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.     

COMPARATIVE STUDIES OF THE β-D-GLUCAN RELEASED INTO SORGHUM AND BARLEY WORTS

Worts prepared from two cultivars of Nigerian grown sorghum six day melts — LI87 end SK5912 had β-D-glucan levels off five to seven times more than that of proctor barley. In contrast to barley, malting of the sorghums results in the release off more β-D-glucan into wort. Apparently, this is due to increasing levels of β-glucan solubilase and (1→3)-β-glucanase during malting with no significant (1→3, 1→4)-β-glucanase activity.     

EXTRACTION OF α-GLUCOSIDASE FROM GERMINATED BARLEY KERNELS

α-Glucosidase activities were extracted from flour of lyophilized, germinated barley seeds. To establish conditions resulting in maximal extraction of α-glucosidase activity from flour of germinated barley, we tested different buffer pH's, extraction times and temperatures, and additions of sulfhydryl reducing reagents, salts, and detergents to the extraction buffer. More enzyme activity was extracted as the extraction buffer pH increased from 3 to 9. The sulfhydryl reagents studied did not significantly increase extractable activity. The extractable activity rose with increased NaCl concentrations up to 2 M. Of the six detergents tested, the zwitterionic detergent CHAPS was most effective in solubilizing α-glucosidase activity, and when combined with NaCl resulted in a doubling in extractable activity as compared with phosphate buffer at pH 8.0. Extraction time and temperature were not critical for the actual solubilization of activity but temperature was quite critical in retaining solubilized α-glucosidase activity in crude extracts.     

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.     

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.     

STUDIES ONα-GLUCOSIDASE IN BARLEY AND MALT*

A specific and sensitive method was used to determine α-glucosidase activity in barley and malt. Reliable results were obtained only after extracts of barley and malt had been dialyzed extensively to remove low molecular weight carbohydrates that interfered with the enzyme assay, α-Glucosidase was present in immature kernels of Bonanza and Ellice barley shortly after anthesis but enzyme levels fell rapidly as the kernels matured. A high proportion of the activity was present in pericarp tissue. Enzyme activity increased rapidly in Bonanza and Klages barley during initial stages of germination and fell only slightly during kilning. A high proportion of enzyme activity was present in the scutellum of 4-day germinated barley with lesser amounts in the aleurone and endosperm.     

ENHANCEMENT OF AMYLOLYTIC POTENTIAL OF SORGHUM MALTS BY ALKALINE STEEP TREATMENT

The effect of alkaline steep liquor on sorghum maltability was investigated using three improved Nigeria sorghum cultivara. Germination was for four days at 30°C after steeping under four different regimes. Grain germinability, root length and malting loss were significantly (P < 0.001) repressed by steeping in alkaline liquor for all the cultivars. However, the extent of this repression seemed in all cases significantly dependent on cultivar and steep regime plus their possible interactions. Similarly, the development of diastatic enzymes activities appeared to be highly significantly dependent on steep liquor, steep regime, cultivar, plus their possible pairwise and three way interactions. For ICSV400, highest diastatic power and α-amylase development were attained on steeping grains in alkaline liquor under a continuous steep regime incorporating final warm steep treatment. Conversely, exposure of KSV8 and SK5912 to a regime incorporating air-resting and final warm steep significantly enhanced diastatic power and α-amylase development. β-Amylase activity was in all cases enhanced by alkaline steeping. In fact, β-Amylolytic activity constituted over 70% of total diastatic activity in most alkaline steeped ICSV400 malts. However, SK5912 exhibited relatively low hot water extract in spite of the improved amylolytic activity.     

DEVELOPMENT OF AMYLOLYTIC ACTIVITIES IN SORGHUM AND BARLEY MALT

A Nigeria-grown sorghum cultivar was observed to develop more Diastatic Power (DP) and β-amylolytic activity than a previously studied sorghum cultivar grown in Australia. Barley malt produced more Diastatic Power and β-amylolytic activity than the Nigeria-grown sorghum cultivar. In general, the kilned barley malt retained less α-amylolytic activity than kilned sorghum malt. The different diastatic potential of sorghum cultivars may reflect differences in genetic character or growth conditions.     

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.     

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.     

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.     

CHANGES IN ACTIVITY OF SORGHUM LIPASE DURING MALTING AND MASHING

Lipase activity was monitored during malting and mashing of sorghum grains. All three sorghum varieties contained detectable lipase activity in the ungerminated form. Lipase activity changed only slightly during steeping for 24 hours but increased several fold in the course of germination. Between 24% and 60% of the lipase activity of the green malt was retained after kilning at 48°C but no activity was detected in the wort after mashing at 65°C. About 68% of the lipase activity of 72 hours old malt was detected in the plumule, while 29% and 3% were detected in the endosperm and radicle, respectively. Optimum activity was observed at pH 7.0.     

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.     

SOME BARLEY GRAIN AND GREEN MALT PROPERTIES AND THEIR INFLUENCE ON MALT HOT-WATER EXTRACT: I. β-GLUCAN, β-GLUCAN SOLUBILASE AND ENDO-β-GLUCANASE

A study has been made of the variation between varieties in some properties of barley and malt and how this variation relates to malt hot water extract (HWE). The development of enzyme activity along the grain during germination was investigated. In this first paper we have examined β-glucan-related characters and found significant varietal variation in maximum enzyme activities and in the activities in different sections of grain during germination. Varietal variation was greater than environmental variation for each character. The fraction of β-glucan soluble in acid was the character most highly correlated with HWE.     

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.     

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.     

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.     

ENZYMIC QUANTIFICATION OF (1→3) (1→4)-β-D-GLUCAN IN BARLEY AND MALT

A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in maltsamples.α-Amylasedoes not interfere with the assay. The method issuitable for the routineanalysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0–1 for barley flour samples containing 3–8 and 4–6% (w/w) β-glucan content.     

TOTAL β-GLUCAN CONTENT OF SOME BARLEY CULTIVARS

The use of cellulase preparations from Trichoderma reesii for measuring the total β-glucan content of barley was examined. The activities of amyloglucosidase and β-glucanase in the cellulase varied considerably between batches, and different heat treatments were necessary to ensure that amyloglucosidase was reduced to an insignificant level while adequate β-glucanase activity was retained. After suitable treatment the cellulase was used to study variation of total β-glucan concentration in some barley cultivars. Significant varietal variation was found in the fifteen genotypes examined. These had β-glucan concentrations in the range 2.7% to 5.2% dry weight.     

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.