Analysis of molecular structure of starch. Physicochemical properties/ Chemical composition...
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Transcript of Analysis of molecular structure of starch. Physicochemical properties/ Chemical composition...
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