Plant Molecular Biology 38: 379–391, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
Expression of enzymatically active, recombinant barley α-glucosidase in
yeast and immunological detection of α-glucosidase from seed tissue
Brian K. Tibbot
, Cynthia A. Henson
and Ronald W. Skadsen
Department of Agronomy, University of Wisconsin, Madison, WI 53706, USA;
Cereal Crops Research Unit,
USDA, Agricultural Research Service, Madison, WI 53705, USA (
author for correspondence)
Received 17 October 1997; accepted in revised form 19 March 1998
Key words: germination, heterologous expression, maltase, Pichia, recombinant protein, starch hydrolysis
An α-glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Es-
cherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae α-factor secretory
peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recom-
binant α-glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme
hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5–6.3
with an optimum at pH 4.3, classifying the enzyme as an acid α-glucosidase. The enzyme had a K
of 1.88 mM
of 0.054 µmol/min on maltose. The recombinant α-glucosidase expressed in E. coli was used to generate
polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley α-glucosidase early in seed germi-
nation. Theirlevelsdeclined sharply later in germination, as an 81 kDa α-glucosidase became prominent.Synthesis
of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by
a 14-fold increase in α-glucosidase enzyme activity.
Abbreviations: AGL, barley seed α-glucosidase; rAGL, recombinant barley seed α-glucosidase; BMGY,
buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; GA, gibberellic acid; UTR,
The hydrolysis of starch in germinating cereal seeds
is vital for the growing embryo and is a critical
process for the malting and brewing industries. Starch
degradation is thought to occur by the concerted ac-
tion of α-glucosidase (α-D-glucoside glucohydrolase,
EC 126.96.36.199), α-amylase (1,4-α-D-glucan glucanohy-
drolase, EC 188.8.131.52), β-amylase (1,4-α-D-glucan
maltohydrolase, EC 184.108.40.206) and limit dextrinase
(debranching enzyme; α-dextrin 6-glucanohydrolase,
EC 220.127.116.11). Barley α-glucosidases (AGLs) initi-
Mention of a trademark or proprietary product does not consti-
tute a guarantee or warranty of the product by the U.S. Department
of Agriculture and does not imply its approval to the exclusion of
other suitable products.
ate degradation of native starch granules in vitro and
function synergistically with α-amylase to hydrolyze
starch granules [31, 41]. Recent studies also suggest
that AGL is important in starch metabolism in vivo.
Starch and maltose hydrolysis in germinating wheat
Bay m 1099, which caused a reduction in glucose
levels and inhibited plant growth .
We previously reported the isolation and charac-
terization of an AGL cDNA clone (pAGL.2752) from
barley seedling tissue . The deduced AGL se-
quence was 877 amino acids in length and had a
calculated molecular mass of 97 kDa and a calculated
pI of 8.2. AGLs have now been cloned from other
plants, including Arabidopsis , spinach , sugar
beet  and potato [GenBank accession AJ001374].
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