ドウ発酵中の物性変化の化学的機作に関する研究

九州大学学術情報リポジトリ

Kyushu University Institutional Repository

ドウ発酵中の物性変化の化学的機作に関する研究

椎葉, 究

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STUDIES ON CHEMICAL MECHANISM OF RHEOLOGICAL CHANGES

DURING DOUGH FERMENTATION

K.ivva..m-u_ SHIIBA

1996

CONTENTS

page PREFACE --- - ---- - --- --- ---- - --- 1

CHAPTER I CHEMICAL CHA GES DURI G DOUGH FERMENTATIO 1. Introduction

2. Materials and Methods 3. Results and Discussion

6 7 18 3-1. Effect of fermentation on dough rheology 18 3- 2. Changes in the component of flour dough 20

during fermentation

3-3. Changes in the constitution of glutenin 3 2 subunits during fermentation

4. Summary

CHAPTER IT PURIFICATION AND CHARACTERIZATIO OF WHEAT LIPOXYGE ASE

39

1. Introduction -- --- - --- 41 2. Materials and Methods --- 42 3. Results and Discussion --- --- --- --- 44

3-1. Purification of lipoxygenase isozymes 44 3-2. Characterization of purified lipoxygenase 52

isozymes

CHAPTER ill EFFECTS OF PURIFIED LIPOXYGE ASE ISOZYMES ON FLOUR DOUGH

1. Introduction --- 2. Materials and Methods --- 3. Results and Discussion --- 4. Summary

CHAPTER N EFFECT OF PURIFIED PE TOSA S 0 DOUGH RHEOLOGY 61 62 64 70

1. Introduction --- 71

2. Materials and Methods 3. Results and Discussion 72 81 3-1. Purification and characterization of two 81 pentosans from WSH 3-2. Rheological properties of flours with --- 90

purified pentosans 4. Summary --- 92

CHAPTER V CO CLUDING REMARKS --- 93

ABSCHLIESSENDE BEMERKUNGE --- 100

ACKNOWLEDGME TS --- 106

REFERE CES --- 107

PREFACE

The minimum ingredients for making dough are flour, yeast, and water. After mixing these ingredients, the resulting dough is usually fermented at 24 - 26 OC for 4.5 hours (at least 4 hours).

As the fermentation needs a long time, almost half of the total bread-making time, reducing fermentation time has been demanded for economical efficiency. In spite of this demand, however, few studies on the mechanism of chemical changes during fermentation have been done. In order to clarify the rheological changes of dough during dough fermentation, physical dough-testing instru­

ments such as mixogram, farinogram, and exotensigram have been usually used to evaluate the rheological properties of fermented dough (Landis and Freilich 1934, Freilich and Frey 193S, Ikezoe and Tipples 1968, Barber et al. 1980, Preston and Kilborn 1982, Kilborn and Preston 1982, Casutt et al. 1984). They demonstrated that as fermentation time increased, the strength of the fer­

mented dough decreased. Thinking about a reason why the dough strength decreases during fermentation, it has been understood that organic acids produced by yeast, or endogeneous wheat protease causes a rheological change of dough (Miller and Johnson 1948, Hoseney and Brown 1983). However, it was demonstrated that organic acids and protease produced during fermentation were not enough to change the rheological properties of dough (Shiiba et al. 1990). From these results, it was suggested that factors other than organic acids and/or proteinase might play a major

role in modifying the rheological properties of the dough.

It was possible that some chemical changes caused by fermen­

tation might affect the gluten structure to decrease in mixing tolerance. In the components constituting gluten, glutenin espe­

cially contributes to maintain the gluten structure. In the present study, therefore, interest was focused on the changing conformation of glutenin during fermentation.

The objective of the present study was to examine the mechanism of chemical changes causing rheological change during fermentation and to reveal factors affecting such changes.

In CHAPTER I ^, studying the chemical changes during fermentation, first attention was on the changes in the composi­

tion of gluten. Gluten was fractionated into five components ac­

cording to the modified Osborn's method (Bietz and Wall 1975) and then these components were fractionated by SDS-PAGE and RP-HPLC.

These analyses revealed that chemical changes during dough fer­

mentation did not depend on the changes in the molecular weight of polypeptides constituting gluten, but depended on changing of the conformational structure of glutenin which resulted to decrease the surface hydrophobicity of glutenin.

Thus, three subunits constituting glutenin from nonfermented or fermented dough were fractionated and compared. The analysis of their compositions showed that the aggregative subunit, one of the glutenin subunits, changed the solubility against 70%

ethanol. However, because molecular weight of aggregative subunit did not change during dough fermentation, it was hyphothesized

that the conformational structure in aggregative subunts might be decomposed by the effect of fermentation on lipids and/or carbohydrates, therefore the activated lipoxygenase and pentosans constituting aggregative subunit were thought to be the real in­

ducers causing rheological changes during fermentation.

To study the above hypothesis, in CHAPTERS (IT - N) ^en­

dogenous wheat lipoxygenase and pentosans were purified and characterized. Futhermore, using these purified materials, ef­

fects on the glutenin constituents and rheological properties were studied.

In CHAPTER IT ^, to reveal the role of wheat lipoxygenase during dough fermentation, isozymes of wheat lipoxygenase were purified from wheat germ and characterized.

In CHAPTER ill , using the purified lipoxygenase isozymes, ef­

fects on the constitution of glutenin subunits was investigated.

Lipoxygenase (EC 1.13.11.12), which catalyzes the oxidation of polyunsaturated fatty acids containing cis,cis-1,4-pentadiene, was first purified and characterized by Wallace and Wheeler

(1979) and Nicolas et al. (1982). However, their reports gave limited information about the enzymatic properties of these isozymes and nothing about effects of the purified lipoxygenase isozymes on wheat flour or flour dough.

In this study, lipoxygenase isozyrnes were not only purified and characterized enzymatically, but also using these purified lipoxygenase isozymes effects of each isozymes on the flour dough were investigated.

Daniels et al. (1970) suggested that soy lipoxygenase causes oxidation of the sulfhydryl group (SH) and consequent structural changes in the dough protein. Also, Frazier et al. (1977) ^inves­

tigated the effects of the soy lipoxygenase on the mechanical development of wheat flour doughs. They suggested that the added soy lipoxygenase caused an increase in mixing tolerance. Using a mixograph, Hoseney et al. (1980) also investigated the mechanism by which soy lipoxygenase increases mixing tolerance. To show the role of lipoxygenase during breadmaking, however, these inves­

tigators used soy flour or partially purified soy lipoxygenase.

No one studied effect of endogeneous wheat lipoxygenase on glutenin and dough properties.

In CHAPTER N ^, in order to determine the properties of pen­

tosans constituting aggregative subunit of glutenin and the ef­

fect of these pentosans on the fermentation, pentosans were purified from wheat bran and then characterized. Wheat pentosan has a fundamental structure chracterized by � -(1,4)-xylan back­

bone with branching a -L-arabinofuranosyl groups (Brillouet et al. 1982, Brillouet and Joseleau 1987). ^However, there are two types of arabinoxylan constituting wheat pentosan (Shiiba et al.

1993), and the relationship between the structure of pentosans and these functions has not been clear.

A number of investigators separated wheat bran into classes of various polysaccharides according

different solvents (Schweitzer and

to their solubilities in Wursch 1979, Anderson and Clydesdale 1980, ^{Ring and} Selvendran 1980, ^{Brillouet et al.}

1982). _{Though such novel} techniques were developed to purify arabinoxylans from wheat bran,

purify the pentosans, characteristics.

these methods were not enough to because of their aggregative

In this study, using DEAE-Sepharose column, pentosans having different structure and different characteristics were separated and purified from wheat bran. Finally, effects of these purified pentosans on the dough properties were investigated. These results revealed the roles of pentosans in gluten aggregative subunits.

CHAPTER I

CHEMICAL CHANGES DURING DOUGH FERMENTATION 1)5)7)

1. Introduction

The effects of dough fermentation on rheological properties were examined by a number of investigators (Landis and Freilich 1934, Freilich and Frey 1939, Ikezoe and Tipples 1968, Barber et al. 1980). Preston and Kilborn ( 1982), Kilborn and Preston ( 1982), and Casutt et al. ( 1984) used the extensigram to measure dough rheological properties during fermentation. They showed that fermentation decreases extensigram length, maximum height, and area. Similarly, Pizzinatto and Hoseney (1980) reported that fermentation of cracker sponges changes the extensibility. Most of these reports are concerned with the effects of fermentation on rheological or physical properties, but only a few studied the chemical mechanism of the change in the rheological properties.

The objective of the present study was to examine effects of fermentation on gluten and factors affecting rheological changes during fermentation.

2. Materials and Methods

A. Wheat Flour

The wheat flour was an unbleached commercial blend milled by the Nisshin Flour Milling Co., Ltd .. Protein content was 13.0%, moisture 14.4%, and ash 0.41% which determined by AACC approved methods (1983).

B. Reagents

Linoleic acid (>99% pure substrate) and cis-parinaric acid were purchased from Sigma Chemical Company (St. Louis, MO.).

Other reagents used were analytical grade.

C. Preparation of Fermented Dough

Wheat flour (300 g) and 6 g compressed yeast were mixed with 185ml water as 57% absorption in the farinograph bowl (24°C ) for 4 min, and the resulting dough was placed in a room at 27°C and relative humidity of 80%. At time intervals (ranging from 0 to 4 hr), the dough was frozen by liquid nitrogen. The frozen dough was lyophilized and pulverized by impact mill (ultracentrifugal mill with the 0.5-mm filter, Retsch).

D. Operation of Mixograph

Mixograms were obtained in an air-conditioned room maintained at 25 ± 1� using a mixograph (National Mfg. Co., Lincoln, E) operating at 87 rpm at spring setting 9 (Johnson et al. 1946).

Thirty gram of flour was mixed with 20 ml of distilled water.

E. Sequential Extraction of Fermented Flour Proteins

The protein contained in the flours from each fermented dough having various fermentation times was sequentially extracted by a modified Osborne solubility fractionation procedure (Bietz and Wall 1975).

F. Gel Filtration of Fermented Dough Proteins

Gel filtration on·Sephacryl S-300 was performed as described by Okada et

(1986).

column (Pharmacia, 570 mm

25 mm i.d. ) was equilibrated with 50 mM Tris-HCl buffer (pH 7.0) con­

taining 0.5% sodium dodecyl sulfate

SD

Protein was extracted by 50 mM Tris-HCl buffer (pH 7.0) containing 0.5% SD S and 1.6 mM

ethylmaleimide. The suspension was stirred for 60 min at room temperature and was then centrifuged at 28,000

g for 20 min at 25°C

The supernatant was collected. Flour protein extract (5 ml) was loaded onto the column. Proteins were eluted at a flow rate of 35 ml/hr with upward flow at 25°C

The effluent was collected in 5-ml fractions. The protein concentration in the effluent was estimated by the difference in absorbance at 280 and 350 nm.

G. Separation of Gluten by Chromatography

The 70% ethanol-soluble proteins from the dried fermented doughs were prepared by the method of Bietz et al. (1984);

glutenin subunits were prepared from the dried fermented doughs

by the method of Burnouf and Bietz (1984). The protein solutions were filtered through 0.45 � m and the filtrates (50 � 1) were directly analyzed at 40°C by reversed-phase high performance liq- uid chromatograpy (RP-HPLC) using a chromatograph (Hitachi model 655A-12) equipped with solvent-delivery systems controlled by a model L-5000LC controller, a model 655A-40 automatic sample injector, and a model 655A-21 ultraviolet monitor set at 210 nm.

The packed column (200 mm x 3 mm i.d.) was a silica-based

0

reversed-phase support column (C18) with 300A pores (GL-Science, Tokyo). Data were recorded and processed with a Hitachi Chromato

Integrator D-2000. Solvent A was 12% acetonitrile with 0.05% tri- fluoroacetic acid (TFA) in distilled water, and solvent B was 80%

acetonitrile with 0.05% TFA. Proteins were eluted at a rate of 1 ml/min by a gradient from 15 to 60% solvent B for 55 min. These conditions were similar to those used by Bietz et al. (1984), ex- cept that the TFA concentration was reduced from 0.1 to 0.05% in an attempt to avoid protein deamination as described by Huebner and Bietz (1985).

H. Amino Acid Analysis

For amino acid analysis, protein was hydrolyzed in evacuated tubes with 6M hydrochloric acid at 110°C for 24 hr. Amino acids were determined on a automatic amino acid analyzer (Hitachi model 835).

I. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The SDS-PAGE was carried out as reported by Ng and Bushuk (1987) to determine the polypeptide subunits in flour protein.

The marker proteins used were � -galactosidase (molecular weight 116 kDa), phosphorylase (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carboanhydrase (30 kDa).

J. Assay of Proteolytic Activity

Proteolytic activities (acid and neutral or alkaline protease activity) of flour were determined by the following methods. By a six-bladed knife homogenizer (Ace type, Nihon Seiki Kaisha), 1 g of flour was dispersed with 20 ml of buffer solution (100 mM acetate buffer pH 4.7, or 100 mM phosphate buffer pH 7.5) for 5 min. The suspension was centrifuged at 14,500 x g for 10 min at 4� and the supernatant was filtered through No. 2 filter paper.

This filtrate was used as proteinase extract. Casein as substrate was prepared by the modified method of Kageyama (1955). Then 1 ml of protease solution was incubated with 2 ml of the substrate solution at 37� for 60 min. The undigested casein was precipitated with 0.4 M trichloroacetic acid (TCA), and the amount of TCA-soluble fractions was determined calorimetrically at 660 nm on a spectrophotometer (Hitachi 220 A) by the Folin­

Ciocalteu method (Folin and Ciocalteu 1927) using tyrosine as a standard. Proteolytic activity unit of flour was expressed as micromole of liberated tyrosine per minute per protein contained in the enzyme extract.

---��---�-----...

... ..

K. Determination of Foaming Activity

The foaming activity was measured by the modified stirring method of Kitabatake and Doi (1982). The flour (3.5 g) and 40 ml of 50 mM acetate buffer (pH 5.5) in a water-jacketed 50-ml stain­

less steel container were agitated at 4oC in a six-bladed knife homogenizer (Ace type, Nihon Seiki Kaisha Ltd.). The rotor speed was adjusted to 10,000 rpm. After stirring for 3 min, all of the foam and liquid was immediately transferred to a measuring cylinder by pouring and pipette, and the volume was measured.

Foaming activity (FA) was defined by the expression

FA (F/L-1) x 100

where F is the volume of foam plus liquid, and L is the volume of the liquid phase of the foam. The specific foaming activity was expressed as the ratio of FA to soluble protein in 50 mM acetate buffer (pH 5.5).

L. Measurement of Surface Hydrophobicity of Acetic Acid Soluble Fraction

The hydrophobicity was determined by the method of Kato and akai (1980) and Kato et al. (1981) using the hydrophobic fluorescence probe, cis-parinaric acid. Ethanolic solutions of 3.6 rnM cis-parinaric acid were purged with nitrogen, and equi­

molar butylated hydroxytoluene was added as an antioxidant. cis­

Parinaric acid solution (10 � 1) was added to 2 ml of sample

solution in 10 mM acetic acid. The parinaric acid-protein con­

jugates were excited at 325 nm, and relative fluorescence inten­

sity was measured at 420 nm in a spectra-fluorometer (Hitachi Fluoro-photometer, F-3000). The fluorescence intensity (FI) was expressed as relative activity, comparing standard of fluores­

cence intensity adjusting to 1.0 when 10 � 1 of cis-parinaric acid solution was added to 2 ml of 10 mM acetic acid in the ab­

sence of sample solution. The hydrophobicity of soluble glutenin was expressed as fluorescence intensity/% soluble protein with 10 mM acetic acid.

M. Extraction and Separation of Lipids from Fermented Dough

The total lipids contained in 30 g of the f l o_{u r}s from each fermented dough having various fermentation times were extracted by the means of 2 hr-shaking with 240 ml of n-butanol at room temperature. After the flour-butanol suspension was filtrated by No. 2 filter paper, lipids-butanol solution obtained was com­

pletely evaporated by rotary-evaporator (Tokyo Rikaki) and nitrogen-jet. The dry lipids obtained were weighed. After 20 mg of the dried lipids were dissolved again with 0.2 ml of buthanol, 5 ml of acetone and 0.1 ml of 10% MgClz ·6H20 in methanol were added into the lipids-buthanol solution and then the mixture was for 1 hr. After centrifugation of the mixture at

2,500 rpm for 5 min, the resulting precipitates were washed 3 times with 1 ml of cold acetone before evaporation to obtain dry matter of phospholipids. The dried phospholipids were weighed

before dissolving with 1 ml of chloroform.

fractions containing simple lipids were

Also all supernatant collected, dried by evaporation, and dissolved with 1 ml of chloroform. The amount of the extracted simple lipids was expressed as the amount of the total extracted lipids minus the amount of phospholipids.

Mono-, di-, and tri-glycerides (MG, DG, and TG) and free fatty acids (FA) in the simple lipids-chloroform solution were separated by thin layer chromatograpy (TLC) using silica gel (Kiesel gel 60 HF254, Merck) with solvent system (petroleum ether (b.p.60 70�) diethylether acetic acid 90:10:1).

Chloroform solution containing phospholipids such as phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), and phosphatidic acid (PA) were separated by the TLC with another solvent system (chloroform : methanol : water ⁼ 65:25:4). The mobility of each lipid on TLC was estimated using a calibration plot of mobility versus standard lipids. The separated each lipid on the TLC plate was developed with vapor-iodine and then quantified spectrophotometrically at 410nm with Shimazu Dentidometric System CS-930.

Free fatty acids in the extracted lipids were methylesterized by addition of diazomethane, and the methylesterized fatty-acids were detected by gas chromatography (GC) with the glass column packed with Unicil 3000 and quantified by the internal standard method using margaric acid methylester. GC condition used; injec­

tion temperature at 250� , column temperature at 210� , nitrogen carrier gas with flow rate at 50 ml/min, FID ditector.

N. Preparations of lypoxygenase from wheat flour

Samples (3.5 g) of lyophilyzed flours prepared from the fer­

mented dough were homogenized with 60 ml of 50 mM acetate buffer (pH 4.5) at 4°C for 5 min at 10,000 rpm by a rotating six-bladed knife homogenizer (Ace type, Nihon Seiki Kaisha Ltd.), and these suspensions were centrifuged at 15,000 x g for 20 min to obtain enzyme sol ution. The protein content of enzyme sol ution was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.

0. Assay of Lipoxygenase Activity Lipoxygenase activity

spectrophotometric method was

of

determined by Surrey (1964) and

the modified Walsh et al.

(1970). The substrate was prepared in nitrogen atmosphere by dis­

solving 100 mg of pure l inoleic acid in a mixture of 0.12 ml of Tween 20, 2.5 ml of 50 mM phosphate buffer (pH 7.0), and 0.32 ml of 1.0 M sodium hydroxide. The mixture was dissol ved with 50 mM phosphate buffer (pH 7.0) up to total 50ml of solution. The resulting solution contained 75 mM of linoleic acid. The sub­

strate was seal ed under nitrogen in a syringe bottle and stored at 5 OC in the dark before being used as a stock solution. Reac­

tion mixtures consisted of 5 � 1 of enzyme solution, 90 � 1 of stock solution, and 2.5 ml of 50 mM acetate buffer (pH 4.5). The progress of the reaction was recorded continuousl y in a double­

beam spectrophotometer at 234 nm (Zimmerman and Vick 1970). One

unit of lipoxygenase was defined as the amount of enzyme that produces a change of one unit of absorbance at 234 nm per minute.

P. Fractionation of Subunits Composed of Glutenin

Soluble-glutenin from a flour (100 g) was prepared as acetic­

acid-soluble protein by a modified Osborne solubility fractiona­

tion procedure (Bietz and Wall 1975). The polypeptide components of soluble-glutenin were fractionated by the method of Danna et al. (1978). The extracted glutenin was solubilized in 0.5% SDS containing 1% 2-mercaptoethanol. The alkylation of the reduced polypeptides in the supernatant was accomplished by addition of two times molar weight more acrylonitrile than 2-mercaptoethanol at pH 7.4 for 40 min. The reduced, cyanoethylated polypeptides were fractionated into three fractions (AF-1, AF-2, and AF-3) by the fractional precipitation with 70% ethanol under controlled conditions in the presence of SDS. Protein in these fractions was determined by the micro Kjeldal method (AACC approved method 1983) (protein: N x 5.7). This fractionation scheme is sum­

merized in Fig. 1.

Q. Determination of Sugar Composition of Carbohydrate in Glutenin Aggregative Subunit

After AF-1 obtained by above fractionation mathod (Fig. 1) was washed 3 times with 90% (V/V) acetone and dialyzed against distilled water to remove SDS, the precipetates were lyophilized.

Ten mg of lyophilized matter was hydrolysed with 2% HCl-rnethanol

Reduced, cyanoethylated glutenin solution (1% protein in ^0.5% SDS, pH 4.0)

addition of ethanol to ^70% (v/v) adjustment to pH ^5.2 with ¹ M NaOH incubation at ^25°C for ³⁰ min

centrifugation at ^28,000 x g for ³⁰ min

Supernatant

adjustment to pH 6.7 with 1 M NaOH standing overnight at 5�

centrifugation at ^5�

Supernatant

evaporation to remove ethanol

Ethanol soluble fraction (AF-3)

Precipitate

dissolution in 0.5% SDS

Ethanol insoluble fraction 1 (AF-1) (aggregative subunit) Precipitate

dissolution in 0.5% SDS

Ethanol insoluble fraction ² (AF-2)

(low molecular weight subunit) (high molecular weight subunit)

Fig. 1. A scheme for ethanol fractionation of glutenin subunits

� ��----�---------------...

.... ...

and sugar composition of the carbohydrate in AF-1 was deter­

minated by gas chromatography

(GC)

3. Results and Discussion

3-1. EFFECT OF FERMENTATION ON DOUGH RHEOLOGY

A. Mixograph Studies of Fermented Dough

Figure 2-A and 2-B showed that

hr-fermented dough decreased the intensity on mixogram, comparing a nonfermented dough. The mixogram after

hr of fermentation showed less resistance to ex­

tension (maximum intensity changed from 5.0 to

and a shorter peak time (changed 4.8 min to 3.6 min) than that of nonfermented dough. Similar effects of fermentation time on the extensigraph properties of fermented doughs were reported previously (Preston and Kilborn 1982). It is possible that lowering of pH by organic acids produced during fermentation could affect the rheological properties of dough, causing the decrease in mixing tolerance.

Hoseney and Brown (1983), investigating the effects of lowering pH on mixograph properties, found only a slight change on the mixogram for pH decrease from 5.58 to 5.20. However, in this study the change of pH was small and similar to those reported by Hoseney and Brown (1983) during fermentation; i.e., the pH value was 5.72 for the nonfermented dough and 5.26 for the

hr­

fermented dough. When the pH value of the nonfermented dough was

adjusted to 5.2 by addition of dilute lactic acid, the mixograrn

changed only slightly. Also, even after adding dilute sodium

hydroxide to the

hr-ferrnented dough to raise the pH to 5.7,

>.

.::: 4---+----'-

c:

22

0 0

Mixin g

0 �- 0

Mixing

5 10

Tim e (min)

. .,. -�

5 10

Time (min)

Fig. 2. Effect of fermentation on rheological properties of dough with mixograph.

A: mixogram of nonfermented dough B: mixogram of 4 hr-fermented dough

demonstrated that factors other than pH-changing played a major role in modifying the physical properties of the dough.

3-2. CHANGES IN TilE COMPONENT OF FLOUR DOUGH DURING FERMENTA- TION

A. Fractionation of Proteins from Fermented Dough

Table I showed distribution result of proteins which was fractionated from fermented doughs by modified Osborne's method (Bietz and Wall 1975). The total recoveries of flour protein by fractionation were 95.4, 96.7, 97.2, and 99.0% at various fermen- tation times (0, 1, 2, and 4 hr, respectively). The results showed almost the same recovery of extraction by this procedure.

As fermentation time increased, 70% ethanol-soluble protein (gliadine fraction) increased and acetic-acid-soluble protein (glutenin fraction) decreased significantly. On the other hand, the amount of protein extracted with water and aCl solution changed only slightly. The fermentation evidently caused a change in the solubility of gluten.

Table _I . Effects of Fermentation on Osborne's Protein Fractions

Percent(%) of Total Protein

Fermentation Water 0.5 M NaCl 70% EtOII 50 mM Acetic Acid Total Time (hr) Soluble Soluble Soluble Soluble Insolul·e Recovery

0 11. 8 2. 7 2 5. 2 1 9. 6 3 6. 1 9 5. 4

1 11. 5 1. 7 2 9. 8 1 6. 2 3 7. 4 9 6. 7

2 11. 8 2. 4 3 0. 1 1 3. 3 3 9. 6 9 7. 2

4 1 0. 6 2. 4 4 2. 6 9. 2 3 4. 3 9 9. 0

Figure 3 and 4 showed changes in the molecular welght of gluten protein during fermentation, which were determined by gel filtration profiles and SDS-PAGE. Although the extracted protein was almost 95% of total flour protein, chromatographic and electrophoretic results indicated small differences between molecular weights of polypeptides from nonfermented dough and from fermented dough. Table IT showed a weak and decreasing acidic proteolytic activity (protease) in the doughs during fermentation. These results suggested that proteolytic activity could hardly change the molecular weight of the proteins. Thus, these results showed that during fermentation the change in molecular weight of proteins contained in gluten does not occur.

Table IT ^. Changes in Proteolytic Activity (Protease) During Fermentation

Fermentation Time {hr)

0 1 2 4

Proteolytic ActivitY*

Acidic Neutral

{pll 4.7) {pH 7.5)

0.181 0.127 0.119 0.101

not detected not detected not detected not detected

Values are the average of two replications.

* Values are expressed as micromoles of librated tyrosine per minute per gram of extracted protein.

RP-HPLC detected differences of surface hydrophobicity among proteins from nonfermented and fermented dough. First of all, 70%

ethanol-soluble proteins (gliadine fraction) of nonfermented and

0.6

0 10 20 30 40 50

Fraction Number

Fig. 3. Elution profiles of gluten from the fermented doughs at various fermentation times by Sephacryl S-300 column.

0 _: 0 hr, 0 : 1 hr, e _: 2 hr, • _: 4 hr.

----

116- 97-

66-·

M

0

T-

X

.

45-

�

:E

30-

+

Fig. 4. SDS-PAGE patterns of gluten polypeptides from fermented doughs at various fermentation times.

a: 0

hr,

hr,

hr,

hr.

The molecular weights (left axis) were estimated. from

fermented dough were subjected to RP-HPLC. Figure 5 showed that 70% ethanol-soluble proteins from nonfermented dough were very similar to those from fermented dough. SDS-PAGE and RP-HPLC analysis on ethanol-soluble protein (gliadine fraction) frac­

tionated by the modified Osborne method (Bietz and Wall 1975), revealed that fermentation does not alter 70% ethanol soluble proteins qualitatively. ext, preparations of the total protein subunits of the reduced and cyanoethyl glutenin were subjected to RP-HPLC. Figure 6 showed that as fermentation time was increased, the amounts of two peaks (Pl and P2) having hydrophilic properties increased gradually. The area ratio of peak Pl plus P2 to total glutenin area from nonfermented dough was 9.5:100, which increased in the 4 hr-fermented dough to 28.6:100.

The protein in these peaks was hydrolyzed with 6M HCl and amino acids were determined on the amino acid analyzer. As shown in Table ill , peak P3 had a typical amino acid composition of gluten with high contents of glutamic acids and proline. Also Table ill showed that peak Pl and P2 had higher amount of glycine and glutamic acid and proline were contained relatively lower in peak Pl. These results suggested that the Pl and P2 polypeptides had been aggregated in the aggregative subunit because their amino acid contents were evidently different from those of high or low molecular weight subunit.

a

E c:

0

,...

N

...,

ro (1) b 0 c:

.0 ro

11...

0

.0

<!

c

d

1 0 2 0 3 0 4 0 5 0

Retention Time (min)

e

E

c:

0

� N

+-' ro

f

Q>

u c:

ro .c

.._

en 0 ..c

<!

g

h

1 0 2 0 3 0 4 0 5 0

Retention Time (min)

Table ill . Amino Acid Composition of Polypeptides (P1, P2, and P3)

Mole Percent of Amino Acid•

Amino Acid Pl P2 P3

Aspartic acid Threonine Serine

Glutamic acid Proline

Glycine Alanine Valine Methionine

Isoleucine Leucine Tyrosine

Phenylalanine Lysine

Histidine Arginine Tryptophan

7.1 4.1 11.8 16.0 8.8 17.8 4.7 4.0 0.4 2.9 6.1 2.8 3.1 3.2 2.6 4.7

not detected

4.1 3.9

3.4 3.6

1.1 8.8

33.1 32.0

11.4 12.1

10.3 6.5

3.6 4.3

3.1 5.5

0.2 not detected

3.1 3.7

5.2 8.1

2.2 1.5

5.3 4.1

2.1 1.5

1.8 2.1

3.0 2.5

not detected not detected

Values are the average of two replications.

* Polypeptides corresponding to peak P1, P2, and P3 shown in Fig.

6 were fractionated from chromatographic elution.

B. Changes in Foaming Activities (FA) During Fermentation

The FA of dough increased with large changes between 1 and 2 hr of fermentation time. As shown in Fig. 7, the ratio (specific foaming activity) of FA to soluble protein in 50 mM acetate buffer (pH 5.5) also increased gradually. These results suggested that the increasing FA was due to a change in hydrophobicity rather than quantity of proteins being solubilized.

�

130

+J ·r-i

>

·r-i

+J ()

�

Ol

120

·r-i -�

�-�

() � 0

110

·r-i �

·r-i o\o �

() ...

Q)� �Pt..

oo-

100 0 1 2 3 4

Fermentation Time (hr)

Fig. 7. Changes in foaming activity during fermentation.

C. Changes in Surface Hydrophobicity of Soluble Glutenin Fraction The surface hydrophobicity of soluble glutenin fraction (acetic acid soluble proteins) from nonfermented or fermented dough was determined using cis-parinaric acid as the hydrophobic fluorescence probe. The surface hydrophobicity of soluble glutenin fraction decreased gradually as shown in Fig. 8. ^This result showed that foaming activity increased as a result of decrease in the hydrophobicity of gluten.

19

0

0

18

ri

:><

-

Q

17

>t ·r-1

� <1)

·r-i +J u 0

16

·r-1 � ,.Q�

0

15

,.C: o\o

�' 0 ^.

� -r-1

14

'0 ^.

>t�

:r:-

13

12

0 1 2 3 4

Fermentation Time (hr)

Fig. 8. Changes in hydrophobicity of glutenin fraction during fermentation.

The hydrophobicity of glutenin is expressed as fluorescence intensity per percent protein.

D. Determination of Lipids Extracted from Fermented Dough

As shown in Table N , the total amount of the extracted lipids slightly increased during fermentation. However, the rate of simple lipids (SL) and phospholipids (PL) to the total ex-

tracted lipids did not so changed. Table V showed changing com- position of the extracted SL and PL during fermentation. Though the composition of PL was almost same between before and after fermentation except decreasing in phosphatidyl choline (PC), the

composition of SL was some difference between them. In particular, it showed decreasing in the ratio of fatty acids (FA) and triglycerides (TG) to the total SL during fermentation. Table m showed that in these fatty acids, particularly linoleic and linolenic acid decreased during fermentation. Decreasing the ratio of these FA and TG to the total SL was almost paralleled with decreasing hydrophobicity of acetic acid-soluble proteins.

These results suggested that lipoxygenase affects unsaturated fatty acids such as linoleic and linolenic acid in the fermented dough to change surface hydrophobicity of glutenin.

Table N . Effects of Fermentation on Lipid Extraction

Fermentation Time (hr)

0 2 4

Total Amounts*

of Extracted Lipids (mg)

110 136 148

Percent(%) of Total Lipids Simple Phospho- Lipids Lipids

41 4 1 45

59 59 55

Values are the average of two replications.

* Values are expressed as mirigram of lipids extracted from 30 g of lyophilized dough.

Table V .

Fermentation Time (hr)

0 2 4

Changes in Composition of Extracted Lipids during Fermentation

% of Simple Lipids % of Phospholipids MG DG TG FA others PC PE PA others

6. 4 14. 2 52. 3 2 7. 1 n. d. * 5 . 1 41. 2 4 2. 5 11. 2 13. 1 2 0. 8 41. 9 19. 6 4. 5 n. d. 4 0. 4 4 2. 7 16. 9 12. 5 18. 6 4 0. 3 18. 1 9 . 7 n. d. 4 4. 7 3 8. 7 16. 6 MG, DG, and TG: mono-, di-, and tri-glycerides, FA: fatty a cids,

PC: phosphatidyl choline, PE: phosphatidyl ethanolamine, PA: phosphatidic a cid

Table m . Changes in Composition of Fatty Acids from Extracted Lipids

Mole Percent of Total Five Fatty Acids

Fermentation Myristic Stearic Oleic Linoleic Linolenic

Time Acid Acid Acid Acid Acid

0 1 9 ⁰ 6 21. 2 12 0 4 4 3 ⁰ 7 3. 1

2 1 9 ⁰ 6 2 9 ⁰ 8 14 0 1 3 4 0 1 2. 4

4 21. 3 3 2 0 8 18 0 1 2 7 0 2 n. d. *

* n. d. ; not detected

Values are the average of two replications.

E. Determination of Lipoxygenase Activity

Lipoxygenase activity was determined because during fermenta- tion the rate of unsaturated fatty acids contained in glutenin decreased. Figure 9 showed that as the fermentation time increased, specific lipoxygenase activity (units per milligram of protein) increased. These results suggested that lipoxygenase af- fects glutenin containing lipids to decrease its hydrophobicity during fermentation, as a resut of that the rheological properties changed. To investigate how lipoxygenase affects hydrophobicity of glutenin, glutenin from doughs having differen- tial fermentation times was separated into subunits and these subunits were compared in the subsequent experiments.

12

+-> �

·r-f

>

10

+->

!C(-

<V ·r-f Q

8

t'Jl (1) ro+->

Q 0

<V H

tn�

6

� �

Ol o a

� ...

·r-f t'Jl ...:1+->

4

·ri u Q

·ri ::s

·ri �- u (1)

� ^tl.l

0

0 1 2 3 4

Fermentation Time (hr)

Fig. _9. Changes in lipoxygenase activity during fermentation.

3-3. CHANGES IN THE CONSTITUTION OF GLUTENIN SUBUNITS DURING FERMENTATION

A. Fractionation of Soluble-glutenin Subunits from Fermented

Dough

The total protein of soluble-glutenin obtained from a flour

(100 g) of the dried fermented dough having various fermentation

times for 0, 2, and

hr was 2278.3, 1013.2, and 678.8 mg

respectively. During fermentation, the amount of soluble-glutenin

decreased. Decrease in glutenin fraction during fermentation was already demonstrated from the result of Table I . The polypeptide components of the reduced and cyanoethylated glutenin from the fermented doughs were fractionated into three soluble fractions.

Seventy percent ethanol insoluble fraction 1 (AF-1) which precipitated at pH 5.2, 70% ethanol insoluble fraction 2 (AF-2) which precipetated at pH 6.7, and 70% ethanol soluble fraction (AF-3) were fractionated by the method of Danno et al. (1978).

The total recoveries of protein in the fractions were 93.0, 88.9, and 91.0% at various fermentation times (0, 2, and 4 hr, respectively). Table VII showed that as fermentation time increased, the amount of AF-1 and AF-3 decreased significantly.

As a result, only about 20% of AF-1 and AF-3 polypeptides of non- fermented dough was maintained in the 4 hr-fermented dough. On the other hand, the amount of AF-2 changed only slightly during fermentation, which resulted increase in the ratio of AF-2 to the total protein from 9.9% to 33.5% after 4 hr-ferrnentation.

Table VII . Effect of Fermentation on Proportion of Ethanol Fractionated Polypeptides

Fermentation Fractionated Protein Total

Time AF-1 AF-2 AF-3 Protein

(hr) (mg) (mg) (mg) (mg)

0 1074.2 210.7 833.9 2118.8

(50.7)* ( 9. 9) (39.4) (100)

2 361.8 199.3 339.8 900.9

(40.2) (22.1) (37.7) (100)

4 233.0 207.1 177.6 617.7

(37.7) (33.5) (28.8) (100)

These three fractions were subjected to SDS-PAGE. As shown in Fig. 10, the AF-1 (line a-d) contained 10-20 bands, AF-2 (lines e-h) contained 5 major bands as high molecular weight polypeptides, and AF-3 (lines i-1) contained 2 broad bands as low molecular weight polypeptides.

Comparing these SDS-PAGE patterns obtained, fraction AF-2, mainly contained high molecular weight subunit and fraction AF-3 coincided low molecular weight subunit, and also fraction AF-1 mainly contained some polypeptides which was similar to fraction A (aggregative subunit) reported by Heubner and Wall (1974) be­

cause of both similar SDS-PAGE patterns. However, the differences among SDS-PAGE patterns from the doughs at various fermentation times were almost negligible except appearance of new polypeptide HMP (shown in Fig. 10) having a low mobility (Rf=0.11) on SDS­

PAGE. The HMP in AF-2 appears to be a result of fermentation. It might have relationship with a band having a same Rf fractionated in AF-1 because increasing HMP in AF-2 during fermentation paral­

leled with the decrease of the band in AF-1, in spite of it being a faint band.

These results suggested that the fermentation caused little change in protein molecular weight, but change in conformation of soluble-glutenin. The fermentation evidently caused a change in the component of soluble-glutenin which was revealed as a result of decrease in the surface hydrophobicity of glutenin.

Particularly, decrease in the ratio of aggregative subunit (AF-1) to the total three glutenin causes decrease in the hydrophobicity

Origin ^a b ^c d ^e f 9 h ^I

J

k I

j

116- 97-

M 0

,...

X

66-

I

�45-.

�

30--

·+

Fig. 10. SDS-PAGE patterns of glutenin subunits from doughs fermented for various times.

a to d: AF-1 fractions (aggregative subunit) from dough fermented for 0, 1, 2, and 4 hr.

e to h: AF-2 fractions (high molecular subunit).

i to 1: AF-3 fractions (low molecular subunit).

The arrow indicates HMP.

The molecular weights (left axis) were estimated from protein standard.

of glutenin because aggregative subunit itself has high hydrophobic property which comes from the large amount of lipids contained with AF-1. In fact, the aggregative subunit especially has a complex structure consisting of lipids (15 - 20%), and car­

bohydrates (20 - 25%), besides polypeptides. When the complex structure of aggregative subunit is decomposed, decrease in the hydrophobicity occurs. The appearance of hydrophilic polypeptides (P1 and P2 shown in Fig.

in the reduced glutenin fraction was caused by the decomposition of the aggregative subunit containing some sorts of hydrophilic polypeptides (Danno 1978). The HMP is also one of the polypeptides composed of aggregative subunit which was released by the decomposition of aggregative subunit.

However, because changing molecular weight of polypeptides in

AF-

does not occur, the decomposition of the aggregative subunit structure causing decrease in surface hydrophobicity of glutenin probably depends on lipids contained in aggregative subunit. Also it suggested that carbohydrates as well as polypeptides released by decompositon of aggregative subunits affects rheological properties of fermented dough.

B. Analysis of Carbohydrates in AF-1 from Fermented Dough

Carbohydrates in fraction AF - 1 from nonfermented and 4 hr­

fermented dough were determined (Table �). This result revealed the presence of arabinose, xylose, and mannose as well as glucose and galactose. Comparing between sugar components of AF-1 frac­

tions from nonfermented and fermented dough, however, there was

little differences among the ratio of sugar components, except decrease in glucose which may be due to consumption by yeasts.

The presence of arabinose and xylose as a major component in AF- 1 suggested a significant role of pentosans (hemicellulose component) on aggregation. In fact, aggregative effect of wheat hemicellulose was reported (Shiiba et al. 1992).

Table _{V ill} . Sugar Composition of Carbohydrate Present in AF-1 from Unfermented and Fermented Dough

Percent of Sugar in AF-1*

Fermentation (The Ratio of Each Sugar to Total Carbohydrates) Time (hr) Arabinose Xylose Mannose Galactose Glucose Others

0 7. 1 7 3. 0 7 1. 18 3. 8 6 5. 4 3 0. 6 6

(33. 6) ( 14. 4) ( 5. 5) (18. 1) (25. 4) ( 3. 1 )

4 6. 7 7 3. 2 2 1. 31 3. 0 4 2. 4 4 0. 8 6

(38. 4) (18. 3) ( 7. 4) (17. 2) (13. 8) ( 4. 9)

Values are the average of two replications.

* Polypeptides corresponding to AF-1 shown in Fig.

1

It is known that the flour contained two types of pentosans, short chained xylan with a few arabinose (Cole 1969) and xylan with highly branched arabinose (Medcalf et al. 1968, D'Appolonia and MacArther 1975, and Lineback et al. 1977) which comes from wheat bran during milling-operation. However, no one investigated how many amount of pentosan was contatined in aggregative subunit and what functions of pentosan in aggregative subunit were. This is first time to investigate about pentosans (arabinoxylan) con- tained in aggregative subunit. By the decomposition of AF- 1 structure during fermentation, it was estimated that almost 80%

of pentosans bound with aggregative subunit is released because as shown in Table W only about 20% of original AF-1 from nonfer­

mented dough maintained after 4hr-fermentation. The total amount of pentosans released reaches almost 0.2% of the flour dough. The released pentosans might be enough to affect change in rheologi­

cal property of dough, which are described in detail in following CHAPTER IV .

4. Summary

Since mixograph studies showed that organic acids produced by fermentation are not enough to change the dough rheology, it sug­

gested that factors other than organic acids played a major role in chemical changes during fermentation. The fermentation caused a change in protein solubility; there was a large decrease in acetic-acid-soluble proteins and an increase in 70% ethanol­

soluble proteins. However, these phenomena were not caused by a change in molecular weight of the polypeptides constituting guluten, because SDS-PAGE and gel filtration on Sephacryl S-300 showed no detectable change of molecular weight of proteins from nonfermented and fermented dough, and also proteolytic activity in the fermented dough was low. The RP-HPLC analysis showed that the conformation of glutenin (acetic-acid-soluble protein) might be changed during fermentation, while that of gliadins (70%

ethanol soluble protein) not be changed. Furthermore the surface hydrophobicity of dilute acetic-acid-soluble proteins (glutenin) decreased gradually as fermentation time increased, and that ten­

dency paralleled with increase in the foaming activity of dough.

These results suggested that the decrease in surface hydrophobicity of glutenin occurs as a result of the conforma­

tional change of glutenin without changing molecular weights.

Futhermore, the determination of lipids extracted from doughs having different fermentation times, showed that the ratio of free unsatudated fatty acids (linoleic and linolenic acid) to the

total lipids decreased during fermentation. Also, because lipoxygenase activity evidently increased during fermentation, it suggested that the oxidation of unsaturated fatty acids affected a fermented dough to decrease surface hydrophobicity of its glutenin.

Soluble glutenin from the fermented dough having various fer­

mentation times was fractionated into three subunits and these constitutions were compared. As a result of that, it was sug­

gested that the ratio of glutenin aggregative subunit (abbr. GAS) and low molecular weight subunit (abbr. LMWS) in the soluble glutenin decreased during fermentation, by which the surface hydrophobicity of glutenin decreases. The HPLC also showed that hydrophilic polypeptides were released from GAS. Thus, these results suggested that the decomposition of GAS structure occurs during fermentation.

As a result of that decomposition, also pentosans bound with aggregative subunit are released too. Particularly, almost 80% of pentosans bound with aggregative subunit is released, and then the released pentosans plays significant role to change dough properties during fermentation.

CHAPTER IT

PURIFICATION AND Cl�CTERIZATION OF WHEAT LIPOXYGENASE 2)

1. Introduction

Lipoxygenase (EC 1.13.11.12) catalyzes the oxidation of polyunsaturated fatty acids containing cis,cis-1,4-pentadiene was first purified and characterized by Wallace and Wheeler (1979) and Nicolas et al. (1982). However, their reports gave limited information about the enzymatic properties of these isozymes and nothing about effects of the purified lipoxygenase isozymes on flour dough.

In a previous Chapter I ^, it showed that the endogeneous lipoxygenase was activated during fermentation and the activated lipoxygenase might catalyze oxidation of polyunsaturated fats to

cause changes in the rheological properties of doughs.

To further reveal the role of wheat lipoxygenase during fermentation, wheat lipoxygenase was completely purified from wheat germ and characterized.

� -

2. Materials and Methods

A. Wheat Germ

Wheat germ, obtained from No.1 Canada western red spring wheat, was a flaked product produced by isshin Flour Milling Co., Ltd.

B. Reagents

Linoleic acid and linolenic acid (>99% pure substrate) were purchased from Sigma Chemical Company (St. Louis, MO). Other reagents used were analytical grade.

C. Assay of Lipoxygenase Activity

Lipoxygenase activity was determined by the same method as described in "Materials and Methods" of CHAPTER I .

D. Analytical Disk SDS-PAGE

Lipoxygenase isozymes purified were examined by SDS-PAGE by the method of Weber and Osborn (1969) with slight modification.

Acrylamide concentration in the gel was 7.5%, and electrophoresis was done at 3 rnA per column (70 mm x 5 mm i.d.) for about 5 hr at 20 oc . After electrophoresis, the gel was stained with 0.5%

Coomassie Brilliant Blue R250 dissolved in 50% methanol contain­

ing 7% (v/v) acetic acid for 1 hr at room temperature and des­

tained with gentle shaking four times with a solution of 5%

methanol containing 7% (v/v) acetic acid. The molecular weight

markers used were aldolase (158 ^kDa), bovine serum albumin (66 kDa), ovalbumin (45 ^KDa), and chymotrypsinogen A (25 kDa), pur­

chased from Wako Pure Chemical Co., Ltd.

E. Amino Acid Analysis

Amino acids were determined by the same method as described in "Materials and Methods" of CHAPTER _{I .}

�-

3. Results and Discussion

3-1. PURIFICATION OF LIPOXYGENASE ISOZYMES

The isolation and purification of lipoxygenase isozymes were carried out at 4 _{OC ,} which scheme is summarized in Fig. 11.

A. Extraction of Lipoxygenase

Fresh wheat germ (2 kg) was defatted by acetone and vacuum dried. The deffated germ (1,745 g) was then added to 10 1 of cold 50 mM acetate buffer (pH 5.0). After stirring for 20 _{min at} 4 oc ,

the suspension was centrifuged at 6,000 _{x g for} 10 min to obtain a crude enzyme solution.

B. Ammonium Sulfate Precipitation

After the first precipitation at 25% _{ammonium sulfate} saturation, the mixture was centrifuged at 6,000 _{x g for} 15 _min.

After the second precipitation up to 40% saturation, the mixture was allowed to stir for 20 min and then was centrifuged for 15 min. After dialyzing against the buffer (pH 7.0), the enzyme solution was applied on the DEAE-Sepharose column.

C. DEAE-Sepharose CL-6B Chromatography

A DEAE-Sepharose CL-6B column (Pharmacia, 300 _{mm x} 100 _mm i.d.) was equilibrated with 10 mM phosphate buffer (pH 7.0).

After enzyme solution was applied, protein was eluted by a linear

Defatted wheat germ (1,745 g)

extraction with 10 1 of 50 mM acetate buffer (pH 5.0) fractionation with 0-25% saturation of ammonium sulfate centrifugation at 6,000 x g for 15 min

Supernatant Precipitate (discard)

fractionation with 25-40% saturation of ammonium sulfate centrifugation at 6,000 x g for 15 min

Precipitate Supernatant (discard)

dissolution in 10 mM phosphate buffer (pH 7.0) dialysis against 10 mM phosphate buffer (pH 7.0) centrifugation at 6,000 x g for 15 min

Supernatant Precipitate (discard)

DEAE-Sepharose CL-6B column

I

equilibration with 50 mM acetate buffer (pH 5.0) CM- Sepharose CL-6B column

equilibration with 100 mM phosphate buffer (pH 7.0) Sephacryl S-200 column

equilibration with 10 mM phosphate buffer (pH 7.0) DEAE-Sepharose CL-6B column

dialysis against distilled water Purified lipoxygenase isozyme solution

Fig. 11. A scheme for purification of lipoxygenase isozymes from wheat germ.

concentration gradient (0 - 0.5 M) of sodium chloride at a flow rate of 1 ml/rnin. Efluent was collected in 5-ml fractions and was continuously monitored by absorbance at 280 nm. Figure 12 showed elution pattern. Active fractions were pooled and dialyzed against 50 rnM acetate buffer (pH 5. 5) overnight and concentrated to about 30 rnl using an ultrafiltration system (Asahipak C5P, Asahi Kasei Kogyo Co., Tokyo) with a membrane having a molecular weight cutoff of 13 kDa.

20

16

Active

Fraction

32

0 12

a:>

N

0

0.5

.-..

:E

.._.,

0.25 u z Ctl

... 16

··· ··· ··· ••··· ··· ····

........

•

- 0

Fraction Number

Fig. 12. Separation of wheat lipoxygenase by DEAE-Sepharose CL-6B column chromatography.

D. CM-Sepharose CL-6B Chromatography

A CM-sepharose CL-6B column (Pharmacia, 500 mm _x 30 mm i.d.) was equilibrated with 50 mM acetate buffer (pH 5. 5) . The enzyme concentrate from the DEAE-Sepharose CL-6B column was loaded on this column. At a flow rate of 0.6 ml/min, a linear concentration gradient of 0 - 0. 5 M sodium chloride eluted active isozymes to separate into three major isozymes (L-1, L-2, and L-3) and a

minor isozyme (L-a) as shown in Fig. 13. Each isozyme in frac- tions was pooled and dialyzed against 100 mM phosphate buffer (pH

7.0) overnight and concentrated to about 20 ml again using ultrafiltration.

10

a

0 8

CX>

N

L-3 L-aL-2 L-1

Fraction Number

20

0.5

-

0.25 (.)

co

z 0

Fig. 13. Separation of wheat lipoxygeanse isozymes by CM­

Sepharose CL-6B column chromatography.

Lipoxygenase isozymes were denoted as L-3, L-a, L-2, and L-1, according to their order of elution.

E. Sephacryl S-200 Gel Filtration Chromatography

A Sephacryl S-200 column (Pharmacia, 1,000 mm x 30 mm i.d.) was equilibrated with 100 mM phosphate buffer (pH 7.0). Each isozyme concentrate from the CM-Sepharose CL-6B column was loaded on this column. At a flow rate of 0.4 ml/min, the active isozyme was eluted as shown in Fig. 14. Active fractions of each lipoxygenase isozyme were pooled, dialyzed against 10 mM phos­

phate buffer (pH 7.0) overnight, and concentrated to about 20 ml by ultrafiltration.

F. DEAE-Sepharose CL-6B Chromatography

At the final step, each isozyme was loaded on a DEAE­

Sepharose CL-6B column (Pharmacia, 100 mm x 10 mm i.d.) equi­

librated with 10 mM phosphate buffer (pH 7.0), as used in the first chromatography step. At a flow rate of 0.4 ml/min, a linear gradient of 0 - 0.2 M sodium chloride eluted active isozymes as shown in Fig. 15. The active fractions were pooled and dialyzed against distilled water and the purified enzyme solution was stored at 4 � before use.

The purification of isozymes summarized in Table � . Compar­

ing the crude extract, a high degree of purification was obtained, but there were different degrees of purification among the isozymes. This might be caused by different properties among the isozymes, especially different isoelectric points. The purified protein corresponding to isozyme L-3 exhibited almost 50% of the total lipoxygenase isozyme proteins, while protein

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