Structure and Chemistry of the Starch Granule

Structure and Chemistry of the Starch Granule

5

KEIJI KAINUMA

I. Introduction II. Structure of Starch Molecules and Granules A. The Use of Starch-Degrading Enzymes for the Structure Determination of Starch B. Fine Structure of Amylopectin C. Fine Structure of Amylose III. Gelatinization and Retrogradation of Starch Granules A. Methods to Determine Gelatinization and Retrogradation B. Structural Studies of Retrograded Starch IV. Chemical and Physical Changes of Starch Granules during Plant Growth V. Enzymic Degradation of Starch Granules VI. Conclusion References

I.

INTRODUCTION

Starch is produced by photosynthesis in green leaves of plants and stored in seeds, roots, and stems as an energy-storing carbohydrate. Starch is normally stored in granular form in storage organization. There are two structurally different polysaccharides in the granules. The first, amylose, accounts for 20-30% of most starches and is largely composed of long linear chains of (1 —» 4)-linked α-D-glucopyranose residues. The second, amylopectin, a major component of normal starch, is a macromolecule consisting of short amylose chains with an average degree of polymerization (d.p.) of 20, linked into a branched structure. Since the late 1960s, the development of enzymic methods for structural analysis of polysaccharides has revealed new features for the structure of amylose and amylopectin. For example, through the results of detailed enzyThe Biochemistry of Plants, Vol. 14 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

141

142

Keiji Kainuma

mic structure analysis, it is now generally accepted that amylose is a branched polysaccharide, not the absolutely linear polysaccharide as earlier thought. Also, the branching pattern of amylopectin has been studied carefully and it has been concluded that there is an extremely heterogeneous localization of branch points in these molecules. This finding has led to the "cluster model" structure of amylopectin molecules. Historical background of the structure and chemistry of starch granules was reviewed by Banks and Muir (1980) in the previous edition of this treatise. In this chapter, the more recent developments in research on the chemistry and structure of starch granules and molecules are described. II. STRUCTURE OF STARCH MOLECULES AND GRANULES A. The Use of Starch-Degrading Enzymes for the Structure Determination of Starch Although the main structure of amylose and amylopectin was determined primarily by chemical methods, particularly methylation, further progress has largely depended on the development of enzymic methods of analysis (Manners, 1985). Since the late 1960s, details of the mechanism of action of some starch degrading enzymes have been studied extensively. The results obtained contributed to the determination of the detailed fine structure of amylose and amylopectin, the general features of which had been determined earlier by chemical methods. The enzymes most frequently used for such structural analyses are α-amylase, /3-amylase, glucoamylase, pullulanase, isoamylase, and isopullulanase, whose action patterns on poly- or oligosaccharides are fairly well known. /. OL-Amylase (EC 3.2.1.1) α-Amylases hydrolyze nonterminal (1 —> 4)-a-D-glucosidic linkages in starch by a partially random action to give a mixture of linear malto-oligosaccharides and branched oligosaccharides called α-limit dextrins. Robyt and French (1970) first determined the frequency distribution of bond cleavage of porcine pancreatic α-amylase (PPA) on malto-oligosaccharides and found that the frequency of bond cleavage was highly regular and the action pattern on short-chain substrates was not random (Fig. 1). Kainuma and French (1969, 1970a) synthesized various glucosyl-stubbed malto-oligosaccharides, which were examined for the positional specificity of action of porcine pancreatic α-amylase. (Fig. 2) They showed that the sites of cleavage by the enzyme were extremely restricted by the existence of glucosyl stubs on the linear chain. The effects of modified glucose residues (Fig. 3) on the action pattern of porcine pancreatic α-amylase are also

143

5. Structure and Chemistry of the Starch Granule 0.66 0.34

CM-O -Φ 0.67

a33

0

0

O 1.00

0

CM-O

0

0

0.67 Q32

o—o-^-o^-o- -o—0 aoi

ηικ

o—o-aoi O

aoi ao5 °0.28f

I

0· 2 8

057 I

T i l 0j

f°

I

a

?°

r

CHK>-M>^-<>^-CMK>

"0

Fig. 1. The frequency distribution of bond cleavage during initial action of porcine pancreatic α-amylase on malto-oligosaccharides. (From Robyt and French, 1970.)

O-O-0

O-O-O-0

Lc^ ^ LL·^

ο-δ-ο-Ο^

_,U L·^

Fig. 2. Positional specificity of porcine pancreatic α-amylase on glucosyl-stubbed maltooligosaccharides. (After Kainuma and French, 1969, 1970a.)

Keiji Kainuma

144 ..G-

G—M—G — G

(MG3) G...

G—G—M—G—G

(MG4)

CH2OH

)—o

M Fig. 3. (a) Schematic binding modes of oxidized-reduced amylose that gives rise to tri(MG3) and tetrasaccharides (MG4) containing modified glucose residues, (b) Structure of glucose and oxidized-reduced glucose residue. (After Kainuma and French, 1982.)

now known (Kainuma and French, 1982; Braun et al., 1985a-c; Chan et al., 1984). In addition to porcine pancreatic α-amylase, bacterial "saccharifying" aamylase (Umeki and Yamamoto, 1975), salivary α-amylase (Bines and Whelan, 1960), and bacterial "liquefying" α-amylase have been used for structural analyses. The bond cleavage patterns of malto-oligosaccharides were qualitatively determined by Okada et al. (1969) as shown in Table I. Analytical results of the structure of α-limit dextrin revealed a very definitive branching pattern structure of amylopectin (Kainuma and French, 1970b; Umeki and Yamamoto, 1975) (see ΙΙ,Β,Ι).

A-chain

o-o O-O-O—

o-o O-O-O-O™-

c^-o-Q

o-o^Q

0 - 0 - 0 - - O-O-O-O---

B-chain

Fig. 4. Branching configuration resistant to sweet potato /3-amylase. (After Summer and French, 1956.)

5. Structure and Chemistry of the Starch Granule

145

TABLE I Summary of the Bond Cleavage Patterns of Various α-Amylases on Malto-oligosaccharides"·0 Enzyme source Bacterial saccharifying a-amylase* Saliva Pancreas Endomycopsis Taka Aspergillus niger Rhizopus niveus Malt Bacterial liquefying amylase Thermostable enzymes

G—G—G—G* * ΐ

ί

G—G—G—G—G* J ♦

G—G—G—G—G—G* |

|

ί ί ί ί ί

t t ί ί ΐ

None None

ί ί ί ΐ it

None None

Τί ί t i t t ι t

None

None

None

t

ί Τ t t

None None

a

From Okada et al. (1969). Long and short arrows indicate the sites which are readily and less readily attacked by the enzymes, respectively. (G: a-glucose, G*: radiolabeled reducing-end of glucose molecule, —: a-1,4-glucosidic bond.) b

2. ß-Amylase (EC 3.2.1.2) jS-Amylases hydrolyze the next to last glucosidic linkage from the nonreducing end of amylose and amylopectin and release, specifically, ß-maltose. The action of the enzyme is blocked by a-D-(l -> 6)-branch linkages, ßAmylase is often used to removed glucose residues from the nonreducing side of the branch point in amylopectin and branched oligosaccharides. The structures of the branched saccharides resistant to hydrolysis by ßamylase are shown in Fig. 4 (Summer and French, 1956). The cleavage sites of some glucosyl-stubbed malto-oligosaccharides are shown in Fig. 5 (Kainuma and French, 1970a).

Fig. 5. Position specificity of sweet potato /3-amylase on glucosyl-stubbed malto-oligosaccharides. (After Kainuma and French, 1970a.)

146

Keiji Kainuma

3. Debranching Enzymes (EC 3.2.1.41, EC 3.2.1.68) Pullulanase (EC 3.2.1.41) and Pseudomonas isoamylase (EC 3.2.1.68) are the most frequently used debranching enzymes for the structural analysis of branched polysaccharides. They are commercially available in crystalline form. These enzymes selectively hydrolyze the interchain (1 —> 6)-a-D-glucosidic linkages of branched polysaccharides but have no action on (1 —> 4)α-D-glucosidic linkages. These two enzymes are significantly different in substrate specificities, and selection of the correct enzyme must be carefully done to obtain satisfactory results. Pullulanase cleaves more easily shortchain branches such as maltosyl- or maltotriosyl- (Abdullah et al., 1966; Walker, 1968) compared with the long-chain branches which Pseudomonas isoamylase cleaves very easily. Table II shows the relative reaction rates of the action of pullulanase and Pseudomonas isoamylase on various branched saccharides (Kainuma et al. 1978a). The minimum substrate requirement of isoamylase seems to be a maltose residue (or preferably larger) in the A-chain and at least a maltotriosyl group in the B or C-chain at the point of cleavage. The susceptibility of branched oligosaccharides to Pseudomonas isoamylase and pullulanase in relation to their structures is shown in Fig. 6 (Kainuma et al., 1978a). Pseudomonas isoamylase is often used, with the combination of gel-filtration (Fig. 7) or TABLE II Relative Reaction Rates of Pseudomonas Isoamylase and Pullulanase on Various Singly Branched Oligosaccharides" Relative reaction rates Oligosaccharides Name 1. Panose 2. Isopanose 3. Oa:-D-Glucosyl-(1 —> 6)-maltose

Structure O

0—0 0—0 1 0 O

I 4. 0-a-D-Glucosyl-(3 —> 6)-maltotriose

0—0 O

5. 0-a-D-Maltosyl-(2 -> 6)-maltose

0—0—0 0—0 1 * 0—0 O I 0—0—0

6. 0-a-D-Glucosyl-(2 —> 6)-maltotriose

4

isoamylase

Pullulanase

147

5. Structure and Chemistry of the Starch Granule TABLE II {continued)

Relative reaction rates

Oligosaccharides Name

Pseudomonas isoamylase

Structure

7. 0-a-D-Maltosyl-(l —» 6)-maltose

0—0

8. 0-a-D-Glucosyl-(3 —> 6)-maltotetraose

0—0

Pullulanase

1

9. 0-a-D-Maltosyl-(3 —> 6)-maltotriose 10. 0-«-D-Maltosyl-(2 —> 6)-maltotriose 0-a-D-Maltotriosyl-(2 —> 6)-maltose 11. 0-a-D-Maltotriosyl-(l —> 6)-maltose

o 0—0 1

2.8

22

0—00—0 -0—0

I o-

8.6

0—0 "0 1 -0 0—0- I 0—0 0—00—0- -0

o- 1 12. 0-a-D-Maltosyl-(3 —» 6)-maltotetraose 13. 0-a-D-Maltotriosyl-(3 - * 6)-maltotriose 14. 0-a-D-Maltotriosyl-(2 —> 6)-maltotriose 0-a-D-Maltotetraosyl-(2 —> 6)-maltose 15. 0-a-D-Maltotriosyl-(3 —» 6)-maltotetraose 16. 0-a-D-Maltosyl-(3 —> 6)-maltopentaose

0—0 "0 I 0—0 0—0- o 0—0-

1 o—o- 0—0—0 o- o 0—0- 1 0—0 0—0

o—o1 o0—0 o- o 0—0- 1

17. 0-a-D-Maltopentaosyl-(2 -> 6)-maltose

0—0- 0—0—0

18. 0-a-D-Maltotetraosyl-(2 —» 6)-maltotriose

0—0- 0—0—0 0—0—0

19. Oa-D-Maltotriosyl-(3 —» 6)-maltopentaose

o—o—<

6.9

43

9.7

162

2.7

56

33

8.3

146 98 26

o

0-a-D-Maltotetraosyl-(3 —> 6)-maltotetraose

1 0—0

0—0- 0—0 1

o—o-

6.8

26

18

86

1

100 15

«

O—O—0 20. Pullulan 21. Amylopectin a

From Kainuma et al. (1978).

o 4 0—0—0

o—o

I O—O—O—0

100

Keiji Kainuma

148

(2)

Ί o-o-o

I CHp (5)

CD O

O-O-j!) (4)

0

O-O-jZS γ

O-O-^

(10) Ys (14)

Y/////////////////////////// O-O ΟΌ . o 0 -o-o-o o-o o-o-o

Ο-Ο-Ο-0 Κ/0-Ο-β (8)

0-0 i-0 (10)

K(9)

0-0-0

CMMHi)

(13)

(12)

V/////////////// / / //

| A

A

O-<>-0-$i)d (15)

/////////////////

Fig. 6. Susceptibility of branched oligosaccharides to Pseudomonas isoamylase and pullulanase in relation to their structures: boxed, susceptible to pullulanase; boxed-shaded, susceptible to pullulanase and Pseudomonas isoamylase; *-susceptibility reported qualitatively in Abdullah and French, 1970. (After Kainuma et aL, 1978.)

high-performance liquid chromatography (Kobayashi et aL, 1985; Hizukuri, 1985) for the determination of chain-length distribution of amylopectin or glycogen, as the enzyme completely debranches these polysaccharides. 4.

Isopullulanase (EC 3.2.1.57)

Isopullulanase is another hydrolase that reacts on pullulan and liberates oligosaccharides (Sakano et al.9 1971, 1972). The enzyme reacts specifically

Elution volume (ml) Fig. 7. Elution profile of debranched starch by Pseudomonas isoamylase and fractionated on a Toyopearl HW-50 column. I, II, and III represent Fractions I, II, and III of debranched starch molecules. Fraction I is mainly from the amylose fraction; Fraction II is composed of Bchain and long A-chains; and Fraction III is mainly from the short A-chains of amylopectin.

149

5. Structure and Chemistry of the Starch Granule

on pullulan at the a-(l —» 4) linkages present at the reducing-end side of the glucose residue, where the C-6 position is connected to another glucose residue. When the C-4 position of the glucose residue is linked by other glucose units, however, the reaction above does not occur. The action of the enzyme on branched saccharides is summarized in Fig. 8 (Sakano et al., 1972). Though 0-a-maltosyl-(2 -» 6)-maltose (Table II) is susceptible to hydrolysis by the enzyme, 0-a-maltosyl-(2 —> 6)-maltotriose (Table II) is completely resistant to its action. The enzyme is particularly useful for determination of the structure of branched oligosaccharides when used in parallel experiments with pullulanase (Umeki and Yamamoto, 1975). 5. Other Enzymes Glucan 1,4-a-maltotetraohydrolase (EC 3.2.1.60) (Robyt and Ackermann, 1971), glucan 1,4-a-maltohexaohydrolase (EC 3.2.1.98) (Kainuma et al., 1972, 1975), and other new enzymes which produce specific oligosaccharides can also be used for structural studies because of their strict product specificity. The combination of enzymes whose action mechanisms are well-known and Chromatographie techniques such as two-dimensional paper chromatography, gel-filtration chromatography, and high-performance liquid chromatography has strongly accelerated the structural studies of amylose and amylopectin. B. Fine Structure of Amylopectin Amylopectin consists of short amylose chains (d.p. in the range of 12-60 or more glucose units, with an average of about 20) linked into a branched

Pullulan

n

Isopanose

±

O/0

Panose

>

0

+

0

Isomaltose

Glucose

+

Q/O-O * 0 0-a-D-Glucosyl-(3—►6)-maltotnose Isomaltose

° — ' ^

0-a-D-Maltosyl-(2—-6)- maltose

Isopanose

CHZ)

Maltose

+

0

Glucose

Fig. 8. Action pattern of isopullulanase on branched oligosaccharides. (After Sakano et al., 1972.)

150

Keiji Kainuma

structure. The component chains of amylopectin are conveniently divided into three categories: (1) The A-chains, short amylose chains unsubstituted except at the reducing end; (2) B-chains, substituted at one or more C6—OH groups by A-chains or other B-chains and also substituted at the reducing end; and (3) C-chains, which are substituted at one or more C6—OH groups but are unsubstituted at the reducing end. There is only one C-chain per amylopectin molecule. The ratio of A-chains to B-chains is considered to be important as is an understanding of their multiplicity in the molecule. The ratio of A: B chains is determined by the amount of maltose and maltotriose liberated from ßamylase limit dextrin by pullulanase. There are several papers where A: B ratios have been determined primarily by two different principles (Peat et ai, 1956; Marshall and Whelan, 1974). Manners (1985) summarized the results of these studies and concluded that the A: B-chain ratio of amylopectin lies within the range of 1: 1 to 1.5: 1. The molecular weight of amylopectin has been determined by physiochemical methods and estimated as 107-108 (Banks and Greenwood, 1975). In this review, amylopectin structure is described from the views of: a) branching pattern, b) structure of the acid-resistant fraction, and c) electron microscopic observation of waxy maize starch. /. Branching Pattern of Amylopectin Molecules a. Structure of α-Amylase Limit Dextrin of Amylopectin. If the branches in amylopectin are sufficiently isolated from each other, α-amylase (Section ΙΙ,Α,Ι) converts each branch point into a singly branched oligosaccharide. If the branches are located densely in some region of the amylopectin, multiply-branched oligosaccharides or "macrodextrins" remain as a resistant structure to α-amylase action. Kainuma and French (1970b), using porcine pancreatic α-amylase, extensively hydrolyzed defatted waxy maize starch as a model compound of amylopectin. After reaching the second stage of hydrolysis, hydrolyzates were chromatographed and further purified by macro paper chromatography. α-Limit dextrins of d.p. 4-10 were isolated by paper chromatography. By sequential treatment with various amylases including porcine pancreatic α-amylase, ß-amylase, glucan 1,4-a-glucosidase (EC 3.2.1.3) (glucoamylase), and pullulanase, they determined the structure of singly and multiply branched oligosaccharides. The structures of singly branched oligosaccharides (d.p. 4-7) are shown in Fig. 9. Sixty-five percent of branch points of waxy maize starch amylopectin were isolated as singly branched oligosaccharides; thirty-five percent of the branch points were located in multiply branched oligosaccharides (shown in Fig. 10). These results strongly suggest a heterogeneous localization of branch linkages in the amylopectin molecule. If the molecule has a homogeneous branching pattern such as shown in the "Ideal Meyer Model" (Meyer and Bernfeld,

151

5. Structure and Chemistry of the Starch Granule

Ö-O-0 0-0 0-0-0-0

6-0-0 0-0-0 o-o-ef

0-6-0—0 0-0-0 0-0-0—0

Fig. 9. Singly branched oligosaccharides formed by action of pancreatic α-amylase on isolated branch points in amylopectin. (After Kainuma and French, 1970b.)

1940), all branched linkages should be isolated in singly branched oligosaccharides as indicated by the substrate specificity of porcine pancreatic aamylase. The findings of Kainuma and French (1970b) did not coincide with the "Ideal Meyer Model." A significant proportion of the branches (35%) were shown to be separated by no more than a single glucose unit. Analysis of the specificity of porcine pancreatic α-amylase has shown that where branches are spaced by two or more glucose units, the interbranch regions are easily cleaved by the enzyme, so that no such configurations would accumulate in the amylase-resistant oligosaccharides. Later, Umeki and Yamamoto (1975) analyzed quantitatively the formation of such branched saccharides. They used bacterial saccharifying a-amylase which trimmed glucose units attached to the branch point and gave glucosyl stubs. Six singly branched oligosaccharides shown in Fig. 9 were isolated as only L-* by the action of bacterial saccharifying α-amylase. Sixty-eight percent of the branched point was isolated in singly branched oligosaccharides. Their results of distribution of singly branched oligosaccharides and multiply branching oligosaccharides agreed well with the results of Kainuma and French (1970b). o-o-o o-o-£f

Q o α-ο-ο-ο-^Γ

Fig. 10. Multiply branched oligosaccharides formed by action of pancreatic «-amylase on amylopectin. (After Kainuma and French, 1970b.)

152

Keiji Kainuma

b. Chain Length Distribution of Amylopectin. Since the discoveries of debranching enzymes such as pullulanase and isoamylases oiPseudomonas and Cytophaga (Bender and Wallenfels, 1961; Harada et aL, 1968; GunjaSmith et aL, 1970), the chain length distribution of branched poly- and oligosaccharides have been analyzed in conjunction with gel-permeation chromatography or HPLC. The chain length distribution of the starch molecule as determined by many investigators (Harada et aL, 1972; Akai et aL, 1975; Mercier, 1973; Kainuma et aL, 1978b; Ikawa and Fuwa, 1980) was analyzed using debranching enzymes with separation of products by gel chromatography (Fig. 11). Peak I in Figure 11 corresponds to the amylose molecule, peak II and III are from the longer and shorter chains of amylopectin, and they are about d.p. 45-60 and d.p. 15-20 in length, respectively. Recently, Hizukuri et aL (1985) and Kobayashi et aL (1985) developed a technique for the fractionation of the debranched chain using HPLC which shortened the time required for analysis and also provided better separation of the fractions. c. Cluster Model for Amylopectin. Combining the results of structural analysis of the branching pattern of amylopectin (Kainuma and French, 1970b) and the formation of Nägeli amylodextrin (see Section II,B,2) by heterogeneous acid hydrolysis (Kainuma and French, 1971, 1972), French (1973) proposed a "cluster model" for the amylopectin molecule. This model also explained the high viscosity of amylopectin and the possibility of building the high molecular weight amylopectin (107-108) with the shortchain amylose of d.p. 20-25. According to the cluster model, it is easy to increase the molecular weight by simply increasing the number of clusters (Fig. 12). Robin and co-workers (1974) proposed a somewhat similar model of amylopectin (Fig. 13) based on the results of a sequential enzymic treatment of amylopectin and determination of chain length distribution of Lintnerized starch. They showed the presence of populations of chains having d.p. 2025, and 45 with a small proportion of chains having d.p. 60. The cluster model was further developed by Manners and Matheson (1981), who claimed the branch points are arranged in "tiers" or clusters and not distributed randomly throughout the macromolecule (Fig. 14). Their results strongly supported the original idea of a "cluster model" by French who had questioned the "Ideal Meyer Model" because a large quantity (35%) of branch points in amylopectin was located in a highly dense region (Kainuma and French, 1970b). This suggested that the branching pattern in amylopectin is extremely heterogeneous. 2. Structure of the Acid-Resistant Portion of Amylopectin Molecules

The amorphous gel phase of starch granules readily absorbs aqueous chemical reagents and is degraded by aqueous acid (e.g., 15% H 2 S0 4 or 7-

5. Structure and Chemistry of the Starch Granule

153

A

60

Waxy maize amylopectin 50 9

DP before fractional ion, 2 3

40

60

30h

60

20h

40

10 h

20

B p-timt dextrin of waxy maize amylopectin 40

80

IFF before fractional ion, 1 1

30 20 10 0

5

10

15

20

25

fraction number Fig. 11. Elution profile of debranched waxy maize amylopectin (A) and /3-limit dextrin (B) on Sephadex G-75. (After Akai et aL, 1975.)

CLUSTER (RACEMOSE) HYPOTHESIS OF AMYLOPECTIN STRUCTURE Fig. 12. Cluster (racemose) model for amylopectin molecule. (After French, 1973.)

154

Keiji Kainuma

0

rfffl rfTH

n-

X

A-chains

Kri rClh

FT1

h

DP = 15 (60 A)

RlU

v.

y.

,/

"cluster"

Fig. 13. Proposed structure for potato amylopectin by Robin et al. (1974). 1 = compact area; 2 = less compact area, arrows indicate branching points; 0 = reducing unit.

10% HCI). The crystalline phase is relatively resistant to such treatment for months. During heterogeneous acid hydrolysis, the initial attack occurs in the gel or amorphous region. This degradation leads to a reduction in the viscosity of starch pastes or sols. The product of brief treatment (such as 2 days at 20°C with 7.5% HCI) is "soluble starch" so-called because it readily dissolves in boiling water without gelatinization or paste formation.

5. Structure and Chemistry of the Starch Granule

155

Fig. 14. Modified cluster model of amylopectin, based on that of French (1973) and Robin et al. (1974). The various A and B chains, which are shown as straight lines, may actually exist in double-helix conformations. The interchain linkages are denoted by the vertical arrows, and R represents the sole potential reducing group in the molecule. (After Manners and Matheson, 1981.)

When the acid treatment is more extensive (e.g., 3 months with 16% H 2 S0 4 ), the residual material is called "Nägeli amylodextrin" (Kainuma and French, 1971; Robin et al.9 1974). The acidic hydrolysis can be accelerated by elevating temperature but caution must be used to avoid gelatinization. Acidic treatment of v/axy maize starch granules gives a highly crystalline residue which consists of three populations of molecules (Watanabe and French, 1980). Umeki and Kainuma (1981) studied the details of the fine structure of Nägeli amylodextrin of waxy maize starch. They employed multiple descending paper chromatography (Umeki and Kainuma, 1978) for the separation of each amylodextrin of different molecular size. The method gave sufficient resolution for separation of components differing in size from a difference of only one glucose unit to d.p. 25. The-distribution of the molecular size of Nägeli amylodextrin during acid treatment is seen in Fig. 15. The peak for the larger molecular weight fraction (fraction number >25) decreased gradually, and then two distinct peaks appeared at Fraction 18-22 and 12-16. These peaks corresponded to the so-called Fraction I, II, and III which Kikumoto and French obtained by precipitation of amylodextrin with organic solvent (1983). The structural characteristics of the fractionated polysaccharides of different molecular size were determined by enzymic analysis. Fraction 12-16, mainly from peak III, were partly hydrolyzed by pullulanase to form maltose, maltotriose, and linear maltosaccharides that were 2 or 3 D-glucose units smaller than the original fraction. Fraction 17-24 (the higher molecular weight fraction corresponding to Peak II) was debranched with pullulanase. Approximately 85-90% of the polysaccharides in this fraction were converted into linear maltosaccharides of d.p. 12-16 and the rest were found to be a very small amount of maltose and maltotriose. These observations indicated that most of the amylodextrin of Fraction 17-24 is composed of two chain molecules of d.p. 12-16, linked to each other by an a-(l —> 6) bond near the reducing end. The formation of the amylodextrins of Peak II and III is schematically illustrated in Fig. 16, where double stranded linear molecules were hydro-

156

Keiji Kainuma

>25

25 23 21 19 17 15 Fraction

13 11

number

Fig. 15. Change in the distribution of molecular size of Nägeli amylodextrin during acid treatment as determined by quantitative descending paper chromatography. "Fraction number" indicates linear and branched maltosaccharides which move on paper chromatography as fast as linear maltosaccharides of "d.p.„" (After Umeki and Kainuma, 1981.)

lyzed at the bonds (indicated as a, b, c, d, and e) near the a-(l —> 6)-branch point which distorts the formation of tight double helices of the chains. These results indicate that a pair of a-(l —> 4)-linked glucose chains are more stable to acid treatment than a single chain, and also suggest that the crystalline regions of amylopectin are packed with a number of pairs of intertwining linear chains of approximately 14 glucose residues. This structural evidence supports the idea of the double-helix model for the crystalline portion of starch, as suggested by the studies of X-ray diffraction, reaction with iodine, and the space filling model of Kainuma and

0-H>-O-H>K>-O--O-K)--(>H>H>H>
Fig. 16. Schematic presentation to show the formation of amylodextrin with short branches. Frequently, one or two glucose residues are linked to the right-hand side of the reducing glucose residue (0). (1 —> 4)-a-D-glucosidic linkages adjacent to branch points are designated as a, b, c, d and e. (After Umeki and Kainuma, 1981.)

5. Structure and Chemistry of the Starch Granule

157

French (1971), who proposed the possibility of a left-handed, double helix having six intertwining glucose units in a turn of 21 A. The course of hydrolysis during preparation of amylodextrin is schematically illustrated in Fig. 17. In the initial stage of reaction, starch molecules are cleaved at random at the single chain and densely branched regions of amylopectin. In the following steps, glucose chains not included in the double helix are removed. At the same time, some of the a-(l —» 4) linkages near the branch point are hydrolyzed. By these processes, the amylodextrins, which do not contain any single-chain regions, remain as an acid-resistant fraction after protracted acid treatment. Since in the previous volume (see Preface), Banks and Muir (1980) reviewed the details of the crystalline nature of starch, the classical view of crystallography of starch is not repeated in this chapter. In 1972, Kainuma and French pointed out that no model system based on a six-fold helix could satisfy the experimental values obtained for the X-ray intensity distribution of B-amylose and postulated the presence of double helices. This suggestion has been examined in detail by Wu and Sarko (1978a). They suggest that the structure of B-amylose is based on double stranded helices. They also propose a structure for A-amylose that is very similar to that which they derived for B-amylose, based on the anti-parallel packing of double helices wound parallel around one another. However, the central cavity (containing water molecules in the case of B-amylose) is occupied in a starch double helix in A-amylose, and the water molecules in the A-amylose

Fig. 17. Schematic explanation of the heterogeneous acid hydrolysis of amylopectin. Arrows indicate the position of acid hydrolysis. (After Umeki and Kainuma, 1981.)

158

Keiji Kainuma

Fig. 18. Comparison of unit cells and helix packing in A- and B-amylose. (After Wu and Sarko, 1978b.) Dots among double helices indicate the location of water molecules.

polymorph are found between the double helices. The model for A- and Bamylose are shown in Fig. 18. The presence of water molecules among double helices of A- and B-amylose structures are ob'served (Wu and Sarko, 1978b). French (1984) also discussed in detail the X-ray diffraction of starch. He emphasized the relation between the dimension observed by electron microscopy (such as a periodicity of 70Ä along the molecular axis) and results of X-ray crystallography (such as the repeat distance for an individual strand of a double helix of 21Ä of six D-glucopyranosyl units). He mentioned that the calculated crystalline dimension along the molecular axis is about 50Ä. 3. Transmission Electron Microscopic Observation of Amylopectin Molecules The method of Transmission Electron Microscopy (TEM) is a powerful and useful tool which provides a bridge between the gross structural features seen by optical microscopy or scanning electron microscopy (SEM) and the finer structures determined by X-ray diffraction and chemical or enzymic structural analysis. With modern equipment, it is possible to obtain sections as low as 200Ä. Work with thin sections of starch was initiated in the early works of Whistler and Turner (1955) and Frey-Wyssling (1953). These studies showed that starch has a microgranular structure, accentuated by acid or enzymic erosion. Using X-ray data, Nikuni and Hizukuri (1957) proposed a spherical micelle for starch (Fig. 19).

5. Structure and Chemistry of the Starch Granule

159

Fig. 19. Spherical starch micelles in which there are regular chains and chain terminations and branches within the micelle and starch chains entering and leaving the micelle to form the amorphous phase of starch granules, as suggested by Nikuni and Hizukuri (1957).

Gallant and Guilbot (1969) and Kassenbeck (1978) stained starch by a very simple method of periodate oxidation by reaction with thiosemicarbazide and staining with silver nitrate. Using advanced staining techniques, Kassenbeck distinguished the following types of organization: 1. a radial arrangement of the amylose molecule 2. amylose in an amorphous arrangement and 3. an arrangement of amylopectin such that the crystallites are in tangential lamellae. In a study of waxy maize starch, Yamaguchi and co-workers (1979) examined the fine structure of the amylopectin molecule using various treatments of waxy maize starch. They examined the molecular dispersion in dimethyl sulfoxide (DMSO), of thin sections (500Ä), of samples mashed with a glass homogenizer, and of a sample that had been extensively degraded with 16% sulfuric acid. The molecular dispersion of waxy maize starch autoclaved in DMSO showed elongated particles and aggregates. The regularity of the elongated particles was approximately 100Ä in diameter and 1200-4000Ä long (Fig. 20). Negatively stained, wet-mashed waxy maize starch granules show fibrillar structures and ripples of 70Ä similar to those described by Kassenbeck (1978). The wormlike rippled particles, presumably the amylopectin molecule, were about 100Ä in diameter and, thus, identical in shape and size to the dispersed molecule in DMSO (Fig. 21). Yamaguchi and co-workers interpreted the transmission electron microscope data as being consistent with an extended cluster model for amylopectin as shown in Fig. 22. From previous work (Kainuma and French, 1971), it is known that treatment of native starch granules with aqueous acid erodes the amorphous

160

Keiji Kainuma

Fig. 20. Waxy maize amylopectin dispersion, negative stain. Note wormlike molecule or molecular segment (x 128,000). (After Yamaguchi et al., 1979.)

portion of the starch granule leaving a highly crystalline low molecular weight residue ("Nägeli amylodextrin"). In the Yamaguchi et al. studies; the waxy maize starch granules were treated with 16% sulfuric acid to the level of 65% dissolution of the original starch. As seen in Fig. 23, the nega-

5. Structure and Chemistry of the Starch Granule

161

Fig. 21. Fragment of waxy maize starch granule obtained by wet mashing. F: radial fibriller structure, R: 70 A ripples, G: growth ring, M: wormlike rippled structure—presumably the amylopectin molecule. (After Yamaguchi et ai, 1979.)

etc.

100 Ä

T"

70 A

Reducing end of molecule

Fig. 22. Extended cluster model for the waxy maize amylopectin molecule. The entire molecule would be about 1200-4000 A long and would contain about 20-60 clusters. (After Yamaguchi et al., 1979.)

Fig. 23. Thin section of acid-treated waxy maize starch granule. The stack of lamellae is about 50 A thick (x 177,000). (Photo by Kainuma.)

5. Structure and Chemistry of the Starch Granule

163

tively stained material appeared to consist of elongated particles about 50Ä thick and several hundred angstroms long. These lamellae are more or less tangential to the growth ring and granule surface. Yamaguchi and co-workers presumed that these lamellae likely originated from the 70Ä rippling seen in wet-mashed native waxy maize starch (Fig. 21). Acidic erosion dissolved about 20A of the amorphous regions of starch and left 50Ä lamellae of the more crystalline, organized, acid-resistant portion relatively intact. Similar lamellae are observed in recrystallized amylodextrin Fraction II and Fraction III. Native and acid-treated waxy maize starch granules as well as recrystallized Fractions II and III exist as parallel-stranded double helices (Kainuma and French, 1972) as evidenced by their A-type X-ray diffraction patterns (Kainuma and French, 1971; French, 1973; Wu and Sarko, 1978a,b). Thus, a molecule of Fraction III, which contains an average of 14 D-glucose units (French et al., 1971; Umeki and Kainuma, 1981), is about 50Ä long, and a pair of such molecules can readily form a double helix (Kainuma and French, 1972). Fraction II or Fraction III, or a mixture of them (such as in the acid-treated waxy maize starch granule) exist as double helices which pack side-by-side to form regular, crystalline lamellae. These interpretations of the packing of starch chains and molecules are illustrated in Fig. 24. 4.

Molecular Arrangement of Amylopectin in Starch Granules

The last great bastion and challenge in starch structure determination is that of the starch granule and the organization of amylose and amylopectin within the granule (Lineback, 1986). It is known that native starch granules show "growth ring" formation when observed by optical microscopy, scanning electron microscopy of eroded granules, and transmission electron microscopy of thin sections. The growth rings may originate during deposition of layers of increasing organization.

Fig. 24. Scheme showing side-by-side association of linear regions (solid lines) from adjacent molecules and within individual molecules. These linear regions form the crystalline, acidresistant portion of the starch granule. The dashed lines represent amorphous, less densely packed regions, which are susceptible to acid hydrolysis. (After Yamaguchi et al., 1979.)

164

Keiji Kainuma

Fig. 25. Schematic presentation of starch granules based on "the unitary theory." (After Nikuni, 1969.)

Even after the details of the branching pattern of amylopectin, the double helix packing in crystalline regions, and the cluster arrangement of amylopectin molecules have been elucidated, very little is known about the arrangement of the amylose and amylopectin molecules in the starch granule The trichitic model for the starch molecule was first proposed by A. Meyer (1895). The structure contained a concentric ringlike organization, even though the concept of extended polymers had not been developed. Nikuni (1969) proposed the "unitary theory" of starch where all the molecules in a starch granule may well be covalently bound (Fig. 25). Both amylose and amylopectin are incorporated with the appearance of concentric ring structures in this model. The molecular weight obtained by this model is far greater than that determined by physicochemical methods. Recently, Lineback (1984) proposed the modified Nikuni model. This model incorporates the concept of a double helix of an outer chain of amylopectin. Amylose could exist in a random or helical configuration without binding to an amylopectin molecule. The details of configuration of amylose in the granules is not known (Fig. 26).

Fig. 26. A modified Nikuni model of the starch granule. (After Lineback, 1984.)

5. Structure and Chemistry of the Starch Granule

165

Growth ring

Growth ring Fig. 27. Schematic representation of arrangement of amylopectin molecules within a growth ring. The individual molecules may be intertwined. (After Kainuma, 1980.)

Kainuma (1980) and French (1984) proposed a possible arrangement of amylopectin clusters in waxy maize starch granules (Fig. 27). This model was based on transmission electron microscopy observations, combined with the results of chemical and biochemical analyses of the branching pattern of amylopectin and the structure of Nägeli amylodextrin. Because the exterior growth rings are always observed to be parallel to the outer surface of the granules, they provide a visible record of the history of the granule morphology during its development. It is always observed that the arms of the polarization cross are perpendicular to the growth rings. This shows that the optic axes of the starch crystallites, and hence the molecular axes of the starch molecules are aligned perpendicular to the growth ring. Since this is the model for waxy maize starch, it is still not clear how amylose molecules are incorporated. C.

Fine Structure of Amylose

Amylose is another polysaccharide present in starch granules. The classical chemical studies on amylose until 1950s established that the amylose

166

Keiji Kainuma

molecule is a linear long chain of (1 —» 4)-linked α-D-glucose residues (Williams, 1968; Banks and Greenwood, 1975). The enzymic hydrolysis of amylose by a crude ß-amylase preparation yielded complete degradation of the molecule to maltose. This experimental result misled researchers to propose that the molecule was a linear chain with unmodified glucose residues linked by (1 —> 4)-a-D-glucose. Peat et al. (1952) reported that the crystalline sweet potato ß-amylase only hydrolyzed approximately 70% of amylose. Studies with crystalline ß-amylase led to the suggestion of the presence of an additional starch hydrolyzing enzyme in the crude ß-amylase preparation. The additional enzyme was called "Z-enzyme," and was determined to be a contaminant in the crude preparation of /3-amylase which hydrolyzed linkages in amylose not susceptible to ß-amylase action. Presently, Z-enzyme has been identified as a trace contaminant of aamylase which enables it to bypass the bonds in amylose nonsusceptible to ß-amylase action. Kjorberg and Manners (1963) reported that pretreatment of potato amylose with yeast isoamylase resulted in a significant increase of the ß-amylolysis limit (>90%). This observation suggested that the anomalous barrier to jß-amylase action in amylose molecules was probably (1 —> 6)α-D-glucosidic interchain linkages. Further studies in this area were carried out using the bacterial debranching enzyme pullulanase (Banks and Greenwood, 1966). Banks and Greenwood treated potato and wheat amylose with pullulanase and /3-amylase by both successive action and concurrent action. From these studies, they confirmed the presence of the (1 —> 6)-a-D-glucosidic interchain linkages reported by Kjorberg and Manners. They also concluded that the (1 —> 6)-a-Dglucosidic branches were long-chain rather than short-chain. Most recently, Hizukuri et al. (1981) reconfirmed the multibranched structure of amylose by using sensitive methods for the determination of reducing end residues and nonreducing end residues. They reported that potato amyloses prepared from two different origins gave an average degree of polymerization (d.p.) values 6340 and 4850. Values of 3390 and 1840 were obtained for the amyloses of tapioca and kudzu {Pueraria hirusuta Matsum) when they calculated the d.p. by the reducing end residue. The d.p. of the same samples was determined based on the number of nonreducing end residues. They obtained values of 520 and 510 for potato amylose and 170 and 180 for the amylose of tapioca. When combined with the results of debranching of the molecules, they concluded that these amylose molecules were composed of 9-20 linear (1 —> 4)-a-D-glucan chains linked by (1 —> 6)-a-D-glucosidic bonds. It is likely that isolated amylose is a mixture of linear and branched molecules. As Greenwood and Thomson (1962) described, low molecular weight linear amylose is leached from starch granules at 70°C which indicates high ß-amylolysis (>96%). As the leaching temperature increases, the /3-amyloly-

167

5. Structure and Chemistry of the Starch Granule

sis limit of the extracted polysaccharides decreases indicating the presence of the branched structure in the molecules. III. GELATINIZATION AND RETROGRADATION OF STARCH GRANULES Heating starch granules in water initiates a series of irreversible changes. First, the native starch granule swells, then it undergoes a rapid gelatinization with a manyfold increase in volume and total loss of the native starch granule organization at a critical temperature. When heating is continued to 100°C or above, the swollen starch granule gradually disintegrates, then is solubilized and forms a colloidal sol. Essentially complete disaggregation of starch in water can be achieved by heating under pressure at 120-130°C for 2-4 hr. Starch granules change by heating and cooling with water in the following ways. limited reversible swelling

Native starch granules , gelatinization

gelatinized

(irreversible)

starch granules .

,

starch gel

heating

standing at low temperature

_

^

.'

* disaggregation, solution A

k

swollen granules cooling

*

, ,

retrograded starch

The series of changes occurring in starch granules affects significantly the enzymic digestibility and colloidal properties of starch. A. Methods to Determine Gelatinization and Retrogradation The changes occurring during gelatinization are observed as phenomena such as swelling of starch granules, increase of transparency, viscosity, and digestibility by amylases, changes in X-ray diffraction, and loss of birefringence. When we discuss the gelatinization process, various phenomenal changes should be observed as the overall physical changes of starch granules during gelatinization. For the determination of the degree of gelatinization (DG) of starch, however, enzymic digestibilities are frequently used, because the determination is simple, there is high reproducibility, and it is easy to represent the degree of gelatinization in numerical values. Enzymic digestibility has been studied historically using either the a-amylase "diastase method," (Mikumo et al., 1954; Watanabe and Hase, 1958) or glucoamylase (Toyama et al., 1966; Shetty et al., 1974). The "diastase method" had shortcomings as it was a complicated determination and raw starch had a high digestibility, thus decreasing the accuracy of the determination.

168

Keiji Kainuma

To improve the imperfection of the "diastase method," Toyama et al. (1966) developed the "glucoamylase method." This enzyme system effectively distinguished the difference between raw starch and gelatinized starch. However, it had a lower sensitivity to determinations of gelatinized starch versus retrograded starch as glucoamylase hydrolyzes retrograded starch much more readily than raw starch. After searching for a sensitive method to characterize the digestibility of raw, gelatinized, and retrograded starch, Kainuma et al. (1981) developed the ß-amylase-pullulanase process, which is abbreviated as the "BAP method." Plant ß-amylase does not hydrolyze raw starch granules and is extremely weak in hydrolyzing retrograded starch. Pullulanase debranches amylopectin when the molecule is completely denaturated by heat or alkali. The combination of the two enzymes composes a more sensitive enzyme system to the determine degree of gelatinization or degree of retrogradation than the conventional enzyme methods. The procedure of the BAP method is schematically illustrated in Fig. 28. This method is particularly useful to determine the change of starch containing foods during the storage. For other determination methods of gelatinization, differential scanning calorimetry, photopastegraphy (Kainuma et al., 1968a,b), viscography, etc. are often employed. Banks and Muir (1980), and French (1984) have reviewed in detail the gelatinization of starch. B. Structural Studies of Retrograded Starch Gelatinized starch molecules become gels or precipitate during storage. This phenomenon is called "retrogradation" of starch, and is frequently considered an undesirable change of starch solution or paste in many uses such as foods, adhesives, and industrial applications. The retrogradation has been considered simply as a reorganization of the dispersed starch molecule in gelatinized starch and very little is understood about its structure. Recently, Matsukura et al. (1983) examined the structure of retrograded starch. They first prepared extremely retrograded samples of starches of normal corn and waxy corn by repeating freeze-thaw cycles. The retrograded starch samples were treated with 16% sulfuric acid to obtain an acidresistant structure in the retrograded starch. The same sample was treated with jß-amylase-pullulanase (BAP system) to obtain the enzyme resistant region. The chain-length distribution of the resistant fractions of 16% sulfuric acid and jß-amylase-pullulanase was determined by gel-filtration with or without debranching by Pseudomonas isoamylase, respectively. Amylose content and X-ray diffraction of residual fractions were also determined. By acid or enzymic hydrolysis, Matsukura and co-workers observed a portion of the retrograded amylopectin molecules being strongly resistant to heterogeneous acid hydrolysis. For this acid resistant structure, they proposed the possibility of the formation of inter- or intramolecular parallel and antiparallel double helices in the retrograded amylopectin.

169

5. Structure and Chemistry of the Starch Granule DEHYDRATED SAMPLE (80 mg) ■ Add 8 ml distilled water Disperse with glass homogenizer reciprocally 10-20 times

ALKALINE GELATINIZED SAMPLE

DISPERSED SAMPLE 2 ml-

Sample

2 ml -Add 0.2 ml 107V NaOH keep at 50°C for 3 min Add 1 ml 2N acetic acid to adjust to pH6.0

- Fill up to 25 ml with — 0.8M acetate buffer solution (pH 6.0)

LANK TEST 4 ml

> Sample

4 ml

-Add 1 ml enzyme solution containing 0.8 IU /3-amylase and 3.4 IU pullulanase

- Add 1 ml of inactivated enzyme solution (10 min boiling) 1 ml

■4 ml

Incubate at 40°C for 30 min ► Sample <

1 ml

1 ml

Inactivate the enzymes by boiling for 5 min Dilute 5-fold

ml for Somogyi-Nelson method a

Dilute 5-fold

1 ml for Somogyi-Nelson method A

0.5 ml for phenol-sulfuric acid method B

1 ml for Somogyi-Nelson method A'

0.5 ml for phenol-sulfuric acid method B'

Fig. 28. Procedure of/3-amylase-pullulanase method to determine the degree of gelatinization (DG) of starch. (After Kainuma et aL, 1981.) DG (%) = [(A - a)/2B/(A' - a)/2B'] x 100

In contrast to the results of acid hydrolysis, the retrograded amylopectin fraction was more susceptible to /3-amylase-pullulanase action. However, the retrograded amylose was extremely resistant to enzyme digestion. After determining various structural characteristics, Matsukura et al. illustrated a feasible structure of retrograded starch as shown schematically in Fig. 29. They proposed three domains in retrograded starch based primarily

170

Keiji Kainuma

Fig. 29. A schematically illustrated structure of retrograded starch. Amylose, Amylopectin. A, B, C-structural domains (see text for discussion), (a) Two unit chains of amylopectin in the same molecule form a parallel double helix structure, (b) A unit chain of amylopectin forms an antiparallel double helix structure with another unit chain of different amylopectin molecules, (c) An amylose chain forms by itself a double helix structure and exists side by side with the double helix structure of amylopectin. (d) One-half of an amylose chain forms a parallel double helix with a unit chain of amylopectin and the other half forms an antiparallel double helix structure with another unit chain of amylopectin. The d.p. of the amylose chain is about 40, which corresponds to the length of two chains of amylopectin unit chain, (e) The combined structure of (c) and (d): one amylose molecule forms a double helical structure with two unit chains of amylopectin and one by itself. The d.p. of amylose to form this structure is, for example, about 60.

on the chain length of residual carbohydrate. Domain A is a region that is relatively resistant to acid hydrolysis but is more susceptible to enzyme attack. They assumed domain A was primarily derived from retrograded amylopectin and partly from a combined form with amylose and seemed to contribute to the recovery of some regularity in the retrograded starch molecule which was observed by X-ray diffraction. They postulated intertwining chains in domain A to form a structure resistant to acid treatment, similar to the structure of crystalline raw starch (Kainuma and French, 1971). There are several possible structures to protect amylopectin chains and shorter chain amyloses of d.p. 40-60 against acid treatment. They assumed several different modes of double stranded chains in domain A (designated as a, b, c, d, and e in the figures). A portion of the water molecules hydrated during gelatinization are strongly expelled from the starch chains and this causes a syneresis of the starch gels in domain A. Domain B is a slightly retrograded structure of gelatinized starch, in which amylopectin molecules are still well-hydrated

5. Structure and Chemistry of the Starch Granule

171

and relatively well-dispersed. Domain B is considered to be a transition state of gelatinization to retrogradation and is widely spread in aged starch gel. This is easily hydrolyzed by acid and enzyme. Domain C is primarily from retrograded amylose molecules which are hydrolyzed by acid but strongly resistant to enzyme action. Retrograded amylose is drawn as a folding model. In 1984, Jane and Robyt proposed a structure of retrograded amylose in which there were crystalline double helical regions of 10 nm in length interspersed with amorphous regions. Retrograded starch is considered as a mixture of Domain A, B and C in various proportions. Recently, Matsunaga and Kainuma (1986) reported the effect of helix forming chemicals on the protection of retrogradation of starch solution during storage. They found that amylose in domain C formed a single helix with chemicals such as 1-butanol or a fatty acid ester of sucrose and this prevented retrogradation. The results described in this section are used to propose a feasible structure of retrograded starch. Further studies should be done in the future to confirm the pertinence of proposed structures. IV. CHEMICAL AND PHYSICAL CHANGES OF STARCH GRANULES DURING PLANT GROWTH In the previous edition, Banks and Muir (1980) reviewed the biosynthesis of starch granules with particular emphasis on (a) the gross changes in granular morphology which accompany growth, and the relation between these changes and amylose content; (b) variation in fine structure of amylose and amylopectin during growth and (c) some properties of starch granules from the mature plant, which are relevant to an understanding of the growth process. They described the starches of pea, potato, barley, wheat and amylomaize. In this section, more recent advances of starch structure and the chemistry of various stages of plant growth are discussed. Recently, Asaoka et al. (1984) reported details of the effects of environmental temperature on the structure, amylose content, and other properties of endosperm starch during the development of rice plants. They raised several cultivars of rice plants (Oryza sativa L.) under controlled temperatures in a growth chamber. The chain length distributions of isolated starch were examined after debranching with Pseudomonas isoamylase and gel filtration on Toyopearl HW-50SF. They raised one cultivar (Nipponbare) at 25°C during daytime and 19°C for nighttime (group 1-1), and another group at 30°C during daytime and 24°C for nighttime (group 1-2). As shown in Table III, they found that the amylose content, which corresponds to Fraction I by gel filtration was different in the various growth conditions. Group 1-2 had significantly lower amylose content (15%) than that (20%) of Group 1-1. It

a

TABLE III Properties of Isoamylase-Debranched Materials of Endosperm Starches from Rice Plants Grown at Different Temperatures;/' Distribution of starch components (%)

Chain length at peak (D.P.)

Group

Xmax (nm)

Blue value (at 680 nm)

Fr. I

Int. Fr.

Fr. II

Fr. Ill

Fr. Ill/ Fr. II

Fr. II

Fr. Ill

Control 1-1 1-2

576 567 561

0.223 0.194 0.176

20.0 ± 0.04 19.7 ± 0.30 15.3 ± 0.32

4.4 ± 0.15 3.9 ± 0.21 4.2 ± 0.45

15.7 ± 0.15 16.1 ± 0.65 17.6 ± 0.30

59.9 ± 0.59 60.3 ± 0.78 62.9 ± 0.80

3.83 ± 0.06 3.75 ± 0.20 3.58 ± 0.11

41 ± 3.3 38 ± 2.2 44 ± 0.3

16 ± 1.0 15 ± 1.5 18 ± 0.3

a b

From Asaoka et al. (1984). Cultivar—Nipponbare.

TABLE IV Properties of Isoamylase-Debranched Materials of Endosperm Starches from Rice Plants Grown at Different Temperatures during Grain Filling Period after Anthesis" *

Group

Xmax (nm)

Blue value (at 680 nm)

572 570 550 553 572 568 554 558

Distribution of starch components (%)

Chain length at peak

Fr. I

Int. Fr.

Fr. II

Fr. Ill

Fr. Ill/ Fr. II

Fr. II

Fr. Ill

0.289 0.268 0.194 0.215

18.4 18.4 10.8

3.4 3.2 3.5 3.9

17.8 19.4 22.2 21.8

60.4 59.0 65.8 63.5

3.4 3.0 3.0 2.9

38 37 38 42

15 15 15 16

0.277 0.270 0.221 0.230

20.5 17.8 11.3 11.3

3.2 3.2 3.3 4.4

18.2 19.7 21.0 20„4

58.1 59.3 64.4 63.9

3.2 3.0 3.1 3.1

39 40 42 42

15 16 17 17

Koshihikari II-A-1*1C II-A-2*2' II-A-3*3' II-A-4*47 Hokuriku 93 II-B-1*1 II-B-2*2 II-B-3*3 II-B-4*4 a

8.5

From Asaoka et al. (1984). Heat summation in a growth chamber is 1.000°C during the experiments. c *i—25°C, 40 days. d *2—25°C, 20 days; then 30°C, 17 days. e *3_3o°C, 17 days; then 25°C, 20 days. /*4—30°C, 34 days. b

173

174

Keiji Kainuma

was also noted that starch granules of group 1-1 had higher onset (T0) and conclusion (Tc) temperatures in differential scanning calorimetry determinations. The results of another observation with different temperature conditions for rice starch growing was described and is shown in Table IV. In these studies, four different conditions were set and endosperm starch granules were isolated. It was concluded that the environmental temperature for the first 3 weeks after heading is linked closely to amylose production in endosperm cells of the rice plant. Other experimental results by the same group showed that the environmental temperature from 5 to 15 days after pollination, when the starch accumulation in endosperm cells was most active, was one of the most important determinants of amylose content in endosperm starch in rice plants. For the determination of the fine structure of amylopectin molecules, the more detailed experiments using waxy and normal rice plants of near isogenic lines of Japonica cultivar were carried out and it was concluded that the higher environmental temperature (30°C) increased the long B-chains of amylopectin, while decreasing mainly the short B-chains and slightly the Achains as compared with the lower temperature (20°C) (Asaoka et al., 1985). V. ENZYMIC DEGRADATION OF STARCH GRANULES Due to the increase in the price of petroleum accompanied by its limited supply, alcoholic fermentation of starch has become of interest in recent years. In the normal saccharification of starch, starch is first gelatinized, then liquefied by the action of α-amylase. Thereafter, glucoamylase is added to produce glucose. The process, however, requires a large amount of energy for gelatinization of starch that precedes saccharification. In an early paper, Leach reported a saccharification of starch granules by various α-amylases for the purpose of an economic saccharification process (1961). To minimize the energy consumption for alcohol fermentation, extensive research has been done in recent years to look for new amylases that can hydrolyze starch granules directly to obtain glucose. Such energy saving starch saccharification technology is now essential for the production of alcohol fuel from various starches. In connection with the alcoholic fermentation of raw starch, the works of S. Ueda (1978), Hayashida and Flor (1981), and Mizokami et al. (1977) and of others are known. S. Ueda and associates have long studied the alcoholic fermentation by the Black Aspergillus, A. awamori (Ueda and Koba, 1980; Ueda et al., 1981) and reported that amylase produced by A. awamori is more effective for the hydrolysis of raw starch than that produced by A. niger or malt amylase. Ueda (1957) and Park and Rivera (1982) described the alcohol fermentation of starch without gelatinization by A. niger or A. awamori.

5. Structure and Chemistry of the Starch Granule

175

Mizokami et al. (1977) found that Streptococcus bovis isolated from bovine rumen produced a strong raw starch digesting amylase. As a result of extensive search for microorganisms which produce a strong raw starch digesting enzyme, several new strains of microorganisms have been isolated,

..j^srp.>

Fig. 30. Corn starch granules degraded by Chalara amylase at prolonged reaction times. (After Kainuma et al., 1985.)

176

Keiji Kainuma

such as Bacillus circulans (Taniguchi et al., 1982a), Chalara paradoxa (Kainuma et al., 1985), and Corticium rolfsii (Takao et al. 1986). The amylase produced by B. circulans F-2, readily digests raw potato starch which is the starch granule most resistant to enzyme attack. Taniguchi and co-workers observed that B. circulans F-2 α-amylase was an extracellular tf-amylase which produced maltohexaose from gelatinized starch. The same enzyme acts on the raw starch granule and solubilizes it (Taniguchi et al. 1982b; Taniguchi and Maruyama, 1985). More recently, Kainuma et al. (1985) isolated a black mold from a pith of the sago palm. The mold was identified as Chalara paradoxa and produced extracellular amylases which act on the starch granule to form glucose as the only product. Normally, the activity of raw starch digestion is expressed by the percentage of the activity on raw starch compared to the activity on gelatinized starch. Compared with the conventionally known glucoamylases which have about 5-10% activity on raw starch compared to gelatinized starch, the crude chalara amylase showed greater than 40% of gelatinized starch digesting activity. General properties of the enzyme are reported by Kainuma et al. (1985) and Ishigami et al. (1985 a,b). It has become clear that the high activity of raw starch digestion of this enzyme can be attributed to the combined action of glucoamylases and α-amylase produced by Chalara paradoxa. Some of the results obtained with this enzyme are shown in Fig. 30 and 31. The raw starch digestion capacity will be afforded much attention relative to

Cfl • H CO

O U

M-i O

CU 0)

u 00
0

2

4

6

8

10

12

14

16 18 20 22 24

Time ( h r ) Fig. 31. Hydrolysis curves of various starches by Chalara amylase. (After Kainuma et al., 1985.)

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potential energy saving technology of starch saccharification and basic research on the binding and catalysis of solid substrate to the amylase. Indeed, a simple calculation shows that 30-40% of the total energy requirement of ethanol fermentation involves the heat of gelatinization of starch and saccharification (Matsuoka et ai, 1982). Therefore, the action mechanisms of raw starch digestion are being studied by several research groups. VI.

CONCLUSION

Recent developments in the structural analysis of starch components are reviewed with the emphasis on the role of various starch-related enzymes. The concept of finer structural characteristics of amylose and amylopectin has changed the classical understandings of these polysaccharides. Amylose is now considered to be a linear polysaccharide with a few branches in the molecule. The Double helix of the outer chain of the amylopectin molecule is also generally an accepted structure. The introduction of enzymic structural analysis has enabled researchers to reveal the finer structure of polysaccharides where organic chemical method had only given us the average features of molecular structure. In this chapter, starch gelatinization and retrogradation, and raw starch digestion are described in details from the point of view of starch utilization and biotechnology. Though gelatinization and retrogradation are very important in the nature of starch utilization in foods and industrial application, very little has been studied with respect to the structural changes of gelatinization and retrogradation because of the lack of appropriate methods to determine them. Raw starch digestion by various amylases have gained attention as a potential energy-saving method of starch saccharification. Some features of the structural characteristics of starch have become understood in recent years. But many basic problems have yet to be answered. For example, how are two different polysaccharides synthesized in the same amyloplast? How are the two polysaccharides located in a starch granle? Many problems still remain to be resolved in the future. REFERENCES Abdullah, M., and French, D. (1970). Arch. Biochem. Biophys. 173, 483-493. Abdullah, M., Catley, B. J., Lee, E. Y. C , Robyt, J. F., Wallenfels, K., and Whelan, W. J. (1966). Cereal Chem. 43, 111-118. Akai, H., Yokobayashi, K., Misaki, A., and Harada, T. (1975). Biochim. Biophys. Ada 2S2, 427-431. Asaoka, M., Okuno, K., Sugiomoto, Y., Kawakami, J., and Fuwa, H. (1984). Stärke 36, 189193.

178

Keiji Kainuma

Asaoka, M., Okuno, K., and Fuwa, H. (1985). Agric. Biol. Chem. 49, 373-379. Banks, W., and Greenwood, C. T. (1966). Arch. Biochem. Biophys. 117, 674-675. Banks, W., and Greenwood, C. T. (1975). In "Starch and its Components," pp. 44-47. Edinburgh Univ. Press, Edinburgh. Banks, W., and Muir, D. D. (1980). Biochem. Plants 3, 321-369. Bender, H., and Wallenfels, K. (1961). Biochem. Z. 334, 79-95. Bines, B. J., and Whelan, W. J. (1960). Biochemistry 76, 253-257. Braun, P. J., French, D., and Robyt J. F. (1985a). Arch. Biochem. Biophys. 242, 231-239. Braun, P. J., French, D., and Robyt, J. F. (1985b). Carbohydr. Res. 141, 265-271. Braun, P. J., French, D., and Robyt, J. F. (1985c). Carbohydr. Res. 143, 107-116. Chan, Y. C , Braun, P. J., French, D., and Robyt, J. F. (1984). Biochemistry 23, 5795-5800. French, D. (1973). Denpun Kagaku (J. Jpn. Soc. Starch Sei.) 19, 8-33. French, D. (1984). In "Starch, Chemistry and Technology" (R. L. Whistler, J. N. BeMiUer, and E. F. Paschall, eds.), pp. 184-247. Academic Press, New York. French, D., Kainuma, K., and Watanabe, T. (1971). Cereal Sei. Today 16, 299. Frey-Wyssling, A. (1953). In "Submicroscopic Morphology of Protoplasm," 2nd English Ed. Els vier, Amsterdam. Gallant, P. D., and Guilbot, A. (1969). Stärke 21, 156-163. Greenwood, C. T., and Thomson, J. (1962). J. Chem. Soc. 222-229. Guilbot, A., and Mercier, C. (1985). In "The Polysaccharides" (G. O. Aspinall, ed.), Vol. 3, pp. 209-282. Academic Press, Orlando. Gunja-Smith, Z., Marshall, J. J., Smith, E. E., and Whelan, W. J. (1970). FEBS Lett. 12, 96100. Harada, T., Yokobayashi, K., and Misaki, A. (1968). Appl. Microbiol. 16, 1439-1444. Harada, T., Misaki, A., Akai, H., Yokobayashi, K., and Sugimoto, K. (1972). Biochim. Biophys. Acta 268, 497-505. Hayashida, S., and Flor, Q. (1981). Agric. Biol. Chem. 45, 2675-2681. Hizukuri, S. (1985). Carbohydr. Res. 141, 295-306. Hizukuri, S., Takeda, Y., Yasuda, M., and Suzuki, A. (1981). Carbohydr. Res. 94, 205-213. Ikawa, Y., and Fuwa, H. (1980). Stärke 32, 145-149. Ishigami, H., Hashimoto, H., and Kainuma, K. (1985a). Denpun Kagaku 32, 189-196. Ishigami, H., Hashimoto, H., and Kainuma, K. (1985b). Denpun Kagaku 32, 197-204. Jane, J. L., and Robyt, J. F. (1984). Carbohydr. Res. 132, 105-118. Kainuma, K. (1980). Chori Kagaku 13, 83-90. Kainuma, K., and French, D. (1969). FEBS Lett. 5, 257-261. Kainuma, K., and French, D. (1970a). FEBS Lett. 6, 182-186. Kainuma, K., and French, D. (1970b). Proc. Amylase Symp. Osaka 5, 35-37. Kainuma, K., and French, D. (1971). Biopolymers 10, 1673-1680. Kainuma, K., and French, D. (1972). Biopolymers 11, 2241-2250. Kainuma, K., and French, D. (1982). Carbohydr. Res. 106, 143-153. Kainuma, K., Oda, T., and Suzuki, S. (1968a). Denpun Kogyo Gakkaishi 16, 51-54. Kainuma, K., Oda, T., Fukino, H., Tanida, M., and Suzuki, S. (1968b). Denpun Kogyo Gakkaishi 16, 54-60. Kainuma, K., Kobayashi, S., Ito, T., and Suzuki, S. (1972). FEBS Lett. 26, 281-285. Kainuma, K., Wako, K., Kobayashi, S., and Suzuki, S. (1975). Biochim. Biophys. Acta 410, 333-346. Kainuma, K., Kobayashi, S., and Harada, T. (1978a). Carbohydr. Res. 61, 345-357. Kainuma, K., Yamamoto, K., Suzuki, S., Takaya, T., and Fuwa, H. (1978b). Denpun Kagaku 25,3-11. Kainuma, K., Matsunaga, A., Itagawa, M., and Kobayashi, S. (1981). Denpun Kagaku 28, 235240. Kainuma, K., Ishigami, H., and Kobayashi, S. (1985). Denpun Kagaku 32, 136-141.

5. Structure and Chemistry of the Starch Granule

179

Kassenbeck, P. (1978). Stärke 30, 40-46. Kikumoto, S., and French, D. (1983). Denpun Kagaku 30, 69-75. Kjorberg, O., and Manners, D. J. (1963). Biochem. J. 86, 258-262. Kobayashi, S., Schwartz, S. J., and Lineback, D. R. (1985). J. Chromatogr. 319, 205-214. Leach, H. (1961). Cereal Chem. 38, 34-46. Lineback, D. R. (1984). Bakers Dig. March, 17-21. Lineback, D. R. (1986). Denpun Kagaku 33, 80-88. Manners, D. J. (1985). Cereal Food World 30, 461-467. Manners, D. J., and Matheson, N. K. (1981). Carbohydr. Res. 90, 99-110. Marshall, J. J., and Whelan, W. J. (1974). Arch. Biochem. Biophys. 161, 234-238. Matsukura, U., Matsunaga, A., and Kainuma, K. (1983). Denpun Kagaku 30, 106-113. Matsunaga, A., and Kainuma, K. (1986). Stärke 38, 1-6. Matsuoka, H., Koba, Y., and Ueda, S. (1982). J. Ferment. Technol. 60, 599-602. Mercier, C. (1973). Stärke 25, 78-83. Meyer, A. (1895). "Untersuchungen über die Stärkekörner," p. 318. Fischer, Jena, Germany. Meyer, K. H., and Bernfeld, P. (1940). Helv. Chim. Acta 23, 875. Mikumo, R., Matsui, J., and Nakano, K. (1954). Koshu Eisei Nenpo 2, 18. Mizokami, K., Kozaki, M., and Kitahara, K. (1977). Nippon Nogei Kagaku Kaishi 51, 299307. Nikuni, Z. (1969). Chori Kagaku 2, 6-14. Nikuni, Z., and Hizukuri, A. (1957). Mem. Inst. Sei. Ind. Res. Osaka Univ. 14, 173-181. Okada, S., Kitahata, S., Higashihara, M., and Fukumoto, J. (1969). Agric. Biol. Chem. 33, 900906. Park, Y. K., and Rivera, B. C. (1982). Biotech. Bioeng. 24, 495-500. Peat, S., Pirt, S. J., and Whelan W. J. (1952). J. Chem. Soc. 705-713. Peat, S., Whelan W. J., and Thomas G. J. (1956). J. Chem. Soc. 3025-3030. Robin, J. P., Mercier, C., Charbonniere, R., and Guibot, A. (1974). Cereal Chem. 51, 389-406. Robyt, J. F., and Ackerman R. J. (1971). Arch. Biochem. Biophys. 145, 105-114. Robyt, J. F., and French, D. (1970). J. Biol. Chem. 245, 3917-3927. Sakano, Y., Masuda, N., and Kobayashi, T. (1971). Agric. Biol. Chem. 35, 971-973. Sakano, Y., Higuchi, M., and Kobayashi, T. (1972). Arch. Biochem. Biophys. 153, 180-187. Shetty, R. M., Lineback, D. R., and Seib, P. A. (1974). Cereal Chem. 51, 364-375. Summer, R., and French, D. (1956). J. Biol. Chem. 222, 469-477. Takao, S., Sasaki, H., Kurosawa, K., andTanida, M. (1986). Agric. Biol. Chem. 50, 1979-1987. Taniguchi, H., and Maruyama, Y. (1985). Denpun Kagaku 32, 142-151. Taniguchi, H., Odashima, F., Igarashi, M., Maruyama, Y., and Nakamura, M. (1982a). Agric. Biol. Chem. 46,2107-2115. Taniguchi, H., Tei, M., Maruyama, Y., and Nakamura, M. (1982b). Denpun Kagaku 29, 107116. Toyama, T., Hizukuri, S., and Nikuni, Z. (1966). Denpun Kogyo Gakkaishi 13, 69-75. Ueda, S. (1957). Proc. Int. Symp. Enzyme Chem. 491-494. Ueda, S. (1978). Denpun Kagaku 25, 124-131. Ueda, S., and Koba, Y. (1980). J. Ferment. Technol. 58, 237-242. Ueda, S., Zenin, C. T., Monteiro, D. A., and Park, Y. K. (1981). Biotech. Bioeng. 23, 291-299. Umeki, K., and Kainuma, K. (1978). J. Chromatogr. 150, 242-245. Umeki, K., and Kainuma, K. (1981). Carbohydr. Res. 96, 143-159. Umeki, K., and Yamamoto, T. (1975). J. Biochem. 78, 897-903. Walker, G. J. (1968). Biochem. J. 108, 33-40. Wallenfels, K., Bender, H., and Rached, J. R. (1966). Biochem. Biophys. Res. Commun. 22, 254-261. Watanabe, H., and Hase, S. (1958). Denpun Kgyo Gakkaishi 5, 110-115. Watanabe, T., and French, D. (1980). Carbohydr. Res. 84, 115-123.

180

Keiji Kainuma

Whistler, R. L., and Turner, E. S. (1955). J. Polymer Sei. 18, 153-156. Williams, J. M. (1968). In "Starch Derivatives" (J. A. Radley, ed.), pp. 91-138. Chapman & Hall, London. Wu, H. C. H., and Sarko, A. (1978a). Carbohydr. Res. 61, 7-25. Wu, H. C. H., and Sarko, A. (1978b). Carbohydr. Res. 61, 27-40. Yamaguchi, M., Kainuma, K., and French, D. (1979). / . Ultrastmct. Res. 69, 249-261.