Cynthia A. Henson

Characterization of high pI α-glucosidase from germinated barley seeds: substrate specificity, subsite affinities and active-site residues☆

Abstract Substrate specificity and subsite affinities of high pl α-glucosidase from germinated barley ( Hordeum vulgare L.) seeds were investigated by kinetics. The enzyme has only one maltose binding site per molecule and shows high activity on small maltooligosaccharides and nigerose. Hydrolysis of isomaltose and p -nitrophenyl α-glucoside is moderate. Trehalose is not hydrolyzed at detectable rates. The ratios of the maximum velocities for maltose, nigerose, isomaltose, p -nitrophenyl α-glucoside and malto-triose, -tetraose, -pentaose, -hexaose, -heptaose are 100:95:21:9:111:116:119:104:111. The K m values for these substrates are 1.91, 1.29, 5.32, 1.04, 1.11, 2.37, 2.92, 5.44 and 7.89 mM, respectively. Based on the rate parameters for maltooligosaccharides, the subsite affinities ( A i s) in the active site of the enzyme were evaluated according to subsite theory. Subsites 1, 2 and 3, having positive A i values ( A 1 , A 2 and A 3 ; 1.34, 5.37 and 0.27 kcal/mol, respectively), were considered to be effective for the binding of substrate to the active site. The different arrangement of subsite affinities among α-glucosidases, glucoamylases and amylases was used to explain their substrate specificities.

A Comparison of Standard and Nonstandard Measures of Malt Quality

The objectives of this study were to compare standard and nonstandard measures of malting quality using simple correlations and to determine whether six highly elite malting barley cultivars could be distinguished from each other using multivariate statistics to analyze 9 standard and 22 nonstandard measures of malting quality. Simple linear regression revealed cultivar differences in the thermostabilities of α-amylase, β-amylase, limit dextrinase, and α-glucosidase that were positively correlated with differences in wort osmolyte concentrations (r = 0.853–0.958, P ≤ 0.05–0.01) and differences in the thermostabilities of α-amylase, β-amylase, and limit dextrinase that were correlated with diastatic power (r = 0.872–0.937, P ≤ 0.05–0.01). Principal component analysis (PCA) of the nonstandard measures of malting quality were considered more useful than PCA of the standard measures because the former was able to categorize the six-row cultivar and two-row cultivar with the lowest real degree of fermentation, an important measure of brewhouse performance, as being different from the other two- and six-row malts. The malt quality traits that distinguished the two lowest performing of the six elite malting barleys from the other malts were α-glucosidase, limit dextrinase, and α-amylase activities, which were lower in these two malts, plus the thermostabilities of α-amylase, β-amylase, and limit dextrinase and wort osmolyte concentrations, which were higher in these two malts.

Osmolyte concentration as an indicator of malt quality

This study was conducted to test the hypothesis that malt osmolyte concentrations can be used as an indicator of barley malt quality. Barley seeds of four six-row and four two-row genotypes were steeped and then germinated for 6 days at 20°C. At intervals of 24 hr over the germination regime, green malt from each cultivar was removed, kilned, and analyzed for osmolyte concentration (OC), malt extract (ME), diastatic power (DP), α-amylase activity, soluble/total protein (S/T), and β-glucan concentration. OC increased most rapidly from days one to three of germination. After 4 days, rates of increase in OC began to slow and, after 5 days of germination, OC had plateaued or declined. In all but three genotypes, ME followed a pattern similar to that of OC, but ME levels plateaued or declined approximately 1 day sooner than OC. This suggests that OC continues to measure storage compound degradation for a longer period than ME and could be a better indicator of malt modification than ME. ME and OC were significantly and positively correlated in days two through four and day six (r = 0.740–0.927, P < 0.0001). For days two and three, OC correlated well with ME for all days (r = 0.740–0.942, P < 0.0001) and α-amylase activity for day two (r = 0.771, P < 0.0001). For day two, OC correlated well with days two through six for β-glucan concentration (r = −0.702 to −0.830, P < 0.0001). No significant correlations were found for DP and OC on any day. These data indicate that OC of malt produced at early time points in germination is a good indicator of several measures of the quality of malt produced at later time points in germination.

Differential expression of two β-amylase genes (Bmy1 and Bmy2) in developing and mature barley grain

Two barley (Hordeum vulgare L.) β-amylase genes (Bmy1 and Bmy2) were studied during the late maturation phase of grain development in four genotypes. The Bmy1 and Bmy2 DNA and amino acid sequences are extremely similar. The largest sequence differences are in the introns, seventh exon, and 3′ UTR. Accumulation of Bmy2 mRNA was examined in developing grain at 17, 19, and 21xa0days after anthesis (DAA). One genotype, PI 296897, had significantly higher Bmy2 RNA transcript accumulation than the other three genotypes at all developmental stages. All four genotypes had Bmy2 mRNA levels decrease from 17 to 19 DAA, and remain the same from 19 to 21 DAA. Levels of Bmy1 mRNA were twenty thousand to over one hundred thousand times more than Bmy2 mRNA levels in genotypes Legacy, Harrington, and Ashqelon at all developmental stages and PI 296897 at 19 and 21 DAA. PI 296897 had five thousand times more Bmy1 mRNA than Bmy2 mRNA at 17 DAA. However, Bmy2 protein was not found at 17 DAA in any genotype. The presence of Bmy2 was immunologically detected at 19 DAA and was present in greater amounts at 21 DAA. Also, Bmy2 protein was found to be stored in mature grain and localized in the soluble fraction. However, Bmy1 protein was far more prevalent than Bmy2 at all developmental stages in all genotypes. Thus, the vast majority of β-amylase activity in developing and mature grain can be attributed to endosperm-specific β-amylase.

Histological analysis and 3D reconstruction of winter cereal crowns recovering from freezing: a unique response in oat (Avena sativa L.).

The crown is the below ground portion of the stem of a grass which contains meristematic cells that give rise to new shoots and roots following winter. To better understand mechanisms of survival from freezing, a histological analysis was performed on rye, wheat, barley and oat plants that had been frozen, thawed and allowed to resume growth under controlled conditions. Extensive tissue disruption and abnormal cell structure was noticed in the center of the crown of all 4 species with relatively normal cells on the outside edge of the crown. A unique visual response was found in oat in the shape of a ring of cells that stained red with Safranin. A tetrazolium analysis indicated that tissues immediately inside this ring were dead and those outside were alive. Fluorescence microscopy revealed that the barrier fluoresced with excitation between 405 and 445 nm. Three dimensional reconstruction of a cross sectional series of images indicated that the red staining cells took on a somewhat spherical shape with regions of no staining where roots entered the crown. Characterizing changes in plants recovering from freezing will help determine the genetic basis for mechanisms involved in this important aspect of winter hardiness.

Metabolic changes in Avena sativa crowns recovering from freezing.

Extensive research has been conducted on cold acclimation and freezing tolerance of fall-sown cereal plants due to their economic importance; however, little has been reported on the biochemical changes occurring over time after the freezing conditions are replaced by conditions favorable for recovery and growth such as would occur during spring. In this study, GC-MS was used to detect metabolic changes in the overwintering crown tissue of oat (Avena sativa L.) during a fourteen day time-course after freezing. Metabolomic analysis revealed increases in most amino acids, particularly proline, 5-oxoproline and arginine, which increased greatly in crowns that were frozen compared to controls and correlated very significantly with days after freezing. In contrast, sugar and sugar related metabolites were little changed by freezing, except sucrose and fructose which decreased dramatically. In frozen tissue all TCA cycle metabolites, especially citrate and malate, decreased in relation to unfrozen tissue. Alterations in some amino acid pools after freezing were similar to those observed in cold acclimation whereas most changes in sugar pools after freezing were not. These similarities and differences suggest that there are common as well as unique genetic mechanisms between these two environmental conditions that are crucial to the winter survival of plants.

Detecting Corn Syrup in Barley Malt Extracts

ABSTRACT Methods for detecting corn syrup in barley (Hordeum vulgare L.) malt extract were evaluated. Twelve samples representative of commercially available 2-rowed and 6-rowed malting barleys were malted. Extracts prepared from the finely ground malts were analyzed for 13C/12C ratios, expressed as δ13C, and concentrations of protein and sugars. The 13C/12C ratios were sufficiently different to distinguish corn syrup from malt extract. By calculating the mean values for the barleys, it was determined that a δ13C > -24.3‰ indicated that the malt extract had been adulterated with corn syrup (99% confidence). Protein concentrations <4.5% (2-rowed malt) or <5.0% (6-rowed malt) of the extracts also indicated probable adulteration with corn syrup, which is devoid of protein. Because of differences in sugar concentrations between the malt extracts and corn syrup, carbohydrate analysis also indicated probable mixtures. These findings were confirmed by analysis of extracts from composite 2-rowed and 6-rowed barle...

Comparisons of Modern U. S. and Canadian Malting Barley Cultivars with Those from Pre-Prohibition: III. Wort Sugar Production during Mashing

ABSTRACT Wort sugars produced during Congress mashing of pre-Prohibition and modern barley genotypes were compared. Four of five modern cultivars were higher (P < 0.0001) in wort glucose production than six pre-Prohibition cultivars. Mean wort glucose levels of the five modern cultivars were higher (P values from 0.0002 to 0.0021) than that of the pre-Prohibition barleys. In contrast, maltose production was variable and there were no significant differences in mean levels in modern versus pre-Prohibition barley worts, although Manchuria (pre-Prohibition) and Tradition (modern) produced higher (P < 0.0001) levels than other genotypes. Wort maltotriose levels were also quite variable among genotypes, although were higher (P < 0.0001) in Harrington (modern) worts than all others at the end of mashing. By the end of mashing, pre-Prohibition and modern genotypes did not statistically differ in their mean levels of maltotriose (P = 0.514). Glucose equivalents from fermentable sugars (i.e., total glucose from glucose plus hydrolysis of maltose and maltotriose) followed almost an identical pattern as that for maltose. Levels of maltotetraose through maltoheptaose were variable in both pre-Prohibition and modern genotypes throughout mashing. However, these maltodextrins usually decreased rapidly in modern cultivars while they increased or decreased more slowly in pre-Prohibition genotypes. In general, maltotetraose through maltoheptaose were higher (P < 0.0001) in Hanna and Hannchen (pre-Prohibition) worts than in other worts. Sucrose levels were relatively constant during mashing of most pre-Prohibition and modern genotypes. Fructose levels increased throughout mashing for all genotypes. Mean wort fructose levels of modern barley genotypes were significantly higher than those for pre-Prohibition genotypes during most of mashing.