Endoproteases of barley and malt

Journal of Cereal Science 42 (2005) 139–156 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: [email protected]. 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

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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 freeze–dried 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,

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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

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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 enzyme’s 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

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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 5.8–6.0 (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.

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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).

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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 Wallace’s enzymes and those studied by Ho, it seems obvious that MEP-1 and EP-B are the same, or very closely

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related, proteases. Among the more important outcomes of Wallace’s 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 30,000–31,000 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.

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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

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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 4.8–5.0 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; To¨rma¨kangas et al., 1991). The enzyme’s 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;

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To¨rma¨kangas 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 3.5–4.5 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 chloroform–methanol 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 7–8 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 Mikola’s 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.

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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.

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SEP-1 was active between pH 4 and 7, with maximal activity at pH 5.5–6.5, and between 50 and 60 8C. The enzyme’s 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.

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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

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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,

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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

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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.

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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

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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

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(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 5.8–6.0 (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 70–80% 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

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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.

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