Endoproteases of Barley Malt PDF

Journal of Cereal Science 42 (2005) 139156
www.elsevier.com/locate/jnlabr/yjcrs
Review
Endoproteases of barley and malt

Berne L. Jones*
RR1, Box 6, Kooskia, ID 83539, USA
Received 24 December 2004; revised 1 March 2005; accepted 1 March 2005
This review is dedicated to Dr Juhani Mikola, whose work laid the bases for many of our later studies on barley and malt endoproteases and their endogenous
inhibitors. I never had the pleasure of meeting him, but if not for his early death, I expect that a very large percentage of the references in this article would have
been to his work.
Abstract
During seed germination several seed biopolymers, including the storage proteins, must be hydrolysed to provide biochemical building
blocks for the growing seedling. This process is particularly important in barley because under the guise of malting, it forms the basis of the
malting and brewing industries. The steps involved in the enzymatic formation of soluble protein during malting and in the mashing phase
of brewing are still not well understood. The barley proteins are initially solubilized by endoproteases and then further degraded by
exopeptidases. The cysteine-class proteases probably play the most important roles, but their contributions are likely not as overwhelming as
was thought previously. The metalloproteases are apparently also important players in protein solubilization, although their contributions
have scarcely been examined. The characteristics of the purified aspartic class proteases imply that they are not important contributors to
protein solubilization, but recent mashing studies indicate that they probably do play a minor role. All indications are that the barley and malt
serine class proteases are not directly involved in storage protein hydrolysis during malting/mashing. More studies are needed to clarify the
roles of the aspartic- and metalloproteases. One important aspect of further studies should be to ensure that appropriate biochemical methods
are used, as well as conditions that are truly appropriate to commercial malting and mashing processes.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Endoproteases; Proteases; Barley; Malt; Mashing; Brewing; Inhibitors; Seed germination; Protease analysis
1. Introduction
During and after the germination of barley seeds, many
of the seed biopolymers must be broken down into their
component subunits for use by the growing plant. One of the
most important of these processes is the hydrolysis of
proteins into peptides and amino acids. In addition to being
a critical step for perpetuating species via seeds, this protein
Abbreviations 2-D, two dimensional; ASBC, American Society of
Brewing Chemists; 2-ME, 2-mercaptoethanol; E-64, (trans-epoxysuccinylL-leucylamido-(4-guanidino)butane); EP-A and -B, cysteine-class endoproteases -A and -B; DTT, dithiothreitol; FAN, free amino nitrogen; HvAP,
Hordeum vulgare aspartic protease; IEF, isoelectric focusing; kD, kilo
Dalton; MEP-1, malt cysteine-class endopeptidase 1; MP, metalloproteases; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; SEP-1, serine endopeptidase-1; SP, soluble protein.
* Tel.: C1 208 926 4429.
E-mail address: bhjones@bmi.net.
0733-5210/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2005.03.007
hydrolysis is critical to the brewing and malting industries,

which are based on the preparation and use of malt. During
the malting process, barley is germinated via a carefully
controlled procedure so that its biopolymers, which cannot
be utilized by yeast for growth and ethanol production, are
degraded to sugars, amino acids and other low Mr
compounds which can be used in the brewing process.
Much of the research in this field has been performed using
various malting procedures, but the findings also apply to
the general germination process.
During malting, enough of the barley protein complement must be degraded to amino acids and small peptides
to provide sufficient nutrients for brewing yeasts to grow
rapidly and to metabolize sugars into alcohol. Presumably,
most of the proteins that are being degraded will be storage
proteins, which are mainly hordeins. There is, however, no
reason to think that various albumins and globulins are also
not degraded during malting. The complete degradation of
all of the barley proteins is not desirable because too little
protein in beer (the main product made from malt) can result
140
B.L. Jones / Journal of Cereal Science 42 (2005) 139156
in a product that has insufficient foaming ability, mouthfeel

and other required characteristics. The American Malting
Barley Association, which is representative of the malting
and brewing industries in the USA, currently suggests that
between 42 and 47% of the protein of a commercially
acceptable malt sample should be solubilised by the end of
the mashing process (American Malting Barley Association,
2004).
Most of the research discussed in this review was
conducted to obtain information about the biochemistry of
malting so that the process of breeding improved malting
barleys can be made more efficient and to enable maltsters
and/or brewers to improve their processing methods.
Some endoproteases from the leaves of barley plants
have been studied in detail (Runeberg-Roos et al., 1994;
Sundblom and Mikola, 1972; Thayer and Huffaker, 1984),
but they will not be discussed because they do not directly
affect grain or malt characteristics.
2. Some definitions
2.1. SP; soluble protein
The amount of malt protein that is dissolved at the end of
the mashing process. This value is determined either by
measuring the nitrogen content of an extract or its UV
absorbance and includes soluble proteins, peptides and
amino acids. A mole of protein will contribute more to this
value than will a mole of amino acid, since it will contain
multiple nitrogen atoms and UV-absorbing groups. The SP
of an extract is normally presumed to indicate its content of
proteins and large peptides, which contribute to the foaming
ability, mouthfeel and other physical properties of beers.
depending on what attributes are wanted in the final wort

and beer. When the mashing is performed experimentally to
measure the characteristics of a malt sample, using
conditions that, in the USA, are strictly specified by the
American Society of Brewing Chemists (American Society
of Brewing Chemists, 1992, method Malt-4), the final
product is called an extract. The process conditions used to
produce extracts and worts differ significantly, although the
terms are sometimes used interchangeably. Generally, the
initial mash temperature is held constant for a time at a
relatively low level, and this step is called a protein rest.
The mash temperature is normally then increased at a
constant rate (ramping) and then held constant, usually at
around 70 8C (conversion), until all of the starch has been
hydrolysed to fermentable sugars.
2.4. Malting
The process of preparing malt. Barley grain is soaked
until germination begins (steeping), is then held under moist
and warm conditions for several days (germination) and
finally is dried in a stream of air whose temperature is
slowly raised (kilning). The method that is normally used at
the USDA Cereal Crops Research Unit is described in Jones
et al. (2000). When grain is germinated in this way, but not
kilned, the product is called green malt. Malting
conditions can be varied, depending on the malt characteristics needed. Barley grain that is germinated on filter paper
or some other medium and air-dried or freezedried is not
malt, but is simply germinated barley. It should be
remembered that the biological germination of barley
begins during the steeping process and is already well
established when what maltsters call germination begins.
2.2. FAN; free amino nitrogen
2.5. Class-specific proteases
The amount of NH2 groups in an extract, as measured

by their interaction with a reagent that reacts specifically
with this group. The reagent detects proteins, peptides and
amino acids, but since the large proteins, intermediate
peptides and small amino acids each contain only a single
terminal NH2 group, a given weight of amino acid will be
detected as having much more FAN than an equivalent
weight of protein. FAN is therefore assumed to denote the
amount of amino acids and small peptides in a wort. Since
brewing yeasts can only metabolize these amino acids and
small peptides, it is an indication of the nutritional value of
an extract to the yeast.
Most endoproteases fall into one of four classes. The

catalytic mechanisms of these classes differ and the
members of each are specifically inhibited by different
chemicals. For example, the cysteine proteases [EC
3.4.22.-] contain the amino acid cysteine at their active
centers and are specifically inhibited by a compound called
E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane). The other protease classes and their commonly used
specific inhibitors are the aspartic proteases [EC 3.4.23.-],
inhibited by pepstatin A, serine proteases [EC 3.4.21.-],
phenylmethylsulfonyl fluoride, or PMSF and metalloproteases [EC 3.4.24.-], 1,10-phenanthroline, or o-phenanthroline, and sometimes EDTA.
Throughout this review the terms protease or endoprotease have been used to cover the terms proteinase and
endoproteinase when refering to enzymes that hydrolyse
internal peptide bonds in proteins. The terms exopeptidase
and peptidase will refer to enzymes that hydrolyse either
the N- or C-terminal peptide bonds of polypeptides.
2.3. Mashing, extract, wort

When ground malt is extracted with water whose
temperature is increased in a carefully controlled manner,
the industrial process is called mashing, and the product
is termed wort. The mashing processes used will vary,
2.6. 2-D IEF!PAGE

A separation method in which the components of extracts
are resolved by isoelectric focusing (IEF), after which the
IEF gels are incorporated at the top of non-denaturing
polyacrylamide gel electrophoresis (PAGE) gel slabs so that
PAGE separations can be performed in the second
dimension (Zhang and Jones, 1995a). To detect proteolytic
activities, the second dimension (PAGE) gel normally
contains a proteinase substrate, most often gelatin.
3. Early studies
Most studies performed on the proteolytic activities of
barley and malt prior to 1980 are not discussed in detail,
because they used preparations that were poorly characterized and contained complex mixtures of proteases. These
studies were, however, quite important, because they laid a
strong foundation for further, more specific studies. For
example, they showed that the proteolytic activity of mature
barley grain was low, but that during malting the activity
increased greatly (Burger and Prentice, 1970; Enari et al.,
1964; Kringstad and Kilhovd, 1957). It was also shown that
barley, and especially malt, contained several proteases
(Burger, 1973; Burger and Prentice, 1970; Enari et al., 1964;
Enari and Mikola, 1968) although the number of malt
proteases was still underestimated. These studies indicated
that the solubilization of barley proteins to SP was catalyzed
by the endoproteases, and that the SP polypeptides were
then further hydrolysed by exopeptidases to yield FAN
(Mikola, 1983). It appeared that the cysteine class proteases
catalyzed most of the SP release (Burger, 1973; Enari et al.,
1964; Enari and Mikola, 1968; Sundblom and Mikola,
1972), but that aspartic- (Morris et al., 1985) and
metalloproteases (Enari et al., 1964; Enari and Mikola,
1968; Sundblom and Mikola, 1972) were also probably
involved. It was found that SP was released during both
malting and mashing, although the contributions of each of
these procedures to the final mash SP values varied
considerably, depending on the processing and analytical
methods used (Barrett and Kirsop, 1971; Burger and
Schroeder, 1976a).
4. Meaningful protease assays

The protease activities of barley and malt have been
measured in many ways in the past, and the results obtained
from a given study can vary greatly, depending on how the
measurements are made. This makes it imperative that, to
get meaningful results, appropriate analytical methods and
conditions must be used. This has not always been the case.
Some of the methods used were: measuring the amino
acids released during mashing (Jones and Pierce, 1967a,b)
or from a hordein preparation (Baxter, 1976; Phillips and
141
Wallace, 1989), measuring the reduction in viscosity of a

gelatin solution (ten Hoopen, 1968), and following the
hydrolysis of either radioactively-labeled proteins (Morris
et al., 1985), synthetic peptides (Suolinna et al., 1965),
derivatized amino acids (Galleschi and Andreoni, 1990), or
even the protein layer on a photographic film (Burger and
Schroeder, 1976b).
Some questions need to be asked about these and similar
methods to ensure that they gave meaningful and relevant
results. Among these are:
(1) Was the right endoproteinase substrate used? For
years researchers have been seeking easier and faster ways
to measure endoproteinase activities. One of the most
common has been to use a small peptide or a peptide
analogue as a substrate instead of a protein. These methods
have the advantage that the hydrolysis products are easy to
quantify, compared to the polypeptide mixtures that are
produced from proteins. However, the results obtained using
these unnatural substrates may not reflect the actual
reactions that occur when the enzymes hydrolyse proteins.
For example, Jones and Poulle (1990) characterized the
hydrolytic specificity of a purified 30,000 Mr green malt
endoproteinase using as substrates two small purified
proteins with known amino acid sequences that occur
naturally in barley. In 1989 Phillips and Wallace isolated a
very similar or identical proteinase, and determined its
specificity using low Mr amino acid esters. The specificities
obtained by these two methods were very different, although
the enzymes appeared to be quite similar otherwise. The
discrepancy was most likely due to the fact that the low Mr
substrates, because of their very small sizes, did not contain
the amino acid residues that really defined the specificity of
the proteinase. It seems obvious that the best experimental
substrate should be the one whose structure is most similar
to that of the enzymes natural substrate; that is, a protein.
But what protein substrate should be used? Not all
proteins are equally useful for measuring proteolytic
activities. This is especially true in the case of malt,
where multiple proteases are present whose specificities
differ. Thus azocasein, for example, is a poor substrate for
green malt proteinase extracts (Phillips and Wallace, 1989)
and we have found that when malt proteases are separated
by 2-dimensional (2-D) electrophoresis only a few of the
separated enzymes hydrolysed either azocasein or haemoglobin, two commonly used substrate proteins. Thus they
are not appropriate for measuring the multiple activities of
barley and malt. We have found that the best substrates for
measuring malt proteases are gelatin or its colored
derivative, azogelatin. These proteins are readily hydrolysed
by serine-, cysteine- and metalloproteases from malt and
other sources. They are more slowly degraded by most
aspartic class proteases (Jones et al., 1998), including those
of malt. These latter enzymes are best analyzed using the
substrate edestin. A major advantage of using gelatin or
azogelatin substrates is that they are soluble at pH values
ranging from 3.0 to 10.5, and thus can be used to measure all
142
of the various malt proteases (other than the aspartic

proteases) whose pH optima cover this entire pH range.
Most of the proteins that have previously been used as
substrates are not soluble throughout this extended pH
range.
(2) When substrates are used in different physical forms,
how does this affect the hydrolysis results? For example,
several studies have been performed with the substrate
gelatin in its soluble form (ten Hoopen, 1968; Jones et al.,
1998), as a layer on a plastic backing (Burger and
Schroeder, 1976b), and as, most likely, a suspension of
molecules entrained in an acrylamide matrix (Zhang and
Jones, 1995a). Little or nothing is known about how these
different forms of gelatin affect its susceptibility to
hydrolysis. We have performed hydrolyses using both
gelatin and azogelatin substrates in solution and also
entrapped inside acrylamide gels and have occasionally
observed differences in the hydrolytic actions of malt
proteases on these substrates (Jones and Budde, 2003).
(3) Can natural proteins be used as substrates? Because
barley storage proteins (hordeins) are the major proteins
hydrolysed during malting and mashing, it would be helpful
to be able to use these proteins as substrates in enzyme
characterizations. However, by definition hordeins are
insoluble in both water and dilute salt solutions, conditions
that are normally used for enzyme studies. The polymeric
hordeins (hordenins) are even less soluble under these
conditions. Thus for hordeins to be used as substrates in
vitro, they must either be rendered soluble or the substrate
must be present as a suspension, rather than in solution. To
solubilize hordein fractions requires very harsh treatments
and after solubilization the proteins are, by definition, no
longer hordeins and thus are no longer truly natural
substrates. Even if the hordeins are simply extracted from
the grain and used as a suspension the extraction process is
still quite vigorous and then the problem arises of whether
the suspended solid hordein is hydrolysed differently from
hordein in solution.
When protein solubilization occurs during malting and
mashing, the initial hordein hydrolysis presumably involves
a depolymerisation of the proteins that are still in their
native states. As the depolymerisation continues (as the
grain is modified) and the internal structure of the grain is
lost, the environment in which the hordeins reside changes,
and their molecular structures may also be modified.
Buchanan and Kobrehel and their collaborators (Besse
et al., 1996) have proposed that during grain modification
some of the disulphide bonds in hordein may become
reduced, rendering the proteins more soluble. However, gel
electrophoresis has indicated that the hordeins remain
relatively unchanged during malting, with the individual
protein bands simply becoming fainter and fainter as
malting proceeds (Marchylo and Kruger, 1985; Poulle and
Jones, 1988; Smith and Simpson, 1983). However to
perform these electrophoretic analyses the hordein samples
had first to be solubilized, which probably also modified
their structures. Although the hordein extracts used as

endoproteinase substrates do not really contain native
hordein molecules, presumably their amino acid sequences
are unaltered, although their secondary, tertiary and/or
quaternary structures have probably been changed. In any
case, when hordein suspensions have been used as
substrates some purified proteases hydrolysed them and
others did not, and this has been used as a major criterion of
whether a given enzyme is involved in solubilizing storage
proteins in vivo.
(4) Have characterizations been performed at the
appropriate pH values? It has been noted since the earliest
studies that the different barley/malt endoproteases showed
maximal activities at different pH values, with the greatest
activity of extracted enzyme mixtures occurring at low pH
levels. Subsequently, many proteases have been studied at
pH values between 3 and 4. However, the pH of the
endosperm of germinating barley is about 4.9 (Mikola and
Virtanen, 1980), and that of a North American mash is
around 5.86.0 (Jones and Budde, 2003), so many of the
data from those early studies are not really relevant to the
events that occur during either malting or mashing (see
Section 7.3). A similar problem has arisen due to the adding
of reducing agents to enzyme extracts and to their analysis
mixtures. It has been shown recently that the enzymatic
hydrolysis of protein substrates by malt enzymes is strongly
enhanced in the presence of reducing agents and lowered by
oxidizing agents (Jones and Budde, 2003). Nearly all earlier
barley/malt proteinase analyses were performed in the
presence of added reducing agents, so many of the results
are probably not indicative of what really occurs during the
processing of barley and malts. Specific examples of these
problems are discussed in Section 7.4. To ensure that their
results are relevant, researchers hoping to apply their
findings to real systems need to be aware of how those
systems operate and to ensure that they use appropriate
conditions and techniques.
(5) Are meaningful data being collected and reported?
One of the most basic aspects of enzymology involves the
measurement of initial rates of enzyme catalyzed reactions,
and holds that this measurement should be made, if at all
possible, while the reaction rate is still linear. To ensure this,
multiple measurements of the concentrations of either the
reactants or products must be made and it must be shown
that these reaction components are utilized or released at a
constant rate throughout the measurement period. Unfortunately, this basic enzymological principle is often overlooked, and the cereal endoproteinase literature is replete
with reports of inappropriately measured activities. The
hydrolysis rates of most endoproteinase-catalyzed reactions
performed in solution are generally linear for 30 min or less
(Jones et al., 1998), so any data obtained using a single
measurement made several hours after a reaction has been
started, without previously proving that the rate is constant
throughout that period, are at best only semi-quantitative.
5. Purified barley/malt endoproteases

Between about 1985 and 1990, there was a burst of
publications that described the purification and characterization of individual proteases from barley tissues or malt.
5.1. Aleurain
In 1985, Rogers and co-workers (Rogers et al., 1985)
isolated and sequenced a cDNA clone from gibberellintreated barley aleurone cells that apparently encoded a 361amino acid protein. Because of the similarity of its amino
acid sequence with those of cathepsin H and two plant thiol
proteases, they concluded that the protein was a thiol
endoproteinase, and named it aleurain. It was speculated
that it might be the proteinase that was being studied
concurrently by Hammerton and Ho (1986). However, when
barley leaf aleurain was finally purified and characterized, it
was shown to be an aminopeptidase, rather than an
endoproteinase (Holwerda and Rogers, 1992), so it is
unlikely to play any major part in solubilizing grain storage
proteins. This is but one of many examples of a
phenomenon that researchers too often forget: that although
amino acid or DNA base sequence homologies can indicate
possible biochemical similarities, until the protein is
isolated and characterized, nothing is proven.
5.2. Cysteine-class proteases
5.2.1. EP-A and EP-B
Hammerton and Ho (1986) showed that gibberellic acidtreated barley aleurone layers synthesized several proteases
with Mr of w37,000, that were inhibited by cysteine
protease inhibitors, and that hydrolysed extracted barley
storage proteins. Two of these enzymes, called endoproteases-A (EP-A) (Koehler and Ho, 1988) and -B (EP-B)
(Koehler and Ho, 1990a) were purified. The EP-A
preparation had a Mr of 37,000, hydrolysed internal peptide
bonds in substrate proteins, had a pH optimum of 5, and
contained three very similar isozymes. The enzyme(s) was
not aleurain, but was apparently closely related to papain, a
papaya (Carica papaya) proteinase. The purified EP-B had a
Mr of 30,000, contained two proteins with pI values of 4.6
and 4.7, and behaved as a cysteine endoproteinase on the
substrate haemoglobin. Its N-terminal amino acid sequence
and properties were similar to those of EP-A, and both EP-A
and -B produced similar polypeptides when they hydrolysed
a hordein preparation (Koehler and Ho, 1990a).
The cDNA cloning of EP-B has been performed and
reported (Koehler and Ho, 1990b). Three clones were
obtained, two of which encoded EP-B isozymes that
were 98% similar and showed large preprosequences.
These were converted via a multistep process into the
mature enzymes. The processing of proEP-A was less
complicated and the final form of the enzyme was secreted
from the aleurone tissue (Koehler and Ho, 1990b).
143
Gibberellin stimulated the aleurone to produce both EP-A

and -B, but EP-B was synthesized more quickly than EP-A.
Not surprisingly, the mRNA for EP-B was essentially absent
from mature barley grain, but increased strongly within 1 or
2 days of germination. Initially, the EP-B was expressed in
the scutellar epithelium and aleurone cells adjacent to the
embryo. Later the mRNA concentration was highest in the
aleurone layer adjacent to the endosperm, and, with time, its
concentration increased along the length of the grain to its
distal end (Marttila et al., 1993). The concentration of newly
synthesized EP-B protein showed a similar distribution.
These findings are all consistent with endoproteinase EP-B
being one of the major enzymes responsible for degrading
barley storage proteins during seed germination. Mikkonen
et al. (1996) found evidence for only two EP-B genes in
barley, both residing on chromosome 3. The hormonal
regulation of one of the barley EP-B genes has since been
described (Cercos et al., 1999).
5.2.2. Malt endopeptidase 1 (MEP-1)
Phillips and Wallace, working at the same time as
Koehler and Ho, but using green (unkilned) malt, found that
the predominant proteinase activity under their assay
conditions, was also a cysteine class protease (Phillips and
Wallace, 1989). They purified and characterized the
enzyme, named it MEP-1, and showed that it hydrolysed
hordein in suspension. The hordein had been solubilized
with 50% alcohol and, as discussed in Section 4, was
presumably not physically the same as native hordein. In
addition, only a single reaction sample, taken after 1 h of
incubation, was analyzed, so it seems likely that the values
reported are not true initial reaction rates. The enzyme also
hydrolysed azocasein and haemoglobin, but again only
single 2 h reaction samples were analyzed. The purified
MEP-1 migrated on SDS-PAGE as a single band of Mr
29,000, but on isoelectric focusing it separated into two
components with pI values of 4.2 and 4.3 (Phillips and
Wallace, 1989).
When MEP-1 hydrolysed a series of N-t-butoxycarbonylL-amino acid-p-nitrophenyl ester substrates containing
various amino acid residues, the hydrolysis rates were:
GlnOAlaOLeuOTyrOTrpOAsnOPhe. The hydrolysis of
hordein was strongly increased by the addition of the
reducing agent 2-mercaptoethanol (2-ME), whereas the
hydrolysis of azocasein and of the synthetic arginine
substrate were only weakly increased. An antibody raised
against MEP-1 also cross-reacted with a Mr 37,000
endoproteinase. It was later reported (Guerin et al., 1992)
that the amino acid sequence of the first 20 residues of MEP1 was identical with that of the EP-B studied by Ho and his
collaborators, and that the gene encoding MEP-1 was
located on the long arm of chromosome 3 of the barley cv
Betzes.
Although there are some discrepancies between
Wallaces enzymes and those studied by Ho, it seems
obvious that MEP-1 and EP-B are the same, or very closely
144
related, proteases. Among the more important outcomes of

Wallaces study were the findings that MEP-1 (EP-B) and,
probably, EP-A were present in germinating barley, as well
as in the isolated aleurone tissue, and that the purified green
malt enzyme could hydrolyse extracted barley storage
proteins.
5.2.3. The 30 kD proteinase of green malt.
At the time the EP-B and MEP-1 enzymes were being
studied, Jones and Poulle independently purified and
characterized a proteinase from green malt that they
designated the 30 kD endoproteinase. The native 30 kD
enzyme had an apparent Mr of 20,000 from gel filtration
analysis, but SDS-PAGE indicated that it was a single
polypeptide of Mr 30,000 (Poulle and Jones, 1988). It had a
pH optimum of 3.8 and was a cysteine class enzyme that
hydrolysed several large proteins. Its amino acid composition was quite different from that of aleurain.
The purified enzyme rapidly hydrolysed all three barley
hordein classes (B, C and D), with the B and D hordeins
being degraded faster than the C proteins (Poulle and Jones,
1988). When a mixture of B and D hordeins prepared by
HPLC was used as substrate, a few discrete, intermediate
sized peptide reaction products were detected by PAGE, but
most of the hordein was hydrolysed to peptides that were too
small to be retained on the acrylamide gel. The changes that
occurred in the SDS-PAGE patterns of a hordein preparation that was hydrolysed by the purified 30 kD protease
were very similar to those seen in hordein samples that were
removed from barley undergoing malting, indicating that
the purified enzyme was behaving like those that were
active during malting. Immunomicroscopy of aleurone
tissues (Marttila et al., 1995) showed that after synthesis
this enzyme was transported to the starchy endosperm,
where the storage proteins are located. From the similarities
shared by the 30 kD, EP-B and MEP-1 enzymes, it seems
apparent that they were the same endoproteinase.
5.2.4. The 31 kD proteinase of green malt.
Zhang and Jones (1996) purified and characterized a
second endoproteinase from 4-day germinated green malt.
It was also a cysteine protease, with a pI of 4.4, was
hydrolytically most active at pH 4.5, and its SDS-PAGE
molecular mass was about 31,000. It hydrolysed gelatin,
azocasein, haemoglobin, edestin and a hordein preparation. The 31 kD proteinase was one of five proteins in a
crude malt extract that reacted with antibodies raised
against the 30 kD proteinase of Poulle and Jones (1988).
Even though they were well separated on non-denaturing
Western blot acrylamide gels, all five cross-reacting
proteins migrated to the Mr 30,00031,000 zone of gels
that contained SDS and reducing agent (Zhang and Jones,
1996). The sequence of the N-terminal nine amino acids
of the 31 kD protein was identical to that of one of the Mr
37,000 EP-A isozymes studied by Koehler and Ho (1988),
but the 31 kD enzyme did not cross-react with antibodies
raised against EP-A. Conversely, its N-terminal amino

acid sequence differed from that of the Mr 30,000 EP-B,
but it did cross-react with antibodies raised against the
30 kD malt proteinase of Poulle and Jones (1988), which
was very similar to EP-B.
5.2.5. Hydrolytic specificities of the cysteine proteases
To evaluate its specificity, purified 30 kD green malt
proteinase was used to hydrolyse two small barley proteins,
the a- and b-hordothionins, into peptides (Jones and Poulle,
1990). As the native forms of the hordothionins were
intractable to hydrolysis by the 30 kD enzyme and other
proteases, they were reduced and alkylated prior to use.
Hydrolyses were performed for varying periods and
analyses of the resultant peptides defined the exact bonds
hydrolysed by the protease and their relative rates of
hydrolysis. It was apparent that the principal specificity of
the 30 kD enzyme was not defined by either of the amino
acids directly involved in forming the hydrolysed peptide
bond. The enzyme specifically cleaved hordothionin peptide
bonds between the P1 and P 0 1 residues of hydrolysis sites
that had the general formula
NH2 /P2 K P1 YKP10 K P20 /COOH
when the amino acid residue located at the P2 site was either
Leu, Val or Tyr, with Leu specifying the fastest hydrolysis.
Because hordothionin substrates contained no Ile or Trp,
and the single Phe was located at the C-terminus, and thus
not available for endoproteolytic hydrolysis, the effects of
these other large aliphatic or aromatic residues could not be
tested. Although the amino acid in the P2 site was the major
factor for determining hydrolysis, the residues occupying
the P1 and/or P10 sites also seemed to have some small effect
on the hydrolyses (Jones and Poulle, 1990).
The specificity of the 31 kD malt proteinase was
determined similarly, using as substrates reduced and
alkylated b-purothionin and several other polypeptides
that were chosen to ensure that one or more of them
contained each of the 20 common amino acids (Zhang and
Jones, 1996). b-Purothionin is a wheat protein that is
homologous to the barley hordothionins. Analysis of the
resultant peptides showed that the specificity of the 31 kD
enzyme was similar to that of the 30 kD enzyme; hydrolysis
was determined mainly by the amino acid occupying the P2
position relative to the bond that was hydrolysed. The
effectiveness of amino acids for specifying hydrolysis was
TrpOPheOLeuOIleOValOTyrOAla, which was identical to that found with the 30 kD protease, except that Ala did
not specify hydrolysis with the 30 kD enzyme. This order is
very nearly the same as that of the hydrophobicities of the
amino acids at pH 7 (Creighton, 1984). As with the 30 kD
enzyme, the presence of the large hydrophobic amino acid
pyridylethylcysteine at the P2 site was not enough to
promote hydrolysis, but hydrolysis did occur when two such
residues were located together at positions P2 and P3.
Davy et al. (1998) confirmed these results by hydrolysing

a recombinant C hordein molecule with EP-B (Mr 30,000),
and a group of synthetic peptide derivatives with both EP-A
and EP-B. The hydrolytic sites were primarily specified by
the amino acids at the P2 position, in the order LeuOPheO
Val[ProOSer. They also found that hydrolysis was
severely restricted when Pro was present at either the P1
or the P10 sites. This imposes a rather strict limitation on the
number of sites on barley storage proteins that are available
for hydrolysis, since they are all proline-rich proteins. Some
secondary hydrolysis sites were detected, but it was not
clear what specified hydrolysis at these locations. In a
second study using a larger and more directed set of
synthetic peptides, Davy et al. (2000) again found that the
major specifying site was at residue P2 for both EP-A and
EP-B. The amino acids listed above for the 31 kD enzyme
were most effective, except that Ala was replaced by Met.
Specificity due to Met might have been overlooked in the
study of the 31 kD enzyme (Zhang and Jones, 1996) because
it was not present in the studied peptides at locations where
its effect would have been readily discerned. The hydrolysis
of peptides that were designed to highlight any specificity
that was due to the amino acids at the substrate P1 site
showed that the residue at this position had only a small
effect on the hydrolysis rates, except that when Pro was
present, no hydrolysis occurred.
When the hydrolyses of wild type and mutated forms of a
recombinant hordein C molecule by EP-B were compared,
the hydrolysis of the wild type protein was again determined
by the residues at the P2 site, while the hydrolysis of the
mutated protein molecule, whose P2 specifying sites had
been removed, occurred at a very slow rate (Davy et al.,
2000). One important finding from this study was that the
hordeins that were present in isolated protein bodies were
hydrolysed by the EP-B, adding credence to the hypothesis
that the malt cysteine proteases really do hydrolyse storage
proteins in vivo.
5.2.6. Cysteine proteinase summary
From a comparison of their characteristics, it appears that
the aleurone-derived proteinase EP-B is probably identical
with the MEP-1 and 30 kD proteases isolated from green
malt. The 31 kD protease apparently differs from both EP-A
and EP-B, but is quite similar to both. The 30 and 31 kD
proteases are probably isoenzymes, and it seems likely that
malt contains at least three other very similar enzymes. All
of these purified endoproteases were reported as being
major activities in either barley or malt, presumably being
the main proteases responsible for degrading storage
proteins. However, this is not necessarily the case, because
in essentially all cases the enzyme purifications and activity
assays were performed in the presence of added reducing
agent, usually 2-ME. As is discussed in Section 7.4, the
addition of reducing agents to these particular enzymes
greatly increases their activities. Thus, the 2-ME addition
would have enhanced their activities relative to those of
145
other enzymes that were present and thereby led to the

conclusion that they play a bigger role in vivo than in fact
they do. In addition, most of the enzyme assays were
performed at pH values lower than the pH 4.85.0 of
germinating barley seeds (Henson, C., personal communication; Mikola and Virtanen, 1980). Because the cysteine
proteases are most active at these low pH values, whereas
the metallo- and serine endoproteases are most active at
higher pH values, this would also make it appear that the
cysteine proteases were relatively more important to seed
germination than they really are. It is, however, probably
significant that the specificities of these enzymes are
admirably adapted for quickly digesting storage proteins
to relatively small peptides. These can then serve as
substrates for the exopeptidases that release the amino
acids that are required by germinating seeds and by brewers
(Zhang and Jones, 1996).
5.3. Aspartic endoproteases
5.3.1. HvAP
Doi et al. (1980) showed that dormant rice seeds
contained an acid protease that was inhibited by pepstatin,
indicating that it was an aspartic proteinase, and Belozersky
et al. (1989) purified and characterized aspartic proteases
from seeds of wheat and buckwheat (Fagopyrum esculentum), a pseudo-cereal (Belozerskii et al., 1984). This led
Sarkkinen et al. (1992) to purify a similar enzyme from
barley seeds. Their preparation contained two proteases; one
was a precursor molecule of Mr 48,000, the other the mature
enzyme of Mr 40,000, each comprising two subunits. The
enzyme was most active at pH around 3.7 and its activity
declined quickly above pH 4.0. It did not hydrolyse either a
barley globulin or a reduced and alkylated purothionin, but
did degrade haemoglobin. Apparently, the ability of the
enzyme to hydrolyse barley storage proteins was not tested.
Incubation with class-selective inhibitors indicated that it
was an aspartic proteinase.
The cloned and sequenced cDNA encoding the enzyme
showed clearly that the two enzyme forms were translated
as a single proenzyme and then processed into the two
forms, the Mr 40,000 form being the final product
(Runeberg-Roos et al., 1991; Tormakangas et al., 1991).
The enzymes amino acid sequence and inhibition characteristics were similar to those of mammalian and yeast
aspartic proteases, especially cathepsin D. The enzyme was
named Hordeum vulgare Aspartic Proteinase (HvAP) and
its specificity was determined using small peptide substrates
(Kervinen et al., 1993). Hydrolysis was maximal when the
substrate contained either aromatic or aliphatic amino acids
in both its P1 and P10 sites. Vacuoles of barley leaves and
roots have an enzyme that is very similar to HvAP that
processes a prolectin (Runeberg-Roos et al., 1994), but it is
not clear whether this enzyme is the same as the one in
seeds. Expression of the expression of HvAP in germinating
and developing barley seeds (Marttila et al., 1995;
146
Tormakangas et al., 1994) showed that the enzyme was

present in aleurone cells but was not secreted into the
starchy endosperm, where most storage proteins reside.
Based on its characteristics, it is clear that HvAP is not
involved in the solubilization of barley storage proteins,
although it would be of interest to know whether the purified
enzyme hydrolyses barley hordeins.
5.3.2. Aspartic proteases in dormant and germinating
barley seeds
Using 2-D electrophoresis, Zhang and Jones showed that
there were several aspartic proteases in germinated barley
(Zhang and Jones, 1995a,b. Section 6.2), and it seemed
likely from their characteristics, their seed locations, and the
changes that occurred during malting that they might play
an important role during germination. This supposition was
tested by comparing the aspartic proteases of barley seeds
and green malt (Zhang and Jones, 1999). Both seeds and
green malt contained multiple aspartic protease forms, four
in malt and six in seeds. These different forms were
separated by pepstatin A affinity chromatography, followed
by 1-D and 2-D IEF and gel electrophoresis. Three of the
four separated malt proteases cross-reacted with antibodies
raised against HvAP. All of the seed and malt proteases
digested edestin, a Cannabis sativa globulin, most actively
at pH 3.54.5 and of all the class-specific inhibitors, only
pepstatin A caused inhibition. The enzymes all had identical
pI values, except for one of the seed forms. SDS-PAGE
showed that the seed proteases had subunit sizes of Mr 8000;
18,000; 31,000; 38,000 and 48,000 and the green malt
subunits were Mr 8000; 11,000; 15,000; 18,000; 31,000 and
38,000. Thus, the barley and malt enzymes were very
similar to each other and to HvAP, and the multiple
forms probably arose by post-translational processing, as
occurs with HvAP. None of the proteases hydrolysed
the components of a hordein preparation, but they did
associate with the barley chloroformmethanol soluble or
CM proteins (Shewry, 1993) during the purification
process and they digested some of the components of a
CM protein mixture. Thus this study reinforces the previous
findings for HvAP, indicating that these particular enzymes
are apparently not involved in storage protein solubilization
during malting (see also Section 7.5).
5.4. Barley metalloproteases
In some of their earliest studies Enari and Mikola
(1968) reported that metalloproteases, as well as cysteine
proteases, were present in barley seeds and in kilned and
green malts. Later they showed that an EDTA-inhibited
protease was synthesized and secreted from GA3-stimulated barley aleurone cells (Sundblom and Mikola, 1972).
Also, Belozersky, Dunaevsky and their associates found a
metalloprotease in dormant buckwheat seeds (Voskoboinikova et al., 1989) and purified it (Belozersky et al.,
1990). The enzyme hydrolysed buckwheat storage
proteins (Dunaevsky et al., 1983) and was inhibited by

an endogenous inhibitor (Dunaevskii et al., 1995). Wrobel
and Jones (1993) then showed that 4-day-germinated
barley seed extracts contained five high M r
metalloproteases
5.4.1. Purification and characterization of malt
metalloproteases (MPs)
Chromatography and chromatofocusing were used by
Fontanini and Jones (2001) to isolate a group of metalloproteases from green malt. The metalloprotease mixture
(MP) was separated by PAGE and 2-D IEF!PAGE on
gelatin-containing gels and its individual components
studied. All the MP enzymes were maximally active at
pH 78 and were inhibited by the chelating agents EDTA
and o-phenanthroline, but not by other class-specific
proteinase inhibitors. The activities of EDTA-inhibited
enzymes were restored by the addition of low concentrations of either Co2C, Mn2C or Zn2C ions, but they were
inhibited by higher concentrations of these same ions. In
nature, they probably contain Zn2C ions at their active sites.
The MP mixture was separated by 2-D electrophoresis into
three major and six minor components, all of which behaved
like metalloproteases.
The MPs were located mainly in the aleurone tissues of
the malt and were present in only very small amounts in
ungerminated barley seeds and during the first day of
malting. The MP hydrolysed the D component of a hordein
preparation in vitro at pH 4 much faster than it did the B or C
hordeins. A purified D hordein preparation and its C
hordein contaminants were hydrolysed at pH 4, with the
D hordein being converted into smaller, although still large,
fragments. The C and D hordeins were degraded quickly at
pH 8 (D, 40 min; C, 120 min) but no hydrolysis occurred in
the presence of either EDTA or o-phenanthroline.
The results obtained with the isolated metalloproteases
differed from those gotten with crude extracts. Enari and
Mikola (1968) reported that 31% of the proteinase activity
of barley was due to metalloproteases, but that in malt
they accounted for only 9% of the activity, whereas none
of the MP enzymes studied by Fontanini and Jones (2001)
were present in unmalted barley. Sundblom and Mikola
(1972) reported that the metalloproteases were synthesized
in, and secreted from, the barley aleurone, and Zhang and
Jones (1995b) detected metalloproteases in the starchy
endosperm of green malt using 2-D electrophoresis.
However, no MP were detected in malt endosperm
tissues. One explanation for these divergent results is
that Mikolas group and Zhang and Jones may have
extracted metalloproteases that differed rather strikingly
from those present in the MP mixture. Alternatively, the
results could be explained if the endosperm and seed
contained endogenous metalloprotease inhibitors, as
reported by Enari and Mikola (1968). In that case, even
if the enzymes were present, they might not have been
detected because they were complexed with the inhibitors.
That such metalloproteinase inhibitors do occur in grains

and that they play an important part in storage protein
hydrolysis was shown by the studies with buckwheat
(Dunaevskii et al., 1995). The presence of metalloproteinase inhibitors in malt might be one reason why this
group of enzymes has been so hard to extract and study in
the past. It could also help explain why only relatively
low metalloproteinase activities are detected in malt
extracts when recent studies (Jones and Budde, 2005)
indicated that these enzymes play a major role in the
release of soluble protein during malting and mashing (see
Section 7.5).
5.5. Barley serine proteases
Enari and Mikola (1968) found that specific serine
protease inhibitors caused no inhibition of the endoproteolytic activity of green malt extracts and concluded that no
serine class proteases were present. However, 2-D electrophoretic separations and analyses of malt extracts revealed
the presence of several serine proteases (Zhang and Jones,
1995a) and showed that one of them was especially
abundant after 2 days germination (Zhang and Jones,
1995b).
5.5.1. Hordolisin
Terp et al. (2000) purified and characterized a serine
endoproteinase from green malt that they called hordolisin.
Its Mr of 74,000, and pI of 6.9, suggested that it was
probably one of the B group proteases identified by Zhang
and Jones (1995a). Its inhibition characteristics indicated
that it was a serine class protease and its N-terminal amino
acid sequence was similar to that of cucumisin, a subtilisinlike melon (Cucumis melo) enzyme. The enzyme had a pH
optimum of 6 and was remarkably heat stable. A thorough
study of its specificity using synthetic peptide substrates
showed that its specificity was similar to that of savinase or
subtilisin BPN. Incubation of hordolisin with barley protein
bodies for 24 h released very little hordein material. No 24 h
controls were shown, so it may be that even the apparent
small loss of hordein that did occur was due to protein
precipitation, as reported by Fontanini and Jones (2001),
rather than hydrolysis. In any case, it appears that this serine
proteinase does not play any appreciable role in solubilizing
hordein proteins during malting.
5.5.2. SEP-1
Fontanini and Jones (2002) purified and studied what was
apparently the major green malt serine proteinase detected
earlier by Zhang and Jones (1995a). The purified enzyme,
named serine endopeptidase-1 or SEP-1, was present in
small quantities, but had a high specific activity for gelatin
and was easily detected on zymograms. Of many protease
inhibitors tested, including those specific for trypsin and
chymotrypsin, only PMSF and APMSF affected the
enzyme, confirming that it was a serine class enzyme.
147
SEP-1 was active between pH 4 and 7, with maximal

activity at pH 5.56.5, and between 50 and 60 8C. The
enzymes N-terminus was blocked, but sequencing of
internal peptides indicated that its primary structure was
similar to those of the cucumisin-like enzymes.
Ungerminated seeds contained no SEP-1 activity or
protein, but both were present after 2 days germination and
persisted through at least 6 days germination. The enzyme
was present only at very low levels in the scutellum/embryo
of dormant seeds, but increased between 2 and 6 days of
germination in all the tested tissues except the starchy
endosperm, where it was never detected.
5.5.3. Neither hordolisin nor SEP-1 is involved with protein
solubilization during germination
The characteristics of both hordolisin and SEP-1
indicated that they are similar to cucumisin (Kaneda and
Tominaga, 1975), but they differed strikingly from each
other in their pI values and temperature stabilities. SEP-1
appears to be the A1 activity of Zhang and Jones (1995a),
whereas hordolisin is probably one of the B activity group.
If this is so, then SEP-1 should have been present in the
endosperm of malt, even if it did not hydrolyse storage
proteins. Since neither SEP-1 nor hordolisin hydrolysed
hordein preparations and SEP-1 was unable to hydrolyse
any of the common barley seed protein classes, it seems
very unlikely that they solubilize storage proteins during
germination. This agrees with the finding that none of the
serine proteases solubilized proteins during mashing (Jones
and Budde, 2005). SEP-1 and hordolisin probably have
protein processing roles like those of other plant serine
proteases. The fact that they increase strongly during seed
germination implies that they do play an important role
during the initiation of growth of new plants.
6. Detecting proteolytic enzymes in barley/malt

Many assays, using several substrates, have been used to
detect endoproteolytic enzymes in barley and malts, and
during the mashing phase of brewing (see Section 4). It is
apparent that most of these methods have major drawbacks.
Two of the most important problems were that each of
substrates used is susceptible to hydrolysis by only a few
proteases and that the methods are unable to distinguish
between the individual proteolytic components in extracts.
To investigate any particular enzyme in an extract, it had to
be purified from other contaminating proteases. Such
purifications are time consuming and costly and whenever
several similar enzymes are present, which is common in
malt (see preceding sections), it is often impossible to obtain
completely pure enzymes. Additionally, there is always a
possibility that the proteases will be altered during the
multiple-step purification processes.
148
6.1. An in solution quantitative assay

We set out (Jones et al., 1998) to define a system that
could be used to study quantitatively individual proteases or
their mixtures and to quickly and efficiently separate the
individual proteases present and to measure their activities,
in at least a semi-quantitative manner. The assay utilized the
colored substrate protein, azogelatin, in an in solution
assay for measuring the activities of samples that contained
either individual enzymes or mixtures. The azogelatin
substrate has several excellent attributes: (1) it is a protein
and thus should give a realistic view of how the enzymes act
on a natural substrate; (2) it is readily hydrolysed by many
proteases, in contrast to several other substrate proteins that
were digested by only a few; (3) it is hydrolysed by enzymes
of all four of the common protease classes, although it is not
as susceptible to attack by the aspartic proteases as it is to
those of the other classes (Jones et al., 1998); (4) its
hydrolysis products are colored red and absorb light at
440 nm, so they can be monitored without derivatization.
This also obviates problems that are associated with
measuring the release of peptides at 280 nm, where
contaminating amino acids, proteins, etc. also absorb. The
hydrolyses are also more reproducible, since only the
hydrolysis of the azogelatin substrate is detected, whereas
the commonly used 280 nm wavelength also detects the
hydrolysis of any contaminating proteins, each of which
might be hydrolysed at a different rate; (5) it is readily
soluble between pH 3 and 10.5, the range of the proteolytic
activity pH optima of the four malt proteinase classes; (6) it
is readily precipitated with trichloroacetic acid; and (7) the
azogelatin derivative is easily and reliably pared from
porcine skin type A gelatin of w300 bloom (Sigma
Chemical Company, Cat No. G2500), by carefully following the protocol in Jones et al. (1998).
It should be noted that most of the enzyme reaction rates
reported in Jones et al. (1998), involving several different
proteinase types, were linear for 30 min or less, so that only
reactions performed for 30 min or less would yield true
initial reaction rates. The main drawback to the method is
that even though azogelatin and gelatin are hydrolysed by
pepsin, an aspartic protease, the hydrolyses proceed at a
much slower rate than they did with proteases of the other
three classes (Jones et al., 1998). This, together with the data
gathered from enzymes separated by 2-D electrophoresis
that were used to hydrolyse azogelatin (see Section 6.2)
indicates that neither gelatin nor azogelatin are particularly
good substrates for measuring the activities of many of the
aspartic proteases. Nonetheless, the susceptibility of
azogelatin to hydrolysis by so many of the malt proteases
makes it the substrate of choice.
It would have been preferable to use underivatized
gelatin as substrate rather than its azo derivative but that was
not practical, because gelatin is not precipitable with TCA
and the reaction products must be measured at or near
280 nm. Therefore, in gelatin hydrolyses controls must be
run for every individual reaction that contains different

amounts of protein.
6.2. A two-dimensional IEF!PAGE analysis system
In many situations where malt proteases are being
assayed more than one active enzyme is present but
measurements of the individual component enzymes, rather
than the overall activity, are required. In this situation, the
component proteases are best separated using a 2-D gel
system, after which the separated fractions can be assessed.
Based on the findings of Wrobel and Jones (1992) that
active enzymes of germinating barley could be partially
separated by non-denaturing PAGE in gels that contained
substrate protein, such a separation system was developed
(Zhang and Jones, 1995a). After the separation, the gels
were developed by allowing the separated enzymes
detected to hydrolyse the incorporated protein substrate
and staining the gel for protein. The areas containing the
separated activities were detected as clear areas, where the
substrate protein had been digested, against a blue background of stained protein. By separating the proteases on an
IEF tube gel, incorporating the IEF gel into the top of the
PAGE gel slab and then performing the gel separation and
development, it was possible to detect many different malt
proteolytic enzymes on a single gel (Zhang and Jones,
1995a).
When gelatin or azogelatin was incorporated into the
PAGE gel the advantages of these substrates, as outlined in
Section 6.1, could be exploited. Incubating the gel slabs in
solutions of varying pH values and/or that contained class
specific inhibitors allowed the pH optima of the separated
enzymes and their hydrolytic classes to be readily
determined (Zhang and Jones, 1995a). For analyses of the
aspartic proteases, PAGE gels containing edestin were used.
Using the gelatin and edestin systems together, over 40
separate protease enzymes were detected in green malt
(Zhang and Jones, 1995a). The 2-D method yielded very
reproducible separation patterns in experiments performed
at different times. Generally, the extent to which the
incorporated substrate was cleared from the gel was
proportional to the activity, and semi-quantitative data
could be obtained. It was very easy to differentiate between
very low, low, medium, strong and very strong activities.
This resolution was, of course, lost if too much extract was
loaded onto the gels, so that the substrate was completely
hydrolysed from some areas.
This method was used to determine when the various
malt endoproteases which were active at pH 4.8 (the
apparent pH of germinating barley seeds) appeared during
malting and where they were located in malted kernels
(Zhang and Jones, 1995b). The results corroborated and
extended those of many earlier experiments, showing that
ungerminated and steeped barleys contained few endoproteases, but that many components appeared within 2 days of
germination. The endosperm, where presumably most of
storage proteins are hydrolysed, contained representatives

of each of the serine-, aspartic- and metalloproteinase
classes and numerous cysteine proteases. The formation and
cellular locations of the various proteinase types and their
significance for malting and mashing could thus be
discerned.
An important initial finding from the 2-D studies was that
within each protease class the individual members generally
had similar pH optima but that between classes there were
major differences. The cysteine and aspartic proteases were
most active at pH values between 3.8 and 4.5, whereas the
serine- and metalloproteases were optimally active at pH
levels from about 6.0 to 8.5 (Zhang and Jones, 1995a).
6.3. Use of barley proteins as substrates
6.3.1. Hordeins
It would, of course, be highly desirable to use barley
storage proteins as substrates in endoproteinase assays and,
as reported above, that has times been done. However, in
addition to having to physically alter the hordeins to extract
them from the grain matrix, other problems also arise. An
in solution assay that used, hordein preparations as
substrates (Baxter, 1976) differentiated exopeptidase and
endoproteases but was very complicated and susceptible to
error. The assays had to be conducted at around pH 3 to keep
the substrate in solutions. These conditions are very
different from those that exist during malting, brewing or
seed germination. The internal pH of germinating barley
seeds is 4.8 and the pH of North American mashes is 6.0. Six
different hordein preparations were used by Baxter, and
their hydrolysis rates varied by nearly 30-fold. This raises
questions about which, if any, of these results is most
indicative of the hydrolysis of the natural hordein substrate
and the reasons for the variable results.
Another approach has been to hydrolyse hordeins in
solution (or in suspension) and then analyze the resulting
hydrolysate by PAGE. This has shown more promise, but
the results obtained are only semi-quantitative and most of
the peptides released are too small to be detected on gels
(Marchylo and Kruger, 1985; Poulle and Jones, 1988). This
method has, however, been used by several researchers
(Davy et al., 1998; Koehler and Ho, 1990a,b; Poulle and
Jones, 1988) to show that their purified proteases can
probably hydrolyse barley storage proteins in vivo.
It would be advantageous to be able to incorporate a
well-characterized hordein into IEF!PAGE 2-D gels, as
has been done with gelatin, so that the individual
components of proteinase extracts could all be tested at
once. This experiment has been attempted, but has never
been performed successfully. There are, however, reports
that 1-D PAGE gels containing incorporated hordeins have
been used to partially separate and analyze enzyme extracts
(Kaneda and Tominaga, 1975; Wrobel and Jones, 1992; Dr
Mark Schmitt, personal communication). In these 1-D gels,
149
the hordein substrate was not hydrolysed nearly as readily as

gelatin.
6.3.2. Highly degradable barley protein fraction (HDBPF)
Osman (2003a) proposed the use of a barley preparation
known as highly degradable barley protein fraction
(HDBPF), as a natural substrate for malt endoproteinase
assays. There are several problems with this substrate,
however. Its hydrolysis rate was not linearly correlated with
the amount of added enzyme and the reaction rates actually
dropped as the substrate levels were increased. In addition,
the HDBPF has none of the characteristics of normal
hordein storage proteins (Mr too low, amino acid composition completely different, etc.). In five malt proteinase
mixtures assayed using this substrate with class specific
protease inhibitors, the results suggested that none of the
enzymes present were either cysteine- or metalloproteases.
All were completely inactivated by the serine class
inhibitor, DIC, and two were also inhibited by 98% by
pepstatin A and may have been aspartic proteases (Osman,
2003b). Remarkably, a number of the enzymes were, in fact,
activated by various inhibitors, by up to 60%. Experiments
with what was apparently the same substrate, now called
glutelin, indicated that about 60% of the endoproteolytic
activity of malt was present after one day of germination,
whereas analyses using gelatin-containing gels or the
hydrolysis of haemoglobin both indicated that only about
10% of the activity was present at that time (Osman et al.,
2002). Compared to the seven characteristics of gelatin
listed in Section 6.1, this substrate does not meet criterion 4,
has not been tested for criteria 2, 3, and 5, and its
compliance with criterion 7 is questionable. It is unlikely
that this substrate will prove useful for protease assays.
7. Understanding in vivo proteolysis

As seen from Section 5, we now have a reasonable
knowledge of the characteristics of several of the barley/
malt proteases, but we still need a better understanding of
what really happens in barley at pH 4.8 and in mashes at pH
6.0. This information is needed by researchers so that they
can rationally alter the barley protein hydrolysis system to
produce improved malting or germinating barleys and by
maltsters/brewers so that they can adjust their processing
methods to prepare improved malts and beers.
7.1. The formation of proteolytic enzymes during malting
and their stabilities to kilning
Since the earliest studies, it has been clear that the overall
endoproteolytic activity of barley grains is quite low and
that proteases were formed and/or activated during the early
phases of seed germination or malting (Harris, 1962). The
real question was, therefore, which proteases were formed
and what did they do? These questions could only be studied
150
after methods had been developed for quickly and efficiently

separating the various enzymes and measuring their
activities under conditions similar to those that occur in
the germinating seed and in mashes. By quantifying the total
endoproteolytic activities of samples using the azogelatin
in solution assay (Jones et al., 1998) and semi-quantitatively analyzing the component enzymes with the 2-D
method, using azogelatin or gelatin as substrate, it became
possible to see how changes in individual enzymes affected
the overall protease activity.
These methods were first applied to the study of barley
undergoing malting (Jones et al., 2000). As expected, under
malting conditions the proteolytic activity at the end of
steeping was very low, but began to rise at germination day
1 and was maximal by day 3. The activities at pH 3.8
(measured for comparison with the results of many earlier
studies), at pH 4.8 (the internal pH of inside germinating
grain) and at pH 6.0 (mashing pH) increased concomitantly.
The assays at pH 3.8 would have measured only the
cysteine- and aspartic proteases and the assays at pH 6.0
mainly the serine- and metalloproteases (Zhang and Jones,
1995a, see Section 6.2), so the enzymes of the different
classes must have increased at the same time.
Sampling of green malt during the kilning phase of
malting showed that there was no diminution of proteolytic
activity during kilning at any of the three measured pH
values, even when the temperature was raised to 85 8C. In
the absence of added cysteine, the kilned malt activities
were even slightly higher than those of green malt (Jones et
al., 2000). 1-D PAGE analyses confirmed these findings and
showed that the same proteases were active throughout the
kilning process, but that completely different sets of
enzymes were active at pH 3.8 and 6.0. Analyses using
the more sensitive 2-D system, however, showed that some
minor changes did occur in some cysteine and serine
proteases during the 68 and 85 8C steps. Apparently, there
was a partial denaturation of the enzymes that resulted in the
enzyme spots on the gel becoming more diffuse. It was
obvious that there was no loss of proteinase activity during
kilning, even upon heating to 85 8C.
7.2. The effect of mashing on proteolytic activities.
Mashing is the first step in the brewing process, when
milled malt is subjected to a carefully controlled extraction
with water whose temperature is gradually raised. The effect
of mashing on the malt proteinase activities was tested using
a mash regime that was based on US commercial methods
(Jones and Marinac, 2002). In solution assays showed that
the overall proteolytic activity was constant throughout a
50 min, 38 8C protein rest phase, but fell rapidly when the
temperature was raised to 72 8C for the conversion phase.
The results were the same at pH 4.8 and 6.0, so the
components of all of the enzyme classes behaved similarly.
These results were confirmed by 1-D PAGE analysis, which
also confirmed that quite different enzymes were active at
pH 4.8 and 6.0. 2-D PAGE analyses verified the 1-D results
and defined which protease types were active at each pH.
All four enzyme classes were active at pH 4.8, but
metalloproteases predominated at pH 6.0, although the
major serine enzyme was also active. During mashing, all of
the enzymes were inactivated simultaneously and the
inactivation of the individual enzymes correlated well
with the loss of overall activity, as measured in solution.
That the enzymes have practically identical heat labilities
implies that it will not be possible to alter the amino acid
compositions of mashes by changing the mash temperature
regime. However, the soluble protein levels of worts can
obviously be increased by extending either the malting
process or the protein rest phase of mashing, but not by
extending the mash conversion time. The great majority of
green malt proteases were much more heat stable in the malt
kernel (stable to 85 8C for over 3 h during kilning) than in
solution (inactivated within 5 min at 72 8C).
7.3. The effect of pH on malt and mash proteases
and on worts
Because the pH optima of the different malt protease
enzyme classes are so different, it should be possible to vary
the compositions of worts by changing the mashing pH.
This was tested by conducting room temperature mashes
at initial pH values from 4.4 to 7.1 (Jones and Budde, 2003).
The mashes had good buffering capacities and their pH
values adjusted quickly towards pH 5.8, the apparent natural
mash pH value, so that the final mash pH values ranged from
4.8 to 6.4.
The wort characteristics of mashes performed at 45 8C
and between pH 5.1 and 6.6 were very different (Jones and
Budde, 2003). At each pH value, the individual endoproteinase activities remained constant throughout the 30 min
protein rest, but the overall activity dropped more than
5-fold as the pH was raised from 5.1 to 6.6. This reflects the
change from cysteine proteases to serine- and metalloproteases as the predominant active enzymes. The wort soluble
protein and FAN levels changed in concert with the
proteolytic activities, in a sigmoidal manner. In addition,
the extract values dropped by 4% points which, for a
commercial malt, is a very large change. The (1/3,1/4)b-glucan levels increased as the pH was raised, but
remained at commercially acceptable levels.
It was thus possible to produce worts with widely varying
compositions simply by changing the mashing pH. It would
be interesting and instructive to see how using worts with
these non-traditional FAN, SP and extract contents would
affect the brewing process. The differences in the proteolytic
activities at pH 4.8 and 6.0 indicate that during malting and
mashing the pattern of hydrolysis of the storage proteins
must be very different, and that measurements made in one
system cannot be presumed to apply to the other.
151
7.4. The effects of reducing and oxidizing agents

on proteases in malts, mashes and worts
The extract (1/3,1/4)-b-glucan levels were unaffected

by any of the redox agents.
Reducing agents (most often 2-ME) have routinely been

added to nearly all barley and malt proteinase preparations
for decades because they allegedly increased the amounts of
extractable enzymes and/or maintained their activities.
Some of this reducing agent was usually transferred into
assay mixtures along with the enzyme and additional
reducing agent was often added directly to the proteolytic
analyses to maximize the activities. These practices raise the
question of whether the results thus obtained provide a true
indication of what really happens during mashing and
brewing.
Apart from the effect of exogenous reducing agents,
Kobrehel et al. (1991, 1999) have proposed that endogenous
oxidation/reduction systems may control the reduction
states of various grain proteins and thereby affect the
operation of some of the grain proteases. The evidence for
this is circumstantial, but it needs to be investigated.
The effects of adding reducing and oxidizing agents to
malt extracts have recently been studied (Jones, 1999; Jones
and Budde, 2003). When considering both the studies of
Jones and Budde and those of Buchanan and his
collaborators (Kobrehel et al., 1991, 1999) it must be
remembered that redox reagents, whether exogenous or
endogenous, may have two distinct effects. They may
activate/inactivate cysteine proteases and/or they may alter
the substrate molecules, making them more or less
susceptible to hydrolysis.
The addition of cysteine to mashes increased their
proteolytic activities by 3- to 4-fold (Jones, 1999; Jones and
Budde, 2003). Other amino acids had no effect, indicating
that the cysteine effect was due to its reducing power. The
addition of weak (diamine) or strong (hydrogen peroxide,
H2O2) oxidizing agents to malt extracts lowered their
proteolytic activities and when diamine or H2O2 was added
to reactions together with cysteine, the cysteine effect was
cancelled, confirming that the effects were due to redox
reactions. With proteinase assays in solution, the addition of
low concentrations (up to 1 mM) of the strong reducing
agent dithiothreitol (DTT) led to an enzyme activation that
disappeared as the DTT concentration was increased
further. On the other hand, the proteolytic activity was not
affected by the addition of 2-ME.
Conversely, for individual proteases that were separated
and analyzed using the 2-D system cysteine, DTT and 2-ME
all strongly activated certain cysteine class proteases, and all
of these activations were negated by diamine (Jones and
Budde, 2003). No serine- or metalloproteases were affected
by any of the redox agents. The addition of cysteine, DTT or
2-ME to ASBC mashes led to increases in their wort SP,
FAN and extract values. The presence of either diamine or
H2O2 in mashes produced some reduction in their SP and
FAN levels, and both oxidizing agents effectively
negated the increases caused by the three reducing agents.
7.5. Which proteases actually release SP and FAN

during malting and mashing?
To better define which endoproteases effect changes in
the SP and FAN levels of brewhouse worts, experiments
were performed in which class-specific proteinase inhibitors
were added to Morex (6-rowed) and Harrington (2-rowed)
barley malt mashes (Jones and Budde, 2005). At pH 6.0,
the efficacy of inhibitors in lowering the wort SP levels
was o-phenanthrolineOE-64Opepstatin AOPMSFZ0,
indicating, surprisingly, that the metalloproteases were
responsible for controlling the solubilization of more
protein during mashing than were the cysteine enzymes,
and that the aspartic proteases also played a significant, if
lesser, role. These results were confirmed with mashes
conducted at pH 3.8 (cysteine and aspartic proteases active)
and pH 8.0 (serine and metalloproteases active), even
though the control SP levels were greatly increased at pH
3.8 and reduced at pH 8.0. These metalloproteases results
were based on inhibition with o-phenanthroline, since
EDTA strongly disrupted the system and, in most cases,
led to increased, not lowered, protease activities, SP levels
and FAN contents. The SP, extract and FAN levels were all
strongly affected by pH, but proteinase inhibitors did not
affect the wort extract values. The presence of inhibitors
generally lowered FAN levels at pH 3.8 and 6.0, but the
effect was not dramatic. The inhibitory effects did not vary
with either malt concentrations or mashing conditions. The
SP, extract and (1/3,1/4)-b-glucan levels varied among
the different pH treatments, but inhibitors had no effect on
wort extract or (1/3,1/4)-b-glucan levels and the
inhibition of the cysteine and metalloproteases most
strongly lowered the wort SP concentrations.
These experiments led to the conclusion that malt
metalloproteases play a much more prominent role in the
solubilization of protein during mashing (and possibly also
during malting) than was previously suspected. It also
appears that, notwithstanding the characteristics of those
aspartic proteases purified and characterized to date, these
proteases also play a significant role during mashing. In rye,
the aspartic proteases apparently play a major role in
hydrolyzing storage proteins during seed germination (Brijs
et al., 2002), so it is not too surprising that they are also
important in barley germination.
7.6. When is protein solubilized during seed germination?
Several attempts have been made to determine the
percentage of the wort SP that is released during the separate
malting and mashing procedures. For mashing, values
reported have varied from 0% (Lewis et al., 1992) to 30%
(Burger and Schroeder, 1976a) and 47% (Barrett and Kirsop,
1971). By mashing ungerminated barley and malt in
152
solutions that contained mixtures of protease inhibitors,

Jones and Budde (2005) measured the SP levels of the barley,
malt and wort, and calculated that, at pH 6.0, 32% of the final
wort SP was already present in unmalted barley, 46% was
solubilized during malting and 22% during mashing. At pH
8.0, where the cysteine and aspartic proteases were inactive,
10% of the total wort SP was solubilized during mashing,
another indication of the major contribution of the metalloproteases to the SP levels. Because this study measured, for
the first time, the SP levels of the unmalted grain and used
relatively benign specific protease inhibitors, the results
should be particularly meaningful. The results obtained
would presumably vary if different malting and mashing
regimes were used.
Of the FAN in wort, 15% was present in unmalted barley,
58% was released during malting and 26% during mashing
(Jones and Budde, 2005). The difference between the FAN
and SP results was expected since the FAN and SP are
released by different sets of enzymes. At pH 8.0, no FAN
was released during mashing, implying that none of the malt
exopeptidases were active at this high pH.
7.7. Variation among barley cultivars
Jones and collaborators used both 6-rowed (Morex) and
2-rowed (Harrington) barleys in their proteinase experiments. Both are very good malting cultivars although they
have significantly different characteristics. Their proteolytic
profiles were the same and they responded identically to
redox compounds and changes in pH (Jones and Budde,
2003), to inhibitors (Jones and Budde, 2005) and during
malting and mashing (Jones et al., 2000; Jones and Marinac,
2002). The proteolytic activities of six Australian and North
American barleys, tested with three different substrates, also
did not differ greatly (Osman et al., 1997, 2002). On the
other hand, analyses of malts made from 43 barley lines
from around the world showed that their proteinase values
varied by 4- to 6-fold, that the cysteine and aspartic protease
activities predominated, and that only the cysteine proteinase activities correlated with their SP values or Kolbach
Indexes (Kihara et al., 2002). For a number of reasons these
latter results are questionable; the pH values of the protease
assays were not specified, 5 mM DTT was added to the
assays, which would have greatly increased both the
cysteine class proteinase activities and the wort soluble
protein levels (Jones and Budde, 2003), and a casein
derivative was used as substrate, even though casein is not
hydrolysed by most malt endoproteases (Jones et al., 1998).
Moreover, initial reaction rates were not used for the kinetic
analyses (see Section 4). From the high aspartic- and low
metalloprotease levels that were reported, it seems likely
that the assays were conducted at pH 5.0 (Zhang and Jones,
1995a), which would have strongly enhanced their overall
proteolytic activities and FAN and SP levels.
7.8. Variations in the proteolytic capacities

of various grain species
Various grain species apparently hydrolyse their storage
proteins differently during germination. For example, in
buckwheat seeds the initial hydrolysis is catalyzed by a
metalloproteinase (Dunaevsky et al., 1983), after which a
cysteine proteinase degradation predominates (Dunaevsky
and Belozersky, 1989), possibly with the assistance of an
exopeptidase. In oats, most of the at proteases pH 6.2 active
present initially were serine and metalloproteases (Mikola
and Jones, 2000a), even though cysteine proteases apparently degraded most of the storage protein (Mikola and
Jones, 2000b). In germinating rye seeds, the protein
breakdown was mostly catalyzed by the cysteine and
aspartic enzyme classes (Brijs et al., 2002).
A comparison of the proteolytic complements in malted
grains of barley, bread and durum wheats, rye, triticale, oats,
rice, buckwheat and sorghums was made by separating their
proteases and analyzing them using the gelatin 2-D system
(Jones and Lookhart, 2005). Their IEF and PAGE migration
characteristics and the effect of pH changes allowed an
estimate to be made of the members of the various enzyme
classes that were present in each species. All of the
germinated grains contained multiple enzymes. The separation patterns and pH characteristics of the bread and durum
wheats, ryes, oats and sorghums were fairly similar to those
of barley, whereas the patterns in other grains showed more
variability. Rice and buckwheat proteases developed very
slowly. In triticale the activity patterns were similar to those
of their wheat and rye parents, but the triticales contained
many more proteases and their overall activities were the
highest of any of the species that were tested.
These results complement previous findings that indicated that cereal grains tend to contain similar proteases, but
that each species may degrade its storage proteins
differently. One important finding was that all of the cereals
except rice exhibited strong metalloproteinase activities,
supporting the proposal that these enzymes play a greater
role in the degradation of grain proteins than has been
previously assumed.
8. The current situation

In overviewing the current state of proteinase studies in
barley and malts, several things stand out. Among these are:
(1) Cysteine proteases are clearly important players in the
hydrolysis of barley proteins during malting and
mashing. However, it seems likely that they do not
play as predominant a role as was attributed to them in
the past. Most earlier studies were performed at pH
values below 4.8 and in the presence of added reducing
agents, conditions that strongly increase the cysteine
(2)
(3)
(4)
(5)
and aspartic proteinase activities while diminishing

those of the metalloproteases.
Conversely, the role of metalloproteases has been
almost completely overlooked, even though Enari and
Mikola showed as early as 1968 that they contributed
substantially to the overall proteolytic activity, even in
reactions that were performed at a low pH and in the
presence of strong reducing agents. Evidence now
points to the metalloproteases playing a very significant
role in solubilizing proteins, especially during mashing
at pH 5.86.0 (Jones and Budde, 2005). Metalloproteases are known to play a crucial role in the
solubilization of buckwheat storage proteins. Due to
problems associated with their purification and
measurement, biochemical studies of these enzymes
have only recently begun (Fontanini and Jones, 2001).
An in-depth investigation of these enzymes needs to be
performed; always bearing in mind the possibility that
malt may contain metalloproteinase inhibitors.
All current evidence suggests that the serine proteases
play little or no direct role in the solubilization of barley
storage proteins (Fontanini and Jones, 2002; Jones and
Budde, 2005; Terp et al., 2000), even though they
comprise one of the most active enzyme forms present
in malt (Zhang and Jones, 1995a). Any studies aimed at
determining how they might affect malting and mashing
should be directed toward understanding whether and
how they might control some overall aspect of seed
germination. Serine class exopeptidases do, however,
play a role in FAN formation, because Mikola et al.
(1971) reported that 7080% of the free amino acid
production of mashes was inhibited by diisopropylfluorophosphate,
an
inhibitor
of
serine
carboxypeptidases.
While none of the barley aspartic proteases that have
been purified and characterized seem to be involved in
hydrolysing the seed storage proteins, it is likely that
other members of this group do participate. Four
aspartic proteases were detected in green malt by 2-D
separations (Zhang and Jones, 1995a) and Zhang and
Jones (1999) showed that at least six forms occurred in
ungerminated seeds, but none of the seed enzymes
hydrolysed barley hordein preparations. However, the
addition of pepstatin A to mashes routinely caused an
intermediate level of inhibition of SP release (Jones and
Budde, 2005), indicating that aspartic proteases were
involved. In addition, both wheat (Belozersky et al.,
1989) and rye (Brijs et al., 1999, 2002) endosperm
tissues contain aspartic endoproteases that hydrolyse
their respective storage proteins. This is an indication
that barley, a closely related species, may also contain
similar enzymes. This aspect is worth researching.
Any new studies of barley/malt proteases should
concentrate on the aspartic and metalloproteinase
enzymes. The studies must determine the roles they
153
play in the overall picture of SP formation and the

enzymes need to be characterized under conditions that
are truly relevant to malting and mashing. For example,
at pH 4.8 and 6.0 in the absence of added reducing
agents and using substrates that meet the criteria listed
in Section 6.1.
(6) The term purified should only be applied to enzyme
preparations whose purity has been demonstrated.
Dividing the multiple malt proteases into five groups
of enzymes without showing that these fractions contain
single components does not constitute a purification.
(7) Finally, there are protein and non-protein molecules in
barley that interact with certain of the proteolytic
enzymes to change, and probably control, their
activities. These interactions must be borne in mind
when attempts are made to measure and purify
proteases from crude mixtures. Two proteins have
been shown to bind very strongly to, and inhibit,
cysteine proteases (Jones and Marinac, 1997, 2000),
several proteins inhibit serine proteases (Jones and
Fontanini, 2003) and there is evidence for inhibitors of
metalloproteases (Enari and Mikola, 1968). Similarly,
those yet unpurified aspartic proteases that hydrolyse
malt proteins may also have complementary
inhibitors that interact with them in extracts to
render them inactive, and thus undetectable, as happens
with the cysteine proteases (Jones, 2001). These
proteinase inhibitors will be the subject of a future
review.
In 1995, Enari (1995) stated that by the end of the 1970s
research .had resulted in a clear picture of the events.
that were involved in proteolysis by barleys and malts.
This clearly was overly optimistic, because our knowledge
of malt proteolysis more than doubled between 1975 and
1995, and has increased by as much again since 1995. We
can only hope that this rate of knowledge increase will
continue for another decade, and that we will then finally
have a clear view of how the biochemical degradation of
storage proteins during seed germination really occurs.
Unfortunately, several of the groups that were carrying out
research in this field have disbanded within the last few
years, so the rate at which this knowledge is accrued may
well drop.
Acknowledgements
I wish to acknowledge the following researchers who
have worked in my laboratory over the years. Without their
stimulating interactions and excellent technical capabilities
my contributions to this field would have been impossible.
Ms Laurie Marinac, Dr Michel Poulle, Dr Radoslawa
Wrobel, Dr Ningyan Zhang, Dr Debora Fontanini and Mr
Allen Budde.
154
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Journal of Cereal Science 42 (2005) 139156

Endoproteases of barley and malt

hydrolysis is critical to the brewing and malting industries,

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

in a product that has insufficient foaming ability, mouthfeel

depending on what attributes are wanted in the final wort

2.2. FAN; free amino nitrogen

2.5. Class-specific proteases

The amount of NH2 groups in an extract, as measured

Most endoproteases fall into one of four classes. The

2.3. Mashing, extract, wort

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

2.6. 2-D IEF!PAGE

4. Meaningful protease assays

Wallace, 1989), measuring the reduction in viscosity of a

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

of the various malt proteases (other than the aspartic

their structures. Although the hordein extracts used as

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

5. Purified barley/malt endoproteases

Gibberellin stimulated the aleurone to produce both EP-A

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

related, proteases. Among the more important outcomes of

raised against EP-A. Conversely, its N-terminal amino

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

Davy et al. (1998) confirmed these results by hydrolysing

other enzymes that were present and thereby led to the

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

Tormakangas et al., 1994) showed that the enzyme was

proteins (Dunaevsky et al., 1983) and was inhibited by

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

That such metalloproteinase inhibitors do occur in grains

SEP-1 was active between pH 4 and 7, with maximal

6. Detecting proteolytic enzymes in barley/malt

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

6.1. An in solution quantitative assay

run for every individual reaction that contains different

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

storage proteins are hydrolysed, contained representatives

the hordein substrate was not hydrolysed nearly as readily as

7. Understanding in vivo proteolysis

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

after methods had been developed for quickly and efficiently

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

7.4. The effects of reducing and oxidizing agents

The extract (1/3,1/4)-b-glucan levels were unaffected

Reducing agents (most often 2-ME) have routinely been

7.5. Which proteases actually release SP and FAN

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

solutions that contained mixtures of protease inhibitors,

7.8. Variations in the proteolytic capacities

8. The current situation

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

and aspartic proteinase activities while diminishing

play in the overall picture of SP formation and the

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

Dunaevskii, Y.E., Pavlikova, E.B., Belyakova, G.A., Belozerskii, M.A.,

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

Mikola, M., Jones, B.L., 2000a. Electrophoretic and in solution analyses

B.L. Jones / Journal of Cereal Science 42 (2005) 139156

Aspartic proteinase from barley seeds is related to animal cathepsin

Zhang, N., Jones, B.L., 1995a. Characterization of germinated barley