Analysis of molecular structure of starch

Physicochemical properties/ Chemical composition Molecular structure Biosynthesis (enzymes) Genes

Molecular Structure of Amylose

Molecular Structure of Amylopectin

Average chain-length and amount (mole %) of the fractions of amylopectin unit chain

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 photometer Refractive 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 sugar Average chain length (CL) = total sugar non-reducing end sugar Average number of chain (NC) = DP/CL

Total sugar: Anthrone-H 2 SO 4 Phenol-H 2 SO 4 Reducing end sugar Modified Park-Johnson’s method Ref; 1. J. Park and M.J. Johnson, J. Biol. Chem., 181 (1949), S. Hizukuri, Y.Takeda, M. Yasuda, Carbohydrate Research, 94 (1981), Non-reducing end sugar Rapid Smith Degradation method Ref; 1. J.K. Hamilton and F. Smith, J. Am. Chem. Soc., 78 (1956), S. Hizukuri and S. Osaki, Carbohydrate Research, 63 (1978),

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 Procedure Amylopectin structure studied by HPSEC Fractionation (selective precipitation) Starch Amylose Debranched Amylopectin Molecular analyses (HPSEC) Chromatographic Techniques with Enzyme Reaction

C B A B A A B Isoamylase or pullulanase. A

Chromatographic Techniques with Enzyme Reaction Figure 6Block diagram showing the component of an HPSEC instrument. Mobile Phase Solvent Delivery System Injector |S| |M| |L| Detectors Recorder Injection of debranched amylopectin Column Chart record Retention time Response L MS

Experimental Procedure Amylopectin structure studied by HPSEC  Column:Zorbax PSM 60S (  2)  MW range:5  10 2 – 10 4  Column dimension:6.2 mm ID  250 mm  Loading size:40 μ l  Eluent:90% DMSO  Flow rate:0.5 ml/min  Pressure: <3,000 psi  Column temperature:50 o C  Standard: maltoheptaose, pullulan6000 and pullulan12000 (MW 1,170, 5,900, 11,800, respectively)

Results & Discussion Figure 7High-performance size exclusion chromatography of maltoheptaose, pullulan6000 and pullulan MW 1,170, min MW 11,800, min MW 5,900, min

Results & Discussion Figure 8Standard curve for Zorbax PSM60S (  2). Log MW = (Retention time; min) R 2 = Maltoheptaose Pullulan12000 Pullulan6000

Results & Discussion Figure 9High-performance size exclusion chromatography of isoamylolyzate of amylopectin from starches. Normal riceWaxy rice Waxy potatoNormal potato Waxy corn Normal corn

Results & Discussion Yuan 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.

Results & Discussion Figure 10High-performance size exclusion chromatography of isoamylolyzate of amylopectin from starches. Normal rice Normal corn Waxy rice Waxy potato Normal 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), 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, 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, System: HPLC Column: For amylose TSK gel G6000PW, G4000PW and G3000PW (7.5  75 mm) (Tosoh Co., Tokyo, Japan), connected in series TSK guard column PWH (7.5  75 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)

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

Chromatograms of Fluorescence-labeled Amyloses Fluorescence RI DP DP sample = (RI/F)sample (RI/F)std. x DP std.

Column for amylopectin (unit chain) Sample: Isoamylolyzate Column: Shodex OHpak SB-803HQ and SB-802.5HQ x 2 (8  300 mm) Eluent: Aq. Me 2 SO (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 Molecule Linear molecule Branch molecule Reducing end Glucose alpha-1,4 alpha-1,6 -amylolysis

Swelling of starch granule Increase viscosity of starch paste

Action of amylase on starch

Phosphorus

Sugar Phosphate