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STUDIES ON BARLEY AND MALT AMYLASE (X) : Salt -solublezymogen-β-amylase in barley (2)(麦芽アミラーゼに関する研究 (X) : 大麦中の塩類可溶性チモーゲン-β-アミラーゼについて (2))

著者Author(s) Shinke, Ryu / Mugibayashi, Narataro

掲載誌・巻号・ページCitat ion 兵庫農科大學研究報告. 農芸化学編,6(2):41-45

刊行日Issue date 1964-12

資源タイプResource Type Departmental Bullet in Paper / 紀要論文

版区分Resource Version publisher

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DOI

JaLCDOI 10.24546/81008239

URL http://www.lib.kobe-u.ac.jp/handle_kernel/81008239

PDF issue: 2021-07-02

STUDIES ON BARLEY AND MALT AMYLASE eX)

Salt-soluble zymogen-8-amy lase 1Tl barley (2)

Ryu SHINKE and Narataro M UGIBAYASH I

Introduction

Since Payen and Persoz recognized the presence of a substance to decompo e starch in malt extra·t in 1833,'J many investigators have been concerned wi th barley /l-a mylases2J-" J. It is now generally reo cognized that there are two types of /l-amylases in ungerminated barley; free·/l·amylase that is water or ~alt soluble and active, and zymogen·/l·amylase that is water or salt insoluble but brought into solution a an nctive form with papain. thioglycollit: acid and other disu lfide bond reducing reagents. In the pre· ceding 1:l.1 per" J, we have al ready reported the fact that the /l.amylase activity in the water or salt ex-tract from g round barley is increased by papain or disul fide bond reducing reagents. That increment in the enzyme activity was thought to be attributable to water or salt soluble zymogen-/l·amylase which was different from the well·known zymogen-/l.amylase.

In this paper a report is made on reaffirmation and separation of the salt soluble zymogen-/l.amylase if m ground barley. At the same time. from the results in extracting /l.amylases from ground barley with variou reagents the possibi li ty is suggested that there may be another type of zymogen-/l-amylase that is activated but not extracted even with papain or disulfide bond reducing reagents. In addition to such an insoluble but act ive type of /l.amylase, some other possible types of the enzyme are discussed from the solubility of enzyme proteins.

Materials and Methods

( I ) E t-tractioJl oj' f3 .alilylases f rom g round barley.

Barley used in the experments was Kallto-Golden-meloll ( two-row bar ley) . The grains were ground in a WI LEY mill (0.5 mm eyes of the sieve). Each 1 g of ground barley was taken to a 50 ml centr ifugal tube, to which 20 ml of deionized water was added and well mixed. After incubation with a few drops of toluene as disinfectant a t 30°C for 20 hrs, it was centrifuged a t 9,000 rpm for 5 min. T he supernatant was moved to a 100 ml measuring flask . The residue was again extracted with 20 ml of deionized water

41

nnd centrifuged. By repeating this procedure the combined supernatant was allowed to fill up to 100 ml. The same procedure was carried out with 5% potassium sulfate. 0.005% thioglycol1ic acid (pH 5.6) and 0.2% papain solutions. T he enzyme activity of these extracts (100 m l) was determined. On the other hand, the remaining enzyme activities in the residues were also determined by suspending them in a contant amount of deionized water and by add-ing them to the reaction mixture as the enzyme solutions.

(2 ) Determinatio/l of /3-amy lase activ ity .

The amylase activities in the extracts and residues were determined by WI LLSTAETTER-SCHUDEL method and presented as maltose mg (as dry matter) which was produced by 1 g of ground barley (as dry matter) at 30°C for 60 min."J

(3) Detenllination of zYJ7logen-/l ·amy lase in ex· tracts.

To the 5% potassium sulfate extract an equa l volume of 0.2% papain, 0.005% thioglycollic acid, O.01M sodi um bisulfite and 2% peptone solutions was respectively added. Each mixture was kept at room temperature for 60 min, and enzyme assayed 111 the same way as described in (2) .

(4) Isolation of salt -soluble zymogell -/l .amy lasc by salting out with a.mmonium SitUate.

Ground ba rley was extracted with about five times volume of 5% potassium sulfate solution at 30°C for 20 Ius a nd centrifuged at 9,000 rpm for 5 min. The supernatant was allowed to be 0.3 saturation with ammonium sulfate. The precipitate fo rmed was collected by centrifugation a t 9.000 rpm for 5 min and washed with 5% potassiu m sultate solution con-taining ammonium sulfate up to 0.3 saturation unti l no free tJ-amylase activity was recognized in the solution. Then, the precipitate was dialyzed with toluene as disinfectant in the cellophane bag against running water for one day and against deionized water for two days. After the dialyzed precipitate was washed with deionized water. it was loyphilized.

Sci. Rep. Hyogo Univ. Agric. Vol. 6 No.2

/Ground Barleyl

extraction with cold 5:' K2SO 4

lResidue / / Solution

0.:5 eat. with (NH4)2 S04

centrifuge at 9,000 rpm tor 5 min

l ppt. I L PH trate I dialyeie against 0.6 eat. with running water and (NH4)2 S04 deionized water

centrifuge at J I 5,000 rpm tor L ppt. I l PH trate 11 5 min

1 I ppt. I l :Piltrate I

washing with deionized water

l :P11 trate II / L ppt. I redissolution wi th 5:' K2SO 4

I I ppt. r l!'iltrate III I

treatment with 0.2~ papain

I ppt. liltrate IV I Fig. 1 Isolation of salt·soluble zymogen-,B-amylase from ground barley.

The isolation scheme above is shown in Fig. 1.

(5) Further isolalioll of salt-solliMe ::ylJlogen-,B-amylase by paper electrophoresis.

The lyophil ized preparation was redissolved with 5% potassium sulfate solution, the residue of which was treated with 0.2% papain solution . To these two solutions and the dia lysis supernatant paper ele-ctrophoresis was applied. The buffer used was com-posed of M i lO KH,PO •. M / 20 Na.B.O,- 10H20 and Verona I buffer (1 :3:2) and pH, 8.8 (1' =0.107). To know the positions of proteins separated at 250V for 2 hrs, a part of paper was cut off and dyed with the

42

color reagent (bromphenol blue), and fractions on paper corresponding to dyed bands were stripped off and extracted with 5% potassium sulfate solution. Each extract was subjected to determination of the amylase activity in the same way_

Results and Discussion

(1) Activation of salt-soLub/e ::YlJlogell. ,B-amylase ill 5% potassiuIJI slIlfate ex/met.

As suggested in the preceding paper,") the ap-parent increase in the amylase activity of the water

XII, 1964 Series: Agricultural Chemistry

Table 1 Activation of salt-soluble zymogen-,a-amylase in 596 potassium suUate extract.

o. Activator

1 596 K ,SO. (as control)

2 0.296 Papain

3 296 Peptone

4 O.01M NaHS03

5 0.00596 HSCH,COOH

,a-Amylase activity

1043

1166

1111

1098

1180

Difference

123

68

55

137

Table 2 ,B-Amylase activity in residues after exhaustive extraction.

No. Reagent Enzyme I Each activity I Total activity Difference --- - ---

Ext.

I

3499 3585 -

Resi. 86 --- ---.

_I Ext. 3481 I 358R 3 Resi. 107 --2 596 K,SO. I

Ext. 4150 H ,COOH 4279 691

Resi. 129 3 0.00596 H C

-Ext. 4185

I 4335 750

Resi. 150 4 I 0.296 Papain

Table 3 1 ewly recognized possible types of ,B-a mylase in barley.

Type Solubility in water or salt

Soluble Active

Insoluble

I Soluble Inactive 1--

Insol uble

or salt extract from ground barley was reafiirmed by using the 5% potassium sulfate extract and 0.2% papain, 0.005% thioglycollic acid, O.OlM sodium bi-sulfite and 2% peptone solutions. As shown in Table I, the amylase activity in the 5% potassium sulfate extract was increased by each reagent and the effects of 0.2% papain and 0.005% thioglycollic acid solutions were higher than those of 2% peptone and O.OlM sodium bisulfite solutions. From these results it may be said that a salt-soluble inactive type of ,a-amylase is contained in the ungerminated barley and activated by disulfide bond reducing reagents, and that it might be called a salt-soluble zymogen-,a-amylase in comparison with the well-known insoluble zymogen-,a.amylase.

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Tentative name

Free ,a-amylase

Insoluble active ,a-amylase

Soluble zymogen-,a-amylase

Insoluble zymogen-,B-amylase

(2) ,a-Amylase activity in residues after exhaustive extraction with some reagents.

The residues after repeated and exhaustive extrac-tion with 5% potassium sulfate, 0.005% thioglycollic acid, 0.2% papain solutions and deionized water were suspended in a certain amount of deionized water and the enzyme assayed in the same way. The results obtained are shown in Table 2.

Table 2 shows that there is little difference in the total enzyme activities of the deionized water and the 5% potassium sulfate extracts but that thioglycol-lic acid and papain can extract a greater amount of the enzyme from groung barley. Furthermore, it should be noted that the enzyme activities in the

Sci. Rep. Hyogo Univ. Agric. Vol. 6 No. 2

residues extracted with thioglycollic acid and papain are considerably higher than those by deionized water and 5% potassium sulfate . This would mean that zymogen.,;.amylase which must be combined with such water or salt insoluble proteins as hordein and glutelin in barley is activated hy thioglycollic acid or papain but not extracted from them because of being still in a binding state. As we suggested in the preceding paper") that free /3-amylase is found in albumin and globulin fractions and that zymogen-/3-amylase is found in such water or salt insoluble protein fractions as hordein and glutelin, ROWSELl. and GOAD also pointed out recently that the latent /3-amylase of wheat is chemically bound to glutenin.") As to such an insoluble but active enzyme, it is of great interest for us to be able to fi nd the reports on the /3-amylase in kaffir corn by NOVELLIE") and on the protease in barley by NISHITAI'9) and KR1NGSTAO'O). From these results it may be thought that there would he four different types of /l-amylases in barley: namely. in addition to hitherto well-known two types of /l-amylases which are soluble and active (free /3-amylase) and insoluble and inactive (zymogen-/l-amylase) , there would be an insoluble but active type and an soluble but inactive one which is de-scribed in this paper as the s,'l.It-soluble zymogen-!3-amylase. These types of barley /l-amylases are summ-ari7.ed in Table 3.

(:3) Isolation afsall-soll/M" .O:Y/l/ogen-{3-Glllylase ji-oJll

grol/nd harley.

According to the scheme shown ill Fig. 1, isolation of the salt·soluble zymogen-/l-amylase was carried out. By salting out with ammonium sulfate the salt-soluble 7,ymogen-/3-amylase was found to be in the precipitate of 0.3 saturated solution. But this fraction ('ontained both the free /l-amylase and the salt-soluble zymogen-/l-amylase. To the solution, from which the precipitate was removed by centrifugation, am-monium sulfate was added up to 0.6 saturation . The precipitate formed in this case did not contain the zymogen-/l-amylase any more. Filtrates I, II, III and IV which are shown in Fig. 1 were put on filter paper (Toyo Filter Paper No. 50) for electro-phoresis. The results obtaincd are displayed in Fig. 2. Filtrates I and II which were composed of free Il-amylase showed the same behavior on paper. Filt-rate IV remained at the starting poin t. But Filtrate III was expectedly separated into three fractions, two of which were, as seen in F ig 3. at the same positions on paper as those of Filtrates I and II, Filtrate IV. Each fraction on paper was stripped off and extracted with 5% potassium sulfate solution for the enzyme assay. Only the middle fraction separated in the case of Filtrate III did show no

44

Filtrate r. II (H 20 )

XII, 1964 Series: Agricultural Chemistry

which was activated by disulfide bond reducing re-agents. (2) The ,8-amlase activites of the residues after ex-haustive extraction with deionized water, 5% potas-sium sulfate, 0.005% thioglycollic acid and 0.2% pa-pain solutions were determined and it turned out that the enzyme activities in the residues by 0.005% thioglycollic acid and 0.2% papain solutions were considerably higher than those by deionized water and 5% potassium sulfate solution. From these results it was thought that there would be another type of ,8-amylase in barley that was insoluble but active. (3) The salt-soluble zymogen-,8-amy!ase in the 596 potassium sulfate extract from ground barley was isolated by salting out with ammonium sulfate and by paper electrophoresis.

(Laboratory of Fermentation Technology, Received Aug. 31, 1964)

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