Annual Progress Report on Malting Barley Research March, 2002

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150 STUDIES OF STARCH DEGRADATION AND FERMENTABLE SUGAR PRODUCTION IN MALTING BARLEY Cynthia A. Henson USDA-Agricultural Research Service - Cereal Crops Research Unit and Department of Agronomy, University of Wisconsin, Madison, 53706 Four carbohydrases (α-amylase, β-amylase, α-glucosidase and limit dextrinase) are involved in degrading starch and producing fermentable sugars during the processes of malting and mashing. α-Amylase is the only one of these enzymes that is sufficiently thermostable to remain active throughout the latter stages of mashing when most of the starch is degraded. Hence, it is often said that the yield of fermentable sugars from adjunct and malt starches is probably limited by the thermolability of some or all of the other three enzymes. Numerous labs around the world are working to enhance the thermostability of two of the three labile enzymes in order to maximize starch degradation and fermentable sugar production during mashing. Most of these labs are either creating better proteins via mutagenesis or trying to find alleles that encode more stable or more active enzymes. The first to succeed was Yoshigi et al. (1995) who engineered the βamy1 gene isolated from barley to produce β-amylases with thermostabilities õ 10°C higher than the wild type enzyme. Alleles encoding β-amylases with enhanced thermostability, albeit less than Yoshigi and coworkers achieved via mutagenesis, have been identified by Ma et al. (2001) in Australian germplasm and by Clark, Hayes and Henson (manuscript in preparation) in North American germplasm. The thermostability of barley ∀-glucosidase has been increased by 10°C relative to the wild type enzyme via site directed mutagenesis of Agl1 (Muslin et al., 2002). To date, no alleles encoding ∀-glucosidases or limit dextrinases with enhanced thermostability or enzymatic activities have been reported. A project, funded by the NABGP program, to screen for variation in ∀-glucosidase alleles has been initiated but will not be reported on here. AMBA supported research in my lab established the basis of the thermolability of αglucosidase which led to the successful engineering of a more thermostable enzyme (Muslin et al., 2002). We have presented preliminary data on the thermostability of this mutant α-glucosidase (2001 AMBA <strong>Progress</strong> <strong>Report</strong>, 2001 Barley Improvement Conference <strong>Report</strong>). In this report I present data showing the impact of the thermostable ∀-glucosidase upon the production of fermentable (DP 1 - 3) and nonfermentable (DP 4 - 7) maltodextrins during mashing and upon the calculated RDF values. The thermostabilities of the recombinant wild type ∀-glucosidase and the recombinant mutated ∀-glucosidase are compared in Figure 1. The temperature at which the wild type enzyme has only 50% of its maximal activity (T50) was 48°C whereas the T50 of the mutant α-glucosidase was 58°C. It is noteworthy that this increase in stability was achieved by mutating only a single base to result in a change in one amino acid. Not only is this result atypical (Yoshigi et al.’s work required changes in 7 amino acids to achieve a 10°C increase in stability), but it is encouraging. Since we showed that α-glucosidases exist in three plant genera that have this base change (Muslin et al., 2002) and since a
151 10°C increase in thermostability was achieved with just this mutation, it is possible that an allele with this single nucleotide mutation exists in the genus Hordeum. Finding this allele, should it exist, could be a significant step in the enhancement of malting barley germplasm. The efficacy of this thermostable α-glucosidase during mashing was tested by adding the mutant enzyme or the recombinant wild type α-glucosidase (control) to mashes at the beginning of protein rest and monitoring the production of appropriate sugars during mashing. The mashing schedule used is shown in Figure 2. The adjunct grain was rice and the malt was Morex. Mashes spiked with the control enzyme or the mutant enzyme were conducted in triplicate in the automated MA-002 mashing apparatus (LG-Automatic APS, Denmark). The sugars produced, shown in Figure 3, were detected and quantified by pulsed amperometry after separation on an anion exchange column by HPLC (Im and Henson, 1995). The specific gravity of the filtered worts was determined with an Anton Paar digital density meter at 20°C. The addition of the recombinant thermostable α-glucosidase to mashes resulted in a significant increase in glucose production that was evident as early as the end of protein rest (Fig. 3A). The only starch present during this stage of mashing is nongelatinized starch in malt. The most important enzyme in hydrolysis of nongelatinized starch is αamylase and the second most important enzyme is α-glucosidase (Sun and Henson, 1991). Furthermore, in vitro studies conducted at lower temperatures than used here for protein rest showed that α-amylase and α-glucosidase function synergistically to hydrolyze nongelatinized starch (Sun and Henson, 1990; Sissons and MacGregor, 1994). The enhanced production of glucose from nongelatinized malt starch likely resulted from both the individual action of α-glucosidase and its synergistic action with α-amylase. The amount of glucose produced at the end of starch conversion was also significantly increased by the action of the thermostable α-glucosidase compared to the amount produced by the recombinant wild type enzyme (Fig. 3B). Since glucose is a sugar preferentially taken up and metabolized by brewer’s yeast, increasing its yield from raw materials is potentially important. The production of sugars other than glucose during mashing was also affected by the thermostable α-glucosidase (Fig. 3). At the completion of starch conversion the production of glucose, maltose, and maltotriose were all significantly increased by addition of the thermostable α-glucosidase relative to the amount of these same sugars produced by an equivalent amount of wild type α-glucosidase. Although the addition of thermostable α-glucosidase did result in differences in the amounts of maltodextrins with DP õ 4 (Fig. 3C,D), these differences were not significant. The additions of thermostable α-glucosidase to mashing also influenced the calculated RDF values of worts (Table 1). When thermostable α-glucosidase was add to the mashes the RDF values were 10.7% greater than the RDF values obtained by adding the wild type α-glucosidase (control) to the mashes and these differences were significant at the P=0.03 level. Because the amount of wild type α-glucosidase activity added to the control mashes was actually slightly greater than the activity of the thermostable αglucosidase, we also calculated the RDF values based on the unit of activity added. The
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Annual Pro
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-i- INTRODUCTION The March 2002 <st
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Barley Transformation G.J. Muehlbau
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1 2000/2001 WINTER NURSERY: BARLEY
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3 2001 Barley Stripe Rust Screening
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5 Triumph/Tyra//Arupo, a selection
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7 Washington (Fossum Cereals and Wa
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9 This project is supported by the
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11 ‘Triumph’, ‘Valier’, ‘
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13 Table 2. Agronomic data for sele
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15 Table 4. Agronomic data for sele
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17 Table 8. Malting quality data* f
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19 Six-rowed barley selections of c
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21 Table 14. Agronomic data for sel
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23 Table 16. Agronomic data for win
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25 Table 19. Malting quality data*
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Garnet/98Ab11865 Garnet/98Ab12895 9
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29 rust resistant NSGC accessions o
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31 DEVELOPMENT OF SIX-ROWED MALTING
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33 backcrossing. The selfed lines w
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35 MINNESOTA BARLEY IMPROVEMENT PRO
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37 ADVANCED LINE EVALUATION Varieti
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39 OTHER BARLEY DISEASES In 2001, r
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41 Wingbermuehle, W. J., K.M. Belin
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43 In 2001 winter/spring, over 300
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45 assessments were made by countin
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47 IDENTIFICATION OF NOVEL DISEASE
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49 Unfortunately, disease levels in
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51 BARLEY TRANSFORMATION G.J. Muehl
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53 We have available the sugarcane
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55 Secondly, we tried to grow F. gr
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57 References: Issac, S. and Jennin
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59 Huntley-dryland) with two replic
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61 Table 2. 1992 thru 2001 Overall
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63 performance for Montana farmers.
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65 Blake, T,. Hensleigh P., Boss D.
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67 settling tower cannot accommodat
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Table 3. Seedling reaction to strip
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71 TWO-ROWED BARLEY IMPROVEMENT PRO
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Table 1. Agronomic trait comparison
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75 mutants reduce plant vigor and g
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77 SIX-ROWED BARLEY IMPROVEMENT PRO
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79 this variety has decreased over
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Table 7. Malt quality comparisons o
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83 • Management studies for malt
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85 STUDIES ON BARLEY DISEASES AND T
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87 With funding from the US Wheat a
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89 MALTING AND BREWING QUALITY OF B
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91 β-glucans, it might be most ins
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10 9 8 7 6 5 4 3 2 1 0 93 Figure 1.
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95 Bolin, P., Schwarz, P., Jones, B
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97 Results Results from field and g
Page 107 and 108: 99 DEVELOPING BARLEY TISSUE CULTURE
Page 109 and 110: 101 trials of plants regenerated fr
Page 111 and 112: 103 Table 2. Agronomic performance
Page 113 and 114: 105 BSMV spread between entries. Ho
Page 115 and 116: 107 involving two susceptible backg
Page 117 and 118: 109 THE OREGON BARLEY IMPROVEMENT P
Page 119 and 120: 111 for traits that are difficult/e
Page 121 and 122: Germplasm Development: 113 Crosses:
Page 123 and 124: 115 • All lines in spring yield t
Page 125 and 126: 117 Table 2. Agronomic data for Ore
Page 127 and 128: 119 Table 6. Malting quality of win
Page 129 and 130: 121 Table 7. Across location summar
Page 131 and 132: 123 MOLECULAR MARKER ASSISTED MODIF
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Page 135 and 136: 127 exchanges. Spring and winter ba
Page 137 and 138: 129 Table 2. 2000-2001 Agronomic Pe
Page 139 and 140: 131 Table 7. Spring 2- and 6-Row #
Page 141 and 142: 133 2. Direct Seeding Cropping Syst
Page 143 and 144: 135 D.M. Wesenberg - spring and win
Page 145 and 146: 137 A STUDY OF THE MALTING QUALITY
Page 147 and 148: 139 Methods. Our malting schedule.
Page 149 and 150: 141 modification measures indicated
Page 151 and 152: 143 CHARACTERIZATION OF THE PROTEIN
Page 153 and 154: 145 Purification of a serine endope
Page 155 and 156: 147 STUDIES ON THE PROTEINASES THAT
Page 157: 149 with the proteinase T will be s
Page 161 and 162: 153 Sissons MJ and MacGregor AW (19
Page 163 and 164: µM Carbohydrate / Unit of α-Gluco
Page 165 and 166: 157 Tissue-specific gene products f
Page 167 and 168: 159 The promoters were subcloned fr
Page 169: 161 Fig. 3. Expression of Trx-Hth f
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Annual Progress Report on Malting Barley Research March, 2002

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