INITIAL STAGES IN α-AMYLOLYSIS OF BARLEY STARCH

The initial stages in α-amylolysis of large and small barley starch granules were followed by gel chromatography. Hydrolysis of gelatinised large and small granules is very similar, indicating a similar structure of the individual components in the two starches. Amylolysis of intact large granules is quite different from that of intact small granules, suggesting differences in the mode of packing of the starch components within the granules. It is concluded that α-amylolysis is of a non-random nature, probably due to the amylopectin component of the starch. On the basis of the intermediate products obtained, a possible structure for the amylopectin molecule is presented, based on a unit cluster with a molecular weight of 3 × 10⁴.     

EFFECT OF pH,TEMPERATURE, AND CALCIUM IONS ON BARLEY MALT α-AMYLASE ISOENZYMES

In this study we have detected the α-amylase isoenzymes in the two barley varieties ‘Ingrid’ and ‘Pomo’. We further report on the effect of different pH and temperatures on the activities of the α-amylase I and II (following the nomenclature of MacGregor & Daussant¹⁸) in ‘Pomo’ malt, as well as the stabilising effect of Ca²⁺ under such different conditions. The importance of enzyme concentration and the inhibition of Hg²⁺ is also considered.     

α-AMYLASE IN DEVELOPING BARLEY KERNELS — A REAPPRAISAL1

The outer layers that can be dissected readily from immature barley kernels were identified by light microscopy. α-Amylase was located in the outer pericarp of developing Bonanza and Himalaya barley. Only low pl α-amylase (α-amylase 1) was detected in the pericarps but only one of the two major low pl components of α-amylase from germinated Himalaya was found in the pericarp tissue. These enzymes appeared to hydrolyze the small starch granules (2–3 μm) present in the outer pericarp of developing barley kernels. The even smaller starch granules (0.25–2μm) present in the inner pericarp were hydrolyzed at a later stage of kernel development.     

THE DETERMINATION OF BARLEY α-AMYLASE ACTIVITY

Starch-iodine colour (S.I.C.) units of α-amylase activity are re-defined. Two improved, alternative methods of calculating results are given, either (a) by a revised graphical procedure used in conjunction with a standard graph, or (b) by a graphical procedure using points calculated from the experimental results with an empirical equation using a programmed electronic calculator. Semiautomation of the measurement of starch-iodine colours usefully reduces the work load involved in this enzyme assay. Enzyme activities may be expressed in S.I.C. units, or as a rate of increase in reducing power.     

SECRETION OF α-AMYLASE BY THE EPITHELIUM OF BARLEY SCUTELLUM

Aleurone-free scutella prepared from barley (Hordeum vulgare L. cv Himalaya) grains after 1 day of germination released α-amylase into the incubation medium at a constant rate for at least 2 hours. The release was arrested by a Golgi inhibitor, monensin which caused accumulation of α-amylase inside the scutella instead of the incubation medium. Immunofluorescence labeling showed that α-amylase in the scutella was located in the epithelial cells, no label was found in the parenchyma cells. These results indicate that the scutellar epithelial cells have a true capacity to synthesize and secrete α-amylase.     

BACTERIAL EXPRESSION OF THE BIFUNCTIONAL α-AMYLASE/SUBTILISIN INHIBITOR FROM BARLEY

The bifunctional α-amylase/subtilisin Inhibitor (BASI) is an endogenous inhibitor of the high pl cereal α-amylases encoded by the amyl genes. Evaluation of the potential role of this protein in malting and brewing would be greatly assisted by the availability of large quantities of the protein. We have produced the protein by expression of the barley gene in bacteria. The barley gene was cloned into a pMAL vector and expressed as a fusion protein. The purified fusion protein was successfully cleaved with a specific protease to release the native BASI protein. The BASI produced by bacterial expression will be a useful source of the protein for studies of interactions with barley α-amylases and studies of the influence of this protein on malting and brewing.     

IMPROVED EXTRACTION AND ASSAY OF β-AMYLASE BROM BARLEY AND MALT

β-Amylase was extracted from barley or malt using four physical techniques to break up grists which had been prepared using a Moulinex coffee grinder. Grinding with a Polytron homogeniser apparently completely disrupted all cells, as determined by transmission electron microscopy, and increased the efficiency of extraction of β-amylase from barley by more than 30%. The other treatments tested were without value . The β-amylase activity in extracts of barley or malt was assayed by measuring the production of reducing sugars from reduced soluble starch, using a PAHBAH reagent. α-Amylase, which interferes with the quantitation of β-amylase in extracts of malt, was not totally inactivated by the chelating buffer used for enzyme extraction or by several other chelating agents. α-Amylase activity was quantified specifically using Phadebas. Using purified α-amylase a calibration was developed which related activity, as determined using Phadebas, to reducing power units. Thus the α-amylase activity present in an extract containing β-amylase could be determined using Phadebas and the reducing power equivalent activity subtracted from the total “apparent” activity to give the actual β-amylase activity. α-Glucosidase and limit dextrinase activities are believed to be too low to have a significant effect on the apparent β-amylase . The soluble and bound β-amylase activities were measured in samples taken from micromalting barley (Alexis). Dry weight losses increased to over 10% after 8 days germination. Antibiotics, applied during steeping, were used to control microbes in one experiment. However, their use checked germination and reduced malting losses to 8.4% in 8 days germination. The soluble enzyme present in extracts from steeped barley and early stages of germination was activated (20–40%) by additions of the reducing agent DTT .     

EVIDENCE FOR THE PRESENCE OF MALTASE AND α-GLUCOSIDASE ISOENZYMES IN BARLEY

An examination of the development of α-glucosidase and maltase activities (as measured by the hydrolysis of p-nitrophenyl α-D-glucoside and maltose respectively) indicated that two genotypes of Glacier barley had the same general pattern of enzyme development. However, the development of α-glucosidase activity followed a different course from that of maltase activity suggesting that separate enzyme proteins are involved in hydrolysing these substrates. Further evidence that separate enzyme proteins were responsible for hydrolysis of maltose and p-nitrophenyl α-glucoside was obtained by column chromatography of extracts of germinated barley which indicated the presence of two maltases and two α-glucosidases. The maltases and the α-glucosidases differed in molecular weight, pH of optimum activity and in thermostability. When isomaltose was used as a substrate the optimum pH and behaviour on gel chromatography were coincident with that of maltase activity but different from the α-glucosidases.     

MULTIPLE FORMS OF α-AMYLASE IN MALTING BARLEY VAR. EMMA.

Multiple forms of α-amylase arise from translation of separate messages and post-translational proteolytic modification during malting. α-amylase activity is tolerant of proteolysis. The enzyme appears to have two domains one of which is associated with starch cleavage. The second site which binds cyclodextrin is destroyed by proteolysis. Cleavage yields a low molecular weight form which retains α-amylase activity.     

α-AMYLASE COMPONENTS IN EXCISED,INCUBATED BARLEY EMBRYOS*

Excised embryos from kernels of Betzes and Bonanza barley were incubated on sand for 3 days in the presence of CaCl₂ (10-3³M), CaCl₂ + gibberellic acid (10-⁴M), CaCl₂ + casein hydrolysate (0·2%) and a mixture of all three. α-Amylase components in these embryos were analysed quantitatively by chromato-focusing. The α-amylase I component predominated in all samples. Addition of gibberellic acid or casein to the incubation medium did not increase α-amylase synthesis but addition of casein did appear to lower the proportion of α-amylase I synthesised.     

ISOLATION,PURIFICATION AND ELECTROPHORETIC PROPERTIES OF AN α-AMYLASE FROM MALTED BARLEY

An α-amylase component from malted barley was isolated and purified using aqueous extraction at pH 8·0, heat treatment of the extract at 70°C, specific precipitation with glycogen and ion exchange chromatography on carboxymethyl (CM) and diethylaminoethyl (DEAE) cellulose. The enzyme preparation was shown to be pure by disc electrophoresis at pH 8·9 and iso-electricfocusing on polyacrylamide gel in a pH 4–8 gradient.     

QUANTITATIVE DETERMINATION OF α-AMYLASE ENZYMES IN GERMINATED BARLEY AFTER SEPARATION BY ISOELECTRICFOCUSING1

Quantitative extraction of malt and germinated barley α-amylases from polyacrylamide gels after isoelectricfocusing was achieved using bovine serum albumin (2 mg/ml) in the extracting medium. Sharp bands of activity were obtained when extracts from polyacrylamide gels were re-focused on another gel. This technique demonstrated that α-amylase III was the major component in malt and germinated barley extracts. This enzyme was converted to α-amylase II when such extracts were heated at 70°C.     

RELEASE AND ACTIVATION OF BARLEY β-AMYLASE

The aim of this study was to determine the role of low molecular weight thiols both in the release and activation of β-amylase during grain germination. In quiescent barley grains (Hordeum vulgare L. cv. Torrent) about 55% of the β-amylase was extracted with buffer, the remaining 45% was in the bound form. During micromalting the bound form was progressively solubilised between germination days 1 and 4. When free β-amylase, extracted from ungerminated grains, was incubated with dithiothreitol the enzymic activity increased by 15%-20%. This activation did not occur when free β-amylase, from grain germinated for 3 days or more, was incubated with DTT. The release of bound β-amylase with thiols was pH dependant, occurring most rapidly at and above pH 8.0. At the onset of germination the embryo released soluble thiol (approximately 5 nmol per embryo) into the endosperm. Degermed grains were dosed with reduced glutathione and incubated for 72 h. The addition of 60 nmol glutathione caused the release of about 80% of the bound β-amylase. When less glutathione was used, 5 nmol (an amount similar to that released by the embryo in vivo) no significant release of the bound enzyme was detected. When degermed grains were dosed with oxidised glutathione (60 nmol), no bound β-amylase was released. However, addition of the disulphide bis-hydroxyethyldisulphide (60 nmol) did cause the release of about 90% of the bound enzyme. The aleurone layer reduced the bis-hydroxyethyldisulphide to a thiol, presumably 2-mercaptoethanol. Oxidised glutathione and cystine were not significantly reduced to thiols by isolated aleurone layers. The aleurone layer did cause the disappearance of cysteine from solution. When preparations of bound β-amylase were incubated with extracts from the endosperms of grains germinated for three days, the bound enzyme was released. This release was due to the high molecu lar weight material (>5 kDa) in the extract and not to low molecular weight thiols. It seems unlikely that simple thiols, such as glutathione, are solely responsible for the release of bound β-amylase.     

EVALUATION OF MALTING QUALITY IN A BARLEY BREEDING PROGRAMME USE OF α-AMYLASE AND β-GLUCAN LEVLES IN MALT AS PRESELECTION TOOLS

Analysis according to the EBC protocol, immunological determination of a α-amylase and estimation of malt β-glucan using the Calcofluor-FIA method, were used to screen 327 barley breeding lines for malting quality. The results obtained with the α-amylase and β-glucan methods are highly correlated to the important malt quality paramters: extract yield and β-glucan content in the wort. It is recommended that either of the two methods, which are simple to perform are used as prescreening tools in breeding programmes for malting barley.     

ASSESSMENT OF BACILLUS LICHENIFORMIS α-AMYLASE AS A CANDIDATE ENZYME FOR GENETIC ENGINEERING OF MALTING BARLEY1

Bacillus licheniformis α-amylase, a thermostable starch-degrading enzyme, has been assessed as a candidate enzyme for the genetic transformation of malting barley. The temperature optimum, pH optimum and thermostability of B. licheniformis α-amylase were compared with those of barley α-amylase. The bacterial enzyme has a higher pH optimum (?9), a higher temperature optimum (?90°C) and much higher thermostability at elevated temperatures than the barley enzyme. The specific activity of the bacterial enzyme under conditions of pH and temperature relevant to the brewing process (pH 5.5, 65°C) is ?1.5-fold higher than that of the barley enzyme. Measurements of α-amylase activity during a micro-mash showed that the bacterial enzyme is at least as stable as the barley enzyme under these conditions, and that a level of expression for the bacterial enzyme corresponding to ?0.5% of total malt protein would approximately double the α-amylase activity in the mash. B. licheniformis α-amylase activity was rapidly eliminated by boiling following mashing as would occur during brewing. The combined results suggest that barley expressing the bacterial enzyme may be useful in the brewing process.     

DETERMINATION OF α- AND β-AMYLASE IN MALT

The Analysis Committee of the European Brewery Convention carried out a collaborative trial on malts using the specific analysis methods for α- and β-amylase activities based on dyed substrates supplied by MegaZyme (Aust.) Pty. Ltd. The repeatability and reproducibility values for the methods were judged to be unsatisfactory and consequently the methods were not recommended for Analytica-EBC.     

THE MILLING ENERGY OF MALTED BARLEY AND ITS RELATIONSHIP WITH HOT WATER EXTRACT AND α-AMYLASE ACTIVITY

Hot water extract is dependant on endosperm structure and its breakdown during malting. These endosperm characteristics can be rapidly assessed on 5 g samples by measuring the milling energy of malted barley. α-amylase determinations on the resultant flour indicate that good malting barleys have either moderate or high levels of the enzyme. High levels of α-amylase will not confer good malting quality where endosperm modification has proceeded slowly.     

PURIFICATION OF BARLET MALT α-AMYLASE BY IMMUNOAFFINITY CHROMATOGRAPHY

Immunoaffinity chromatography was used to purify the high pl α-amylase (α-amylase II) in a one step procedure after fractionation of the whole barley malt extract on Sephadex G25. The immunoglobulin G (IgG) fraction of an immune serum specific for the malt α-amylase II was immobilized on Ultrogel. A mild desorption procedure was used, combining distilled water elution with an interrupted elution. The purification was achieved within half a day including kernel extraction. The quality of the purification was assayed by SDS polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and isoelectric focusing. For the second technique, an immune serum was used which was polyspecific for malt proteins including the high pl α-amylase (α-amylase II). The effect of this procedure on the specific activity of the enzyme and on its antigenicity was evaluated. The results underline the efficiency of the purification procedure and indicate that α-amylase II accounts for a few percent of the total soluble protein in malts. However, the α-amylase II fraction was not completely free from α-amylase I. The procedure resulted in a partial loss of the enzymatic activity but not of the antigenicity.     

DIFFERENT RATES OF DEVELOPMENT OF α-AMYLASE IN DISTAL ENDOSPERM ENDS OF GERMINATED (MALTED) CHARIOT AND TIPPER BARLEY VARIETIES

Chariot Barley had a faster malting rate than Tipper. Even when the total levels of the aleurone-produced enzyme α-amylase were similar in both Chariot and Tipper, the distal ends of the grains of Chariot developed α-amylase at a faster rate than the corresponding endosperm ends of Tipper. The excised aleurone layers of Chariot and Tipper had similar potentials to produce α-amylase. Therefore the faster rate of development of α-amylase in the distal ends of the grains of Chariot may reflect faster transport of active gibberellic acid through the aleurone layer. Differences in the rates of transport of gibberellic acid through the plasmodesmata of the aleurone layer may determine the efficiency of production and distribution of endosperm-degrading enzymes during malting. The transport of gibberellic acid in the aleurone layer was facilitated by high moisture levels.     

OPTIMIZED McCLEARY METHOD FOR MEASUREMENT OF TOTAL β-AMYLASE IN BARLEY AND ITS APPLICABILITY

The McCleary method for determination of β-amylase in malt has been modified in order to allow determination of total β-amylase in barley as well as malt. A ruggedness test, performed on the modified method, demonstrated that the method is quite robust and highly reproducible. When the variables α-amylase, β-amylase and diastatic power were measured in 90 malt samples, only β-amylase was significantly correlated to diastatic power (r² = 0.85 and p < 0.0001). The same high correlation was found between total β-amylase in 20 barley samples and diastatic power in the corresponding malts. The validity of this relationship was tested by predicting diastatic power in malt from total β-amylase in barley. Predicted values correlated highly to measured values (r² = 0.95). In breeding material a positive relationship was found between total β-amylase in barley and protein content. This relationship must be considered when evaluating new barley lines.