Annual Progress Report on Malting Barley Research March, 2002

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STUDIES OF STARCH DEGRADATION AND FERMENTABLE SUGAR
PRODUCTION IN MALTING BARLEY
Cynthia A. Henson
USDA-Agricultural Research Service - Cereal Crops Research Unit
and Department of Agronomy, University of Wisconsin, Madison, 53706
Four carbohydrases (α-amylase, β-amylase, α-glucosidase and limit dextrinase) are
involved in degrading starch and producing fermentable sugars during the processes of
malting and mashing. α-Amylase is the only one of these enzymes that is sufficiently
thermostable to remain active throughout the latter stages of mashing when most of the
starch is degraded. Hence, it is often said that the yield of fermentable sugars from
adjunct and malt starches is probably limited by the thermolability of some or all of the
other three enzymes.
Numerous labs around the world are working to enhance the thermostability of two of the
three labile enzymes in order to maximize starch degradation and fermentable sugar
production during mashing. Most of these labs are either creating better proteins via
mutagenesis or trying to find alleles that encode more stable or more active enzymes. The
first to succeed was Yoshigi et al. (1995) who engineered the βamy1 gene isolated from
barley to produce β-amylases with thermostabilities õ 10°C higher than the wild type
enzyme. Alleles encoding β-amylases with enhanced thermostability, albeit less than
Yoshigi and coworkers achieved via mutagenesis, have been identified by Ma et al.
(2001) in Australian germplasm and by Clark, Hayes and Henson (manuscript in
preparation) in North American germplasm. The thermostability of barley ∀-glucosidase
has been increased by 10°C relative to the wild type enzyme via site directed mutagenesis
of Agl1 (Muslin et al., 2002). To date, no alleles encoding ∀-glucosidases or limit
dextrinases with enhanced thermostability or enzymatic activities have been reported. A
project, funded by the NABGP program, to screen for variation in ∀-glucosidase alleles
has been initiated but will not be reported on here.
AMBA supported research in my lab established the basis of the thermolability of αglucosidase
which led to the successful engineering of a more thermostable enzyme
(Muslin et al., 2002). We have presented preliminary data on the thermostability of this
mutant α-glucosidase (2001 AMBA <strong>Progress</strong> <strong>Report</strong>, 2001 Barley Improvement
Conference <strong>Report</strong>). In this report I present data showing the impact of the thermostable
∀-glucosidase upon the production of fermentable (DP 1 - 3) and nonfermentable (DP 4 -
7) maltodextrins during mashing and upon the calculated RDF values. The
thermostabilities of the recombinant wild type ∀-glucosidase and the recombinant
mutated ∀-glucosidase are compared in Figure 1. The temperature at which the wild type
enzyme has only 50% of its maximal activity (T50) was 48°C whereas the T50 of the
mutant α-glucosidase was 58°C. It is noteworthy that this increase in stability was
achieved by mutating only a single base to result in a change in one amino acid. Not only
is this result atypical (Yoshigi et al.’s work required changes in 7 amino acids to achieve
a 10°C increase in stability), but it is encouraging. Since we showed that α-glucosidases
exist in three plant genera that have this base change (Muslin et al., 2002) and since a