Annual Progress Report on Malting Barley Research March, 2002

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adjusted RDF value obtained for mashes with added thermostable α-glucosidase were
69% greater than those obtained for mashes with wild type α-glucosidase added.
This study shows that the yield of fermentable sugars from the degradation of adjunct and
malt starches can be increased by the presence of thermostable α-glucosidase.
Additionally, this work shows that the thermostable α-glucosidase produced significantly
more glucose when nongelatinized starch from the malt was present and when the
starches from the malt and adjunct were gelatinized. In other words, the thermostable αglucosidase
was a significant contributor to the production of fermentable sugars and
maltodextrins with DP 4-7 throughout mashing as long as starch and/or large
polysaccharides were present.
Additional research funded by AMBA supports the production of transgenic barley
containing the mutated α-glucosidase gene imparting thermostability. To meet this goal
we are subcloning the mutated barley α-glucosidase DNA into the pAHC25 plasmid
(Ubi:Gus:Nos/Ubi:Bar:Nos) which will later be used to transform barley calli using
particle bombardment. Site directed mutagenesis was used to add a start codon and a
Sma1 restriction site to the 5' end of the T340P/PTZ18U construct. During this stage of
the research the vendor that supplied our preferred mutagenesis kit and reagents
discontinued their production. Replacement sources of helper phage and the enzymes
required for mutagenesis have now been found and the research is again progressing.
The T340P was cut out of PTZ18U at both the 5' and 3' Sma I sites and purified using
GENECLEAN II. pAHC25 was digested with Sma I and Sac I, thus removing the GUS
gene, and purified using GENECLEAN II. The sticky end of the pAHC25 plasmid
caused by cutting with the SacI will be made blunt-ended with T4 DNA polymerase. The
T340P DNA will then be ligated into the pAHC25 vector using T4 DNA ligase. Upon
complete generation of this construct it will be sent to Dr. Lynn Dahleen who has agreed
to collaboratively produce barley (cv. Conlon) containing this gene for thermostable αglucosidase.
Publications
Clark SE, Hayes PM, Henson CA (2002) A comparison of β-amylase alleles from two
North American barley cultivars. Manuscript in preparation.
Im H and Henson CA (1995) Characterization of high pI α-glucosidase from germinated
barley seeds. Carbohydrate Research 277:145-159
Muslin EH, Clark SE, Henson CA (2002) The effect of proline insertion on the
thermostability of a barley α-glucosidase. Protein Engineering 15:29-33
Sun Z and Henson CA (1990) Degradation of native starch granules by barley αglucosidases.
Plant Physiology 94:320-327
Sun A and Henson CA (1991) A quantitative assessment of the importance of α-amylase,
β-amylase, debranching enzyme and α-glucosidase in starch degradation. Archives of
Biochemistry and Biophysics 28:298-305