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6 Immunochemical Approach to Questions Related to αDownloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 28, 2018 | https://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch006

and ß-Amylases in Barley and Wheat J. DAUSSANT C.N.R.S., Physiologie des Organes Végétaux, 92190 Meudon, France

I· I n t r o d u c t i o n I n food and beverage processing o( - and £ - amylases have an important f u n c t i o n i n two main areas : beer and bread making. I n these i n d u s t r i e s , there i s s t i l l a need f o r d e f i n i n g the source and the q u a l i t y o f barley, malt, and wheat. Among the biochemical parameters used f o r c h a r a c t e r i z i n g these products, Λ - n d (3 - amylases are of great i n t e r e s t : the a c t i v i t i e s are used f o r d e f i n i n g the seed q u a l i t y and a b e t t e r understanding o f the isoenzymic contents seems promising f o r cha r a c t e r i z i n g seeds. However, Çt- amylase a c t i v i t i e s and Q - amyl a s e zymograms are d i f f i c u l t t o d e f i n e when OK - amylase i s present. The immunochemical c h a r a c t e r i z a t i o n o f oK - and Q- amylases which provided new i n f o r m a t i o n on b a r l e y and wheat amylases i n p h y s i o l o g i c a l s t u d i e s may a l s o be u s e f u l i n the above mentioned characterizations. This a r t i c l e w i l l present r e c e n t l y developed techniques : 1) f o r studying q u a l i t a t i v e l y and q u a n t i t a t i v e l y ^ - amylases i n presence o f θ(- amylases, and 2) f o r i d e n t i f y i n g the o r i g i n o f C ( - amylase sometimes pre sent i n small amounts i n mature and apparently ungerminated wheat seeds. a

I I . Q u a n t i t a t i v e and Q u a l i t a t i v e Techniques f o r @- Amylase Inves t i g a t i o n s i n the Presence o f o Ç - Amylase A. The problem. The c h a r a c t e r i z a t i o n o f amylases i s p a r t i c u l a r l y important f o r e v a l u a t i n g malt used i n brewing. The a c t i v i t i e s o f both C l - and amylases and t h e i r r a t i o c h a r a c t e r i z e malt ; the data are o f major importance f o r the brewing process. E l e c t r o p h o r e t i c a n a l y s i s o f CK- and @- amylase c o n s t i t u e n t s may prov i d e means o f g e t t i n g a deeper i n s i g h t i n t o the q u a l i t y and the o r i g i n o f the malt. However, the measurement and the d e t e c t i o n of these a c t i v i t i e s remain approximate. I n f a c t , OC- amylase may be c h a r a c t e r i z e d i n the presence o f amylase by using s t a r c h

80 Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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preincubated with β - amylase* T h i s procedure i s used f o r quan t i t a t i v e as w e l l as f o r q u a l i t a t i v e techniques such as e l e c t r o p h o r e s i s . Nevertheless, f o r q u a n t i t a t i n g |3- amylase a c t i v i t y by measurement of reducing sugars that appear upon i n c u b a t i o n of s t a r c h with the enzyme, the e v a l u a t i o n i s approximate when 0C~ amylase i s present. In f a c t , θζ - amylase a l s o produces reducing sugars by i n c u b a t i o n w i t h s t a r c h , and there i s a s y n e r g i s t i c e f f e c t between oC~ and β - amylases. On the other hand, i n e l e c t r o p h o r e t i c a n a l y s i s of β - amylase c o n s t i t u e n t s , some of them may be hidden by OC- amylase components. An i n h i b i t i o n o f OC- amylase and not of (3 - amylase could then be used : 0L- amylase r e q u i r e s Ca f o r i t s a c t i v i t y which i s not the case f o r β - amylase. Thus, the treatment with p o l y phosphates, c i t r a t e or EDTA which i n h i b i t s the OC- amylase a c t i v i t y ( l ) could solve the problem although amylases which may not r e q u i r e C a have been reported (2). We s h a l l d e s c r i b e here techniques i n v o l v i n g the s e l e c t i v e absorption o f 0 ( - amylase cons t i t u e n t s by using an immune serum s p e c i f i c f o r θ{- amylase ; these techniques permit q u a l i t a t i v e as w e l l as q u a n t i t a t i v e a n a l y s i s of (i - amylases i n presence of 0( - amylases. For q u a n t i t a t i o n of Λ - amylase, we have developed a d i f f u s i o n technique s i m i l a r t o a d i f f u s i o n technique developed f o r OC- amylase determination (3)· Ρ - amylase and, to a smaller extent, OC - amylase are damaged by the r o a s t i n g process of malting ; both enzymes being a l t e r e d in their activities. The r e s u l t s of previous immunochemical s t u d i e s on b a r l e y and malt β- amylases i n d i c a t e d that malting, and i n p a r t i c u l a r r o a s t i n g , do not a f f e c t much the a n t i g e n i c s t r u c t u re of the enzyme. An immunochemical measurement of the enzymatic p r o t e i n contents i n b a r l e y and malt e x t r a c t s could thus be c a r r i e d out (4)· On the b a s i s of immunochemical c h a r a c t e r i z a t i o n of b a r l e y β - amylase, techniques f o r q u a n t i t a t i n g the enzyme are mentioned. Data concerning ^ - amylase a c t i v i t y , even i n presen ce o f θ(- amylase, and {3 - amylase p r o t e i n content provide a means of o b t a i n i n g β - amylase s p e c i f i c a c t i v i t y . T h i s parameter could than serve f o r f u r t h e r c h a r a c t e r i z i n g e f f e c t s of r o a s t i n g i n the malting process. + +

B. Combined immunoabsorption and QÇ - amylase determination i n the same g e l medium (5> 6 ) . amylase s o l u t i o n s were poured i n t o w e l l s cut i n an agarose g e l c o n t a i n i n g 1,5 % s t a r c h p r e i n c u bated with ft- amylase. The enzymes were allowed to d i f f u s e f o r 2.4 h at 20°C and the g e l s were s t a i n e d with an i o d i n e s o l u t i o n . The Q( - amylase a c t i v i t y was r e f l e c t e d by white spots occuring on a red background. There i s a l i n e a r r e l a t i o n s h i p between the diameter of the r e a c t i o n spots and the logarithm o f the OC- amyl a s e c o n c e n t r a t i o n (3)· The immune serum a n t i 0C- amylase of germinated seeds was the one d e s c r i b e d i n s e c t i o n I I I , B. OC- amylase p u r i f i e d by an a f f i n i t y technique (7) from e x t r a c t of germinated barley seeds was used f o r immunization.

Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


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For immunoabsorption the w e l l s were f i r s t f i l l e d with the immune serum and when the serum was sucked i n by the g e l they were r e f i l l e d with the malt e x t r a c t . During d i f f u s i o n the c< - amylase meets the a n t i b o d i e s which are a l l around the w e l l s and p r e c i p i t a te, o r are i n h i b i t e d by an excess o f a n t i b o d i e s . F i g . l shows the r e s u l t s o f the immunoabsorption o f - amylase e x t r a c t e d from 7 days germinated b a r l e y seeds. D i f f e r e n t d i l u t i o n s o f the immune serum were t e s t e d f o r the immunoabsorption o f o( - amylase from the malt e x t r a c t . D i l u t i o n 3 o f the immune serum absorbed p r a c t i c a l l y a l l o f the - amylase i n the malt e x t r a c t . This d i l u t i o n o f the immune serum was then used f o r e l i m i n a t i n g amylase from the malt e x t r a c t , i n order t o measure i t s |3 - amyl a s e a c t i v i t y . I t i s noteworthy that, i n order t o e l i m i n a t e the small - amylase-like a c t i v i t y present i n the immune serum, the l a t t e r was heated at 50°C f o r 30 min before use. C. D i f f u s i o n technique f o r fi- amylase determination i n agarose g e l i n the absence of QÇ - amylase. This technique i s s i m i l a r to the one developed by B r i g g s ( 3 ) f o r - amylase determination ; s t a r c h i s used as substrate i n s t e a d of l i m i t d e x t r i n s (Fig.2 upper p a r t ) . D i f f e r e n t d i l u t i o n s o f barley e x t r a c t were poured i n t o

F i g . l - Immunoabsorption o f Q4- amylase from malt e x t r a c t and eval u a t i o n o f the remaining a c t i v i t y a f t e r absorption. This experiment shows that d i l u t i o n 3 o f the a n t i 0^ - amylase immune serum absorbs p r a c t i c a l l y a l l o(,- amylase present i n the malt e x t r a c t . Upper and middle p a r t : w e l l s were c u t i n a 1.2 % agarose g e l prepared i n 0.2 M acetate b u f f e r , pH 5·7> c o n t a i n i n g 1.5 % s t a r c h preincubated with amylase. Upper p a r t : w e l l s were f i l l e d with d i f f e r e n t concentrations o f o(- amylase ( d i l u t i o n s e r i e s ) . I n the i n i t i a l malt e x t r a c t , the oC- amylase concentration was a r b i t r a r i l y designated as 100. Middle p a r t : For OC- amylase immunoabsorption, d i f f e r e n t d i l u t i o n s of the immune serum were f i r s t depos i t e d i n the w e l l s . A f t e r these s o l u t i o n s were sucked i n by the gel the i n i t i a l malt e x t r a c t (cone. 100 o f the enzyme) was deposited i n the w e l l s . A f t e r 24 hr d i f f u s i o n a t 20°C, the g e l was s t a i n e d with i o d i n e . Note that the r e a c t i o n spot corresponding t o the immunoabsorption o f the malt e x t r a c t with d i l u t i o n 3 o f the immune serum (lS/3) i s much smaller than the r e a c t i o n spot corresponding to concentration 0.2 o f the malt Ck- amylase. Lower p a r t o f the f i g u r e : R e l a t i o n s h i p s between diameter o f r e a c t i o n spot and logarithm o f amylase concentration. The p o i n t s represent the mean values o f two measurements made on each o f three r e a c t i o n spots corresponding t o one experiment. Absorption with d i l u t i o n s 27 and 9 o f the immune serum r e s u l t e d i n the absorption o f only p a r t o f the i n i t i a l a c t i v i t y ( r e s p e c t i v e l y about 40 % and 75 °/°) · D i l u t i o n 3 o f the immune serum absorbs more than 99*8 % o f the i n i t i a l activity.




DAUSSANT

Figure 1



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3 mm diameter w e l l s c u t i n agarose g e l , c o n t a i n i n g 0& % s t a r c h (see legend o f F i g . 2 f o r d e t a i l s ) . The enzyme was allowed t o d i f fuse i n t o the g e l f o r 24 h. at 20°C. The g e l was then s t a i n e d with i o d i n e s o l u t i o n . The amylase a c t i v i t y i n the barley ex t r a c t s o l u t i o n s was r e f l e c t e d by pink spots o f d i f f e r e n t s i z e s , o c c u r r i n g on a blue background. The diameter o f the r e a c t i o n spots and the logarithm o f the corresponding f*> - amylase concen t r a t i o n s c o r r e l a t e i n a l i n e a r r e l a t i o n s h i p ( F i g . 2 , lower p a r t ) . p> - amylase may e x i s t under d i f f e r e n t forms of aggregation which are d i s r u p t e d by reducing agents (see (4) f o r review). I n order t o convert these aggregate forms o f \*> - amylase i n t o t h e i r b a s i c u n i t s , 0.03 % mercapto ethanol was used i n the s o l u t i o n s . When X - amylase i s present, as i s the case i n the malt e x t r a c t s , the r e a c t i o n spots are white and there i s no p o s s i b i l i t y o f measuring - amylase a c t i v i t y without having s p e c i f i c a l l y removed o ( - amy l a s e from the s o l u t i o n ( F i g . 2 , upper p a r t , M a l t ) . D. Combined immuno-absorption o f - amylase, and ft - amy l a s e determination i n the same g e l medium.The w e l l s cut" i n the gel c o n t a i n i n g s t a r c h were f i r s t f i l l e d with d i l u t i o n 3 o f the heated antiCX, - amylase immune serum. When the s o l u t i o n was sucked i n by the g e l , the d i f f e r e n t d i l u t i o n s o f barley e x t r a c t and d i l u t i o n 2 o f the malt e x t r a c t were poured i n t o the w e l l s (Fig.2 mid dle p a r t ) . The a c t i v i t y o f the enzyme was r e f l e c t e d f o r the ex-

F i g . 2 - Π>- amylase a c t i v i t y estimation using a d i f f u s i o n t e c h n i que which a l s o i n v o l v e s the s p e c i f i c e l i m i n a t i o n o f *X - amylase present i n malt e x t r a c t s by immunoabsorption. Upper and middle p a r t s : wells were c u t i n a 1.2 % agarose g e l prepared i n 0.1 M phosphate b u f f e r pH 6 c o n t a i n i n g 0.8 % s t a r c h . Upper part : The w e l l s were f i l l e d with d i f f e r e n t d i l u t i o n s o f the barley e x t r a c t ( P>- amylase concentration i n the i n i t i a l e x t r a c t was a r b i t r a r i l y designated as 100), o r with the malt e x t r a c t d i l u t e d twice. Mid dle p a r t : The w e l l s were f i r s t f i l l e d with d i l u t i o n 3 o f the ant i C X - amylase immune serum. When the s o l u t i o n s were sucked i n by the g e l , the wells were r e f i l l e d with the d i f f e r e n t d i l u t i o n s o f the b a r l e y e x t r a c t * o r with the malt e x t r a c t d i l u t e d twice. A f t e r 24 hr d i f f u s i o n a t 20°C, the g e l was s t a i n e d with i o d i n e . Note that the immune serum has no e f f e c t on the diameter o f the reac t i o n spots corresponding t o the d i l u t i o n s o f the b a r l e y e x t r a c t . With the malt e x t r a c t , the absorption allows one t o get a reac t i o n spot corresponding t o fi - amylase and not t o (A- amylase. Lower p a r t : R e l a t i o n s h i p between diameter o f r e a c t i o n spot and logarithm o f /3- amylase concentration i n barley e x t r a c t s . Each p o i n t represents the mean value o f two measurements made on each of the three r e a c t i o n spots corresponding t o one experiment. fi> amylase a c t i v i t y i n the twice d i l u t e d malt e x t r a c t corresponds t o 60 °/o o f the a c t i v i t y i n the b a r l e y e x t r a c t .




DAUSSANT

Figure 2


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t r a c t s of b a r l e y and malt by pink spots on a blue background. The presence of the immune serum d i d not modify the s i z e of the spot diameters corresponding to the d i l u t i o n s of the b a r l e y ex t r a c t . This i n d i c a t e s that the a c t i v i t y measurement of β» - amyla se was s t i l l p o s s i b l e under these c o n d i t i o n s , β - amylase a c t i v i t y i n the twice d i l u t e d malt e x t r a c t was found to be n e a r l y 60 % of the a c t i v i t y i n barley e x t r a c t . E. Combined immunoelectroabsorption of o< - amylase and e l e c t r o p h o r e s i s of p> - amylase i n the same g e l medium (8). The p r i n c i p l e of t h i s technique i s the same as the preceeding technique, except that enzymatic antigens are brought i n t o contact with the antibodies by means of e l e c t r o p h o r e s i s , i n s t e a d of d i f f u s i o n . The technique aims at p r o v i d i n g from a mixture of amylase^ electropherograms of β - amylase alone by e l i m i n a t i n g s p e c i f i c a l l y o{ - amylase c o n s t i t u e n t s : the immune serum a n t i amylase of germinated b a r l e y seeds was poured i n t o w e l l s cut i n the agarose g e l ; when i t was sucked i n by the g e l , the w e l l s were r e f i l l e d with the e x t r a c t s of e i t h e r b a r l e y or malt. During e l e c t r o p h o r e s i s , o 24> 25)· X - amylases o c c u r r i n g i n c e r e a l s during germination are known t o be synthesized i n the aleurone l a y e r , the enzymes found i n developing seeds, however, have been l o c a l i z e d i n the p e r i c a r p (13, 19, 20, 26, 27, 28). I t i s worth n o t i n g that - amylase found i n endosperm and aleurone during l a t e stages of development was a s s o c i a t e d with damaged areas of the k e r n e l (26). One may speculate t h a t amylase secreted i n the aleurone l a y e r may, i n v i v o s l i g h t l y d i f f u s e i n t o the starchy endosperm. Thus, i t could mo d i f y p a r t of the s t a r c h , whereas the (χ- amylase l o c a t e d i n the p e r i c a r p would have l e s s opportunity to attack i n v i v o the s t a r chy endosperm. The d i f f e r e n c e i n the l o c a l i z a t i o n of the two t y pes of X- amylases and the d i f f e r e n c e s i n the s p e c i f i c a c t i v i t y of these enzymes may e x p l a i n some cases concerning t r i t i c a l e , whe re both f a l l i n g number and a c t i v i t y l e v e l s were found to be high (29)· The disappearance of χ - amylase during maturation seems more probably due to a d e s t r u c t i o n of the enzyme than to an i n a c t i v a t i o n or to an i n s o l u b i l i z a t i o n (30, 31)· Further evidence was provided, i n d i c a t i n g that the g r e a t e s t p r o p o r t i o n of (X - amylase which appears during germination and i s i d e n t i c a l to c

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