Extraction, Isolation, and Characterization Process of Phytoglycogen from Varieties of

SU-1 Maize and Rice Grain

Prepared by: ChE-5201
Rationale
Phytoglycogen is a highly branched soluble -glucan found in plants, particularly those
with decreased activity of isoamylase-type starch debranching enzyme. Owing to its unique
structure and properties, the glucose dendrimer phytoglycogen is gaining interest for medical and
biotechnology applications. Although many maize variants are available from commercial and
academic breeding programs, most applications rely on phytoglycogen extracted from the
common maize variant, sugary-1. An improved technique has been designed to extract and
isolate phytoglycogen from the grain of su-1 maize and rice with minimal degradation for
structural characterization. The structures of extracted phytoglycogen samples were analyzed
using size-exclusion chromatography (SEC, also termed GPC) and transmission electron
microscopy (TEM) and compared with the structure of pig liver glycogen. The SEC weight
molecular size distributions indicate that the extraction procedure with protease is most effective
in obtaining pure phytoglycogen from grain. The extracted and purified phytoglycogen samples
from grain contain wide distributions of molecular sizes (analyzed by SEC and TEM), with the
smallest being “individual” particles, which collectively form larger particles; the latter are
dominant in the phytoglycogen samples examined here. The results show that phytoglycogen is
similar to liver glycogen in both the range of molecular size distribution and in the presence of
particles.

Objectives
 To be able to extract phytoglycogen from mutated SU-1 strand of maize using enzymatic
process
 To be able to apply the methodology to the rice strain having the same variant of mutated
SU-1
 To be able to characterize the extracted phytoglycogen

Materials and Method

Some of the kernels were ground and used for phytoglycogen extraction from grain. The
kernels were ground to a fine powder in a cryo-mill; 1 min precooling followed by 5 min
grinding. This cryogrinding technique has been shown to minimize the mechanical and heat
degradation on starch molecules that can occur during dry grinding at ambient temperatures
(Syahariza et al., 2010).
Glycogen was extracted from pig livers and purified as described in a previous study
(Sullivan et al., 2012). The structure of the liver glycogen is used here as a comparison to the
structure of phytoglycogen extracted from leaves and grain.
Extraction of Phytoglycogen from Grain
Kernel flour (100 mg) was weighed into a centrifuge tube. Phytoglycogen from each
sample was collected as water-soluble extract after the kernel flour had been incubated in 2.5 mL
aqueous solution for 30 min. Incubation treatment conditions used was protease (2.5 units/mL;
bacterial type XIV) in tricine buffer (pH 7.5, 250 mM) at 37 ◦C. These extraction method was
modified from that used to extract starch molecules from grain flour (Syahariza et al., 2010).
Although the protease treatment may hydrolyze the binding between particles, potentially
mediated by protein or peptide bonds, in forming the larger particles of phytoglycogen, the
results from a previous study (Sullivan et al., 2012) indicate that this protease treatment does not
significantly affect the molecular size distribution of liver glycogen containing particles. The fact
that particles are not noticeably degraded by the protease treatment can be clearly observed from
the molecular size distributions of phytoglycogen with and without the protease treatment. An
additional 2.5 mL ice-cold tricine buffer was added to each sample after incubation followed by
centrifugation at 4000 × g for 10 min, and the supernatant containing the water-soluble solids
was collected. The phytoglycogen in the supernatant was precipitated using approximately four
volumes of absolute ethanol then centrifuged at 4000 × g for 10 min, and the precipitate was
dissolved in 1.5 mL dimethyl sulfoxide solution containing 0.5% (w/w) lithium bromide
(DMSO/LiBr) overnight in a water bath at 80 ◦C to yield a complete dissolution of the
phytoglycogen molecules (Schmitz, Dona, Castignolles, Gilbert, & Gaborieau, 2009). The
dissolved sample was centrifuged, and the supernatant was collected. Ethanol precipitation and
centrifugation were repeated, and the phytoglycogen precipitate was re-dissolved in 0.5 mL
DMSO/LiBr solution in a thermomixer at 80 ◦C for 2 h and occasionally inverted by hand. The
sample was then centrifuged once more, and the supernatant was collected and stored in a vial
for SEC analysis. Samples were dissolved in DMSO/LiBr solution as this solvent, coupled with
heating at 80 ◦C and shaking, has been shown to completely dissolve starch molecules with
minimal degradation (Schmitz et al., 2009; Syahariza et al., 2010).

Reference:
 Prudence O. Powell, Mitchell A. Sullivana, Michael C. Sweedmana, David I. Stapleton,
Jovin Hasjim, Robert G. Gilbert, September 2013 from Extraction, Isolation and
Characterization of Phytoglycogen from SU-1 Maize Leaves and Grain
 Min-Soo Yun, Takayuki Umemoto, and Yasushi Kawagoe, May 2011 from Rice
Debranching Enzyme Isoamylase3 Facilitates Starch Metabolism and Affects Plastid
Morphogenesis
 Renjie Liu, Susan K. Boehlein, William F. Tracy, Marcio F. R. Resende, Jr. and Gregory
A. Hudalla, January 2020 from Characterizing the Physical Properties and Cell
Compatibility of Phytoglycogen Extracted from Different Sweet Corn Varieties