Mechanism of amylase action on glucoside starch bonds]

Functional groups of glucoamylase and alpha-amylase from Asp. awamori, alpha-amylase from Asp. oryzae and alpha- and beta-amylases from barley malt are identified. Kinetic curves of the activity dependency on pH, values of ionization heats and photooxidative inactivation draw to the conclusion that carboxyl-imidazole system enters into the active site of the enzymes. A hypothetic mechanism of hydrolysis of alpha-1,4-glucoside bond in starch molecule by alpha- and beta-amylases and of alpha-1,4- and alpha-1,6-glucoside bonds by glucoamylase is given. A theory of induced correspondence of enzyme and substrate satisfactorily explains the specificity of the enzyme action and the cause of complete starch convertion into glucose under glucoamylase action and of terminal starch hydrolysis by alpha- and beta-amylases.     

Thermal stability of starch degrading enzymes of teff (Eragrostis tef) malt during isothermal mashing

Thermal stability of starch degrading enzymes varies from one source to another. This research was aimed to study thermal stability of starch degrading enzymes of teff malt. Isothermal mashing at temperatures ranging between 40 and 75 °C with sampling in 15 min interval for a total of 90 min was conducted. The study showed that deactivation rate constants of alpha- and beta-amylases ranged from 0.0003 to 0.0409 min^?1, and 0.002 to 0.032 min^?1, respectively. Rate of deactivation of limit dextrinase was not significant at temperatures lower than 60 °C but showed high deactivation at higher temperatures with rate constants ranging from 0.02 to 0.1 min^?1. The thermal deactivation energies of alpha-amylase, beta-amylase, and limit dextrinase were found to be 148, 82, and 144 kJ/mol, respectively. The present findings have significant applications in commercial processes where determination of the upper temperature limits for these enzymes is required.     

糖化过程中阿拉伯木聚糖溶解及内切木聚糖酶随机进攻预测模型的建立

建立了糖化过程中阿拉伯木聚糖溶解及内切木聚糖酶随机进攻的预测模型,希望通过此模型能预测在不同初始条件和参数设置下糖化过程中阿拉伯木聚糖的浓度,以减少其在酿造过程中的负面作用。结果显示,此模型预测麦汁中阿拉伯木聚糖浓度的误差在-9.5%到+13.6%之间。工业验证模型的误差要大于实验室条件下的误差,分别为16.8%和17.9%。仿真结果表明,麦汁中阿拉伯木聚糖的浓度随糖化初始温度的升高而增加,而延长糖化初始时间能降低阿拉伯木聚糖的含量。并且麦芽中内切木聚糖酶活对麦汁中阿拉伯木聚糖的浓度的影响要远远小于麦芽中阿拉伯木聚糖的初始值。     

Mathematical modeling for prediction of endo-xylanase activity and arabinoxylans concentration during mashing of barley malts for brewing

A mathematical model describing the degradation of arabinoxylans by endo-xylanase during mashing process was developed. Endo-xylanase activities and arabinoxylans concentrations in laboratory scale mashing process at different temperature profiles were measured and then used for identifying the model parameters for Harrington barley malt. The modeling errors range for the final concentration of arabinoxylans in wort was -4% to +11.9%. The model developed was also used for predicting the other three different malts mashing processes in laboratory scale, and the prediction errors ranged from -9.5% to +13.6%. The model prediction accuracy for industrial scale mashing process was lower than that in laboratory scale. The simulation results showed that, a lower concentration of arabinoxylans could be achieved when maintaining the mashing-in at 45 degrees C and prolonging the mashing-in time.     

Iteration model of starch hydrolysis by amylolytic enzymes.

An elaborate computer program to simulate the process of starch hydrolysis by amylolytic enzymes was been developed. It is based on the Monte Carlo method and iteration kinetic model, which predict productive and non-productive amylase complexes with substrates. It describes both multienzymatic and multisubstrate reactions simulating the "real" concentrations of all components versus the time of the depolymerization reaction the number of substrates, intermediate products, and final products are limited only by computer memory. In this work, it is assumed that the "proper" substrate for amylases is the glucoside linkages in starch molecules. Dynamic changes of substrate during the simulation adequately influence the increase or decrease of reaction velocity, as well as the kinetics of depolymerization. The presented kinetic model, can be adapted to describe most enzymatic degradations of a polymer. This computer program has been tested on experimental data obtained for alpha- and beta-amylases.     

Antisense downregulation of the barley limit dextrinase inhibitor modulates starch granule size distribution, starch composition and amylopectin structure

The barley protein limit dextrinase inhibitor (LDI), structurally related to the alpha-amylase/trypsin inhibitor family, is an inhibitor of the starch debranching enzyme limit dextrinase (LD). In order to investigate the function of LDI, and the consequences for starch metabolism of reduced LDI activity, transgenic barley plants designed to downregulate LDI by antisense were generated. Homozygous antisense lines with reduced LDI protein level and activity were analysed and found to have enhanced free LD activity in both developing and germinating grains. In addition the antisense lines showed unpredicted pleiotropic effects on numerous enzyme activities, for example, alpha- and beta-amylases and starch synthases. Analysis of the starch showed much reduced numbers of the small B-type starch granules, as well as reduced amylose relative to amylopectin levels and reduced total starch. The chain length distribution of the amylopectin was modified with less of the longer chains (>25 units) and enhanced number of medium chains (10-15 units). These results suggest an important role for LDI and LD during starch synthesis as well as during starch breakdown.     

The effect of milling parameters on starch hydrolysis of milled malt in the brewing process

The effect of milling parameters on the hydrolysis of starch during the mashing process was investigated. Hammer milling was compared against roll milling. Roll gap settings, roll speed, speed differential were also analysed, as well as comparing four- to six-roll milling. The parameter of differential speed was also studied through grist particle size distribution. Employing a 65 °C infusion type mashing process for the wort, the glucose and maltose concentrations of malts milled in different ways were analysed. Results showed that the glucose concentration in the wort after 45 min of mashing, obtained using a hammer mill, was the same as that achieved from roll milling in 60 min. For roller mill gap settings the 0.8 mm gap grist required 60 min of mashing to reach a glucose concentration of 3.46 g l⁻¹, whereas the 0.1 mm gap grist achieved the same level of starch hydrolysis in almost half the time, around 30–35 min of mashing. The results regarding roll speed showed that the 300 and 700 rpm mashes required roughly 50 and 40 min, respectively. Comparable sugar concentrations in the 50 rpm mash were obtained in 60 min. Finally, the comparison between simulated four- and six-roll milling showed the latter yielded higher glucose concentrations.     

The biodiversity of predominant lactic acid bacteria in dolo and pito wort for the production of sorghum beer

AIM: To quantify and identify the predominant lactic acid bacteria (LAB) in dolo and pito wort processing, and to examine their biodiversity at strain level. MATERIALS AND RESULTS: The processing of dolo and pito wort was studied at four production sites in Burkina Faso and Ghana. The succession of dominant micro-organisms, pH and titratable acidity were determined from sorghum malt through mashing and acidification to final wort. In the sorghum malt and during mashing, the LAB counts were 5.7-7.5 log CFU g(-1). Similar levels of yeasts and gram-negative, catalase-positive bacteria were observed. These levels decreased to 3.7-4.5 log CFU g(-1) and相似文献   

Removal of the four C-terminal glycine-rich repeats enhances the thermostability and substrate binding affinity of barley beta-amylase

Barley beta-amylase undergoes proteolytic cleavage in the C-terminal region after germination. The implication of the cleavage in the enzyme's characteristics is unclear. With purified native beta-amylases from both mature barley grain and germinated barley, we found that the beta-amylase from germinated barley had significantly higher thermostability and substrate binding affinity for starch than that from mature barley grain. To better understand the effect of the proteolytic cleavage on the enzyme's thermostability and substrate binding affinity for starch, recombinant barley beta-amylases with specific deletions at the C-terminal tail were generated. The complete deletion of the four C-terminal glycine-rich repeats significantly increased the enzyme's thermostability, but an incomplete deletion with one repeat remaining did not change the thermostability. Although different C-terminal deletions affect the thermostability differently, they all increased the enzyme's affinity for starch. The possible reasons for the increased thermostability and substrate binding affinity, due to the removal of the four C-terminal glycine-rich repeats, are discussed in terms of the three-dimensional structure of beta-amylase.     

Bioregulation of carbohydrate metabolism in relation to source-sink operation during grain-filling phase of growth in wheat

Mobilization of free sugars from vegetative tissues to grain and their transformation to starch in relation to activities of some relevant enzymes during growth and development were investigated in wheat (Triticum aestivum L.). Vegetative tissues, viz. flag-leaf, flag-leaf sheath, nodes and internodes contained high concentration of free sugars from 70 DAS to 18 DPA and that was in the order of accumulation--flag-leaf sheath> flag-leaf and internodes > nodes. In these tissues, major portion of 14C appeared in endogenous sucrose, irrespective of the nature of (U-14C]-sugars supplied. In photosynthetic structures above flag-leaf node, namely peduncle, rachis and bracts, the free sugar make-up was maximum at anthesis (90 DAS). Activity of soluble acid invertase (EC 3.2.1.26) was high in these tissues during early stages of grain growth but reverse was true for soluble neutral invertase (EC 3.2.1.27) activity. In apical and basal portions of grain, free sugars were more or less similarly distributed in concentration. Linear and rapid accumulation of starch in endosperm paralleled with a decline in accumulation of this polymer in pericarp-aleurone. In the latter tissue, the activities of starch hydrolyzing enzymes, i.e alpha- and beta-amylases (3.2.1.1 and 3.2.1.2) were high during initial stages of grain growth. During active grain-filling, alkaline inorganic pyrophosphatase (EC 3.6.1.1) seemed to play a vital role during starch accumulation in endosperm, whereas the involvement of 3-PGA phosphatase (EC 3.1.3.38) was almost confined to pericarp-aleurone. Impairement of ear head photosynthesis by shading depressed starch synthesis (approximately 50%) indicating, thereby, the significant role of current photosynthates during grain-filling. The results suggested that grain growth in wheat was influenced by an efficient operation of source as well as regulatory factors, including enzymes, constituting intrinsic potential of grain sink.     

Beta-AMYLASE4, a noncatalytic protein required for starch breakdown, acts upstream of three active beta-amylases in Arabidopsis chloroplasts

This work investigated the roles of beta-amylases in the breakdown of leaf starch. Of the nine beta-amylase (BAM)-like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable beta-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized beta-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active beta-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total beta-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that beta-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.     

Three alpha-amylases from malted finger millet (Ragi,Eleusine coracana,Indaf-15)--purification and partial characterization

Three alpha-amylases (E.C. 3.2.1.1) were purified to apparent homogeneity from 72 h finger millet malt by three step purification via fractional acetone precipitation, DEAE-Sephacel ion exchange and Sephacryl S-200 gel permeation chromatographies with a recovery of 6.5, 2.9, 9.6% and fold purification of 26, 17 and 31, respectively. alpha-Nature of these amylases was identified by their ability to rapidly reduce the viscosity of starch solution and also in liberating oligosaccharides of higher D.P. and were accordingly designated as amylases alpha-1((b)), alpha-2 and alpha-3, respectively. These amylases, having a molecular weight of 45+/-2 kDa were found to be monomeric. The pH and temperature optima of these alpha-amylases were found to be in the range of 5.0-5.5 and 45-50 degrees C, respectively. K(m) values of these amylases for various cereal starches varied between 0.59 and 1.43%. Carbodiimide (50 mM) and metal ions such as Al(3+), Fe(2+), and Hg(2+) (5 mM) have completely inhibited these enzymes at 45 degrees C. Amino acid analysis of these enzymes indicated high amounts of glycine which is an unusual feature of these enzymes.     

啤酒生产酵母全循环新工艺的研究

以改善浅色啤酒质量,降低生产成本,提高经济效益的目的,提出一种新颖的浅色啤酒的酿造方法。采用煮－浸法糖化工工艺及５０％麦芽和５０％大米作为啤酒酿造的原料及辅料。在糖化过程中添加啤酒酵母提取物作为补充氮源,不仅使所酿造的成品啤酒色泽浅,口味淡爽,纯正,泡沫洁白细腻,持久挂杯,而且具有较显著的经济效益和社会效益。     

Short communication: Brewing properties of Nigerian white and yellow malted sorghum varieties mashed with external enzymes

Both white and yellow varieties of Nigerian sorghum have good brewing properties although the white are better. Thermamyl, the industrial enzyme used by the breweries in Nigeria in mashing unmalted sorghum, influences the brewing properties of both varieties, and yields of extract are increased when a combination of malt and Thermamyl is used. The mashing of sorghum with added enzymes is therefore recommended to improve both malt and beer quality.     

Effects of cultural conditions on the mycelial growth of healthy and virus-infected cultivated mushroom, Agaricus bisporus

There was a significant inverse correlation (P= 0=001) between concentrations of mushroom viruses 1 and 2 in sporophores and amounts of mycelial growth on malt agar of isolates taken from them. Increasing virus concentrations decreased linear growth of one mushroom strain from 76 mm (healthy) to 35 mm (mildly infected) and 7 mm (severely infected) when incubated at 25°C for 3 wk. Mycelial growth rates of isolates from healthy and from virus-infected mushrooms were compared on eleven agar media. All media clearly differentiated between healthy and severely infected isolates, but fewer separated healthy from mildly infected isolates. Those that did contained maltose, sucrose or starch as carbon source. Media containing peptone usually gave better differentiation than those with other sources of nitrogen, but the best differentiation was obtained with malt agar. Growing healthy and infected isolates on a range of media affected their subsequent growth on malt agar, the growth of some isolates apparently being changed permanently after 2 months on some of the different media. Whereas none of the infected isolates grew less rapidly after this treatment, the growth of some of the mildly infected isolates improved to such an extent that they could no longer be distinguished from healthy isolates. After heat-treatment (1–6 wk at 33°C), mycelial growth rates of infected isolates were increased, but viruses 1 and 2 were not always eliminated unless the heat-treatment was begun immediately after subculture. Mycelial growth rate and colony characters are not infallible criteria of the presence or absence of virus, a feature of particular significance when checking the health of mushroom spawn.     

THE STUDY OF THE INFLUENCE OF TEMPERATURE AND INITIAL GLUCOSE CONCENTRATION ON THE FERMENTATION PROCESS IN THE PRESENCE OF Saccharomyces cerevisiae YEAST STRAIN IMMOBILIZED ON STARCH GELS BY REVERSED-FLOW GAS CHROMATOGRAPHY

The technique of reversed-flow gas chromatography (RFGC) was employed for the determination of the alcoholic fermentation phases and of kinetic parameters for free and immobilized cell systems, at different initial glucose concentrations and temperature values. In addition to this, due to its considerable advantages over other techniques, RFGC was used for the characterization of a new biocatalyst, yeast cells immobilized on starch gel, and especially wheat starch gel. Immobilization of wine yeast Saccharomyces cerevisiae AXAZ-1 was accomplished on wheat and corn starch gels in order to prepare new biocatalysts with great interest for the fermentation industry. The RFGC led with great accuracy, resulting from a literature review, to the determination of reaction rate constants and activation energies at each phase of the fermentation processes. A maximum value of rate constants was observed at initial glucose concentration of 205 g/L, where a higher number of yeast cells was observed. The increase of glucose concentrations had a negative influence on the growth of AXAZ-1 cells and rate constants were decreased. The decrease of fermentation temperature caused a substantial reduction in the viability of immobilized cells as well as in rate constant values. Activation energies of corn starch gel presented lower values than those of wheat starch gel. However, the two supports showed higher catalytic efficiency than free cell systems, proving that starch gels may act as a promoter of the catalytic activity of the yeast cells involved in the fermentation process.     

THE STUDY OF THE INFLUENCE OF TEMPERATURE AND INITIAL GLUCOSE CONCENTRATION ON THE FERMENTATION PROCESS IN THE PRESENCE OF Saccharomyces cerevisiae YEAST STRAIN IMMOBILIZED ON STARCH GELS BY REVERSED-FLOW GAS CHROMATOGRAPHY

The technique of reversed-flow gas chromatography (RFGC) was employed for the determination of the alcoholic fermentation phases and of kinetic parameters for free and immobilized cell systems, at different initial glucose concentrations and temperature values. In addition to this, due to its considerable advantages over other techniques, RFGC was used for the characterization of a new biocatalyst, yeast cells immobilized on starch gel, and especially wheat starch gel. Immobilization of wine yeast Saccharomyces cerevisiae AXAZ-1 was accomplished on wheat and corn starch gels in order to prepare new biocatalysts with great interest for the fermentation industry. The RFGC led with great accuracy, resulting from a literature review, to the determination of reaction rate constants and activation energies at each phase of the fermentation processes. A maximum value of rate constants was observed at initial glucose concentration of 205?g/L, where a higher number of yeast cells was observed. The increase of glucose concentrations had a negative influence on the growth of AXAZ-1 cells and rate constants were decreased. The decrease of fermentation temperature caused a substantial reduction in the viability of immobilized cells as well as in rate constant values. Activation energies of corn starch gel presented lower values than those of wheat starch gel. However, the two supports showed higher catalytic efficiency than free cell systems, proving that starch gels may act as a promoter of the catalytic activity of the yeast cells involved in the fermentation process.     

The barley protein degradation: Mechanism of protein solubilization during barley mashing with neutral proteinase

Kinetics of protein solubilization during barley mashing with neutral proteinase were studied. By plotting the kinetics data in Foster-Niemann coordinates for barley concentration range of 10-30% linear relationships with high correlation coefficients (r >/= 0. 999) were obtained. The slopes of straight lines were very close to corresponding reciprocal initial insoluble nitrogen concentrations. Barley proteolytic inhibitors affected the ordinate intercept; by their addition the values of ordinate intercept decreased. The data suggest that the modified Foster-Niemann equation can be proposed to interpret kinetics of insoluble barley protein degradation. The proteolytic activity decay was studied as well. The enzyme decay was faster in buffer solution than during barley mashing, but in both cases first-order kinetics can be applied. A mathematical model describing protein solubilization and enzyme decay kinetics were developed. The results of computer simulation were in good agreement with experimental data.     

The proteinaceous inhibitor of limit dextrinase in barley and malt

Barley limit dextrinase catalyses hydrolysis of alpha-1,6-D-glucosidic bonds in branched poly- or oligosaccharides from starch. A specific inhibitor of this enzyme is found in mature barley kernels, but disappears after several days of germination. Two forms of this proteinaceous inhibitor, identical in amino acid sequence, have been isolated and characterized. They differ in attachment of cysteine or glutathione to a sulfhydryl group, possibly that of cysteine residue 59 of the inhibitor. They can form a 1:1 complex with limit dextrinase and are believed to interact specifically with the enzyme active site. The inhibitor present in mature barley can effectively reduce enzyme activity in barley germinated for a short time and in commercial malt.