSEVIER

& ta

Biochimica et Biophysics Acta 1289 (19961315-321

Substrate specificity and transglucosylation P-glucosidase

catalyzed by cycad

Fumio Yagi *, Kenjiro Tadera Biochetnisr~

nnd Biotechnology,

Fucul~ ofAgriculturr,

Kagoshima

Urzir~ersity,I-21-24 Korimoto. Kagoshima

890. Japan

Received 14 June 1995; accepted 13 September 1995

Abstract Initial rates of transglucosylation with diglucosides and diglucose-azoxyglycosides as acceptor by cycad P-glucosidase were tentatively obtained. The formation of p-1,3 glucosidic linkage was predominant, except for neocycasin A ( /3-laminaribioside of methylazoxymethanol, MAM) as an acceptor. With neocycasin A as an acceptor, p-l.4 and p-1.6 glucosidic linkages were formed but p-l,3 linkage was not. Whereas with laminaribiose as acceptor, laminaritriose and triose with /3-I ,6 linkage were formed. but triose with p-I.4 linkage was not. On the other hand, with other diglucoses and neocycasin B ( P-gentiobioside of MAM) as acceptor, all the linkages formed were p-l.3 glucosidic. The aglycone of azoxyglycosides, MAM. affected the kind of linkages formed in the trisaccharides. When initial rates of the linkage formation of the transglucosylation at 100 mM acceptor were compared with the hydrolysis rates obtained by Lineweaver-Burk plot, the order of formation rates of the di- and tri-glucosides by transglucosylation was the same as obtained for the hydrolysis parameter, kcat/K,,. K, values for various substrates could be grouped according to the kind of the linkages ( p-1,3, p-1,4: and p-1.6) first split by the enzyme. &words:

P-Glucosidase; Cycad; Azoxyglycoside: Cycasin; Transglucosylation

1. Introduction Many glycosidases

have high transglycosylation

activity

when the sugars and other molecules serve as acceptor molecules instead of water. Cycad @-D-glucosidase was known as cycad emulsin to have a transglucosylation activity [ 1.21. We have purified this enzyme in a homogeneous state [3] and showed that three trisaccharide-azoxyglycosides were formed by transglucosylation of this enzyme [4]. p-l,4 and -1,6 glucosidic linkages were formed with neocycasin A. laminaribioside of methylazoxymethanol (MAM). as a transglucosylation acceptor, and p-1,3 glucosidic linkage was formed with neocycasin B, the gentiobioside of MAM, as a transglucosylation acceptor. Thus, azoxyglycosides-isomers with multiple linkages were formed by the enzyme and the formation of the linkages seemed to be dependent on the acceptor glycosides. Nilsson [5-71 reported that the formation of various linkages sometimes depended on the aglycone of the acceptor glycosides (regioselectivity). However, most of the studies on transglycosylation by exoglyand

the

activity

is observed

. Corresponding author. Fax: +81 992 8.58525or 8632. 03043165/96/$15.00 SSDl

0304.4165(95)00149-2

0 1996 Elsevier

Science

B.V.

All

rights

reserved

cosidases were restricted to the formation of disaccharides and the formation of trisaccharides were scarcely reported. Recently. Christakopoulos et al. [S] reported that trioses were synthesized from cellobiose and gentiobiose using fi-glucosidase from Fusarium nxysporum but their structure were not elucidated. One purpose of this study is to reveal the regioselectivity of cycad enzyme in the formation of triglucosides with or without aglycone MAM. A purpose of transglycosylation is generally the production of biologically active saccharides [9]. Therefore, optimal conditions for producing and accumulating final products are regarded to be important, and in many cases the products in the course of reaction and the transglucosylation rate were not analyzed. Theoretical analysis of transglucosylation have been carried out for some glycosidases. Subsite theory [ IO,1 1] is undoubtedly the most successful one for analyzing transglycosylation. However, the application of the theory to the analysis of transglucosylation of azoxyglycosides seem to be difficult because of the formation of multiple p-linkages in a single oligosaccharide molecule. In transglycosylation. the product oligosaccharide formation depends on the partition of enzyme-glycosyl inter-

F. Yagi, K. Tadera / Biochimica et Biophysics Acta 1289 (1996) 315-321

316

mediate between hydrolytic and transglycosidic reactions. If the reaction time is long and/or enzyme concentration is high, the hydrolysis of oligosaccharides proceeds extensively. However, at the initial stage of the reaction, where the hydrolysis of the transglycosylation product is very low, we can tentatively estimate the rate of the formation of product oligosaccharides in order to a analyze the transglycosylation. In this paper, we analyzed initial rate of each unit reaction of transglucosylation (transfer of one glucose unit from enzyme-glucosyl complex to one acceptor molecule) with appropriate concentrations of donor and acceptor glucosides to estimate the transglucosidic formation of various linkages of azoxyglycosides (diglucosides and triglucosides) and some oligosaccharides, and further obtained hydrolytic parameters of some glycosides to compare the transglucosidic reactions with the hydrolytic reactions.

sulfate, and acetic acid according to the method of Nagahama et al. [ 121. O-P-D-Glucopyranosyl-(1 -+ 6)-O-p-~glucopyranosyl-(1 -+ 3)-0-P-D-glucopyranoside (III) and + 3)-0-/3-D-glucopyranosyl0-P-D-glucopyranosyl-(1 (1 + 4)-o-p-D-g1 ucopyranoside (IV) were gifts from Professor Ken’ichi Takeo, Kyoto Prefectural University. O-j?D-Glucopyranosyl-(1 + 3)-0-/3-D-glucopyranosyl-(1 -+ 6) (V) was prepared by deacetylation 0-/3-D-glucopyranoside with sodium methoxide from the peracetylated form of the trisaccharide, which was kindly supplied by Dr. Martin Rychener, University Bern, Switzerland. The P-glucosidase used in this study was component III that was obtained as a crystalline preparation from seeds of Japanese cycad, Cycas revoluta Thunb [3]. One unit of activity was defined as the amount of enzyme which liberated 1 pmol of o-nitrophenol per min in 0.1 M sodium acetate buffer, pH 5.0 at 30°C with 10 mM onitrophenyl /3-D-glucopyranoside as the substrate. Specific activity of the enzyme was 148.3 units/mg.

2. Materials

2.1. Enzyme reactions.

and methods

All azoxyglycosides were from our laboratory collections. Laminaritriose and laminaritetraose were purchased from Seikagaku Co. (Tokyo, Japan). O+D-Glucopyranosyl-(1 -+ 4)0-P-D-glucopyranosyl-(1 + 3)O-p-D-glucopyranoside (I) and 0-P-D-glucopyranosyl-( 1 + 6)-[ O-pD-glucopyranosyl-( 1 + 3)]-0-P-D-glucopyranoside (II) were prepared from neocycasin H and I, respectively, by the decomposition of aglycone, MAM, with zinc, cupric

Neocycasin

1) Kinetics of hydrolysis

Preliminary experiments were carried out for all the substrates for setting the concentrations of substrates and enzyme, and reaction times. The enzyme reaction was started by the addition of enzyme solution to otherwise complete reaction mixture and quenched at 0°C by the addition of 0.1 M HCl. Hydrolysis of azoxyglycosides was estimated by the production of MAM and azoxyglycosides with smaller sugar units, using HPLC systems according to

B

HOCH2 I-‘\,

0-CH, Neocycasin

Neocycasin

A

I

Neocycasin

HOCH2 @‘a”z:.

Fig. 1. Transglucosylation

products from cycasin.

H

F. Yagi, K. Tadera/Biochimica

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et Biophysics Acta 1289 119961315-321

Yagi et al. [4,13]. Hydrolysis of disaccharides and laminari-oligosaccharides were measured by Somogyi-Nelson method [ 141, and glucose oxidase method (Glucotest Wako, Wako Pure Chemicals, Tokyo, Japan) or hexokinase-glucase-6-phosphodehydrogenase method (F-kit, Boehringer Mannheim, Mannheim, Germany). Hydrolysis products of laminari-oligosaccharides were analyzed by HPLC system with a column of Shodex S-614 and a differential refractometer (Knauer Model No. 98.00, Berlin, Germany) as described in a previous paper [4]. 2.2. Transglucosylation

reactions

-0.2

-0.1

0

0.1 l/S

Transglucosidic products were analyzed as described for hydrolytic products. Fig. 1 shows the transglucosidic formation of azoxyglycosides with cycasin as a stating material. The formation of neocycasin species was already confirmed [4]. One unit reaction means, for example, the formation of neocycasins A and B from cycasin. The oligosaccharides formed by transglucosylation with cellobiose, gentiobiose, and laminaribiose as acceptors were identified by analyzing their partially methylated alditol acetates [15] after HPLC separation on a column of Shodex S-614.

3. Results 3.1. Hydrolysis of azoxyglycosides

and some oligosaccha-

rides

Table 1 shows the parameters for the hydrolysis of azoxyglycosides and some oligosaccharides. In the hydrolysis of diglucose-azoxyglycosides, substrate was assumed to be first hydrolyzed into cycasin and glucose, and then cycasin into MAM and glucose. The products by the

Table 1 Kinetic constants

of cycad

P-glucosidase

Substrate ;&I cycasin neocycasin A neocycasin I neocycasin F laminaribiose laminaritriose laminaritetraose neocycasin B neocycasin G gentiobiose neocycasin E neocycasin H cellobiose sophorose

1.7 5.8 18.0 5.0 14.9 35.7 86.9 80.2 100 83.1 12.5 3.2 21.7 220

Hydrolysis rates for diglucoses amount was divided by 2.

L (mm- ‘1 4820 6700 5940 2.6 8640 1120 121 134 550 220 27 13 34 4940 were obtained

L /K, (min~‘.mIC’) 2870 1150 330 0.52 580 31.3 1.4 1.7 5.5 2.7 2.2 4.2 1.6 22.5 after the released glucose

0.2

0.3

0.4

0.5

(m&w1

Fig. 2. Double-reciprocal plot for hydrolysis of neocycasin A by cycad P-glucosidase. Hydrolysis rate of neocycasin A was estimated by the summation of released cycasin and MAM.

hydrolysis were cycasin, MAM and glucose. The hydrolysis rate for neocycasin A was estimated by the total amounts of released MAM and cycasin. Fig. 2 shows the linear double reciprocal plot for the hydrolysis of neocycasin A. When neocycasins G and H were hydrolysed, similarly, neocycasin A, cycasin, MAM and glucose were produced in the reaction mixture. However, when enzyme concentration was very low, neocycasin A was predominant as an initial product. The hydrolysis of di- and tri-glucoseazoxyglycoside was stepwise and no release of di- or tri-glucose from azoxyglycosides was found. When neocycasin B was hydrolyzed, main product was MAM and glucose. Neocycasin B was first hydrolyzed into cycasin and glucose, but hydrolysis parameter (k,,,/K,) of cycasin was much higher than that of neocycasin B. Accordingly, cycasin was scarcely accumulated in the reaction mixture. Exactly, one glucose release from each substrate should be measured (e.g., neocycasin A from neocycasin G) for estimating the hydrolysis of oligoazoxyglycosides. However, such a reaction condition was impossible to be selected. All the values in Table 1 were tentative for the hydrolysis, and the further hydrolysis of initial products were not taken into consideration. In Fig. 2, at 2 mM neocycasin A, 20% of cycasin was further hydrolyzed into MAM and glucose, whereas at 10 mM, further hydrolysis of cycasin was lower than 5%. On the basis of cycasin amount, K, was estimated to be 7.0 mM. Therefore, these hydrolysis parameters in Table 1 seemed not to be far from the true hydrolysis rates for the release of first one glucose from the diglucose-azoxyglycosides (eg. neocycasin A into cycasin and glucose). Hydrolysis parameters could be grouped according to the kind of linkages hydrolyzed between the nonreducing glucose and the next glucose. The K, values for neocycasin A, F and I were in the range of 5 N 18.0 mM and were comparable to 14.9 mM for the hydrolysis of laminaribiose. k,,,/K, values of these glycosides were higher than 330 except for 0.52 of

318

F. Yagi, K. Tudera /Biochimica

et Biophysics Acra 1289 (19961315-321

r

0

20

40

60

0

20

Reaction Reaction

Time

neocycasin F. The hydrolysis rate of these substrates were attributed to the cleavage of p-1,3 linkages. The K, values for neocycasins B and G were 80.0 and 100.0 mM, respectively, and were comparable to the values for gentiobiose (i.e. p-1,6 linkage). k,,,/K, values for these azoxyglycosides and gentiobiose were in a similar range. values for the hydrolysis of /3- 1,4 K, and k,,,/K, linkage of neocycasin E ( /3-cellobioside of MAM), neocycasin H and cellobiose were also in the same ranges. Thus, the parameters for the hydrolysis of respective linkages show their characteristics. There are some exceptions. The K, values for laminaritriose and laminaritetraose were higher than the values for p-1,3 linkages of other substrates and with the increase of glucose unit, the hydrolysis rates of laminari-oligosaccharides decreased. with cycasin as a substrate

When cycasin was used as a substrate at high concentration, neocycasins A and B were formed as the initial products by the transglucosylation. Fig. 3 shows the time course of the reaction at 150 mM cycasin and 0.19 U/ml of enzyme. The release of MAM and the formation of the two azoxyglycosides proceeded linearly under the reaction condition. When cycasin was 150 mM and reaction time was 10 min, the two azoxyglycosides were formed in proportion to 0.2 _ 0.8 units/ml of enzyme concentration. When reaction time was 10 min and enzyme concentration was 0.19 U/ml, the formation of the two azoxyglycosides were proportional to cycasin concentration (50-200 mM). 3.3. Transglucosylation

(min)

(min)

Fig. 3. Time course of neocycasins A and B formation from cycasin. Reaction was carried out pH 5.0 at 30°C in the presence of 150 mM cycasin and 0.19 U/ml of enzyme. Open circle, neocycasin A; closed one, neocycasin B.

3.2. Transglucosylation

60

40

Time

Fig. 4. Time course of neocycasins G and H formation from neocycasin A. The reaction mixture (150 mM neocycasin A and 0.38 U/ml of enzyme) was incubated at 30°C. Open circle, neocycasin G: closed one, neocycasin H; A, cycasin.

served as both donor and acceptor in a similar manner with cycasin. During the course of the reaction, the amount of cycasin did not increase linearly. The released cycasin might be further hydrolyzed by the enzyme and/or be used as an acceptor for transglucosylation. However, the formation of neocycasins G and H proceeded linearly within 30 min. Fig. 5 shows the dependence of the formation of the two triglucose-azoxyglycosides on the concentration of neocycasin A. The formation of neocycasin G (i.e. formation of p-1,6 linkage) was proportional to the concentration of neocycasin A, but the formation of neocycasin H was not, and was very low at the lower concentration of neocycasin A. 3.4. Transglucosylation tors

with other neocycasins

as accep-

As neocycasin B was a poor substrate and macrozamin ( P-primeveroside of MAM) was not a substrate for cycad fi-glucosidase, donor substrate was necessary in order to

0.3

0

0.05

0.1

0.15

Neocycasin

A (M)

0.2

with neocycasin A as a substrate

Fig. 4 shows the time course of transgiucosylation with neocycasin A as a substrate. In the reaction, neocycasin A

Fig. 5. Dependence of neocycasins G and H formation on the concentration of neocycasin A. Neocycasin A (150 n&l) was incubated with 0.50 U/ml of enzyme for 15 min at 30°C. Open cicle. neocycasin G; closed circle, neocycasin H.

F. Yqi. K. Tadera/Biochimica

JO.4

-0.2

o-o 0

e .r(

/CC? - 0.3

3 0 5 g z

7

RI

40

Reaction

x4

A6

- 0.1

20

40

50

10

20

30

4

0

60

Time

Elution

(min)

Fig. 6. Time-course of neocycasins I and F formation from neocycasin B. Reaction mixture contained 0.38 U/ml of enzyme, 60 mM cycasin and 100 mM neocycasin B. Open circle, neocycasin I: closed circle, neocycasin F.

form an enough amount of enzyme-glucosyl intermediate for transglucosylation. Irrespective of the concentration of cycasin (60- 100 mM), the formation rates of neocycasins I and F were almost constant at 100 mM neocycasin B. Fig. 6 shows the time course of the formation of two neocycasins with neocycasin B as an acceptor. With macrozamin as an acceptor, the formation rate of neocycasin J, O-~-DI + 6)1-Oglucopyarosyl-( 1 -+ 3)[ 0-/3-D-xylopyranosyl-( @-o-glucopyranoside of MAM, was almost the same as that of neocycasin I. 3.5. Transglucosylation

319

et Biophvsica Acta 1289 (1996i315-321

with diglucoses

Volume

(ml)

Fig. 8. A typical HPLC chromatogram of a reaction mixture with gentiobiose as acceptor. An elution profile of the reaction mixture (30 ~1) obtained after 2-h incubation of the enzyme (1.5 U/ml) with 100 mM cycasin and 90 mM gentiobiose. As the eluting solvent, 85% aqueous methanol was used instead of aqueous acetonitorile in Fig. 7. 1, cycasin; 2, neocycasin A; 3, glucose; 4, laminaribiose; 5, gentiobiose; 6 and 7; specimens of triose II and V.

mixture. The triglucoses formed were identified to be trioses V and II. Finally, cellobiose and cycasin were used as an acceptor and a donor, respectively. Fig. 9 shows the HPLC chromatogram. The peak eluted at 37.5 min was identified as triose IV from its elution position and by analysing its partially methylated alditol acetates. Fig. 10 shows the time course of the transglucosylation with 100 mM of the respective diglucosides as acceptors.

as acceptors

When laminaribiose was used as a substrate, two trioses were found on HPLC chromatogram (Fig. 7). They were identified as laminaritriose and triose III. When gentiobiose was used as an acceptor, cycasin was used for forming sufficient enzyme-glucosyl intermediate. Fig. 8 shows the HPLC chromatogram of the reaction

3.6. Comparison ous acceptors

of the transglucosylation

rate with uari-

The formation rates of the transglucosylation products at 100 mM acceptor are compared in Table 2. Assuming that the hydrolysis velocity was proportional to the amount of enzq ;ne-glucosyl intermediate, the calculated values of

2

1

60

50

40 Elution

30

20

Volume

(ml)

10

0

Fig. 7. HPLC chromatogram of a reaction mixture with laminaribiose as a substrate. An elution profile of the assay mixture (10 ~1) obtained after l-h incubation of the enzyme (0.38 U/ml) with 100 mM laminaribiose. Glucosides were eluted from a column of Shodex S-614 with acetonitorile/water (75:25, v/v) at a flow rate of 0.8 ml/min. 1, glucose; 2, laminaribiose; 3, 4 and 5, specimens of laminaritriose, triose III and I.

50

40 Elution

10

20

30 Volume

0

(ml)

Fig. 9. A typical HPLC chromatogram of a reaction mixture with cellobiose as acceptor. An elution profile of the assay mixture (10 ~1) obtained after 7.5-h incubation of the enzyme (0.5 U/ml) with 80 mM cycasin and 120 mM cellobiose. The elution condition was the same as in Fig. 9. 1, cycasin; 2, neocycasin A; 3, glucose; 4, cellobiose; 5, triose IV and 6, unknown.

F. Yagi, K. Tadera/Biochimica

320

1.0 f 0.8 0.6 z o 0.4

.r( b

0.2

601

-0

c

i5

2

Reaction

4

6

Time

8

(h)

Fig, 10. Time courses of transglucosylation products with diglucosides as acceptors. A, Laminaribiose (100 mM) and enzyme (0.38 U/ml) were incubated. Open circle, laminaritriose; closed circle, triose III; A, glucose. B, Gentiobiose (100 mM1, cycasin (60 mM1, and enzyme (0.50 units/ml) were incubated. Open circle, triose II; closed circle, triose V; A, glucose. C, Cellobiose (100 mM1, cycasin (60 mM), and enzyme (0.50 U/ml) were incubated. Open circle, triose IV; A, glucose; 0, neocycasin A.

et Biophvsica Acta 1289 (1996) 315-321

The effect of aglycone MAM were observed in the formation of trisaccharides from laminaribiosides and gentiobiosides. From neocycasin A, neocycasins G and H were formed, whereas laminaritriose and triose III were formed from laminaribiose. Triose I which had the same sugar moiety of neocycasin H was not formed from laminaribiose. In the presence of aglycone, the structure around substrate binding site is not favourite for accommodation of the laminaritrioside. Similar results of aglycone effect were observed for the trisaccharides from neocycasin B and gentiobiose. The two trioses formed from gentiobiose had the same sugar moieties as neocycasins I and F, and the linkage formed in this unit reaction was only p-l,3 glucosidic linkage. However, the formation rate of these hydroxyl linkages were different between with and without the aglycone. Without the aglycone, the formation rate of two trioses were similar, whereas neocycasin I was formed much faster than neocycasin F. In the transglycosylation of galactose, Nilsson [5-71 reported regioselectivity of transglycosylation and the influence of aglycone structure of acceptor molecule. The formation of p-1,3 linkage was also found with cellobiose as an acceptor. These results suggested that laminaribiosyl moiety of saccharides was easily recognized by the enzyme but with the increase of p- 1,3 glucosyl chain length, the binding affinity of sugars to the enzyme decreased. Laminaritriose and laminaritetraose were poor substrates of this enzyme (Table I). These results coincided with the fact that this enzyme had no action on P-1,3-glucans (unpublished results).

Table 2 Formation rates concentration Acceptor

transglucosylation rates were obtained at the same hydrolysis velocity as that of cycasin.

Product

Cycasin Neocycasin

4. Discussion

Laminaribiose

Cycad P-glucosidase formed three types of /?-linkages by transglucosylation but the kind of linkages formed depended on the acceptor molecule. The mode of transglucosylation action of several P-glucosidases were reported but formation of various hydroxyl-linkages of trisaccharides were not reported. When k,,,/K, and transglucosylation rate at 100 mM of acceptor were compared, the orders of their values with various substrates were apparently related. k,,,/K, values decreased as tallows; neocycasin A, neocycasin I, neocycasin G, neocycasin H, neocycasin B and neocycasin F (Table I). The same order was found for transglucosylation formation of azoxyglycosides (Table 2).

Neocycasin Gentiobiose Cellobiose

of transglucosylation

A

B

neocycasin A neocycasin B neocycasin G neocycasin H laminaritriose triose III neocycasin I neocycasin F triose II triose V triose IV

products

at 100 mM acceptor

Formation rate ( pmol/min per pm01 enzyme) Observed

Calculated

1608 29 195 53 128 101 363 13 233 221 60

146 40 80 64 379 14 243 230 68

a

The concentration of all the acceptors was 100 mM. 60 mM of cycasin was used for the formation of enzyme-glucosyl intermediate for cellobiose, gentiobiose and neocycasin B a Calculated values were obtained assuming that the amount of enzymeglucosyl intermediate was propotional to the hydrolysis velocity and the velocity was equal to that for cycasin. With cellobiose, gentiobiose, and neocycasin B, as acceptor, respectively, the hydrolysis rates were obtained by use of the equation: u = (V; A + V,, B)/(l+ A + B), where V, and V, are the velocity maximum for cycasin and acceptor, and A and B are the substrate concentration/K, for cycasin and acceptor.

F. Yagi, K. Tadera/Biochimica

et Biophysics Acta 1289 (1996) 315-321

Previously, Ajisaka et al. [16] reported the four diglucases were formed by condensation reaction of almond P-glucosidase. In the reverse, almond enzyme can hydrolyse all the four diglucosides; laminaribiose, gentiobiose, cellobiose and sophorose. Similarly, cycad P-glucosidase could hydrolyze the four diglucoses (Table 1) and the formation of the four diglucoses from glucose were also observed (unpublished result). With cycasin as a substrate, neocycasin A and B were formed by transglucosylation reaction but neocycasin E was not found in the reaction products of initial enzyme reaction. During the long reaction period, it gradually accumulated. The formation of neocycasin H seemed to be similar to neocycasin E. As shown in Fig. 4, the formation of neocycasin H was not proportional to the concentration of neocycasin A, whereas neocycasin G increased linearly with the concentration of neocycasin A. Each neocycasin has p-1,4 glucosidic linkage at the nonreducing end. The low concentration of neocycasin A was not convenient for the formation of neocycasin H. When enzyme-glycoside (containing cellobiosylmoiety) complex was formed, the release of the glycoside might not be as good as that for other enzyme-glycosyl complex. Freer [ 171 recently reported the hydrolysis of cellodextrins by Candida wickerhamii P-glucosidase. He described that the enzyme hydrolyzed cellopentaose sequentially from the non-reducing end. The enzyme showed the very high K, values for various cellodextrins. On the contrary, K, values of cycad enzyme toward various substrates were relatively low and k,,,/K, values were very different. Enzyme-glycosyl complex might not completely dissociated for some substrates. Especially in the case of transglucosylation formation of neocycasin E and neocycasin H, such mechanism may be conceivable. Tanaka and Takeda [ 181 reported that subsite affinities of glucoamylase toward nonreducing glucose of maltooligosaccharides and isomaltooligosaccharides were different. The difference of linkage seemed to be reflected on the interaction of glucoamylase and the oligosaccharides.

321

Related with the difference of linkage in the substrates, the large difference of K, values in Table 1 may be found. Some of these trisaccharide structure with heterolinkages are found in various P-glucans, but their possibility as bioactive substances are not revealed. Production of these saccharides may open the way for investigation of their usefulness. Furthermore, azoxyglycosides are known as toxic glycosides with labile aglycone MAM and its decomposition by chemicals can produce the trisaccharides. This can be considered as the alternative way for the trisaccharide production. References 111Nagahama,

T., Nishida, K. and Numata, T. (1960) Bull. Agric. Chem. Sot. Jpn. 24, 322-323. 121Nishizawa, K. and Hashimoto, K. (1970) in The Carbohydrate Chemistry and Biochemistry (Pigman, W. and Horton, D., eds.), Vol. HA, pp. 241-300, Academic Press, New York and London. [31 Yagi, F., Hatanaka, M., Tadera, K. and Kobayashi, A. (1985) J. Biochem. 97, 119-126. 141 Yagi, F., Tadera, K. and Kobayashi, A. (1985) Agric. Biol. Chem. 49, 2985-2990. 151 Nilsson, K.G.I. (1987) Carbohydr. Res. 167, 95-103. 161Nilsson, K.G.I. (1989) Carbohydr. Res. 188, 9-17. 171 Nilsson, K.G.I. (1990) Carbohydr. Res. 204, 79-83. 181Christakopoulos, P., Kekos, D., Marcris, B.J., Goodenough, P.W., and Bhat, M.K. (1994) Biotech. Lett. 16, 587-592. [91 Ichikawa, Y., Look, G.C., and Wong, C-H. (1992) Anal. Biochem. 202, 215-238. [lOI Hiromi, K. (1970) Biochem. Biophys. Res. Commun. 40, 1-6. 1111 Suganuma, T., Matsuno, R., Ohnishi, M. and Hiromi, K. (1978) J. B&hem. 84, 293-316. 1121 Nagahama, T.. Numata, T. and Nishida, K. (1959) Bull. Agric. Chem. Sot. Jpn. 23, 556-557. [131 Yagi, F., Tadera, K. and Kobayashi, A. (1980) Agric. Biol. Chem. 44, 1423-1425. [I41 Somogyi, M. (1952) J. Biol. Chem. 195, 19-23. H51 Soloneker, J.H. (1972) Methods Carbohydrate Chem. 6, 20-24. [I61 Ajisaka, K, Nishida, H. and Fujimoto, H. (1987) Biotechnol. Lett. 9, 243-248. [171 Freer, S.N. (1993) J. Biol. Chem., 268, 9337-9342. [I81 Tanaka, A. and Takeda, S. (1994) Biosci. Biotech. Biochem. 58, 1809-1813.