SITE OF α-AMYLASE IN DEVELOPING BARLEY KERNELS

Dissection studies of two cultivars of barley showed that the pericarp of developing barley kernels contained α-amylase as well as starch granules. Shortly after anthesis, the starch granules disappeared completely and the α-amylase activity of the pericarp fell rapidly to a low level that was maintained until the kernels reached maturity. The α-amylase was isolated, partially purified by ion-exchange chromatography on carboxymethyl (CM) cellulose followed by selective precipitation with glycogen, and characterized by analysing the end-products of its action on amylose.     

RELATIONSHIPS BETWEEN β-AMYLASE POLYMORPHISMS IN DEVELOPING,MATURE AND GERMINATING GRAINS OF BARLEY

A β-amylase polymorphism (electrophoretic forms Sd1 and Sd2) in barley malt is shown to be closely associated with the proportion of free to total (free plus latent) β-amylase, but not with the level of total β-amylase in the mature grains of 46 cultivars. All of the cultivars with Sd1 malts have a proportion of free β-amylase less than 50% (usually from 30% to 40%) of total whereas Sd2 types have free to total β-amylase (F/T) ratios greater than 50% (usually between 62% and 78%). These polymorphisms are also correlated with forms of β-amylase in the developing grain, although, in the latter, Sd1 cultivars can be divided into two types, Sd^d and Sd^e which cannot be distinguished either in malts or on the basis of F/T ratio. Unusual F/T ratios of an intermediate type (approximately 50%) and a very low type (under 30%) also occurred in these experiments. These may result from environmental effects or may be new genetic types.     

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.     

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 .     

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.     

FACTORS AFFECTING THE PRODUCTION OF α-AMYLASE IN BARLEY GRAINS

The production of α-amylase by the endosperm of barley varieties, both commercial and non-commercial, in normal germination and after incubation with gibberellic acid, has been measured. The level of activities produced differed considerably between varieties. All varieties tested produced considerably more enzyme when supplied with a source of exogenous gibberellic acid than under normal germination. In general the differences between varieties were less in the de-embryonated treatment.     

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.     

TISSUE DISTRIBUTION OF α-AMYLASE AND PHOSPHORYLASE IN DEVELOPING BARLEY GRAIN

Approximately half the total α-amylase and phosphorylase activity detectable in crude homogenates of the tissues of developing barley grain was localized in the pericarp. This tissue is particularly active in the early stages of grain development. It is suggested that this activity may influence the starch type and content of the mature barley grain.     

SIMPLIFIED METHOD FOR CALCULATING THE RESULTS OBTAINED WITH BRIGGS' α-AMYLASE ASSAY

A theoretical basis for Briggs' starch iodine colour α-amylase assay is presented and, from this, a simple method for calculation of α-amylase concentration is derived.     

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 FROM IMMATURE BARLEY: PURIFICATION AND PROPERTIES

α-Amylase, extracted from Conquest barley harvested during the period 7 to 10 days after anthesis, was purified using acetone fractionation, glycogen complex formation and ion exchange chromatography. The specific activity of the enzyme was increased 750-fold during purification. The purified α-amylase was homogeneous and free of other starch-metabolizing enzymes. Changes in enzyme activity and stability with pH and temperature were studied and the molecular weight and isoelectric point of the enzyme were determined.     

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.     

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.     

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.     

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.     

PRODUCTION OF α-AMYLASES IN GERMINATING WHOLE AND INCUBATING DE-EMBRYONATED BARLEY KERNELS*

α-Amylases produced in germinated barley and incubated de-embryonated barley kernels (cv. Bonanza), in the absence and presence of gibberellic acid (GA₃), were analyzed qualitatively by polyacrylamide gel isoelectric focusing (PAG-IEF) and quantitatively by chromatofocusing. Identical patterns of α-amylase components were obtained for both germinated barley and incubated de-embryonated barley kernels at each germination/incubation stage, in the absence or presence of GA₃. Total α-amylase increased rapidly in the germinating whole seed whereas in the incubating de-embryonated grain the α-amylase activity increase was much slower. Addition of exogenous GA₃ did not induce production of higher levels of α-amylase in either the germinating whole or incubating de-embryonated barley kernel. Quantitative chromatofocusing analysis revealed that the proportion of α-amylase III to α-amylase II activity decreased linearly with germination time in the whole grain but remained constant in the incubating de-embryonated grain in the absence or presence of GA₃. The major proportion of α-amylase activity in the germinating whole grain and incubating de-embryonated grain was synthesized in the form of α-amylase II components. However, α-amylase I represented a larger proportion of the total α-amylase activity produced in the incubating de-embryonated grain, as compared to the germinating whole seed in the absence or presence of GA₃. These results suggest that embryo excision differentially affects production of α-amylase II as compared to α-amylase I.     

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.     

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.     

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.     

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.