BIOCHIMIE, 1976, 58, 875-877.

the The use of cross-linked amylose in establishing

or S-amylase activity of an amylolytic preparation.

M. A. MATEESCU, H. D. SCHELL, l ~. MIHAILESCU * a n d I r i n a CORNOIU Insti tute of Biological Sciences, Laboratory of Enzymologg,

sir. Docentilor 20, 7000-Bucharest 2, Romania. • Insti tute of Cereals and Technical Plants-Fundulen.

Bd. Marasti 61, 7000-Bucharest 2, Romania. (29-1-1976).

u - A m y l a s e ( a - l , 4 - g l u e a n - 4 - g l u c a n o h y d r o l a s e , EC 3.2.1.1) is k n o w n to differ f rom B-amylase ~ct-l,4-glu- can mal tohydro lase , EC 3.2.1.2) in the i r m e c h a n i s m of action. Thus , a - a m y l a s e breaks the 1,4-u-glycosydic bounds ins ide the polysacchar ide chains , whi le ~-amy- lase is directed towards the same bounds only at the non- reduc ing end of the macromolecule .

Since the final product is the ma l tose in bo th cases, there are ser ious difficult ies in es tab l i sh ing the a- or ~-amylase na tu re of an enzyme prepara t ion exhib i t ing amylo ly t i c activity.

Several a t t empt s have been reported in the l i tera- ture to d i s t i ngu i sh between the two enzymes . One me thod consis ts f rom specifically inh ib i t ing ~-amylase by heavy meta l ions like Hg2+, Cu2÷, Ag ÷, or is based upon the fact t h a t a - a m y l a s e requires Ca2÷ ions for its ca ta lyt ic act ivi ty [1]. Other me thods are based on the differences between the opt imal condi t ions for acti- vi ty (pH, t empera ture ) of the t'wo enzymes [21. But these me thods are not a lways accurate enough. Thus , recent da ta show t h a t a - a m y l a s e i tself is sensi t ive to heavy meta l ions, supposed to block the s u l f h y d r y l groups essent ia l for its catalyt ic act ivi ty [3]. a - A my - lase ~ a s also found to re ta in about 30 per cent of i ts ini t ia l act ivi ty follo~ving a hea t ing at 70°C, and I~-amsqase loses about ha l f of i ts act ivi ty at pH 3.4 [4]. On the other hand, the opt imal condi t ions of act ivi ty for these two enzymes are different enough, depending of thei r source.

For the above reasons specific subs t ra tes were looked for, capable to make a sharp d is t inc t ion between the two enzymes . Based on the fact t h a t 1,6-a-glycosidic bounds (branching points) stop the ~-amylase action, b u t do not h inde r the u - a m y l a s e one, ~- l imit ing dext r ines suscept ible only to the a - a m y l a s e a t tack were used in order to d i s t ingu i sh between the two enzymes [4, 5, 6].

These subs t ra tes are difficult and expensive to obtained, as bo th long- t ime dialyses of large vo lumes of so lu t ions and large a m o u n t s of reagents are involved. The p resen t work describes the behav iour of the two enzymes aga ins t an amylose derivat ive obta ined by i ts c ross- l ink ing ~vith ep ich lorhydr ine [7].

MATERIALS AND METHODS.

The cross- l inked amylose was obta ined by a pa ten- ted method, p rev ious ly described [7], by t rea t ing the amylose in al;kaline m e d i u m 'with epichlorhydr ine . The product is a g ranu la ted gel tha t can be stored un t i l

To w h o m all correspondence shou ld be addressed.

use e i ther suspended in dist i l led water , or dried a f te r a previous dehydra t ion with acetone [8]. Crys ta l l ine (electrophoretic pure) pancreat ic ¢~-amylase (Karl Ro th -Kar l s ruhe) and B-amylase f rom bar ley ma l t (Fhlka) were used.

The study of ct- and ~-amylase action on cross- linked amylose. Two samples of amylose X-40 (*) deposited gel bed of 25 ml each (i.e. 2.75 g dry pro- duct) are treated in Berzel ius flasks 'with 5 mg of a - a m y l a s e and 5 mg B-amylase, respectively, dissolved in 5 ml dis t i l led water, and magne t i ca l ly s t i r red for 24 hours . Between whi les a l iquots of 0.5 ml each are taken off the s u p e r n a t a n t to a s say the reducing sugars released by the hydro ly t ic act ion of enzymes , me a s u - r ing every t ime the vo lumes of gel, too. Reducing pro- ducts were reduc tomet r ica l ly de te rmined by the me- thod of Noelt ing and Bernfeld [9].

The influence of enzyme concentrations upon the rates of hydrolytic breakdown of the substrate. Ali- quots of 0.35 ml each (0.04 g dry product) of cross- l inked amylose gel qX-40), previous ly t rea ted ~ ' i th B-amylase to remove the mal tose un i t s of the non- reducing ends of the amylose inacromolecule located outside the glyceric bridges, are t reated w i t h incre- as ing a m o u n t s of u- and B-amylase, respectively, rang ing f r o m 0.25 to 2.50 m g / m l . The reagent m i x t u r e is b rough t up to 3.00 ml wi th dist i l led water. The samples are s t i r red for 30 m i n u t e s at room tempera - ture , t hen the re leased reducing products are assayed.

In all cases, the act ion of the two enzymes on na- tive amylose was tested as control.

RESULTS AND DISCUSSIONS.

Our previous a t t empts to separate u- and B-amylase by aff ini ty c h r o ma t o g r a p h y on the cross- l inked a my - lose, revealed a different behav iour of the two enzymes aga ins t th i s subs t r a t e [10]. Thus , B-amylase pract ical ly w a s not re ta ined by the amylose deriva- tive, and released w h e n passed t h r o u g h the co lumn (1.5 × 18 era) only a smal l a m o u n t of mal tose resul - t ing f r o m the hydro ly t ic b r eakdown of the ma l tose un i t s f rom the non- reduc ing ends of the polysaccha- ride located outs ide the glyceric bridges (scheme 1) (less 1 per cent of the a m o u n t of mal tose released by a - a m y l a s e in the same condi t ions) , whi le u - a my l a s e was re tarded by the suppor t and released large a m o u n t s of reducing sugars together wi th a marked

( ' ) The X indicates tha t a inylose is cross- l inked, and the n u m b e r indicates the a m o u n t of epichlorhy- dr ine expressed in t e rms of g r a m m e s used to cross- l ink 100 g amylose.

25

M. A. Mateescu, H. D. Schell, F. MihaiIescu and I. Cornoiu.

decrease of the gel bed in the c o l u m n f r o m 18 cm to 3 cm dur ing the exper imen t .

I I

- o

SCHEME I SCHEME 1. --- Schematical diagram of amylolytic

action of a-amylase and iB-amylase on cross-lin'ked amylose by glgceric bridges.

0 ....... © = mal tose . [ : glycerie bridge.

• = a - a m y l a s e action. ........ )- _-- B-amylase action.

This different b e h a v i o u r of the two enzymes aga ins t ou r s u b s t r a t e suggested the idea to s tudy the even tua l poss ib i l i t i e s to use th i s ma t e r i a l as a specific sub- s t ra te to d i s t i n g u i s h be tween the t~vo amylo ly t i c enzymes .

W i t h th i s a im we first s tudied the t ime course of the ac t ion of u- a~d IB-amylase, respect ively, on the

• --'A . . . . . A . . . . . & . . . . . . . . . " ~ . . . . . . . - A - - --

.o ~

0 0 0 - 0 -0

0.5 1.0 1.5 2.0 2.5

Enzyme c o n c e n t r a t i o n ( m g l m l ) .2O

• 15 E

2J

-I0 ~ >

(.9

5

100-

90-

80-

70-

: 6o- 50-

E 40.

%-~ 30. b #

s 20.

:~ 1.o: 0.6- 0.4- 0.2

0 0 2 4 6 8 10 12 1~ 16 18 20 22 24

Time ( h )

Fro. l. - - Time course of maltose amount released from the cross-linked amylose follolwing a- ( 0 - - - - 0 ) and iB-amylase (0 . . . . ©) action, respectively, and time course of gel volume variation of cross-linked amylose treated Iwith a- (4 i ) and B-amylase (A . . . . A), respectively.

cross-lii~ked amylose . A release of ma l t o se was f o u n d in b o t h eases w i t h i n the first six h o u r s (h igher in the ease of a - a m y l a s e ) , and a qui te s l ight increase was noticed thereaf te r , so t ha t the reduc ing p rodnc t s level appea red prac t ica l ly unchanged (fig. 1).

BIOCHIMIE, 1976, 58, n ° 7.

The a m o u n t of ma l t o se re leased u n d e r the act ion of ~-a lnylase r ep re sen t s only 0.65 per cent of the r edu- eing suga r s yielded by s - a m y l a s e .

Record ing the v a r i a t i o n in the gel vo lume (fig. 1), a - a m y l a s e a t tack resu l ted in to a m a r k e d decrease of the gel bed du r ing the first six hour s , then the vo lume r ema ined prac t ica l ly unchanged , whi le p - a m y l a s e is concerned, no change of the gel v o l u m e is observed.

The changes in the vo lume of the gel are corre la ted w i th the a m o u n t s of ma l tose re leased as a r e su l t of the a - and ~B-amylase action, respect ively. The cons t an t bed vo lume in the case of B-amylase, reflects the pre- s e rva t ion of gel t h r e e - d i m e n s i o n a l s t ruc ture . The li t t le a m o u n t of ma l t o se re leased by B-amylase r e su l t s on ly f r o m the pe r iphe r i e ma l to se uni ts , ex is t ing outs ide of the glycerie h o u n d s (scheme 1).

u - A m y l a s e fai ls to h y d r o l y s e comple te ly cross- l inked amylose . The p h e n o m e n o m m a y be supposed to be s im i l a r to t h a t of B-amylase a t tack on amy lo -

O

18] o

~ 12 E

E

~ 6 >

~ O ~ 0

r r

876

FIG. 2 . - The influence of enzyme eoncenlration on hgdrohJsis rate of cross-linked am~.llose treated with ~- (0 0) and ~-amglase (0 . . . . ()), respeetiveh.I.

pectin, w h e n the release of ma l to se res idues s tops at the b r a n c h i n g po in t s ( l ,6-a-glyeosidie l inkages) , r e su l - t ing in to so called << l imi t dex t r ins >>. S imi la r ly , u - a m y l a s e may be s topped in i ts hydro ly t i c ac t iv i ty at the level of glucose res idues involved in en ter - cha in glycerie br idges .

Tes t ing the influence of va r i ous concen t r a t i ons of the two enzymes on c ross - l inked amy lose p r ev ious ly subjec ted to iB-amylase action, i t can be seen t h a t th i s enzyme re leases no more ma l to se even at h igh concen- t r a t i o n s (fig. 2), whi le u - a m y l a s e re leases reduc ing p roduc t s a m o u n t s inc reas ing 'with the enzyme concen- t r a t ion .

The n o n - l i n e a r increase of the reac t ion ra te as a func t ion of a - a m y l a s e concen t ra t ion m a y be due to some r ea sons depending on the m a c r o m o l e c u l a r n a t u r e of the inso lub le subs t ra te , as w o u l d be on the one h a n d the enzyme dif fus ion t h r o u g h o u t the th ree-d i - m e n s i o n a l n e t w o r k of the gel, and on the o the r h a n d the s i m u l t a n e o u s or successive h y d r o l y s i s of more b o u n d s in the subs t r a t e molecule , in a s im i l a r m a n n e r w i t h the p ro teases ac t ion on pro te ins . I t is k n o w n

a- and (j-amylases differentiated by cross-linked amyIose. 877

tha t in the case of proteolytic enzymes attack on natural substrates, the react ion rates do not exhibi t a l inear dependence against the enzyme concentrat ion ; they obey to more complicated kinetic rules [11, 12, 13].

Summaris ing the data f rom above, the following conclusions are resul ted :

1. the amylose cross-lir~ked by epichlorhydr ine and previously treated ~vith ,(j-amylase to spli t the peri- pherie maltose units, is a specifical subst ra te for a-am,ylase only, and not for ~-amylase.

2. Treat ing ~vith .a-amylase a kno~cn quant i ty of cross-l inked amylose gel, the volume of the gel bed is considerably reduced by the enzymatic hydrolysis , mean'while releasing reducing products. Under the same condit ions (J-amylase shoves no reactions.

Accordingly, the absence of maltose and preserva- t ion of gel volume when treat ing the amylose deri- vative ~vith an amylolyt ie preparat ion, points out to its (j-amylasic nature, whi le the presence of reducing products and the reduct ion of gel volume shows an a-amylas ie activity if the prepara t ion is pure, or else the presence of ~-amylase if it is not homogeneous.

REFERENCES.

1. Frydenberg, O. & Nielsen, G. (1965) Heredilas, 54, 123-140.

2. Dure, L. S. (1960) Plan t Physiol . , 35, 925-934. 3. Alexandrescu, V. & Mihaileseu, F. (1975) Rev. roum.

Biochim., 12, 3-6. 4. Mc.Gregor, A. W., La Berge, D. E. & Meredith, W.

O. S. (1971) Cereal. Chem., 48, 255-269. 5. Kruger, J. E. (1972) Cereal. Chem., 49, 379-390. 6. Strumeyer, D. H. (1967) Anal. Biochem., 19, 61-71. 7. Serban, M., Schell, H. D..& Mateescu, M. A. (1975)

Rev. roum. Biochim., 12, 187-191. 8. Ghetie, V. a Schell, H. D. (1971) Experient ia , 22,

1384-1385. 9. Noelting, G. & Bernfeld, P. (1948) Helu. Chim. Acta,

31, 286-290. 10. Schell, H. D. a Mateescu, M. A., Unpubl ished data. 11. Anson, M. L. (1939) J. Gen. Physiol . , 22, 79-89. 12. Bergman, M. & Fruton, J. S. (1961) Advan. Enzy -

tool., 1, 63. 13. Dixon, M. z, Webb, E. C. (1964) Enzymes , 2 e Ed.,

p. 69, Longmans, Green and Co., London.

BIOCHIMIE, 1976, 58, n ° 7. 59