Analysis of molecular structure of starch

Physicochemical properties/ Chemical

composition

Molecular structure

Biosynthesis (enzymes)

Genes

Molecular Structure of Molecular Structure of AmyloseAmylose

Property Thai green canna

Thai purple canna

Mung bean

Number average DP, DPn

Colorimetric method 1650 1590 2200

Labeling method 1550 1440 2110

Average chain length, CL 471 420 350

Average number of chain,NC 3.5 3.8 6.3

Linear amylose (mole %) 84 84 84

Branched amylose (mole% ) 16 16 16

NC of branched amylose 17 17 34

Molecular Structure of Molecular Structure of AmylopectinAmylopectin

Property Thai greencanna

Thai purplecanna

Mungbean

Average chain length, CL

I soamylolysis 26 28 24

Labeling method 25 27 23

Source Average chain - length Unit chain (mole %)

A B1 B2+B3 A B1 B2+B3

Thai green canna 14 52 84.3 15.7

Thai purple canna 15 53 84.0 16.0

Mung bean 10 21 48 59.1 31.4 9.5

Average chain-length and amount Average chain-length and amount (mole %) (mole %)

of the fractions of amylopectin unit of the fractions of amylopectin unit chainchain

Molecular characterization

Amylose

Number average degree of polymerization (molecular size, DP)

Average chain length (CL)

Average number of chain (NC)

Linear amylose fraction (mole%)

Branched amylose fraction (mole%)

Molecular characterization

Amylopectin

Average (branch) chain length, overall

Unit chain (A, B1, B2, B3,..) fraction (mole%)

Average (branch) chain length, of A-chain

Average (branch) chain length, of B1-chain

Average (branch) chain length, of B2-chain

Methods of Analysis

Colorimetric methods

chemical reaction

chemical reaction + enzyme reaction

Chromatographic Techniques

without enzyme reaction

with enzyme reaction

Detector

Low-angle laser-light-scattering photometerRefractive index detector

Pulsed amperometric detector

Fluorescence detector

Colorimetric methods (chemical reaction)

Determine: Total sugar/ Reducing end/Non-reducing end

Average degree of polymerization = total sugar (molecular size, DP) reducing end sugarAverage chain length (CL) = total sugar

non-reducing end sugar

Average number of chain (NC) = DP/CL

Total sugar:

• Anthrone-H2SO4

• Phenol-H2SO4

Reducing end sugar

Modified Park-Johnson’s method

Ref; 1. J. Park and M.J. Johnson, J. Biol. Chem., 181 (1949), 149-151.

2. S. Hizukuri, Y.Takeda, M. Yasuda, Carbohydrate Research, 94 (1981), 205-213.

Non-reducing end sugar

Rapid Smith Degradation method

Ref; 1. J.K. Hamilton and F. Smith, J. Am. Chem. Soc., 78 (1956), 5907-5909.

2. S. Hizukuri and S. Osaki, Carbohydrate Research, 63 (1978), 261-264.

Colorimetric methods

(chemical reaction + enzyme reaction)

Branch chain length (CL) = total sugar

non-reducing end sugar

Isoamylase/pullulanase

Hydrolyze -1,6 by isoamylase/pullulanase

Determine reducing end sugar by Modified Park & Johnson’s method

Experimental ProcedureAmylopectin structure studied by

HPSEC

Fractionation(selective precipitation)

Starch

Amylose

Debranched

Amylopectin

Molecular analyses(HPSEC)

Chromatographic Techniques with Enzyme Reaction

Chromatographic Techniques with Enzyme Reaction

C

B

A

B

A

A

B

Isoamylase or pullulanase.

A

Chromatographic Techniques with Enzyme Reaction

Figure 6 Block diagram showing the component of an HPSEC instrument.

Mobile Phase

Solvent Delivery System

Injector

|S| |M| |L|

DetectorsRecorder

Injection of debranche

d amylopecti

n

Column

Chart record

Retention timeR

esp

on

se

L M S

Experimental ProcedureAmylopectin structure studied by

HPSEC Column: Zorbax PSM 60S ( 2) MW range: 5 102 – 104

Column dimension:6.2 mm ID 250 mm Loading size: 40 μl Eluent: 90% DMSO Flow rate:0.5 ml/min Pressure: <3,000 psi Column temperature:50oC Standard: maltoheptaose, pullulan6000

and pullulan12000 (MW 1,170, 5,900, 11,800, respectively)

Results & Discussion

Figure 7 High-performance size exclusion chromatography of maltoheptaose, pullulan6000 and pullulan12000.

6 8 10 12 14 16 18 20 22 24Retention time (min)

Refractiv

e in

dex

resp

ond

Pullulan12000Pullulan6000Maltoheptaose

MW 1,170,17.845 min

MW 11,800,12.639 min

MW 5,900,13.727 min

Results & Discussion

Figure 8 Standard curve for Zorbax PSM60S ( 2).

2.0

2.5

3.0

3.5

4.0

4.5

5.0

8 10 12 14 16 18 20 22

Time (min)

Log M

W

Log MW = -0.1867(Retention time; min) + 6.3882

R2 = 0.9906

Maltoheptaose

Pullulan12000

Pullulan6000

Results & Discussion

Figure 9 High-performance size exclusion chromatography of isoamylolyzate of amylopectin from starches.

8 10 12 14 16 18 20

Refra

ctiv

e in

dex

resp

ond

1

10

100

1000

10000

100000

8 10 12 14 16 18 201

10

100

1000

10000

100000

Mole

cul

ar w

eig

ht (dalto

n)

8 10 12 14 16 18 201

10

100

1000

10000

100000

Mole

cul

ar w

eig

ht (dalto

n)

8 10 12 14 16 18 20

Refra

ctiv

e in

dex

resp

ond

1

10

100

1000

10000

100000

8 10 12 14 16 18 20

Retention time (min)

1

10

100

1000

10000

100000

Mole

cul

ar w

eig

ht (dalto

n)

8 10 12 14 16 18 20

Retention time (min)

Refra

ctiv

e in

dex

resp

ond

1

10

100

1000

10000

100000

Normal rice Waxy rice

Waxy potatoNormal potato

Waxy cornNormal corn

Results & DiscussionYuan et al. (1993)

Refractive index response is

proportional to the mass of the eluted

material.

The relative mole was derived by

dividing the relative mass (RI

response) by the corresponding

molecular weight. weightmolecular ingCorrespondresponse) (RI mass Relative

mole Relative

150 100 80 60 40 30 20 10 5

8 10 12 14 16 18 20

Refractiv

e in

dex

resp

ond

Degree of polymerization

RI detector responseMole

Results & Discussion

Figure 10 High-performance size exclusion chromatography of isoamylolyzate of amylopectin from starches.

Normal rice

150 100 80 60 40 30 20 10 5

8 10 12 14 16 18 20

Refractiv

e in

dex

resp

ond

Mole

(RI r

esp

onse

/MW

)RI detector responseMole

150 100 80 60 40 30 20 10 5

8 10 12 14 16 18 20

Mole

(RI r

esp

onse

/MW

)RI detector responseMole

150 100 80 60 40 30 20 10 5

8 10 12 14 16 18 20

Retention time (min)

Refractiv

e in

dex

resp

ond

Mole

(RI r

esp

onse

/MW

)RI detector responseMole

150 100 80 60 40 30 20 10 5

8 10 12 14 16 18 20

Retention time (min)

Mole

(RI r

esp

onse

/MW

)RI detector responseMole

150 100 80 60 40 30 20 10 5

8 10 12 14 16 18 20

Degree of polymerization

Mole

(RI r

esp

onse

/MW

)RI detector responseMole

Normal corn

Waxy rice

Waxy potatoNormal potato

Waxy corn

Ref: 1. Koizumi K. and Fukuda M., Estimation of the distributions of chain length of amylopectins by HPAEC-PAD, J. of Chromatography, 585 (1991), 233-238.

2. Hanashiro, I., Abe, J., & Hizukuri, S. (1996). A periodic distribution of the chain length of amylopectin as revealed by high-performance anion-exchange chromatography. Carbohydrate Research, 283, 151-159.

High performance anion-exchange chromatography with pulsed amperometric

detection (HPAEC-PAD)

System: Model 4000i Dionex BioLC system

Column: Dionex HPIC-AS6 (now called CarboPac PA-1) 250 4 mm

(10 µm) with AG6 guard column (50 4 mm)

Detector: Model 2 PAD system

Individual members of the components can be obtained

Cannot determine the individual glucans directly by use of their peak areas in the chromatogram, as the responses of a pulsed amperometric detector to glucans having different DPs were different.

High performance size-exclusion chromatography (HPSEC) with fluorescence

detector

Ref: Hanashiro, I., & Takeda, Y. (1998). Examination of number-average degree of polymerization and molar-based distribution of amylose by fluorescent labeling with 2-aminopyridine. Carbohydrate Research, 306, 421-426.

System: HPLC

Column: For amylose

TSK gel G6000PW, G4000PW and G3000PW (7.575 mm) (Tosoh Co., Tokyo, Japan), connected in series

TSK guard column PWH (7.575 mm)

Temp. 37 C, Eluent: 0.1 M phosphate buffer (pH 6.1) containing 0.02% sodium azide

Detector: Fluorescence Detector

Refractive index detector

Fluorescent reagent: 2-aminopyridine (aromatic primary amine)

Std. amylose: AS-110 (DP 521), AS-320 (2320), AS-1000 (4400)

Fluorescent labeling of Fluorescent labeling of amyloseamylose with 2with 2--aminopyridineaminopyridine

DPn = RI response (RI) fluorescence response (F)

Chromatograms of Fluorescence-labeled Chromatograms of Fluorescence-labeled AmylosesAmyloses

FluorescenceFluorescence

RIRI

DPDP

DPDPsample sample = = (RI/F)sample(RI/F)sample

(RI/F)std.(RI/F)std.xx DPDPstd.std.

Column for amylopectin (unit chain)Sample: Isoamylolyzate

Column: Shodex OHpak SB-803HQ and

SB-802.5HQ x 2 (8300 mm)

Eluent: Aq. Me2SO (50%) containing 50 mM NaCl

Column Temperature: 50 C

Std. amylose: G6, AS-10 (52), AS-30 (141), AS-70 (440)

Beta-Amylolysis of Amylose MoleculeBeta-Amylolysis of Amylose Molecule

Linear moleculeLinear moleculeBranch moleculeBranch molecule

Reducing endReducing endGlucoseGlucosealpha-1,4alpha-1,4alpha-1,6alpha-1,6

-amylolysis-amylolysis

Swelling of starch granuleSwelling of starch granule

Increase viscosity of starch pasteIncrease viscosity of starch paste

Action of amylase on starch

Property Thai greencanna

Thai purplecanna

Mungbean

Phosphorus (P)

Total organic P (ppm) 397 420 100

Link to C- 6 (ppm) 275 299 15

Phosphorus at C- 6 (%) 69 71 15

PhosphorusPhosphorus 11

2233

44

55

66

Sugar Phosphate