Annual Progress Report on Malting Barley Research March, 2002

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CHARACTERIZATION OF THE PROTEIN HYDROLYZING SYSTEMS OF
MASHES AND MALTS
Berne L. Jones, Debora Fontanini and Laurie Marinac.
Cereal Crops Research Unit, USDA, ARService and
Department of Agronomy, University of Wisconsin, Madison, WI
Overall Objectives
To further define the biology and biochemistry of the protein hydrolysis processes that
occur during the malting and mashing operations of brewing. The hydrolysis of proteins
into metabolizable low-molecular-weight nitrogenous compounds is one of the most
important aspects of malting and brewing, yet there are still many aspects of this process
that are not known. This is an ongoing study to determine which proteolytic enzymes are
involved in these processes, what barley and malt endogenous inhibitors can repress the
activities of these proteinases, and which of the proteinases and inhibitors really affect the
release of ‘soluble protein’ during malting and mashing. Knowing these things will make
it possible to more scientifically and efficiently design malting barleys that are even
better suited for their particular end uses.
Purification and characterization of three cysteine proteases from green malt
Laurie Marinac and Berne L. Jones
Objective
The objective of this study is to purify and characterize some of the green malt cysteine
class proteases that have not been investigated previously. The enzymes being studied
have gelatinase activities that are stable throughout kilning and they maintain their
activities during mashing until they are inactivated during the ‘conversion’ step, when the
temperature is increased to 72°C. One of these enzymes hydrolyzes the substrate protein
gelatin in vitro at a very high rate during incubation at 40 o C. The others digest gelatin at
about the same rate as the two cysteine proteases that we have studied previously; the 30
and 31 kiloDalton proteases that were purified and characterized by Poulle (Plant
Physiol., 88: 1454_1460, 1988) and Zhang (Planta 199:565-572, 1996) respectively. All
three of the enzymes being studied, however, digest significant amounts of gelatin within
30 min of incubation at 40 o C after separation with a 2-D, IEF x PAGE analysis method,
and thus are some of the most active ones that occur in green malt extracts. The cysteine
proteases of barley are apparently the main enzymes that are responsible for breaking
down the insoluble storage proteins into low molecular weight compounds during
malting. The characteristics of the enzymes that are now being studied differ
significantly from those studied previously, and understanding the roles of the various
proteases is fundamental to understanding how the protein hydrolyzing system in barley
functions. This, in turn, is critical for gaining a more complete understanding of what
happens to the barley proteins during malting and mashing and how this affects brewing.