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α-Amylase Purificationand Separation fromGluco-Amylase by AffinityChromatography on Cross-Linked Amylose(CL-Amylose)Horst D. Schill a , Mircea A. Mateescu a , TraianBentia a & Aurora Jifcu aa Institute of Biological Sciences, Laboratory ofMolecular Biology , Splaiul Independentei 29677748, Bucharest, 17, ROMANIAPublished online: 24 Feb 2007.

To cite this article: Horst D. Schill , Mircea A. Mateescu , Traian Bentia &Aurora Jifcu (1981) α-Amylase Purification and Separation from Gluco-Amylaseby Affinity Chromatography on Cross-Linked Amylose(CL-Amylose), AnalyticalLetters, 14:17-18, 1501-1514

To link to this article: http://dx.doi.org/10.1080/00032718108081476

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ANALYTICAL LETTERS, 14 (BS.7&18), 1501-1514 (1981)

Key words : orose-linked amylose, ar-aqylase, gluco-

amylase, affinity ChrO1nRtQ&r8phy, sepa-

ration

Rorst D. Schell, Yfrcea A, Mateesoo, 'Praia Bentia

and Aurora Jifcu

Institute of Biological sciences, Idoratory @f Yofsoulu Biology

Splaiul hdependsntsi 296, 77748 Buchareet 17,RoludRIA

ABSTBlaCT

Using a CL-amylose colurtn it is poseible t o pu-

rify d-amylase from other protein contaminants (0.g.

albumin, haomoglobin, e tc . ) and from interforfng oxo-

a p Y l Q B O S 9 by its spociffa retention on OEa~ayloso.

~x-Amylaso is olutod off tho oolwm in tho aocond

stop by a mhtaro containing 0.1 Y HaCl in acetic a- cid 0.01 Y and CaC12 0.015 Y. The ~ecovorirs of glu-

coaqlaao md ~x-amylam war0 74% md 65% rospoctire-

1501

Copyright 0 1981 by Marcel Dekker, Inc.

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1502 SCHELL ET AL.

ly. The retontion capacity of the stationary phaao $6

0.5 mg of d-emylase/ml bed volumo.

INTRODUCTION

The ac t iv i ty o f cx-amylase ( oc-1,4-gluc*13 4-

glucanohydrolase, $C 3.2.1.1) and glucoamylare (amy-

loglucosidase, a-1,4-glucanglucohydr61*e., EC 3.2.1.

3) which act on the samo substrates (starch, amyloro,

amylopoctine, glycogen) at a-1,4-gluc.s linkrgoa wf-

thin tho polysaccharido chains, are dotermined by .a-

mo methods (reductometric, amyloclaetic and chromogo-

nic) and solnotines occw together in some raourco~ . These enzymes interfere one another, as thoy hydrol l -

ze Identical substrater. Although, tho bwo onzpoa

a m different; in terms of t h d r reaction uechaniaau:

a-amylaso ac ts on polysacchuido chaim and ro lou~ea

malt000 and doxtrina as f i n a l products (ondo- action).

wheroa.11 glucoamylase s p l i t s the c%-1,4=glucm l h k a -

ge froa the nonroducing end of tho Chain, and role-

asoo glucoso as f i n a l product (exo- action).

1-4

!J?ho sepmation of the t w o associated anzymoa

from c a i n o sora was successfully performed by 0'

Donnoll and IlcGeonoy3 using ge l f i l t r a t i o n chromato- eaphy on Sephadex G-200.

I n this paper, the purification and separation

oc-amylase f r o a glucoamylase w i n g CGamylose arr of

a stationary phase is described. CL-Amyloae waer shown

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cc -AMYLASE PURIFICATION 1503

t o selectively retain oc-amylase , whereas p-amylase

(exo-amylase, By: 3.2.1.2) is excluded from the co-

l ~ n r n ~ ’ ~ . I n the same time, Weber and coworkers have

reported an eff ic ient method f o r the purification of

&-amylase and Its separation from @-amylase by

af f in i ty chromatography on cross-linked starch6. CL-

Amylose can a l a o be used as a specific substrate for

simultaneous qualit a t ive5 and pan tit ative7 de t ermi-

nation of both oc- and /-amylases, as well as of

the associated a- and glucoamylase . 8

Varioue methods for the purification o f @-a-

mylase by af f in i ty chromatography have been repor-

ted using glycogen-AEI-Sepharo se-4Bg, wheat albumin-

Sepharose-2B1* ’ cross-linked dextr ins”, cyclohepta-

amylose bound t o epoqy-Sepha.rose-6B12, as stationary

phasers.

In the suggested conditions propoaed in this

gaper, it is possible t o obtain glucoamylase free from the contaminant oc--agorla8e, that may be of in-

t e m s t f o r some analytical and preparative procedu-

res and f o r some theoretical points of view, too.

MATERIAL AND METHODS

CL-Amylaee-X45 waa prepared by treating 100 g

of amyloee with 45 g of epIoblorohydE5ne In alkaline

medium 13,14. Before m e , the oross-linked gel w a e

incubated overnight w i t h 20 mg glucoaaiylase t o re-

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SCHELL ET AL. 1504

move the terminal glucose residues, that m i g h t l e d

t o some undesirable effects upon the aepa.vation pro-

~ e d u r e ~ ~ ~ .

The a f f in i ty chromatography experiments wema

performed in 2 x 16 cm columns, w i t h a bed

50 m l , pre-equilibrated w i t h 15 mib CaC12. The follo-

wing samples were passed through the oolum, la va-

rious experiments : 10 mg bovine serum albumin, a

mixture of 10 mg glucoamylaee and 10 nrg bovine s e m

albumh and a laixture of 10 rng a-amylase, 1.0 mg al-

bumln and 10 mg glucoamylase, a l l solved in 15 Idl CaO12 solution. Two pll of each sample were loaded in

each separation pun. The f low rate of 40 ml/h w a a

controlled by a pe r i s t a l t i c pump. Five m l fractAana

were collected. The column was f i r a t washed w W h

15 sbl CaC12. After the appearance of first protiein

peak, the column was further washed with 40 m l of

15 mM CaClz and then with a mixture containing

0.1 l ac1 i n acetic acid 0.01 A¶ and CaC12 0.015 M

solutions.

volume of

The eluted peaka w i t h aslylolytSc acrtlviby

rere located by a modified iodomecrlcal technique.

d-Amylaae ac t iv i ty was determined by Inetabating

2.5 m l 0.- atarch solution r f t h 1 m l ample for 5 ain, and then 0.5 m l of 1% I.+ reagent WQB

added, The optical densitqy was mad a% 600 nm. lbo-

sps unit wae expressed ae 1 rag o t u a h hyUolya.q/

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a-AMYLASE PURIFICATION 1505

ain/ml. Gluooamylam act ivi ty was determined by

the reductometric technique uaing 3,5-dinitrooali-

cylate reagent 15d6 and one unit was expreseed aa 1 p o l e glucooe releaeed/ntin/dl.

280 m.

The protein ooncent ra t iw were estimated a t

The ct-amylase and glucoamylase eluted rere confirmed by the method recently reported In this

journal8, baaed on the slmultaneoure action of the

colleoted samples on amylase (a substsate for both

enzyme) and on Cbamylose ( a substrate fom oc-amp

lase only).

Amylose (Koch-Light) and c r p t a l l i n e d-w-

lase ( K a r l Roth - Karloruhe) and glucoamylase

(Yerak-Daraetadt) were wed.

E?lmums 'Phe elution pattern8 obtadped when albumin aad

associated albumin an8 gluaoawlaoe, Eeegectively,

were passed thaaugh the CL-tw@ose column, show that

glucoamylase I s excluded fzom the column, being elu- t;sd tn the void volume (V,) aleng with albumin(fig.1).

'Ph6 V, fraction contained more than 80% of the

glucsaaylaee ac t iv i ty o i i g b a l l y fntiodutled in the

column. Oluaoamylaoe was ercluded in the above men-

tioned oonditiona - fmt confbmed

in the f i r o t peak of Sop from the total. numbex ef

the recovem

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1506 SCHELL ET AL.

)nm n” n c

0

r

; C LI

I . 5

I .o

0.5

Fraction number

Fig. 1. The elution pattern of aamples containing

bovine sexum albumin (fig. 1 a) and a nix- t u r e of albumin and glucoamylase (fig. 1 b) i

OD28O nm (-e), glucoamylase activity

A 1. ( A-.-.-

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a -AMYLASE PURIFICATION 1507

glucoamylase units introduced In the c o l m . On the

other hand, t h i s supposition was supported by the

fact that the elution with acid medium and more con-

centEated elution eystema did not show that gluco-

anylam was adeorbed by the column (no additional

glucoamylase peak was observed). Albumin and gluco-

aeylase were almost completely recovered fmom the

column, as fa.r as protein was concerned.

When a mixture of d-amylase and albumin was

passed Chrough CLamylose, &-amylase was cornple-

te ly retained by the column, whereas albumin was e-

luted In the vo id volume (Vo). &-Amylase was de-

sorbed from the column only when a solution contal-

ning 0.1 lil RaC1, 0.01 YI acetic acid and 0.015 P

CaC12 was used (flg.2).

When a three component mixture of a l b u m i n ,

&-amylase and glucoamylase was gassed through the

column, glucoamylase was not retained, and it was e-

luted in the void volume (VJ, along with albumin,

whereas a-arpylase oame wt only when the elution

solution WBP changed (fig. 3). The anslyais of the eluted fractions sharrs that

peak I contains glucoamylase exclusively (without

a-amylam), whereas peak II conmrists of b-amylaoe

( r l b h no interference of glucoaaylase).

The recoverlee were 74% f o r glucoamylase and

6596 for a-amylaee, respeatively. The values repor-

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1508 SCHELL ET AL.

E280nrn

2'oi 1

1.0-

I 2 0 0

c1 6

N

U 0

I

0 X 0 0

X

I 0

c3

-

Um c;' - 0 I

0.8

0.6

0.4

0.2

0 K) 20 50 Fraction number

Fig. 2. The elution pattern of samples containing

bovine serum albumin and a-amylase;

OD280 nm (-01, a-amylase activitcy

0 ) . ( 0 -.-.-.

bed for the separations in similar systems ranged

betmeen 334 and 6W '*'**''. Tkachuk has obtainea a

separation yield of 85% when he separated OC-myla-

me by using a oomplex stationary phaoe coneiratlng of Sapharose derivatives 9 .

Proteins were almost comple,tely recovered fro8

the column.

The retaining (binding) capacity of the Qt

anylosre column waa 27 m g oc-amylase (0.5 gel).

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a -AMYLASE PURIFICATION 1509

E

6 N

w 2.0

1 .o

0

2 tr! 9

3

0 N

0 V - 8

10 20 30 So 60 I 70 Fmction number

Fig . 3. The elution pattern of samples containing

a mixture of bovine serum albumia, gluco-

amylase and a-amylase; ODzm n,,,(-O);

glucoamylase activity ( b- - -A 3 ;

a-aqylase activity (o--.---o I*

When higher a a m r i ~ s of u-amylase wepa loaded,

the entyPe appeared in the void volwae, as well, so

thali a redromatography of the peak I should be

I! e quiz ed.

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1510 SCHELL ET AL.

proteins, by a f f in i ty chromatography on CZ-amglose

column.

The described procedure allows also the purifi-

cation o f glucoamylase, f r e e f rom a-amylase, in a

s i m i l a r way i n which Weber et a16 have reported the

preparation of p-anylase f ree o f cc-aqlase, by

af f in i ty chromatography on mixed cross-liaked stareh

and Sephadex (2-10 column.

As i t is well known the pxinciple of stationary

phases preparation in a f f in i ty chromatography is 8

STATIOXARY PHASE = SUFPORT + LIGMOD

where the ligand is the group giving 8pecificit;y t o

the stationary phase.

The u8e of CGaBlylose as stationary phase i s a

pare'icular case of a f f in i ty chromatogzqphy, where the

insoluble support i t s e l f I s a specific s t a t l o n a y

phase, due t o i t 6 characterist ic structure crompoel-

tion. Amylose, although cross-linked, contains a cop-

siderable amount o f glucose residues linked by oc-1,4-

glacan bonds; they a m sti l l recognized by c%-amyla60

i n case of a proper three dimensional strucBure of

CL-amylose (scheme I).

The separation pfocedure depends on the degree

of cross-linking of amylose. When the cross-lhking

is t o o hi&, the thme dimensional 8 t r U C t a W J I s t o o

ti&$, SO thati a-amylase is par t ia l ly allowed t o

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Sche

me I.

The

me

chan

iama

act

ion

of

or-a

myl

ase

and

gluc

oam

ylas

e upon cross-linked

amylose;

&-a

myl

ase

acti

on (

- );

glW

O~m

yla8

0 act

ion

( - -

+ )

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15x2 S C H E U ET AL.

reach the sequences o f glucose residues linked by

a-1,4-glucan bonds. In this case a lower retaining

capacity resu l t s , as we have found when amylose X d O

was used f o r the enzyme separation. On the other

hand, when a loose t h e e dimensional structure were

used (amylose X-20, or anylose 13-7.5), higheE re ta i -

ning capacities w e m obtained, but the ct-amylase

retained i n the columns induced a significant hydro-

lys i s of the stationary phase, result ing fn a decreaee

of the bed volum, and the release of reducing pro-

ducts. Under the conditions described above, we have

jbtained the highest retaining capacity, w i t h no appa-

rent variation of hydrolysis of bed volume, by using

amyloae X4$.

The solution used foz &-amylase elution con- tiabed coroponents (la+, Ca2*) that do not impair the

enzyme act ivi ty , but they even show an activation

e f fec t upon the a-amylase and a s tabi l izat ion (Ca2+)

of the protein macromolecule assambly.

We may conclude that the procedure descEibbd

above is a rapid one, and it allows an easy way t o

purify d-amylase and separate i t from the aseociated

exo-amylases (glucoamylase and p-amylase).

ACKHOWIZDGbnllIBLBTS

The authors gratefully thank Mrs. Blena Iaporb

f o r her excellent technical assistance.

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a -AMYLASE PURIFICATION 1513

1.

2.

3.

4.

5.

7.

8.

J.A. Thoma, J.E. Spradlin, S. Dygert, in The

Bnzymee (Boyer, R.D., ed.) 3rd edn., vol.5,

Academic Press, Hew Yorlr, 1971, p. 115.

M.D. O'Donnell, K.P. YcGeenej, 0omp.Biochem.

PhySiOl. L B , 269 (1975).

J.J. Marshall, Anal.Biochem. z, 466 (1970). 111.1. Mateeacu, H.D. Schell, F. MihUlescu,

I. Cornoiu, ~ioch~mfe 48, a75 (1976).

Y. Weber, M.J. BoglieQbI, F. Pemheion,

Biochimle 58, 1299 (1976).

Y.A. Matreeaota, Bimhimie 60, 535 (1978).

M.A. Hateemu, I. Cornoiu, H.D. &hell, Anal. Iabders 13, 1567 (1980).

9. B. Tphachuk, FEBS Letter8 z, 66 (1975).

11. H. Yeler, P. Henkel, Freaeniua 2. Anal. Chem.

m, 187 (1978). 12, Y.P. Silvanovich, BcD. Hill, bal.Bioahem. a,

430 (1976).

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1514 SCHELL ET AL.

13. M . Serban, H.D. Schell, M.A. Mateescu, Bev.Eoun.

Biochim. 3, 187 (1975).

14. Y. Serban, M.A. Mateescu, H.D. Schell, Romanian

patent 61524/1976.

15. G. Boelting, P. Bernfeld, Helv,Chim.Aota 2, 286

(1948)

16. A, Dahlqulst, Biochem,J. 80, 547 (1961).

Received June 30, 1981 Accepted September 9, 1981

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