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 .     

Die Wirkung von α-Amylasen auf β-Cyclodextrin und der Nachweis von α-Amylasefremdaktivitäten in ausgewählten Enzympräparaten

The action of α-amylases on β-cyclodextrin and the evidence of foreign activity of α-amylase in selected preparations of enzymes The interaction between cyclodextrins and α-amylases taken from different sources is discribed contradictious in the literature. Some α-amylases e.g. isolated from Aspergillus oryzae, porcine pancreas and saliva hydrolized cyclodextrins to glucose. The hydrolysis of cyclodextrins catalysed by α-amylase from Bacillus species have been described conflicting. In this paper the action of hydrolysis of different preparations of α-amylases on β-cyclodextrin have been investigated. It has been shown that Rohalase M3 (α-amylase from Aspergillus niger) cleaves the ring of β-cyclodextrin. 2 α-amylases from Bacillus subtilis are not able to hydrolyse β-cyclodextrin. The reasons for the different actions of hydrolysis have been discussed with size and structure of the active centre of the enzymes. Moreover, different preparation of hydrolysis have been tested on secondary activity of α-amylase. 2 glucoamylases from Aspergillus oryzae have been shown secondary activity of α-amylase. With the hydrolases α-glucosidase from fungies, β-amylase from malt, saccharase from yeast, invertase from S. cerevisiae and pullulanase from Aerobacter aerogenes no cleavage of the ring of β-cyclodextrin could be detected.     

Genotypic and environmental variation in barley beta-amylase activity and its relation to protein content

β-Amylase activities of barley cultivars collected from various areas of China, and as well as from Canada and Australia, were assayed. Meanwhile a multi-location trial was conducted to determine variation of β-amylase activity in eight barley cultivars and the relationship between β-amylase activity and protein content. For 56 cultivars in study, β-amylase activity ranged from 458 to 1024 U/g, with a mean of 738 U/g. There was significant variation in both β-amylase activity and protein content for eight barley cultivars grown in four locations. No significant correlation was found betweenβ-amylase activity and protein content surveyed in 56 cultivars.     

Amylase, β-Glucanase and Protease Activities from a Mutant of Bacillus subtilis

Enzymes in the shake culture of a mutant of Bacillus subtilis developed mainly during the stationary phase of growth. Extracellular α-amylase and protease activities increased and then declined to relatively low levels after 48 h of incubation while the β-glucanase activity remained high. The production of extracellular proteolytic activity commenced only when the low molecular weight nitrogen source in the medium was completely consumed. Enzymes were fractionated by ionexchange chromatography and the molecular weights of β-glucanase, α-amylase, protease I and protease II were estimated by gel filtration to be 1.3 × 10⁴, 3.2 × 10⁴, 6.3 × 10³ and 2.0 × 10⁴, respectively. The β-glucanase and α-amylase showed maximum activities at around pH 7, but the two proteases had different pH optima (pH 6–7 and 8.5, respectively). The presence of proteolytic activity in the enzyme preparation had a significant effect on the stability of the β-glucanase and the α-amylase at 65°C.     

脉冲电场对啤酒大麦萌发性质的影响

在啤酒麦芽制备过程中引入脉冲电场(PEF)技术,对浸渍后的大麦种子进行不同电场强度的处理,以了解PEF对大麦萌发性质的影响。结果表明,PEF处理有利于提高大麦萌发活力和相关理化性质。大麦籽粒经电场强度1.5、3.0、4.5、6.0、7.5 kV/cm处理后,萌发大麦的芽长、根长、根数、鲜重、干重、发芽势、发芽率、简化活力指数、还原糖含量、β-葡聚糖酶活力、α-淀粉酶活力、β-淀粉酶活力、可溶性蛋白质和氨基态氮含量等指标与对照组比较均有明显提高,发芽势、根长、β-葡聚糖酶活力、α-淀粉酶活力和β-淀粉酶活力提高的幅度分别为38.96%、43.33%、21.46%、26.48%和23.57%。对14项指标进行主成分分析的结果表明,当电场强度为6.0 kV/cm时,脉冲电场对大麦萌发的促进效果最好。     

THE CHROMATOGRAPHIC PROPERTIES OF BARLEY AND MALT β-AMYLASES

Barley β-amlyase occurs as a heterogeneous, polydisperse enzyme in thiol-free extracts of Conquest barley. During malting, the polydisperse enzyme is altered, resulting in the formation of four distinct enzyme components which increase in activity as germination progresses. Addition of thioglycerol to a thiol-free extract of barley, or initial extraction with thioglycerol, produces extracts containing two discrete β-amylase enzymes. β-amylase I is the major component of the extract; β-amylase II occurs as a minor component. Similarly, malt extracts containing thioglycerol have two β-amylase enzymes, β-amylase III and IV. Barley β-amylase II and malt β-amylase III have similar chromatographic properties on CM-cellulose but it is not known whether these enzymes are identical. During the early stages of germination, barley β-amylase I disappears and cannot be detected in extracts of 1-day malt; β-amylase III is the major β-amylase enzyme in this extract. Malt β-amylase IV cannot be detected in barley extracts. It develops during germination until it becomes the major β-amylase in malt extracts.     

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 RELEASE OF BOUND β-AMYLASE BY MACROMOLECULES

The aim of this study was to clarify the roles of endogenous proteases and higher molecular weight thiols in the release of bound β-amylase, which occurs during barley germination. In resting barley grains (Hordeum vulgare L. cv. Torrent) five major β-amylase monomers were found in the thiol reduced, salt soluble extract. Only the two smallest monomers were present in large amounts in the bound form of the enzyme that had been released with 2-mercaptoethanol. In vitro, bound β-amylase was solubilised by “releasing factors” extracted from the endosperm of decorticated grains germinated for three days. The releasing factors were in the higher molecular weight fraction (>5 kDa) and their formation was induced in degermed grains by gibberellic acid. When an endosperm extract, containing only higher molecular material (>5 kDa) prepared from three day germinated grain, was incubated with a preparation of bound β-amylase, about 75% of the release of the enzyme could be prevented by a mixture of proteolytic inhibitors. The dominant class of proteases in malt were the sulphydryl proteases. These were not fully active when extracted, but could be activated approximately six fold by the addition of 2-mercaptoethanol. Heating the extract to a limited extent destroyed all the proteolytic activity, but 10% of the bound β-amylase could still be released by the extract.     

Comparative immunological and chromatographic study of some plant β-Amylases

A comparative study has been made of the β-amylases of barley, wheat, rye, oats and sweet-potato by means of exclusion chromatography and immunochemical analysis. The reactivity of barley malt and wheat β-amylase was compared with different anti-barley and anti-wheat sera. In exclusion chromatography on Sephadex G100, barley β-amylase yielded four, and both wheat and rye, two active components, whereas oat and sweet-potato had only one active component. During the storage of barley, wheat and rye β-amylases the large-molecule components were split into smaller ones; no changes occurred in oat and sweet-potato β-amylases. On analysis against a specific barley β-amylase antiserum, wheat and rye β-amylase gave a reaction which indicated that they were immunologically partly identical with barley β-amylase, and identical with each other. This serum induced no reaction in β-amylases of sweet-potato and oats. The rye β-amylase precipitation line did not display enzymic activity after reaction with this antiserum. Analyses with different antisera of barley and wheat confirmed the partial immunological identity of barley malt and wheat β-amylase. With some barley antisera, partial inhibition of wheat β-amylase activity was observed. A similar phenomenon was apparent when barley malt β-amylase was precipitated with some wheat antisera.     

Relationship Between β-amylase Activity,Steeliness, Mealiness,Nitrogen Content and the Nitrogen Fractions of the Barley Grain

Steely grains tend to have a higher total nitrogen than corresponding mealy grains. β-Amylase activities of different barley varieties appeared to be associated, to different degrees, with steeliness, high total nitrogen content, and high levels of total salt soluble proteins and hordein proteins. The consistent relationship between β-amylase activity and these parameters suggests that β-amylase contents of barley not only reflect genetic differencies in varieties, but also differences in protein type and grain structure. Notwithstanding, the β-amylase content of the grain may not be indicative of the potential of the grain to develop β-amylase activity during malting.     

不同品种甘薯的干燥特性及对全粉品质的影响

以9个甘薯品种为原料,研究不同品种甘薯在相同工艺条件下的干燥特性,分析甘薯品种与其全粉品质及得率的相关性。结果表明:鲜薯成分中影响熟化甘薯热风干燥速率的因素主要是可溶性糖含量,而影响颗粒全粉得率及品质的因素主要是水分、淀粉、可溶性糖、粗蛋白含量及多酚氧化酶、β-淀粉酶活性。鲜薯的水分含量及可溶性糖含量越低,淀粉含量越高,加工成颗粒全粉的产品得率越高。     

酿酒酵母表面展示β-淀粉酶及其酶学性质研究

通过PCR技术扩增来源于大麦的β-淀粉酶基因,将其与酿酒酵母细胞壁蛋白α凝集素基因在读框内融合,构建得到表面展示载体pBA-AG,进一步将该重组质粒通过遗传转化,整合到酿酒酵母W303-1A的染色体中,获得了β-淀粉酶经过α凝集素锚定信号结合到细胞壁上的重组酵母。重组酵母表面展示的β-淀粉酶活力为131U/g干细胞。对展示的β-淀粉酶酶学性质研究表明,其最适反应温度为50℃,最适作用pH为5.0,与游离酶相比,其温度稳定性和pH稳定性均得到提高。本研究利用α凝集素系统首次将β-淀粉酶成功展示在酿酒酵母表面,为以酿酒酵母为基础的全细胞催化剂研究与应用打下了一定基础。     

超高麦芽糖浆的生产

本文研究了超高麦芽糖浆的生产，结果发现，普通β－淀粉酶和脱支酶共同作用适于制造普通非结晶性麦芽糖浆，组成大致为８０％左右的麦芽糖和１０％左右的麦芽三糖，而外切型α－麦芽糖－α－小淀粉酶（Ｍａｌｔｏ－ｇｅｎａｓｅ）由于价格及水解方式两方面的原因，不适合用于此类麦芽糖浆的生产。但生产用于制造结晶麦芽糖的超高麦芽糖浆时，则宜用β－淀粉酶、Ｍａｌｔｏｇｅｎａｓｅ和脱支酶共同作用，通常的组成为８０％以上的麦芽糖，４％以下的麦芽三糖。     

Über einige aktuelle theoretische und praktische Probleme der Stärkeenzymologie

Some Actual Theoretical and Practical Problems of Starch Enzymology. Some recent results of traditional starch research carried out in our institute have been presented. In the field of theoretical research, the probability of splitting in the case of exo-enzymes (amyloglucosidase, β-amylase) and α-amylase as an endo-enzyme has been studied. Based on theoretical research, some enymological problems of industrial concern have also be investigated; such as isoamylase and β-amylase degradation of starch carried out for the preparation of maltite; the splitting with isoamylase and glucose isomerization for the preparation of invert sugar and examination of immobilized amyloglucosidase. With these examples it should be demonstrated that successful results can be attained both in theoretical basic research and applied research only by close cooperation between these two fields.     

Immunoelectrophoretic identification of a heterodimer beta-amylase in extracts of barley grain.

Gel filtration followed by immunoelectrophoretic characterisation showed that approximately 30% of the β-amylase activity in salt extracts of Emir barley grains was localised in a dimei complex (mol. wt. 95 000) between β-amylase (mol. wt. 56 000) and a non-active protein Z (mol. wt. 40 000). This complex seemed to be the only polymer β-amylase present in 1 mM β-mercaptoethanol extracts of barley and barley green malt. The complex was split by 100 mM β-mercaptoethanol.     

兰州百合鳞茎冷藏保鲜过程中碳水化合物含量及淀粉酶活性的变化

以兰州百合为试材,采用紫外-可见分光光度法测定了-2 ℃冷藏保鲜过程中百合鳞茎内可溶性糖、还原糖、淀粉的含量与淀粉酶活性的动态变化,分析了碳水化合物含量与淀粉酶活性之间的关系。结果表明,在-2 ℃、60 d的冷藏保鲜期内,随着冷藏时间的延长,兰州百合鳞茎内淀粉含量明显下降,其下降幅度达62.29%,可溶性糖与还原糖含量呈上升趋势,分别上升了2.22%和1.39%。β-淀粉酶是兰州百合鳞茎中的主要淀粉酶,其活性随冷藏时间的延长而有所降低,α-淀粉酶的活性随冷藏时间的延长先降低后升高,总淀粉酶在高活性的基础上整体呈现降低的趋势。相关性分析表明,鳞茎中碳水化合物含量及淀粉酶活性之间的相关性差异水平有所不同。总淀粉酶、β-淀粉酶活性与可溶性糖、还原糖含量具有极显著负相关性,而与淀粉含量具有极显著正相关性。可溶性糖和还原糖含量的增加与低温诱导下淀粉在β-淀粉酶的作用下分解有关。     

Immobilised β-Amylase in the Production of Maltose Syrups

In order to have a full continuous starch hydrolysates production process, the use of continuous conversions catalysed by immobilised enzymes is necessary. With respect to this, immobilisation of β-amylase on anion exchange resins and continuous conversion of maltodextrins to maltose syrups is investigated in packed bed column reactors at lab-scale. It is observed that the type of carrier and the source of the β-amylase are important parameters for the required capacity of the continuous process. Results show that, from the investigated enzymes, the commercial Spezyme BBA 1500L® β-amylase preparation immobilised on Duolite A568® as preferred carrier gives the best results in terms of initial performance and stability under realistic conversion conditions (50°C, pH 4.5, 50% d.s. substrate). On basis of the present results, the process is promising in terms of applicability at plant-scale after optimisation.     

3种酶制剂对生产面包的体积影响

研究真菌木聚糖酶、β-葡聚糖酶对面粉粉质及拉伸性能的影响,并考察真菌α-淀粉酶、真菌木聚糖酶和β-葡聚糖酶3种单酶对面包的作用效果。通过正交试验确立20 mg/kg真菌α-淀粉酶、50mg/kg真菌木聚糖酶和50 mg/kgβ-葡聚糖为加工面包的最优化工艺条件。在上述条件下,面包平均体积为885 mL,该体积比不使用酶制剂的平均体积提高了17%。     

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.     

MALT DIASTATIC ACTIVITY. PART II. A MODIFIED EBC DIASTATIC POWER ASSAY FOR THE SELECTIVE ESTIMATION OF BET A-AMYLASE ACTIVITY,TIME AND TEMPERATURE DEPENDENCE OF THE RELEASE OF REDUCING SUGARS

Ammoniumcitrate, sodium EDTA and ammonium oxalate were tested as possible inhibitors of α-amylase in the EBC diastatic power assay. A contact time of 90 min at a concentration of 0·1 M ammonium oxalate was shown to inactivate α-amylase completely in the enzymic malt extract while only very slightly enhancing β-amylase activity. The selective release of reducing sugars by β-amylase in the diastatic power assay was evaluated as a function of time using a p-hydroxybenzoic acid hydrazide assay and compared with that of the diastatic power procedure with the same colourimetric technique. The diastatic power results of eight different malts are well correlated with those of the β-amylase activity (r² = 0·98). In a linear regression model the diastatic power results at 20°C of the same malts correlated better with results obtained at 55°C (r² = 0·77) than with those obtained at 60°C (r² = 0·63). For the results of β-amylase activities we found r² = 0·86 for the relation between the activities at 20°C and at 55°C and r² = 0·92 for the relation between the activities at 20°C and 60°C. Since the temperature conditions (20°C) of the EBC-diastatic power assay do not correspond to those of brewing practice it is suggested that the activity of β-amylase as well as the malt diastatic power be evaluated at 55°C.