Revie

Endoproteases of b

Berne L. J

skia,

d 1 M

many

th, I e

o his

Journal of Cereal ScienceE-mail address: bhjones@bmi.net.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

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 Mrcompounds 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 comp-

lement 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,

Abbreviations 2-D, two dimensional; ASBC, American Society of

Brewing Chemists; 2-ME, 2-mercaptoethanol; E-64, (trans-epoxysuccinyl-

L-leucylamido-(4-guanidino)butane); EP-A and -B, cysteine-class endo-

proteases -A and -B; DTT, dithiothreitol; FAN, free amino nitrogen; HvAP,

Hordeum vulgare aspartic protease; IEF, isoelectric focusing; kD, kiloRR1, Box 6, Koo

Received 24 December 2004; revise

This review is dedicated to Dr Juhani Mikola, whose work laid the bases for

inhibitors. I never had the pleasure of meeting him, but if not for his early dea

been tw

arley and malt

ones*

ID 83539, USA

arch 2005; accepted 1 March 2005

of our later studies on barley and malt endoproteases and their endogenous

xpect that a very large percentage of the references in this article would have

work.

42 (2005) 139156

www.elsevier.com/locate/jnlabr/yjcrsproteins, 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 result0733-5210/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jcs.2005.03.007

fonyl fluoride; SEP-1, serine endopeptidase-1; SP, soluble protein.* Tel.: C1 208 926 4429.most of the proteins that are being degraded will be storageDalton; MEP-1, malt cysteine-class endopeptidase 1; MP, metalloprote-

ases; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsul-

2.2. FAN; free amino nitrogen

until germination begins (steeping), is then held under moist

catalytic mechanisms of these classes differ and the

members of each are specifically inhibited by different

eal SThe 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.

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 productin 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.

B.L. Jones / Journal of Cer140is termed wort. The mashing processes used will vary,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)bu-

tane). 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 metallopro-

teases [EC 3.4.24.-], 1,10-phenanthroline, or o-phenanthro-

line, and sometimes EDTA.

Throughout this review the terms protease or endopro-

tease 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 eitherand 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 character-

istics 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.

2.5. Class-specific proteases

Most endoproteases fall into one of four classes. Thedepending 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 beenhydrolysed to fermentable sugars.

2.4. Malting

The process of preparing malt. Barley grain is soaked

cience 42 (2005) 139156the N- or C-terminal peptide bonds of polypeptides.

real S4. 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)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 character-

ized 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).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.

B.L. Jones / Journal of Ceor from a hordein preparation (Baxter, 1976; Phillips andWallace, 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 Mrsubstrates, 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 haemo-

globin, 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

cience 42 (2005) 139156 141ranging from 3.0 to 10.5, and thus can be used to measure all

eal Sof 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

B.L. Jones / Journal of Cer142had first to be solubilized, which probably also modifiedtheir 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. Unfortu-

nately, this basic enzymological principle is often over-

looked, 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

cience 42 (2005) 139156throughout that period, are at best only semi-quantitative.

real S5. Purified barley/malt endoproteases

Between about 1985 and 1990, there was a burst of

publications that described the purification and character-

ization 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 gibberellin-

treated barley aleurone cells that apparently encoded a 361-

amino 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 acid-

treated barley aleurone layers synthesized several proteases

with Mr of w37,000, that were inhibited by cysteineprotease inhibitors, and that hydrolysed extracted barley

storage proteins. Two of these enzymes, called endopro-

teases-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

B.L. Jones / Journal of Cefrom the aleurone tissue (Koehler and Ho, 1990b).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 Mr29,000, 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-butoxycarbonyl-

L-amino acid-p-nitrophenyl ester substrates containing

various amino acid residues, the hydrolysis rates were:

GlnOAlaOLeuOTyrOTrpOAsnOPhe. The hydrolysis ofhordein 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 MEP-

1 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

cience 42 (2005) 139156 143obvious that MEP-1 and EP-B are the same, or very closely

cal to that found with the 30 kD protease, except that Ala did

eal Srelated, 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 compo-

sition 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 prep-

aration 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 prep-

aration. 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 Mr37,000 EP-A isozymes studied by Koehler and Ho (1988),

B.L. Jones / Journal of Cer144but the 31 kD enzyme did not cross-react with antibodiesnot 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 suchraised 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 P01 residues of hydrolysis sites

that had the general formula

NH2 /P2 KP1YKP01 KP

02 /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 P01 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 polypeptidesthat 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 ishomologous 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 P2position relative to the bond that was hydrolysed. The

effectiveness of amino acids for specifying hydrolysis was

TrpOPheOLeuOIleOValOTyrOAla, which was identi-

cience 42 (2005) 139156residues were located together at positions P2 and P3.

real SDavy 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 LeuOPheOVal[ProOSer. They also found that hydrolysis wasseverely restricted when Pro was present at either the P1or the P01 sites. This imposes a rather strict limitation on thenumber 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

B.L. Jones / Journal of Cewould have enhanced their activities relative to those ofother 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 communi-

cation; 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 esculen-

tum), 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 charac-

teristics 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 P01 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

cience 42 (2005) 139156 145and developing barley seeds (Marttila et al., 1995;

eal STormakangas 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-stimul-

ated barley aleurone cells (Sundblom and Mikola, 1972).

Also, Belozersky, Dunaevsky and their associates found a

metalloprotease in dormant buckwheat seeds (Voskoboi-

nikova et al., 1989) and purified it (Belozersky et al.,

B.L. Jones / Journal of Cer1461990). The enzyme hydrolysed buckwheat storageproteins (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 Mrmetalloproteases

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 metallo-

proteases from green malt. The metalloprotease mixture

(MP) was separated by PAGE and 2-D IEF!PAGE ongelatin-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 concen-

trations 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

cience 42 (2005) 139156detected because they were complexed with the inhibitors.

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

during the mashing phase of brewing (see Section 4). It is

real SThat 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 metalloprotei-

nase 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 endoproteo-

lytic activity of green malt extracts and concluded that no

serine class proteases were present. However, 2-D electro-

phoretic 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 subtilisin-

like 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

B.L. Jones / Journal of Ceenzyme, confirming that it was a serine class enzyme.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 theappears 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, andSEP-1 was active between pH 4 and 7, with maximal

activity at pH 5.56.5, and between 50 and 60 8C. Theenzymes 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

cience 42 (2005) 139156 147multiple-step purification processes.

eal S6.1. 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 (SigmaChemical Company, Cat No. G2500), by carefully follow-

ing 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

B.L. Jones / Journal of Cer148280 nm. Therefore, in gelatin hydrolyses controls must berun 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 back-

ground 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 endopro-

teases, but that many components appeared within 2 days of

cience 42 (2005) 139156germination. The endosperm, where presumably most of

hydrolysate by PAGE. This has shown more promise, but

the peptides released are too small to be detected on gels

2003b). Remarkably, a number of the enzymes were, in fact,

real S(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, ashas 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; Drthe results obtained are only semi-quantitative and most ofstorage 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

B.L. Jones / Journal of CeMark Schmitt, personal communication). In these 1-D gels,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 formedthe 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 compo-

sition 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,

cience 42 (2005) 139156 149and what did they do? These questions could only be studied

teinase activities remained constant throughout the 30 min

eal Safter 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-quantitat-

ively 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. Inthe 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, therewas 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 thetemperature 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

B.L. Jones / Journal of Cer150also confirmed that quite different enzymes were active atprotein 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 metallopro-

teases 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, butremained 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 onepH 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 endopro-

cience 42 (2005) 139156system cannot be presumed to apply to the other.

real S7.4. The effects of reducing and oxidizing agents

on proteases in malts, mashes and worts

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

B.L. Jones / Journal of Cenegated the increases caused by the three reducing agents.The extract (1/3,1/4)-b-glucan levels were unaffectedby any of the redox 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 amongthe different pH treatments, but inhibitors had no effect on

wort extract or (1/3,1/4)-b-glucan levels and theinhibition 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,

cience 42 (2005) 139156 1511971). By mashing ungerminated barley and malt in

malting and mashing (Jones et al., 2000; Jones and Marinac,

7.8.

of v

V

prot

metalloproteinase (Dunaevsky et al., 1983), after which a

and Jones, 2000a), even though cysteine proteases appar-

ently degraded most of the storage protein (Mikola and

Jones, 2000b). In germinating rye seeds, the protein

eal Science 42 (2005) 1391562002). 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 protein-

ase 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 overallsolutions 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 metallo-

proteases 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 exper-

iments. 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

B.L. Jones / Journal of Cer152proteolytic activities and FAN and SP levels.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 separ-

ation 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 indi-

cated 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 reducingcysteine 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 (MikolabucVariations in the proteolytic capacities

arious grain species

arious grain species apparently hydrolyse their storage

eins differently during germination. For example, in

kwheat seeds the initial hydrolysis is catalyzed by aagents, conditions that strongly increase the cysteine

(2)

(5)

Dividing the multiple malt proteases into five groups

of enzymes without showing that these fractions contain

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B.L. Jones / Journal of Cereal SAny new studies of barley/malt proteases should

concentrate on the aspartic and metalloproteinasebeen 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 prepara