PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ENDO-β1,3-GLUCANASE FROM GREEN MALT

An endo-β1,3-glucanase from a green malt extract was purified by DEAE- and CM-cellulose ion exchange chromatography followed by molecular sieve chromatography on BioGel P-100. A final enzyme preparation had two protein components on disc electrophoresis, one of which was in-active. The enzyme had a pH optimum of 5·0 for activity on laminarin and 5·8 on carboxymethyl pachyman. The activity was stable up to 60°C and was stimulated by NaCl. The isoelectric point of the enzyme was 9·8.     

THE INSTITUTE OF BREWING ANALYSIS COMMITTEE THE DETERMINATION OF ENDO-β-GLUCANASE ACTIVITY IN MALT

Various substrates for the determination of malt endo-β-glucanase activity have been tested in conjunction with the β-glucanase procedure advocated by Bourne and Pierce. It is recommended that one universal standard substrate should be used and that each determination should be measured against a standard check malt. Furthermore, great care must be exercised in the dissolution of the substrate to obtain consistent results.     

THE ACTION OF ENDO-β-1,3-GLUCANASES ON BARLEY AND MALT β-GLUCANS

The β-glucan extracted from ungerminated barley with water at 40 °C has a much lower specific viscosity than the corresponding material isolated from a wort prepared at 65 °C from a two-day germinated barley malt. Both glucans are similar in that they are polymers of β-D-glucose, with approximately 74% of the linkages in the β-1,4 configuration and 26% in the β-1,3 configuration. However, the two glucans are not hydrolysed to the same extent either by a partially purified bacterial endo-β-1,3-glucanase or by a homogeneous endo-β-1,3-glucanase from malted barley. The malt glucan is readily hydrolysed, causing a rapid decrease in specific viscosity but with no measurable increase in reducing power, whereas barley glucan undergoes only limited hydrolysis under similar conditions. Thus, different β-glucan preparations from barley or malt may be identical in the proportion of β-1,3 to β-1,4-linkages but the overall arrangement of linkages, and hence susceptibility to enzyme attack, differs according to the source and the method of extraction of the glucan. The molecular weights of both β-glucan preparations and the products of their enzyme hydrolysis have been determined by agarose gel permeation chromatography. A simple model which illustrates the underlying structural relationships of the β-glucans from barley and malt is suggested.     

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.     

STUDIES ON CARBOHYDRATE METABOLIZING ENZYMES PART XXII. THE β-GLUCANASE SYSTEM OF MALTED BARLEY

Extracts of malted barley have been shown by molecular sieve chromatography to contain at least five enzymes which hydrolyse β-glucosidic linkages. In order of decreasing molecular weight, these are two β-glucosidases, an endo-β-1,4-glucanase, an endo-barley-β-glucanase and an endo-β-1,3-glucanase. This last-named enzyme was purified about 60-fold and shown to be specific for substrates containing only β-1,3-glucosidic linkages. This enzyme was activated by calcium ions, and deactivated by EDTA, but was not affected by chloride ions or a sulphydryl reagent. The endo-β-1,4-glucanase, which was purified about 50-fold, hydrolysed glucans containing only β-1,4- or a mixture of β-1,3- and β-1,4-linkages. The two β-glucosidases were shown to be true glycosidases and not exo-β-glucanases. The changes which occur in the levels of the various enzymic activities during the malting process have been measured.     

裂褶菌产内切β-1,3-葡聚糖酶的分离纯化

采用不同饱和度的硫酸铵溶液对内切β-1,3-葡聚糖酶粗酶液进行分段盐析,以确定最佳盐析条件,将盐析处理后的酶液经透析浓缩、DEAE-Sephadex A-50离子交换层析、Sephadex G-75凝胶过滤层析等进一步分离纯化,浓缩纯化后的含酶组分,经过SDS-PAGE电泳分析其纯度并初步确定其分子量。结果显示:经过纯化后的内切β-1,3-葡聚糖酶的比活力由20.90U/mg提高到933.37U/mg,纯化倍数为44.7倍,酶活回收率为11.6%,电泳分析呈单一条带,分子量近似为45ku。     

THE DEVELOPMENT OF β-GLUCAN SOLUBILASE DURING BARLEY GERMINATION

β-Glucan solubilase in either germinating barley or in endosperm slices treated with gibberellic acid is synthesized before endo-β-glucanase, α-amylase and protease. In common with these enzymes, β-glucan solubilase is synthesized much sooner in endosperm slices than in whole grain. Gibberellic acid stimulates β-glucan solubilase synthesis in endosperm slices and most of the activity is rapidly released into the surounding medium, irrespective of whether the hormone is present. Inhibitors of RNA and protein synthesis block the formation of β-glucan solubilase. Unlike β-glucanase, α-amylase and protease, β-glucan solubilase is present in significant quantity in untreated barley where it is concentrated in the embryo-containing half of the grain. The only β-glucan solubilase activity in barley is due to an acidic carboxypeptidase. Malt contains a small amount of a second solubilizing enzyme which appears to be an endo-β1, 3-glucanase.     

裂褶菌产内切β-1,3-葡聚糖酶的特性研究

对裂褶茵所产内切β-1,3-葡聚糖酶进行有效分离纯化并用电泳法对其纯度进行鉴定,进而研究其酶学特性.结果表明:经过DEAE-Sephadex A-50离子交换层析和Sephadex G-75凝胶过滤分离纯化得到电泳纯分子量约为45 kD的内切β-1,3-葡聚糖酶,其最适pH为5.0,最适温度为45℃;Fe<'2+>、B...     

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.     

AN APPROACH TO THE IDENTIFICATION OF A ß-GLUCAN SOLUBILASE FROM BARLEY1

A ß-glucan solubilase activity was demonstrated in barley extract. This enzyme catalyzed the dissolution of barley ß-glucan by releasing a product having a narrow molecular weight distribution and a molecular weight of about 20,000. The enzyme was partially purified by ion exchange chromatography on DEAE-cellulose and gel permeation chromatography on Bio-Gel P-100. Although carboxypeptidase activity was present in the crude extract of barley flour the partially purified ß-glucan solubilase did not hydrolyse N-CBZ-Phe-ala. Examination of extracts from different barley tissues indicated that the ß-glucan solubilase activity was associated with the husks only; a large portion of the activity was extractable from whole barley kernels. About 85% of the enzyme activity in crude extracts from barley flour was retained after 40 min at 62°C. However, the enzyme was much more heat-labile in extracts of whole barley kernels. The pH of maximal activity was found to be about pH 5.7 and results from column chromatography suggested that the enzyme had a low pl value and a MW between 5 × 10⁴ and 6 × 10⁴.     

THE DEGRADATION OF β-GLUCAN DURING MALTING AND MASHING: THE ROLE OF β-GLUCANASE

Significant β-glucanolysis takes place during mashing and is catalysed by a β-glucanase which is specific to mixed-linkage β-glucans. The enzyme develops during the germination of barley, but is rapidly and extensively destroyed in kilning. Partially-purified preparations of β-glucanase are protected from denaturation by heat if their solutions are adjusted to pH 4 or if bovine serum albumin is added. However the most effective stabiliser of the enzyme is reduced glutathione. Oligosaccharides containing three and four glucosyl units are produced by the action of β-glucanase and they are further converted during malting and mashing by a different enzyme(s) to disaccharides and glucose.     

The (1–3, 1–4)‐β‐Glucanases in Malting Barley: Enzyme Survival and Genetic and Environmental Effects

Eighteen barley genotypes used in Brazilian malting barley breeding programs were characterized in relation to (1–3, 1–4)‐β‐glucanase activity in green and kilned malt. They were tested to determine the loss of enzyme activity during kilning in the malting process and the environmental effects on enzyme activity were measured. The genotypes analyzed showed great variation regarding the enzyme activity in both kinds of malt, in a range from 531.94 to 934.31 U/kg in green malt, and from 187.02 to 518.40 U/kg in dry malt. The mean enzyme activity loss during kilning was close to 60%, very similar to the results obtained in other studies. The loss among genotypes varied from 8.04% to 71.54%. The enzyme activity varied significantly under the different environments tested, showing existence of environmental effects on the genotypes analyzed. Embrapa 127 was the genotype that exhibited the highest enzyme activity in finished malt although it had shown a low activity in green malt, reflecting a negligible loss of activity during kilning. The data indicate promising results to malting barley breeding due to the wide variability exhibited by genotypes as to enzyme activity and levels of isoenzyme with high thermostability.     

ENZYMATIC DETERMINATION OF 1,3:1,4-β-GLUCANS IN BARLEY GRAIN AND OTHER CEREALS†

A direct and specific enzymatic method is described for the determination of 1,3:1,4-β-glucans in barley grain and other cereals. In the procedure, purified, amylase-free, bacterial 1,3:1,4-β-glucan hydrolase is used to depolymerize the 1,3:1,4-β-glucan in autoclaved and ethanol-extracted flour prepared from whole grain. The liberated oligoglucosides are extracted with 80% (vol/vol) ethanol and, following acid hydrolysis, measured by the glucose oxidase method. The method can be used to measure total β-glucan and β-glucan in 65°C water-soluble and water-insoluble fractions of cereal grains. The procedure has been applied to barley grains allowed to develop under controlled environmental conditions and to a series of barleys of different geographical origins. The results for Canadian cultivars are compared with estimates based on viscometric data for the same samples. The 1,3:1,4-β-glucan content of a number of other cereals has also been measured.     

β-GLUCANASE: THE SUCCESSFUL APPLICATION OF GENETIC ENGINEERING

The now well established principles of genetic engineering are described in relation to the solution of problems associated with β-glucans in beer. The application of these techniques has enabled the isolation of a Bacillus subtilis endo-1, 3–1, 4-β-D-glucanase gene which expresses a biologically active enzyme in yeast.^15,16 Although this enzyme is capable of hydrolysing beer β-glucans during fermentation, thereby enhancing beer filtration, insufficient β-glucanase is produced in yeast to enable successful commercial implementation. The requirements for the efficient production of β-glucanase in genetically manipulated brewing yeast are described.     

THE RELATIONSHIP BETWEEN β-GLUCAN SOLUBILASE,BARLEY AUTOLYSIS AND MALTING POTENTIAL

There is a correlation between the autolysis of barleys and their β-glucan solubilase activities. There is no correlation between autolysis and nitrogen content, β-glucan level, Milling Energy or Zeleny sedimentation value of the barley. Activities of endo-β-glucanase are inversely related to coarse-grind Hot Water Extract obtained from malts grown for 4 days. Whilst β-glucanase is not involved in the early stages of autolysis, β-glucan solubilase, present in large amounts in untreated barley, has a role both in extracting and degrading β-glucan. Barleys with low β-glucan content or high β-glucan solubilase modify more rapidly.     

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.     

THE EFFECTS ON MALT OF HIGH TEMPERATURES DURING THE LAST DAY OF GERMINATION

Raising the temperature from 16°C to 40°C on the last day of germination shows no deleterious effects on standard analyses of a malt. Amylolytic activity is not affected by the treatment, so that the malt produced would be suitable for brewing using standard infusion mashes. However, endopeptidase and endo-β-glucanase levels are reduced by the 40°C treatment and the low levels of activity of these enzymes may become limiting and cause problems in some cases, for example when brewing with large quantities of adjuncts such as flaked barley.     

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 .     

TOTAL AND INDIVIDUAL BARLEY (1–3), (1–4)-β-D-GLUCANASE ACTIVITIES IN SOME GREEN AND KILNED MALTS

Total β-glucanase activity was measured in extracts of malts prepared from three winter and three spring cultivars of barley. Samples were taken at intervals during modification and, after 116 hr, from malt kilned to 65°C. Good malting varieties showed highest levels of total β-glucanase activity, with a high correlation (r = 0.926) with HWE. Angora had the highest activity in the intermediate stages of malting, least loss of activity after heating extracts to 48°C for 10 mins and least loss of activity on kilning. Separation of isozymes by HPLC⁹ confirmed the greater heat stability of isozyme II, but, unlike previous studies on Australian cultivars, we found considerable activity of isozyme I after kilning, even up to 85°C. However, total β-glucanase activity was destroyed by heating extracts of all varieties to 60°C. Angora showed the highest proportion of total activity as isozyme II after kilining. Cultivar differences suggested some scope for breeding varieties with increased total activity by combining high activities of each isozyme. The high correlation of total activity with HWE suggests β-glucanase activity as a rapid test of malting quality.     

FAILURE OF MERCURIC CHLORIDE TO SELECTIVELY INHIBIT β-AMYLASE IN SORGHUM MALT

Mercuric chloride has been reported to be a suitable reagent for the determination of α-amylase activity in sorghum malt, based on its ability to selectively inhibit β-amylase. In this re-investigation, the α- and β-amylase activities of eight sorghum malts were determined after treatment of malt extracts with various concentrations of mercuric chloride. At a malt: mercuric chloride ratio of 8.3 × 10³: 1, incomplete inhibition of β-amylase activity, as measured by the Betamyl assay, occurred in all extracts. However, this concentration resulted in significant inhibition of α-amylase activity in all extracts, as measured by both the Ceralpha assay and the Phadebas assay. In addition, α-amylase activity was found to be significantly inhibited at malt: mercuric chloride ratios as low as 1.0 × 10⁵: 1, when measured by the AmyloZyme assay. These findings do not support the original report that a malt: mercuric chloride ratio of 4.0 × 10³: 1 will selectively inhibit β-amylase in sorghum malt. Furthermore, in this context it should be emphasised that the original report was based upon inhibition studies conducted on β-amylase derived from barley, not sorghum malt .