Characterization of Oat Endoproteinases that Hydrolyze Oat Avenins

During oat seed germination, the insoluble storage proteins must be solubilized and transported to the embryo for use by the developing plantlet. We showed earlier that pH 6.2 active serine and metalloproteinases were the predominant gelatin‐hydrolyzing enzymes of oats, while the oat globulins were degraded by pH 3.8 active cysteine proteases. The pH of the endosperms of germinating oats is 6.2. We have continued our characterization of the germinated oat proteinases by determining which hydrolyze avenins, the oat storage prolamins. Avenins of resting seeds were purified and hydrolyzed with proteinases that were extracted from oat seeds that were germinated for various periods. The peptides released were analyzed using SDS‐PAGE. The α‐avenins were hydrolyzed at pH 3.8 by cysteine proteinases from four‐day germinated seeds and the β‐avenins were hydrolyzed by similar enzymes from eight‐day germinated seeds. At pH 6.2 or pH 5.0, the avenins were not degraded by any of the germinated oats endoproteinases. It is probable that some kind of pH compartmentalization occurs within germinating oat seed. After four days of germination, either new proteinases form or some preexisting proteinases are activated. The cysteine proteinases are apparently responsible for the majority of the storage protein hydrolysis that occurs during oat germination.     

Proteolytic Enzymes in Germinating Rye Grains

The proteolytic activities during rye (Secale cereale L. ‘Humbolt’) grain germination were monitored using in‐solution methods and one‐ and two‐dimensional PAGE with gels that contained incorporated substrate proteins. The total proteolytic activity increased during the first three days of germination, but not after that. The proteinase activity was measured at pH 3.8, 6.0, and 8.0 in the presence and absence of class‐specific proteinase inhibitors. This indicated that enzymes from all four proteinase classes were present during the germination process. Germinated rye grain contained mainly aspartic and cysteine proteinase activities that are especially active at pH 3.8. Serine‐ and metallo‐proteinases were less abundant. Overall, the pattern of hydrolysis was very similar to that observed during barley and wheat germination.     

Endogenous Action of Cysteine Endopeptidase and Three Carboxypeptidases on Triticale Prolamins

Quantitative and qualitative changes occurring in the prolamin fraction in the starchy endosperm of triticale grains were analyzed by SDS‐PAGE on consecutive days of germination. The most intensive hydrolysis of prolamins was observed after the second day of the process. The high molecular weight fractions of prolamins were degraded with the highest rate. Endopeptidase EP8 was capable of hydrolyzing all fractions of prolamins isolated from dry triticale grains, but the high molecular weight fractions were the most rapidly degraded by the enzyme. Carboxypeptidases I, II, and III isolated from triticale grains hydrolyzed prolamins proteolytically modified by endopeptidase EP8, whereas intact prolamins were degraded slightly. Differences in the activity of the studied carboxypeptidases against crude prolamins indicate that carboxypeptidase II may be involved in the initiation of the hydrolysis process and, together with carboxypeptidases I and III, participates in the later stages of degradation of prolamins to amino acids. Experiments with exogenous GA₃ demonstrated that the synthesis of EP8 is induced by this hormone and takes place in the aleurone layer. Mass spectrometry analysis showed the enzyme to be a homologue of barley endopeptidase EP‐A. Both enzymes belong to the cysteine class endopeptidases.     

Qualitative Zymographic Studies of Major Proteinases from Malted Sorghum (Sorghum bicolor L.) and Effects of Cultivar

Proteolysis during cereal germination is vital both to seedling growth and the success of commercial malting and brewing. In this study, proteinases in proteolytic extracts from seeds and germinated grains of 11 Botswana sorghum cultivars were analyzed and partially characterized by one‐dimensional electrophoresis on SDS‐PAGE gels containing incorporated gelatin. Proteinase polymorphism was detected in both germinated and ungerminated sorghum grains. Fifteen distinct proteinase bands, with M_r values of 27,000–100,000 were detected in sorghum malt extract, while ungerminated sorghum displayed a maximum of four bands (M_r ≈ 78,000–100,000). Band numbers and identity varied markedly according to cultivar. More proteinase bands were detected at pH 4.6, than at pH 6.2 and 7.0, suggesting pH optima considerably below neutrality. Cysteine‐proteinases constituted a higher proportion (9 of 15) of the detected sorghum malt proteinases and were most detectable at pH 4.6. Multiple representatives were also detected for both serine‐ and metallo‐proteinases, although these were more active at pH 6.2 and 7.0. 1‐10 Phenanthroline inhibited malt metallo‐proteinase more strongly than EDTA, suggesting that these enzymes were most probably zinc‐dependent. Aspartyl‐proteinases were not detected, probably because of the substrate employed. Results indicate that the sorghum proteinase system is complex.     

Comparison of Endoproteinases of Various Grains

Two‐dimensional isoelectric focusing (IEF) × PAGE gels were used to compare the endoproteolytic (gelatinase) activities of germinated barley with those of bread and durum wheat, rye, triticale, oat, rice, buckwheat, and sorghum. Barley was used as the standard of comparison because its endoproteinase complement has been studied previously in the greatest detail. The characteristics of the grain proteases were appraised from their migration patterns and by how they were affected by pH levels. All of the germinated grains contained multiple enzyme activities and their separation patterns and pH levels were at least similar to those of barley. The proteinases of the bread and durum wheats, rye, oat, and sorghum were most similar to those of barley, whereas the other grains provided more varied patterns. The rice and buckwheat proteinases developed much more slowly than those of the other grains. The activity patterns of the triticale resembled those of the parents, wheat and rye, but the triticale contained many more activities and higher overall proteolytic activities than any of the other species. These results should be applied to scientific or commercial procedures with caution because grains contain potent endogenous proteinase inhibitors that could inactivate some of these enzymes in various tissues or germination stages.     

Study of metallopeptidase isozymes from malted barley (Hordeum vulgare cv. Morex)

It has been reported that germinated barley contains peptidases that are sensitive to metal-chelating agents; however, none of these enzymes have been isolated, nor have their roles in germinated barley been investigated. Anion-exchange chromatography and chromatofocusing have been used to isolate a group of peptidases from barley (Hordeum vulgare cv. Morex) green malt that are sensitive to metal-chelating agents. Their activities were studied using one- and two-dimensional polyacrylamide gel electrophoresis. When analyzed on two-dimensional PAGE gels that contained gelatin as substrate, the enzymes separated into three major and approximately six minor activity spots with acidic pI values. The enzymes were optimally active against the gelatin substrate at pH 8.0 and were completely inhibited by 1,10-phenanthroline and EDTA, indicating that they belonged to the metallopeptidase class (EC 3.4.24.x). After the enzymes were inhibited with EDTA, the activities were recovered in the presence of low concentrations of metal ions. The hydrolysis of gelatin substrate was also impaired by the presence of reducing agents. The metallopeptidases readily digested, in vitro, the barley prolamine D hordein, indicating that they may be involved in degrading storage proteins during barley germination.     

Quantitative study of the formation of endoproteolytic activities during malting and their stabilities to kilning

The proteinases of germinating barley (Hordeum vulgare L.) hydrolyze storage proteins into amino acids and small peptides that can be used by the growing plant or, during brewing, by yeast. They are critical for the malting and brewing processes because several aspects of brewing are affected by the amounts of protein, peptide, and amino acids that are in the wort. This study was carried out to quantitatively measure when endoproteinases form in green malt and whether they are inactivated at the high temperatures that occur during malt kilning. Little endoproteolytic activity was present in ungerminated barley, but the activities began forming 1 day into the "germination" phase of malting, and they were nearly maximal by the third germination day. Quantitative studies with azogelatin "in solution" assays showed that the green malt endoproteolytic activities were not inactivated under commercial kilning conditions that use temperatures as high as 85 degrees C but that some actually increased during the final kilning step. Qualitative (2-D, IEF x PAGE) analyses, which allow the study of individual proteases, showed that some of the enzymes were affected by heating at 68 and 85 degrees C, during the final stages of kilning. These changes obviously did not, however, decrease the overall proteolytic activity.     

Protein metabolism in rice seedling

The relationship between protein synthesis and degradation in germinating rice seed were studied with protein synthetic inhibitors. Both DNP and 8-AG inhibited the degradation of glutelin, the major storage protein in rice seed, while the inhibitors had no direct effect in the activity of rice seed proteinllse in vitro. The prevention can be partly ascribed to the Inhibition of proteinase synthesis because the inhibitors depressed the increase in proteinase activity during germination. When DNP treatment was started at the onset of germination, the degradation of glutelin in the endosperm was seriously inhibited and the endosperm remained rigid over 9 days of incubation at 30°C. In contrast, the inhibition was less efficient clent when the treatment was started in the later stage. It is suggested that the degradation of the storage protein in rice seed depends on the synthetic process of the hydrolytic enzymes which increase during germination. disintegrate the compartmentation of the endosperm and allow the storage proteins to come in contact with the existing proteinases     

Polymorphism of Aspartic Proteinases in Resting and Germinating Barley Seeds

Both resting and germinated barley seeds (Hordeum vulgare L. ‘Morex’) contain aspartic endopeptidase activities, and the activities increase during germination. We have extracted and partially purified aspartic endopeptidases from both resting seeds and green malt (four-day germinated barley). Six aspartic proteinase activities were found in resting barley seeds while only four activities were detected in green malt. All of the aspartic proteinases had similar pH activity optima (pH 3.5–4.5) and pI values (≈4.5). The purified green malt aspartic proteinases selectively digested a group of barley seed proteins, postulated to serve as defensive proteins, that are coded by the amylase-trypsin inhibitor super gene family. The aspartic proteinases that bound to a pepstatin A affinity column at pH 4.5 cross-reacted with antiserum raised against aspartic proteinases purified from barley seed. However, those that did not bind the affinity column also did not cross-react with the antiserum, indicating that there are two distinct groups of aspartic proteases in germinating barley.     

Thermostability of Barley Malt Proteases in Western Canadian Two‐Row Malting Barley

During the malting process, barley is germinated via a carefully controlled procedure so that its components are degraded to sugars, amino acids, and other low molecular weight compounds that can be used for subsequent fermentation. One of the most important of these processes is the hydrolysis of proteins into peptides and amino acids. During seed germination, proteases hydrolyze insoluble reserve proteins into soluble peptides that are subsequently hydrolyzed into free amino acids. During kilning, green malt is initially air dried at 40–60°C, and then the temperature is gradually increased to 85–95°C. Although most proteases are denatured during kilning, the malt contains a small proportion of heat‐stable protease enzymes able to further break down protein in the subsequent mashing process. In this study, protocols were developed and standardized to measure the activity of different proteases. These protocols were then used to study protease thermostability in Canadian two‐row spring malting barley lines. We found a wide range in protease activity under controlled conditions. Upon heat treatment, several lines exhibited significant protease thermostability. These thermostable enzymes were purified by ammonium sulfate precipitation and Sephadex columns for further study.     

Improved Methods for High-Throughput Extraction and Assay of Green Barley Malt Proteinase Activity Facilitating Examination of Proteinase Activity Across Large-Scale Barley Populations

We report efficient sample extraction and assay methods allowing quantitative determinations of proteinase activities from barley malt. The improved methods are used to assay >2,200 developmental lines of malting barley for two subsets of proteinase activity. The distributions of the resulting activities suggest differences in population structures between the two types of proteinases. Comparison of the activities of the green malt proteinases with standard malting quality measurements show highly significant correlations that differ between the proteinase subsets. The pH 4.5 hydrolysis of the artificial substrate Z-Phe-Arg-AMC correlates well with the traditional malting quality measurements, supporting the role of cysteine-class proteinases in mobilization of grain reserves during malting and mashing. Results from assays of gelatin hydrolysis at pH 6.0 suggest that these proteolytic activities may be involved in other aspects of seed C and N dynamics also linked to malting quality measurements. The differences between the pH 4.5 and 6.0 activities assayed here and their association with malting quality measurements suggest different physiological roles for the two proteinase activities in several aspects of seed germination. Either assay could be useful for population surveys, depending on the particular facet of seed metabolism under study.     

Plant seed cystatins and their target enzymes of endogenous and exogenous origin

Cystatins are protein inhibitors of cysteine proteinases of the papain family, and those of animal origin have long been studied from medical and physiological aspects. In the meantime, oryzacystatin cloned from rice seeds in 1987 was recognized as the first well-defined cystatin of plant origin. Cloning studies followed to disclose various plant cytstatins including those of corn and soybean origin, their similarities to and differences from animal cystatins being analyzed in detail. Plant seed cystatins are now understood as factors controlling germination by inhibition of endogenous cysteine proteinases. They can also recognize insect midgut proteinases as exogenous target enzymes to control. This paper discusses chemical and phytophysiological relationships between cystatins and their targets.     

油菜花粉谷蛋白酶解物的制备

以陈放1年的油菜蜂花粉为原料,对破壁脱脂后的花粉中各组分蛋白含量进行了研究,其中谷蛋白的含量约占总蛋白含量的55.7%,清蛋白为39.O%,球蛋白和醇蛋白只有3.2%和2.1%.以谷蛋白为原料,在单因素试验的基础上,利用响应面分析法对油菜花粉谷蛋白最佳酶解条件进行了研究.结果表明,Alcalase碱性蛋白酶水解油菜花粉谷蛋白的最佳水解条件为底物浓度6%,pH值9.0,水解温度60℃,酶底比1460U/g,水解时间2h,谷蛋白酶法改性后抗羟基自由基的活性能达到70.0%.     

Degradation of gluten by proteases from dry and germinating wheat (Triticum durum) seeds: An in vitro approach to storage protein mobilization

Vital gluten was used as an ideal substrate to investigate the role of some proteases in storage protein degradation. Aspartic proteinase and carboxypeptidase were identified as endogenous enzymes adsorbed on gluten and their optimum pH values determined. SDS-PAGE of soluble products released by gluten digestion revealed that the activity of these proteases plays a minor role in protein mobilization, whereas cysteine proteinase, purified from wheat seeds at the fourth day of germination, is extremely effective, producing a remarkable protein degradation in short times. Synergistic effects of aspartic and cysteine proteinase were not observed. Spin labeling of the sulfhydryl groups of gluten proteins enabled a comparative EPR investigation of the consequences of proteolytic degradation on gluten elasticity. It was found that storage protein mobilization brings a loss of elasticity to the polymeric network of gluten, which is particularly marked when the hydrolysis is performed by cysteine proteinase.     

Degradation of HMW Glutenins During Wheat Sourdough Fermentations

Bakeries use sourdoughs to improve bread properties such as flavor and shelf life. The degradation of gluten proteins during fermentation may, however, crucially alter the gluten network formation. We observed changes that occurred in the HMW glutenins during wheat sourdough fermentations. As fermentation starters, we used either rye sourdough or pure cultures of lactobacilli and yeast. In addition, we incubated wheat flour (WF) in the presence of antibiotics under different pH conditions. The proteolytic activities of cereal and sourdough‐derived proteinases were studied with edestin and casein. During sourdough fermentations, most of the highly polymerized HMW glutenins degraded. A new area of alcohol‐soluble proteins (≈30.000 MW) appeared as a result of the proteolytic breakdown of gluten proteins. Very similar changes were observable as WF was incubated in the presence of antibiotics at pH 3.7. Cereal and sourdough‐derived proteinases hydrolyzed edestin at pH 3.5 but showed no activity at pH 5.5. An aspartic proteinase inhibitor (pepstatin A) arrested 88–100% of the activities of sourdough enzymes. According to these results, the most active proteinases in wheat sourdoughs were the cereal aspartic proteinases. Acidic conditions present in sourdoughs create an ideal environment for cereal aspartic proteinases to be active against gluten proteins.     

Effects of Different Lipase Inactivation Treatments on Physicochemical Properties of Naked Oat Globulins

Lipase inactivation is an essential treatment for oat processing, because of the negative effects of lipase on nutrient preservation and storage extension. The effects of different lipase inactivation treatments including hot air roasting, infrared roasting, normal‐pressure steaming, and high‐pressure steaming on the physicochemical properties of oat globulins were investigated. Results showed that normal‐pressure steaming had little effect on solubility of oat globulins; hot air roasting increased foaming capacity of oat globulins but did not change their foaming stability; and all the inactivation treatments increased the surface hydrophobicity and content of total sulfhydryls of oat globulin but decreased exposed sulfhydryl groups. In addition, oat globulin granules from the hot air roasting treatment were distributed more evenly in oat globulin powder compared with the control group. All treatments except normal‐pressure steaming changed the molecular weight of oat globulin subunits, which made the bands of 66,000 and 45,000 disappear from SDS‐PAGE. These results indicated that normal‐pressure steaming was ideal to maintain good solubility of oat globulins, and hot air roasting was ideal to maintain relatively good foaming properties. The treatments changed physicochemical properties of oat globulins by influencing protein aggregation and subunit composition that resulted in different content of sulfhydryl groups and surface hydrophobicity.     

Trypsin/alpha-amylase inhibitors inactivate the endogenous barley/malt serine endoproteinase SEP-1

Barley (Hordeum vulgare L.) malt contains endoproteinases belonging to all four of the commonly occurring classes, including serine proteinases. It also contains low molecular weight proteins that inhibit the activities of many of these endoproteinases, but it had never been shown that any barley or malt serine proteinases could be inhibited by any of these endogenous proteins. It is now reported that some proteins that were concentrated using an "affinity" method inhibited the activity of a malt serine endoproteinase. Two-dimensional electrophoretic and in vitro analyses showed that the inhibited enzyme was serine endoproteinase 1 (SEP-1) and that the inhibition could be quantified using a semipurified preparation of this enzyme. Amino acid sequencing and MALDI-TOF MS were used to identify the components of the partially purified inhibiting fractions. Only the "trypsin/alpha-amylase inhibitors" or chloroform/methanol (CM) proteins, most of which had truncated N and C termini, and one fragment of beta-amylase were present in the inhibitory fractions. When a CM protein fraction was prepared from barley according to traditional methods, some of its component proteins inhibited the activity of SEP-1 and some did not. This is the first report of the purification and identification of barley malt proteins that can inhibit an endogenous serine proteinase. It shows that some of the CM proteins probably play a role in controlling the activity of barley proteinases during germination, as well as possibly protecting the seed and young plant from microbes or pests.     

The effect of mashing on malt endoproteolytic activities

During malting and mashing, the proteinases of barley (Hordeum vulgare L.) and malt partially hydrolyze their storage proteins. These enzymes are critical because several aspects of the brewing process are affected by the soluble proteins, peptides and/or amino acids that they release. To develop improved malting barleys and/or malting and brewing methods, it is imperative to know whether and when the green malt endoproteinases are inactivated during malting and mashing. These enzyme activities are totally preserved during kilning and, in this study, we have determined when they were inactivated during mashing. Samples were removed from experimental mashes that mirrored those used in commercial breweries and their endoproteolytic activities were analyzed. The malt endoproteinases were stable through the 38 degrees C protein rest phase, but were quickly inactivated when the mash temperature was raised to 72 degrees C for the conversion step. All of the proteinase activities were inactivated at about the same rate. These findings indicate that the soluble protein levels of worts can be varied by adjusting the protein rest phase of mashing, but not by altering the conversion time. The rates of hydrolysis of individual malt proteins probably cannot be changed by altering the mash temperature schedule, since the main enzymes that solubilize these proteins are affected similarly by temperature.     

Seed phosphorus remobilization is not a major limiting step for phosphorus nutrition during early growth of maize

Phosphorus (P) is the least mobile nutrient in the soil as compared to other macronutrients and therefore frequently limits crop growth. During germination and early growth, seed‐phytate hydrolysis and seed‐P remobilization is the major P source for developing seedlings. The objective of this paper was to investigate whether seed‐P hydrolysis and remobilization of nonphytate P are sufficient for seedling P nutrition during early growth stages of maize. A large part of initial maize endogenous seed P reserves are mainly in the form of phytate. Till 70 cumulated degree days after sowing, nearly all the phytate (98%) was hydrolyzed and caused an increase in nonphytate P in seeds. Phytate hydrolysis and remobilization of nonphytate P was the main source of P supply for the newly growing seedlings and was not a limiting step for seedling P nutrition during the first four weeks of early growth.     

Germination‐Improved Ethanol Fermentation Performance of High‐Tannin Sorghum in a Laboratory Dry‐Grind Process

A high‐tannin sorghum cultivar with 3.96% tannin content was used to study the effects of germination on its ethanol fermentation performance in a laboratory dry‐grind process. High‐tannin sorghum sample was germinated for 3 and 4 days. Original and germinated samples were analyzed for tannin, starch, protein, free amino nitrogen (FAN), and glucose content. Endosperm structures and flour pasting properties of germinated and nongerminated sorghum samples were examined using a scanning electron microscope (SEM) and rapid visco analyzer (RVA). Germination reduced tannin content from 3.96% to negligible levels. The free fermentable sugars (glucose, maltose, and maltotriose) in the germinated samples were significantly higher than those in the nongerminated control. Judged by the starch (starch plus dextrin) and free amino nitrogen contents in the mashed samples, germination improved degree of hydrolysis for starch by 13–20% and for protein by 5‐ to 10‐fold during mashing. Germination significantly shortened the required fermentation time for ethanol production by 24–36 hr, increased ethanol fermentation efficiency by 2.6–4.0%, and reduced the residual starch content in the distillers dried grain with solubles (DDGS) compared to the nongerminated control. Ethanol yield for the 3‐day germinated samples was 2.75 gallons/bushel, which was 3.1% higher than the 2.67 gallons for the nongerminated control. Ethanol yield for the 4‐day germinated sorghum was 2.63 gallons/bushel due to excessive loss of starch during germination.