Comparative Enzyme Kinetics of Two Allelic Forms of Barley (Hordeum vulgare L.) Beta -amylase

The barley (Hordeum vulgare L.) varieties, Franklin and Schooner, contain two different allelic forms of beta -amylase (EC 3.2.1.2) encoded on chromosome 4H by the Bmy 1-Sd1 and Bmy 1-Sd2L alleles, respectively. The corresponding enzymes, referred to as Sd1 and Sd2L, were purified from both mature barley grain and germinated barley (green malt), and their physical and kinetic properties studied. Approximately 4 kDa were cleaved from both Sd1 and Sd2Lbeta -amylases after germination. The K_mvalue for green malt beta -amylase was less than that of mature grain beta -amylase for both varieties when potato starch was used as a substrate, although V_maxwas similar. This indicated that proteolysis after germination increased the affinity of beta -amylase for potato starch. No significant kinetic differences were observed between beta -amylase from mature grain and green malt of the two barley varieties when amylose (degree of polymerisation 100 and 18) and maltopentaose were used as substrates. Kinetic differences were also observed between the two allelic forms of beta -amylase. Sd1 beta -amylase from green malt exhibited a lower K_mvalue for potato starch than Sd2L beta -amylase, demonstrating that at non-saturating starch concentrations Sd1 beta -amylase is better able to hydrolyse starch than Sd2L beta -amylase. As the degree of polymerisation of the substrates decreased from approximately 740 (potato starch) to 5 (maltopentaose), the K_mvalues for beta -amylase increased, whereas V_maxvalues decreased. Maltose, the hydrolytic product of beta -amylase, was found to be a weak competitive inhibitor of both Sd1 and Sd2L green malt beta -amylases with respect to potato starch and amylose. Taken together the kinetic observations for bet a-amylase suggest that the allelic differences and C-terminal proteolysis might be exploited to improve the efficiency of starch hydrolysis during the mashing stage of the brewing process.     

Thermostability variation in alleles of barley beta-amylase

Thermostability assays in conjunction with IEF and molecular mapping were used to identify three beta-amylase alleles (Bmyl-Sd1, -Sd2L, -Sd2H) in cultivated barley and an additional allele (Bmy1-Sd3) in an accession of wild barley Hordeum vulgare ssp. spontaneum. The four forms of beta-amylase exhibit different rates of thermal inactivation in barley extracts. This variation was shown to persist after the proteolytic processing of the enzyme that occurs during germination. Three forms of beta-amylase representing the range of thermostabilities were purified and shown to have T₅₀ temperatures of 56·8°C for the Sd2L enzyme, 58·5°C for the Sd1 enzyme, and 60·8°C for the Sd3 beta-amylase from wild barley. Analysis of the relationship between beta-amylase thermostability and fermentability, i.e. the yield of fermentable sugars obtained from starch hydrolysis during brewing in 42 commercial malt samples suggests that increased thermostability results in more efficient starch degradation. Screening for specific beta-amylase alleles is proposed as a method for increasing fermentability in malting barley.     

Development of proteolytic activities during barley malting and their localization in the green malt kernel

The water-insoluble storage proteins of barley seeds reside in the starchy endosperm tissue. This tissue, when expressed from germinating barley, has a pH of 4·8. The hydrolysis of storage proteins during germination (malting) occurs mainly in the endosperm, so proteinases that are located in endosperm and are active at pH 4·8 are probably important to the storage protein hydrolytic process. This study reports our continued investigations of the endoproteinases of germinating barley (Hordeum vulgare L., cv. Morex) with a two-dimensional gel separation method that uses isoelectric focusing (IEF) and non-denaturing polyacrylamide gel electrophoresis (PAGE) in gels containing incorporated substrate protein. We identified the endoproteinases that were active at pH 4·8 and determined when they appeared during germination and where they were located in 4-day germinated barley (green malt). A total of nine cysteine, four aspartic, and two serine class proteolytic activities that were active at pH 4·8 were extracted from the endosperm tissue of green malt. It seems probable that some or all of these endosperm endoproteinases, especially proteinases C7, C8, C11, D3, E3 and E4, are the ones most intimately involved in hydrolyzing the storage proteins during malting.     

Characterization of germinated barley endoproteolytic enzymes by two-dimensional gel electrophoresis

Germinating barley proteolytic enzymes hydrolyze insoluble seed storage proteins into soluble proteins, peptides and amino acids. This process is of vital importance to both seed germination and the commercial malting process. This study reports the development of a two-dimensional IEF × PAGE separation method utilizing protein substrates incorporated into the PAGE gel to analyze and partially characterize the endoproteinases of germinating barley grain. The method separated 42 different activities, which fell into five groups on the basis of their pI values, PAGE mobilities and biochemical characteristics. Multiple representatives of each of the four proteinase classes were present, but about 64% of the enzymes were cysteine proteinases. The majority of the endoproteinases had pH optima considerably below neutrality, but those of the serine proteinases were generally eight or higher. Most of the endoproteinases hydrolyzed gelatin faster than edestin, but the four aspartic class proteinases and one of an undetermined class only hydrolyzed edestin. These results show that the protein-hydrolyzing system of green malt (4-day-old germinated barley) is very complex.     

Measurement ofBeta-amylase in Malting Barley (Hordeum vulgareL.). I. Development of a Quantitative ELISA forBeta-amylase

A double antibody, sandwich enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal antibodies specific tobeta-amylase to estimate the amount of ‘free’ (soluble in aqueous saline solution) or ‘combined’ (extracted with saline solution including reducing agent)beta-amylase protein in barley grain and malt. This ELISA was used to quantify the amount ofbeta-amylase in barley grain and malt from four varieties grown at nine sites in South Australia in 1993. The antibody used to develop the ELISA reacted differently withbeta-amylase depending on whether the source was barley grain or malt, and on thebeta-amylase band pattern in isoelectric focussing (IEF) of the barley variety. On the basis of their IEF band patterns barley varieties were divided into two types, designatedBmy1-Sd1 andBmy1-Sd2. Malting resulted in proteolytic cleavage of thebeta-amylase peptide with a reduction in the apparent molecular weight of up toM_r4000 and the appearance of new maltbeta-amylase IEF bands that were more basic. The new maltbeta-amylase IEF band patterns still allowed the identification of theBmy1-Sd1 andBmy1-Sd2 IEF types despite the change in molecular weight and pI. The data obtained using thebeta-amylase ELISA were highly correlated withbeta-amylase activity for both the free and combined fractions when the IEF band pattern and its source, barley grain or malt, were taken into account.     

Effect of hydrogen peroxide and ozone treatment on improving the malting quality

The effect of hydrogen peroxide (HP) and ozone (O₃) treatment during barley steeping on the quality of malt produced from two barley varieties (GrangeR and AC Metcalfe) by micro-malting was investigated. The two steeping oxidation treatments that was observed to promote barley acrospire growth. Ozone treatment improved the malt enzyme activity of endo-protease, α-amylase, free beta-amylase and total limit dextrinase to differing extents, with GrangeR improving to a greater degree. HP treatment contributed to the increase of α-amylase, β-glucanase and endo-protease. Surprisingly, HP or ozone oxidation during malting resulted in different and novel outcomes for total beta-amylase in GrangeR and AC Metcalfe. In GrangeR, total beta-amylase activity reduced with respect to the control in both treatments. In comparison with AC Metcalfe there was a substantial increase of 78% with HP and 90% O₃ in total beta-amylase activity. Malt quality including wort free amino nitrogen, β-glucan, turbidity and diastatic power was differentially increased by the oxidation induction treatment during steeping in malting. Gene expression analysis indicated that the effects of the steep oxidation treatments on enzyme and malt quality were putatively linked with the up-regulation of certain genes involved in GA synthesis (GA20ox1) and ABA catabolism (ABA8′OH). Barley grain germination assay results also showed that moderate HP induction could improve barley germination tolerance to the ABA effect. Malting including steep oxidation induction was shown to be beneficial to malt quality by improving the resultant wort quality and the efficiency of the beer brewing process. These observations point the way towards improving malt quality and the efficiency of the malting process.     

Fusarium Species Synthesize Alkaline Proteinases in Infested Barley

Barley (Hordeum vulgare L.) that is infested with Fusarium head blight (FHB, ‘scab’) is unsuitable for malting and brewing because it may contain mycotoxins and has unacceptable malting quality. Fungal proteinases are apparently often involved in plant-microbe interactions, where they degrade storage proteins, but very little is known about the enzymes that the fungi produce in the infected grain. We have shown previously that one plant pathogenic fungus, Fusarium culmorum, produced subtilisin- and trypsin-like enzymes when grown in a cereal protein medium. To establish whether these proteinases were also synthesized in FHB-infested barley in vivo, field-grown barley was infested as the heads emerged. Extracts were prepared from the grain as it developed and matured and their proteolytic activities were measured with N-succinyl-Ala-Ala-Pro-Phe p -nitroanilide and N-benzoyl-Val-Gly-Arg p -nitroanilide. The heavily infested barleys contained both subtilisin- and trypsin-like activities. These enzymes reacted with antibodies prepared against each of the two F. culmorum proteinases, indicating that those produced in the laboratory cultures and in the field-infested barley were the same. The presence of these proteinases correlated with the degradation of specific buffer-soluble proteins in the infested grains. These enzymes readily hydrolyzed barley grain storage proteins (C- and D-hordeins) in vitro. The presence of these Fusarium proteinases in the barley indicates that they probably play an important role in the infestation, but exactly how and when they function is not clear.     

Developmental Expression of Amylases During Barley Malting

Amylase activity and qualitative changes in amylase isoenzymes as a function of barley seedling age were investigated in 10 Brazilian barley cultivars. All cultivars showed few isoenzymes in early germination. An increase in general activity ensued in the following days when new isoenzymes were detected and those already observed since early germination had their activity increased. All cultivars disclosed increase in amylase activity until the third or fourth day of germination. Some cultivars maintained this high activity until the last day analysed. Other cultivars presented a decrease in activity in the fifth or sixth day. No electrophoretic pattern or allelomorph responsible for a higher amylase activity were detected. Beta -amylase activity was always superior to alpha -amylase activity. High beta -amylase activity was already observed on the second day of germination while alpha -amylase activity began to increase only from the third day on. The results obtained suggest that, at least for the cultivars analysed, there is a high general amylase activity around the fourth day of germination, indicating that germination could stop at this moment, ensuring that hydrolitic enzyme activity required in the brewing process is met. Beta -amylase was lightly correlated with diastatic power (r=0·565) but no correlation was observed between alpha -amylase and diastatic power (r=-0·128), or neither betweenalpha - and beta -amylase with malting quality (r=0·153 andr =−0·348, respectively). These results indicate that beta -amylase activity in barley grains, more than alpha -amylase, can be a good predictor of diastatic power.     

Variation of Beta -Amylase Activity in Barley as Affected by Cultivar and Environment and its Relation to Protein Content and Grain Weight

The cultivar and environmental variation of beta -amylase activity was studied using two barley cultivars with contrasting growth properties. There was a significant difference in beta -amylase activity between the two cultivars used, 92-11 being significantly higher than Xiumai 3. A significant variation in beta -amylase activity was detected between grains at different positions within a spike. The two cultivars showed the same pattern, with top grains showing the highest and bottom ones the lowest activities. The relative difference within a spike varied between the cultivars, with 92-11 being larger than Xiumai 3. Both seeding rate and timing of N application dramatically affected the beta -amylase activity. With N application at the booting stage, beta -amylase activity increased, mainly due to the significantly increased beta-amylase activity in the topmost grains. The bottom grains showed a lower response to timing of N application. The variation in protein content and grain weight between cultivars and among the various treatments was also examined. The possible influence of these factors on beta -amylase activity are discussed.     

Mapping of Barley (Hordeum vulgare L.) Beta -amylase Alleles in which an Amino Acid Substitution Determines Beta -amylase Isoenzyme Type and the Level of Free Beta -amylase

The three beta -amylase genes (Bmy1, 2 and 3) in cultivated barley were mapped to chromosomes 4HL, 2HL And 4HL respectively using RFLP analysis. No recombinants between Bmy1 andBmy3 were detected among 264 DH lines. Polymorphism of the Sd1 and Sd2 isoenzymes of beta -amylase co-segregated with the Bmy loci on chromosome 4HL in a doubled-haploid population of the cross Chebec (Sd2)×Harrington (Sd1). This locus also explained 90·5% of the variation in the level of free enzyme between the two parents. Two cDNAs ofbeta -amylase were isolated by RT-PCR from the developing grains of Harrington (Sd1) and Galleon (Sd2). Alignment of the deduced amino acid sequences identified three amino-acid substitutions between the Sd2 and Sd1 forms of beta -amylase (Arg115 – Cys, Asp165 – Glu, and Val430 – Ala). Three allele-specific PCR primer pairs based on the three amino acid substitutions were used to amplify the beta -amylase genes in genomic DNA of sixteen barley cultivars/lines. Only the Arg115(Sd2)/Cys(Sd1) substitution was consistent with the isoenzyme form. This amino acid replacement reduced the pI of the Sd1 beta -amylase consistent with the fact that the Sd2 form is more basic than the Sd1 form when separated by IEF. The mutation from Arg115 to Cys in the Sd1 form also provides one more -SH group to form S-S-bridges. As bound beta -amylase is linked to the insoluble proteins of the endosperm and its inhibitor via disulphide bridges this could explain the higher level of binding exhibited by Sd1 vs Sd2. Thus a single amino acid substitution determines both the isoenzyme type and beta -amylase binding.     

Modelling the Contribution ofAlpha-Amylase,Beta-Amylase and Limit Dextrinase to Starch Degradation During Mashing

Response surface methodology was used to determine the levels ofalpha-amylase,beta-amylase and limit dextrinase enzymes required for efficient conversion of starch to fermentable sugars during mashing. Micro-scale mashes with purified barley starch and malt enzymes were performed in a Brewing Research Foundation mash bath, and mash liquors were analysed for solubilised starch, reducing sugars (neocuproine assay) and fementable sugars (anion exchange HPLC). Fermentable sugars in the mash liquor were positively correlated with reducing sugars (R²=0·94) and the percentage of starch solubilised during mashing (R²=0·68). A multiple regression equation relating the levels of the three starch degrading enzymes to the percentage of starch hydrolysed to fermentable sugars gave a good fit to the second order response surface (R²=1·00, RMSE=1·37%). Addition of limit dextrinase to the mashes resulted in a substantial increase in levels of fermentable sugars, and limit dextrinase showed a synergistic effect in increasing levels of maltose in the mash liquor when combined with high levels ofbeta-amylase. The efficiency of any one starch degrading enzyme in a mash is influenced by the presence of other starch degrading enzymes. Commercial malts contain excess levels ofbeta-amylase and below optimal levels of limit dextrinase. Malt extract may not be a good indicator of the level of fermentable carbohydrates produced during mashing.     

Genetic and environmental variation in the diastatic power of australian barley

Variation in the diastatic power of Australian barley, and the relationships between diastatic power and the starch-degrading enzymes contributing to diastatic power, were investigated in 11 cultivars of barley grown at six diverse locations in Australia. Diastatic power varied with genotype and location, with the levels ranging from 3·1 to 16·5 U/kg. For alpha-amylase activity, levels across cultivar and location ranged from 52 to 214 U/g, for beta-amylase activity they ranged from 201 to 1550 U/g; and, for limit dextrinase activity, they ranged from 56 to 636 U/kg. Alpha-amylase (r = 0·64) and beta-amylase (r=0·77) activities were correlated more strongly with diastatic power than was limit dextrinase (r=0·37). Grain nitrogen content was correlated positively with diastatic power (r=0·71), largely because of the relationship between nitrogen content and beta-amylase activity (r=0·82). High grain nitrogen contents were also associated with small grain sizes (r=−0·76) and low hot-water extracts (r=−0·75). The levels of alpha-amylase activity were correlated more closely with limit dextrinase activity (r=0·65) than with beta-amylase activity (r=0·28). The results indicate the need to select barley cultivars separately for alpha-amylase and beta-amylase activities to achieve high levels of diastatic power.     

The endogenous endoprotease inhibitors of barley and malt and their roles in malting and brewing

Endoproteases play an important role in barley germination by controlling the hydrolysis of the grain's storage proteins into peptides and amino acids that are needed by the young plant. During malting, the commercial version of this process, many high M_r barley biopolymers are converted into malt nutrients that can be utilized by yeasts during brewing. However, barley and malt both contain endogenous proteins that inhibit the enzymatic activities of these proteases. High levels of these inhibitors can cause brewing problems by preventing the proteases from producing optimal levels of soluble proteins and amino acids. Both high and low M_r inhibitors of cysteine proteases occur in barley and malt. Two of the high M_r inhibitors, lipid transfer protein 1 (LTP1) and LTP2, have been purified and studied. Recently, members of the trypsin/alpha-amylase inhibitor protein family (CM proteins) have been shown to inhibit the activity of SEP-1, a purified serine class barley protease. No inhibitors of aspartic proteases or metalloproteases have yet been purified, but it has been reported that endogenous metalloprotease inhibitors do exist. The inhibitors of the cysteine proteases and metalloproteases are probably the ones most important for brewing, because members of these two protease classes apparently catalyse most of the protein hydrolysis that occurs during malt mashing and, presumably, also during malting. More biochemical studies are needed to clarify how these proteins interact with the proteases to control protein hydrolysis during germination.     

The endogenous endoprotease inhibitors of barley and malt and their roles in malting and brewing

Endoproteases play an important role in barley germination by controlling the hydrolysis of the grain's storage proteins into peptides and amino acids that are needed by the young plant. During malting, the commercial version of this process, many high M_r barley biopolymers are converted into malt nutrients that can be utilized by yeasts during brewing. However, barley and malt both contain endogenous proteins that inhibit the enzymatic activities of these proteases. High levels of these inhibitors can cause brewing problems by preventing the proteases from producing optimal levels of soluble proteins and amino acids. Both high and low M_r inhibitors of cysteine proteases occur in barley and malt. Two of the high M_r inhibitors, lipid transfer protein 1 (LTP1) and LTP2, have been purified and studied. Recently, members of the trypsin/alpha-amylase inhibitor protein family (CM proteins) have been shown to inhibit the activity of SEP-1, a purified serine class barley protease. No inhibitors of aspartic proteases or metalloproteases have yet been purified, but it has been reported that endogenous metalloprotease inhibitors do exist. The inhibitors of the cysteine proteases and metalloproteases are probably the ones most important for brewing, because members of these two protease classes apparently catalyse most of the protein hydrolysis that occurs during malt mashing and, presumably, also during malting. More biochemical studies are needed to clarify how these proteins interact with the proteases to control protein hydrolysis during germination.     

Protein and hordein content in barley seeds as affected by nitrogen level and their relationship to beta-amylase activity

Two barley cultivars differing in grain size and protein content were used to investigate the effects of nitrogen nutrition, cultivar and their interaction on grain protein content, hordein content and beta-amylase activity and the relationship between hordein content and beta-amylase activity during in vitro spike culture. The content of protein and hordein fraction, and beta-amylase activity in barley grains increased as the nitrogen level in culture solution increased. Grain protein content was significantly affected by nitrogen treatment and cultivar, and there was no significant interaction between nitrogen treatment and cultivar. Hordein content and beta-amylase activity were significantly affected by nitrogen treatment and cultivar as well as their interaction. Beta-amylase activity was positively correlated with grain protein and hordein contents, and the ratio of hordein B:C was negatively correlated with total protein content and beta-amylase activity.     

LTP is not a Cysteine Endoprotease Inhibitor in Barley Grains

Several different protease inhibitors have been identified in the mature barley grain, which are proposed to play a defensive role against potential barley pathogens. Cysteine protease inhibitors have been detected in mature grain and in the early stages of germination. The nature of these inhibitors has recently been investigated, and barley lipid transfer protein (LTP1) has been identified as an effective inhibitor of both cysteine and serine endoprotease activity expressed in germinating grain. We show that barley LTP1, in its native state, is not a cysteine protease inhibitor, but in a denatured state becomes a preferred substrate for the barley endoprotease EP-B and, as such, behaves as a competitive inhibitor for poorer substrates of EP-B. The presence of significant amounts of LTP1 in barley malt beer suggests that this very compact protein is highly resistant to proteolytic attack during malting and mashing and its denaturation during wort boiling coincides with inactivation of the malt endoproteases. Analysis of the cleavage products of denatured LTP1, generated by EP-B, provides further evidence for the cleavage site specificity of this barley cysteine endoprotease, where a hydrophobic residue in the P₂position is strongly preferred.     

Beta -Amylase Activity and Thermostability in Two Mutants Derived from the Malting Barley cv. Triumph

The malting barley cultivar, Triumph, and two mutants derived from it with higher (TL9) and lower (TL43) dormancy, respectively, were grown in replicated trials at Lleida, Spain and Dundee, Scotland, in 1999. Measurement ofbeta -amylase in the mature grain showed both mutants to have higher enzyme activity than the parental type with Spanish-grown samples higher in beta -amylase than Scottish-grown. There were no genotypic differences in beta -amylase thermostability or in the portion of the enzyme that was water-soluble. However, for all three genotypes, Scottish-grown samples had a higher proportion of water-soluble beta -amylase and total beta -amylase thermostability was also higher in the Scottish-grown samples. Data from the Spanish-grown samples suggested that water-soluble beta -amylase was slightly more thermostable than the portion of the enzyme released by papain.     

Associations between caryopsis dormancy, α-amylase activity,and pre-harvest sprouting in barley

Pre-harvest sprouting (PHS) is a concern for barley (Hordeum vulgare L.) producers, grain processors, and researchers worldwide. Pre-harvest sprouting has been mainly attributed to low dormancy, which is determined by genotype, stage of plant maturation, and environmental conditions during caryopsis development. Fourteen barley genotypes were sown in field experiments at two sites in North Dakota in 2004 and 2005. Spikes were harvested at four different stages: ≈500 g kg⁻¹ moisture content, physiological maturity, harvest maturity, and post-harvest maturity. Results indicated that barley genotypes were released from dormancy at different rates. The 14 barley genotypes were divided into three classes based on their dormancy loss rate during caryopsis development. C93-3230-24 was highly dormant, and ‘Stander’ and ‘Legacy’ were highly susceptible to PHS due to lack of dormancy from as early as 20 d after heading date. All other genotypes fell into the third group that had intermediate dormancy loss rate. No significant correlation was detected between barley α-amylase activity and germination percentage. A moderate association between malt α-amylase activity and caryopsis dormancy suggested that cultivars with increased malt α-amylase activity tend to have low dormancy and may be more prone to PHS.     

CerealBeta-Amylases

Cerealbeta-amylases are perhaps best known in terms of the vital role they play in releasing easily fermentable sugars from cereal grain starch to fuel the production of alcohol by yeast in brewing. The extent to which they have been investigated is indeed largely due to their significance in this economically important industry. However, cerealbeta-amylases are also, or could be, employed in many other aspects of the food industry and the analysis of starch, and they constitute valuable markers in cereal assessment and breeding studies. Quite apart from their practical significance, they are rewarding objects of biochemical and physiological research. They are interesting models for the study of enzyme polymorphism, post-translational modification and the differential expression of isoenzymes. In spite of their often high activitiesin situand all that is known about their generation, they are an enigma in that their physiological function, or even necessity, remains unclear. It has been recently recognised that there are two different categories of cerealbeta-amylases which exhibit different tissue and taxonomic specificities and physiological developmental patterns. The «classical»beta-amylases present at high activities in cereal seeds appear to be limited to the endosperm of the species of the Triticeae tribe of the Festucoideae subfamily of the Gramineae (wheat, barley and rye), whereas all cereals exhibit a different, tissue-«ubiquitous» form of the enzyme which is present at much lower activity levels. The physiological phenomenology and the usage of cerealbeta-amylases are discussed in relation to these two categories of enzyme.