Variation in α-amylase and α-amylase inhibitor activities in barley malts

Journal of Cereal Science 8 (1988) 39-46

Variation in a-Amylase and a-Amylase Inhibitor Activities in Barley Malts C. A. HENSON*t and J. M. STONEt *USDA-ARS Cereal Crops Research Unit, 1575 Linden Drive, Madison, WI 53706 and tDepartment of Agronomy, University of Wisconsin, Madison, WI53706, U.S.A. Received 4 November 1987 Malts of barley genotypes exhibiting a wide range of IX-amylase activities were used to determine if these differences resulted from the absence of functional products of one of the two gene families which encode barley IX-amylase. Additional studies were conducted to determine if IX-amylase activities reflect differences in activities of a specific proteinaceous IX-amylase inhibitor. These malts had a 43-fold variation in in vitro IX-amylase activities. When examined by isoelectric focusing and activity staining, all genotypes exhibited the same IX-amylase isozyme patterns. In vitro amylase inhibitor activities varied only three-fold. The correlation coefficient between inhibitor and IX-amylase activities was -0,92 (P ~ 0,01). Examination of two of these malts by isoelectric focusing followed by inhibitor activity staining, showed that both malts had six bands of activity with one very prominent band. We conclude (I) that no gross absence of functional IX-amylase isozymes occurs, thus both gene families coding for IX-amylases are expressed, and (2) that levels of IX-amylase inhibitor may be an important factor, but probably not the only factor, in regulating malt IX-amylase activities.

Introduction Malting barleys have been subjected to extensive selection for malting performance during their development. As many as 15 different malt quality factors 1 are considered, in addition to numerous agronomic characteristics, when evaluating a barley line of potential interest to malting and brewing industries. This emphasis on malt quality has resulted in a narrow germplasm base from which selections are made. Eslick and Hockett 2 have suggested that less than twelve introductions of new germplasm to the North American malting barley germplasm base have been made since 1900. Even though the gene pool is narrow, closely related cultivars are released which vary significantly in one or more specific traits. An example of such divergent gene expression is the different levels of a-amylase in two commercially available cultivars, Morex and Robust. Morex malt possesses high levels of a-amylase activities 3• Malt of Robust, the

t

To whom correspondence should be addressed. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. Abbreviations used: BASI = barley amylase/subtilisin inhibitor; IEF = isoelectric focusing. 0733-5210/88/040039 +08 $03.00/0

© 1988 Academic Press Limited

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C. A. HENSON AND J. M. STONE

progeny of a cross between Morex and Manker, has greatly reduced ex-amylase levels. Two factors which could result in differences in ex-amylase activities between cultivars are: (I) differential expression of ex-amylase isozyme activities, and (2) differences in levels of the proteinaceous inhibitor of ex-amylase. The two groups of ex-amylases, separable by their isoelectric points, are encoded by two different gene families 4 · 6 • Both the high and low pI ex-amylases are heterogeneous groups of isozymes. The high pI examylase activities are decreased by binding ofa proteinaceous inhibitor 7 • 8 • This protein does not bind to or decrease the activity of low pI a-amylases. The objectives of this study were to elucidate whether differences in barley malt examylase activity result from the absence of functional products of one of the two gene families or reflect differences in activities of the specific proteinaceous ex-amylase inhibitor. The approach taken was to examine 12 different malted barleys (two commercial cultivars and ten breeding lines) for the presence or absence of functional products of both gene families and to quantitate the activities of ex-amylase and its inhibitor. Experimental

Tissue sources Breeder's barley samples submitted for malt quality analysis were micro-malted. Steeping (16°C) was from 6 to 40 h, depending on initial kernel weight. Germination (4 days) was at 16°C, 100 % relative humidity and in complete darkness. Kilning (from 35 to 85°C over a 24 h period) was performed in a constantly rotating drum. Final moisture of malts is less than 4 %. Malts of Morex, Robust, 7016,7017, M82-335, M82-457, M82-409, M82--430, 4228, 6367, M81-255 and M81-31 were from barley grown in Crookston, MN during 1985. The malt numbers are the permanent entry numbers used by the Department of Agronomy, University of Minnesota. All malts were of six-rowed barley.

Extractions Malted kernels were de hulled by hand and ground for I min in chilled mortars containing sand (l g/20 kernels). Extraction buffer (40 mM Tris, pH 8·0, containing I mM CaCI 2) was added (5 ml/g malt) and grinding continued for 2 min. Extracts were centrifuged (4°C) at 15000 g for 15 min. Activities of a·amylase and inhibitors were determined on the supernatants.

Assays a-Amylase actIVItIes were determined on heated (70°C for 15 min) supernatants by the spectrophotometric technique of Doehlert and Duke using starch azure as the substrate 9 . The assay buffer was 50 mM succinate (pH 6'0) containing 3 mM CaCI 2 • Heating was used to reduce or eliminate the amylase inhibitor, thereby allowing apparent maximal activities of a-amylase to be determined. a·Amylase inhibitor activities were determined on non-heated supernatant aliquots that were acid treated to eliminate endogenous a-amylase activity. Acid inactivation of a-amylases was done by equilibrating the aliquots to 30°C then lowering the pH to 3'5. Acidified aliquots were incubated at 30°C for 1'5 h. The pH was then adjusted to 6,5-6,8. This treatment was checked for elimination of a-amylase activities with in vitro a-amylase assays and by electrophoresis on IEF gels followed by staining for amylase activity. This technique resulted in

(X-AMYLASE INHIBITOR IN MALTED BARLEYS

41

no ct-amylase activity being detected on IEF gels and less than 1·5 % of the original ct-amylase activity was detected with the in vitro assay. Inhibitor activities were measured as reduction in activity of known levels of commercial ct-amylase (70°C treated barley malt extracts, Sigma A-2771) and were detected by the technique of Doehlert and Duke D • Inhibitor and commercial ex-amylase were mixed then incubated at room temperature for 10 min prior to addition of substrate.

Isoelectric focusing For determination of ex-amylase isozyme patterns, malted barley extracts (30 ml extraction buffer Ig malt) were heated (70°C for 15 min), centrifuged (4°C) and isozymes separated by electrophoresis (10 0c) on polyacrylamide gels (1'6 mm thick) with a pH gradient of3'5-IO (LKB* ampholines) or 3-10 (Pharmacia ampholines)lO. Electrode buffers were 0·04 M glutamate and I M NaOH. Gels were prefocused for 500 Vh and focusing continued for an additional 7000 Vh after sample application. A surface electrode was used to determine pH gradient of gels. Gels were incubated in a buffered 0'5 % soluble starch (Lintner potato starch) solution (50 mM ethylenediamine, pH 6'0, containing 3 mM CaCI 2 ) at 37°C for I h then stained for ct-amylase activity with KI-I 2 (I mM 12 and 0'5 MKI). For determination of inhibitor activities, non-heated malt extracts (10 ml extraction bufferI g malt), were electrofocused for 9700 Vh beyond prefocusing on gels containing Pharmacia ampholines pH 3-10 and detected with a procedure similar to that of Weselake et al. 7 Inhibitor activities were detected by incubating the gel for 20 min at room temperature with commercial barley malt ct-amylase in extraction buffer. The gel was then incubated with I % soluble starch in extraction buffer for 20 min at room temperature. After thorough rinsing in distilled Hp, the gel was stained with KI-I 2 •

Results and Discussion

Analysis of 12 barley genotypes for in vitro activities of a-amylase and a-amylase inhibitor revealed large differences in a-amylase activities and much smaller differences in inhibitor activities (Table I). The highest a-amylase activities were found in M82-409 and were 43 times greater than activities in the lowest amylase line, Robust. Morex had TABLE I. Activities of barley malt Q(-amylase and ex-amylase inhibitor o:-AmyJase activities are expressed as !-lmol maltose equivalents produced/min/g fresh wt. Inhibitor activities are expressed as J.lmol maltose equivalents inhibited/min/g fresh wt.

n

Cultivarn

Inhibitor activity

Amylase activity

Morex Robust M82-355 M82-457 6367 4228 7017 7016 M82-430 M81-255 M81-31 M82·409

292± 16 326±28 114± 14 92±3 310±23 285±26 307± 14 249± 15 305+21 288± 17 239±31 11O±11

98±3 12±8 416± 14 418±20 14±4 16±3 88±5 91 ±4 104+5 16±0 16± I 532± 19

Minnesota permanent entry number or cultivar name.

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C. A. HENSON AND J. M. STONE

eight times more extractable a-amylase activity than did Robust. Activities of the specific proteinaceous a-amylase inhibitor, termed BASI for barley amylase/subtilisin inhibitor l l , varied three-fold. Inhibitor activity of Morex malts was similar to the level of Robust malts. The correlation coefficient between inhibitor and a-amylase activities from these malts was -0,92 (P::::; 0'01). Munck et aZY have screened mature kernels of 14 European barley varieties and found a 2'5-fold range in BASI protein content. Weselake et aZ. found a 2'5-fold range in inhibitor activity among three barley cultivars 12 • The two heterozygous groups of barley a-amylases encoded for by two multigene families are referred to as Group I and Group II. Consistent with the recommendations of the Biochemical Society13, the most acidic form is isozyme one or Group I. The pIs of barley Group I a-amylases have been reported from pH 4·4 to 5·2. Group II aamylase pIs are reported to range from 5·7 to 6-4. Group III a-amylases are the result of Group II a-amylases binding the amylase inhibitor protein known as BASI (due to its capability to inhibit both barley a-amylases and subtilisin proteases). Group III aamylases typically have pIs ranging from 6·1 to 6·9. An isoelectric focusing gel (LKB ampholines, pH 3'5-10) stained for a-amylase activities is shown in Fig. 1. Isozyme patterns are similar for all eight heated malt extracts. The dominant group of a-amylase activity, Group II, had pIs ranging from pH 5·9 to 6·4. Group III isozymes are seen in Fig. 1 as light bands of amylase activity between pH 6·5 and 7·0. Group I a-amylases are between pH 4·7 and 5·5 (Fig. 1). The LKB ampholine-containing gels consistently resulted in the maximal number of isozyme bands and were therefore used to detect differences in banding patterns between the malt lines studied. The eight malt genotypes shown in Fig. 1 represent lines of intermediate

-7.0 -6.5 -6.0

- 5.5 -5.0 1

2

3

4

5

6

7

8

FIGURE 1. Isoelectric focusing gel, pH 3'5-10, stained for amylase activity. Lanes contain heat-treated barley malt extracts of plant lines: I, Morex; 2, Robust; 3, Manker; 4, 6367; 5, M81-2SS; 6, 3178; 7, M81-31; 8, Robust. Equal volumes of extracts were loaded in all lanes.

a-AMYLASE INHIBITOR IN MALTED BARLEYS

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and low ex-amylase activities 003·6 to 12'2 !-lmol maltose equivalents producedjminjg fresh wt.). Although not shown here, plant lines with high ex-amylase activities (M82355, M82-437, and M82-409) exhibited the same banding patterns as seen for intermediate and low ex-amylase lines. The ex-amylase banding pattern found in all plant lines examined in this study, corresponds to the Type III isozyme pattern documented by Takano and Takeda l 4, who found most of the six-row malting barleys examined to exhibit this pattern. When heated malt extracts were separated on IEF gels containing Pharmacia ampholines, pH range 3-10, apparent differences in Group II and Group III ex-amylases were detected (Fig. 2). In this gel system, Group II ex-amylases were detected between pH 5·8 and 6·1 and Group III ex-amylases between pH 6'3 and 6'5. Extracts of genotypes with high, intermediate, and low ex-amylase activities are shown in Fig. 2. The plant lines with highest ex-amylase activities (lanes 1 and 2) exhibited strongest activity stains in the Group II amylase area. These plant lines also contained in vitro inhibitor activities (Table I) and some activity was detected in the Group III area of this gel. However, the other four gel lanes exhibited more activity staining in the area of Group III amylases and all had greater in vitro inhibitor activities than the high ex-amylase lines (Table I). All genotypes examined by IEF exhibited nearly identical qualitative banding patterns for Group I, II and III ex-amylases (Figs 1 and 2). We conclude that all isozymes are functionally expressed in each of the genotypes examined. Thus, absence of expression of one or the other ex-amylase groups cannot account for the observed differences in amyloytic activities (Table I). This type of electrophoretic analysis cannot exclude the

-6.5 -6.0

-5.5

1

2

3

4

5

6

-5.0

FIGURE 2. Isoelectric focusing gel, pH 3-10, stained for amylase activity. Lanes contain heat-treated barley malt extracts of plant lines; 1, M82-457; 2, M82-355; 3, Morex; 4, Robust; 5,4228; 6, 6367. Lanes were loaded with approximately equal aamylase activities and contain the following levels of a-amylase activity/inhibitor activity, both expressed as Ilmol/min; 1,0·48/0'11; 2, 0'5/0,2; 3,0'55/1'6; 4, 0'49/13; 5, 0'46/8·2; 6, 0'5/11. a-Amylase activities were determined on heated extracts whereas inhibitor activities were from non-heated extracts.

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C. A. HENSON AND J. M. STONE

possibility of differences in amounts of isozyme proteins present or differences in their activity. Our data allow us to conclude that no gross absence of functional a-amylase isozymes occurs in any of the genotypes examined. Group III Cl-amylases were not completely eliminated by heat treatments at 70°C for 15 min (Figs I and 2). As soluble starch was the substrate in activity-stained IEF gels, heat treatments were used to eliminate malt p-amylase activities. These heat treatments have been suggested to eliminate the BASI-Group II complexB ; hence, inhibitor activity in the heated extracts was unexpected. Others have also detected Group III amylase activity in heated malt extracts separated by isoelectric focusing 15 • 16 • Although some Group III a-amylase activity does survive this heating regime, MacGregor and Bal1ance 16 have shown that reduced Group In amylase activities are present in heated malt extracts. The inhibitor is more heat-labile than the a-amylase; thus heat treatments result in reduced Group III amylase activities and increased Group II activities. When we heated aliquots of three malt extracts that had been acid treated (pH 3'5 at 30°C for 1·5 h) to eliminate endogenous a-amylase activities, no in vitro inhibitor activity was detected (data not shown). Data in Fig. 2 clearly show that these three malt extracts (Morex, Robust, and 6367) have inhibitor activity that survived heating when a-amylase had not been previously acid inactivated (see lanes 3, 4 and 6). The presence of Group II a-amylase apparently results in greater heat stability of the inhibitor. Even though some inhibitor clearly survives kilning, it probably does not affect starch conversion during mashing. Munck et alY have shown inhibitor levels to be virtually undetectable by the time that malt conversion is maximal.

-7.5 -7.0

-6.5 1

2

FIGURE 3. Isoelectric focusing gel, pH 3-10, stained for Cl-amylase mhlOltOr activity. Lanes contain non-heat-treated barley malt extracts of 1, Robust and 2, Morex. Approximately equal inhibitor activities were loaded in both lanes.

a-AMYLASE INHIBITOR IN MALTED BARLEYS

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Studies of the BASI protein were continued using only malt extracts of Morex and Robust. These two cultivars were chosen because of their commercial importance in malting and brewing. Isoelectric focusing gels that were incubated with
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C. A. HENSON AND J. M. STONE

References 1. Burger, W. C. and LaBerge, D. E. in 'Barley' (D. C. Rasmusson, ed.), American Society of Agronomy Publications, Madison, Wisconsin (1986) pp 367-401. 2. Eslick, R. F. and Hockett, E. A. Agric. Meleorot. 14 (1975) 13-23. 3. Wych, R. D. and Rasmusson, D. C. Crop Sci. 23 (1983) 1037-1040. 4. Muthkrishnan, S., Chandra, G. R. and Maxwell, E. S. J. Bioi. Chern. 258 (1983) 2370-2375. 5. Rogers, J. C. and Milliman, C. J. Biot. Chern. 259 (1984) 12234-12240. 6. Brown, A. H. D. and Jacobsen, J. V. Genet. Res., Camb. 40 (1982) 315-324. 7. Weselake, R. J.• MacGregor, A. W. and Hill, R. D. Plam Physiol. 72 (1983) 809-·812. 8. Mundy, J., Svendsen, I. and Hejgaard, J. Carlsberg Res. Cornmun. 48 (1983) 81-90. 9. Doehlert, D. C. and Duke, S. H. Planl Physiol. 71 (1983) 229-234. 10. MacGregor, A. W. Cereal Chern. 53 (1976) 792-796. 1 L Munck, L., Mundy, J. and Vaag, P. Am. Soc. Brew. Chern. J. 43 (1985) 35-38. 12. Weselake. R. J., MacGregor, A. W. and Hill, R. D. Cereal Chern. 62 (1985) 120-123. 13. Biochemical Society. 'Enzyme Nomenclature', Elsevier, New York (1972) pp 23-25. 14. Takano, T. and Takeda, G. Jpn J. Breed. 35 (1985) 9-16. 15. MacGregor, A. W. and Daussant, 1. J. Insl. Brew. 87 (1981) 155-157. 16. MacGregor, A. W. and Ballance, D. J. Insi. Brew. 86 (1980) 131-133. 17. Sadowski, J., MacGregor, A. W. and Daussant, J. Eleclrophoresis 7 (1986) 176-179.