Isolation and Characterization of a-Amylases from Endosperm of Germinating Maize

Cereal Chern. 68(4):383-390 Amylases from germinating maize (cv. B73) were fractionated by affinity chromatography, anion exchange chromatography, and chromatofocusing. Two groups of amylase enzymes were separated by affinity chromatography. About one half of the total amylase activity was bound on a cycloheptaamylose-Sepharose 6B column. Bound proteins were fractionated by anion exchange into four major a-amylase fractions, then further separated by chromatofocusing into eight fractions with apparent isoelectric point (pI) values ranging from 5.70 to 4.06. All affinity-bound fractions were confirmed as a-amylases by their action on 13-limit dextrin. The affinity-bound a-amylases with lowest and highest pI values produced The study of cereal a-amylases is important from both fundamental and applied perspectives. The status of research on cereal a-amylases is the subject of a recent review (Hill and MacGregor 1988). Cereal grains synthesize multiple forms of a-amylase during germination to supply soluble carbohydrates for the developing seedling. Heterogeneity of starch-degrading enzymes in germinating seeds enhances the conversion of insoluble granules to soluble starch and dextrins (Beck and Ziegler 1989). The presence of multiple forms of a-amylase suggests that each form may have a particular .metabolic function in situ, as the individual forms act cooperatively to degrade starch during germination. The presence of multiple enzyme forms makes it difficult to assess reaction rates and biochemical parameters of individual forms. Each form must be isolated to accurately characterize its kinetic and regulatory properties, so that its role in the starch degradation process may be determined. a-Amylases from different cereal grains have different biochemical properties, as do different forms that occur within a species (Frydenberg and Nielsen 1966, Kruger and Tkachuk 1969, Chao and Scandalios 1971, Goldstein and Jennings 1975, Okamoto and Akazawa 1979, Mundy 1982). Different forms within a species usually have similar molecular weights in the 42-45 kDa range (Tkachuk and Kruger 1974, Scandalios et al 1978, Jacobsen and Higgins 1982, Mundy 1982) but differ in electrophoretic mobility. Germinating barley and wheat each contain two groups of a-amylases, each of which contains several individual enzymes (Jacobsen and Higgins 1982, Callis and Ho 1983, Marchylo and MacGregor 1983, Kruger and Marchylo 1985). The two groups are products of two different gene families (Nishikawa and Nobuhara 1971, Gale et al 1983, Khursheed and Rogers 1988) and differ in biochemical and antigenic properties (Jacobsen and Chandler 1988). Two forms of a-amylase in germinating sorghum have partial immunological identity and different kinetic properties (Mundy 1982, Lecommandeur and Daussant 1989). The study of amylases in germinating corn is less advanced than in other cereal grains. Polymorphism for a-amylase in germinating maize was first reported by Chao and Scandalios (1969, 1971, 1972), who identified eight amylase isoforms, two 'Seed Biosynthesis Research Unit. National Center for Agricultural Utilization Research. Agricultural Research Service. u.s. Department of Agriculture. 1815 N. University Street. Peoria. IL 61604. Mention of firm names Or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. American Association of Cereal Chemists, Inc., 1991. reaction products from soluble starch containing large amounts of dextrins with degrees of polymerization (DP) 2 and 6, with lesser amounts of intermediate oligosaccharides. Intermediate pI fractions produced primarily DP2, large amounts of DP3-5, and litile DP6. Enzymes not bound by cycloheptaamylose affinity chromatography were purified by hydroxylapatite chromatography, then resolved by chromatofocusing into four subgroups of a-amylase, plus l3-amylase. Among the affinity-unbound fractions, the lowest pI a-amylase had a unique action pattern, producing primarily D P7 and 8 oligosaccharides after exhaustive hydrolysis of soluble starch. of which (aand f3-amylases) they classified as primary gene products. They recognized that maize amylases are different in many respects from those in wheat, barley, and rye; e.g., only 10% of the total maize amylase activity is f3-amylase. Two allelic forms of a-amylase with isoelectric points (pIs) of 4.8 (Scandalios et a11978) and 4.35 (Chao and Scandalios 1971) exhibit differential expression in developing kernels and germinating seedlings of maize (Chao and Scandalios 1969, 1971, 1972). Goldstein and Jennings (1978) separated three forms of a-amylase from deembryonated sweet corn (cv. Seneca Chief). These forms vary in their sensitivities to Hg+2 and heating in the presence of Ca+2. Recently, MacGregor et al (1988) used a combination of isoelectric focusing (IEF) and chromatofocusing to identify a-amylases of several germinating cereal grains. Grains were classified into two groups: those that contain both a high (>5.8) and a low «5.5) pI component (barley, wheat, rye, and triticale) and those with only low pI components (oats, maize, millet, sorghum, and rice). However, maize (cv. Sunnyvee) has intermediate (5.5-6.0) pI amylases as well as low pI components and does not strictly fit either classification. More recently, extracts of germinating maize (cv. Dea) were found to contain six bands on IEF gels with three distinct amylase antigens (Lecommandeur and Daussant 1989). Because relatively little fundamental study has been made of maize a-amylases, and no attempt has been made to use the endogenous amylases commercially, we have undertaken to isolate and characterize the enzymes found in germinating maize and to determine whether any of these might be commercially employed to hydrolyze granular starch at ambient temperatures, in a manner analogous to malting of barley. Our goals in this project are to isolate the enzymes that hydrolyze granular starch during seed germination. We intend to determine the relative abundance and activity of these enzymes, to establish optimum conditions for their action, and to identify the reaction products formed by them. This report describes our initial efforts at isolation and characterization. MATERIALS AND METHODS Seed Germination and Enzyme Extraction Samples of 100 kernels of inbred maize, cultivar B73, were surface sterilized with 1% NaOCI, steeped overnight in 10 mM CaCI2, spread on filter paper saturated with steeping solution, sealed with plastic wrap in glass trays, and allowed to germinate in the dark for seven days at 25° C. The pericarp, scutellum, and embryonic axis were removed and discarded. Endosperm was ground in a mortar and pestle with 25 ml of 20 mM sodium acetate buffer, pH 4.5 or 5.0 (see note below), containing I mM Vol. 68, No.4, 1991 383 CaCI2• The mixture was centrifuged at 25,000 X g for 15 min, and the supernatant was decanted. The residue was washed with a second portion of buffer and recentrifuged. The combined supernatants were treated with two volumes of cold acetone on ice to precipitate proteins and centrifuged at 11,000 X g for 10 min. The pellet was redissolved in 10 ml of acetate buffer and centrifuged at 11,000 X gfor 10 min to sediment insoluble material; the resulting solution thus contained enzyme from 10 kernels in each milliliter. Affinity Chromatography All purification steps and liquid chromatography were performed on ice or at 4°C. Liquid column chromatography was performed using a Pharmacia fast protein liquid chromatography (FPLC) system. Affinity chromatography of the acetoneprecipitated proteins was performed on cycloheptaamylose (CHA)-epoxy Sepharose 6B at either pH 4.5 or 5.0 according to the method of Silvanovich and Hill (1976). (Initial separations were done using pH 4.5. Investigation of the effect of pH on affinity binding [see Results] indicated that the binding was somewhat higher at pH 5.0-5.5; therefore, later separations were done at pH 5.0.) Ten milliliters of sample (i.e., enzyme extract from 100 kernels) in 20 mM acetate buffer containing I mM CaCI2 was applied to a column with a bed volume of 7.5 m!. The sample was eluted at a flow rate of I mIl min. Fractions of I ml were collected. Elution was monitored by measuring absorbance at 280 nm. Elution continued until no protein was detected. Bound proteins were then eluted with a solution of CHA (10 mgl ml) in acetate buffer. The eluate and the retentate were tested for amylase activity using a soluble starch substrate. Anion Exchange Chromatography: Affinity-Bound Enzymes Amylases bound by the affinity column were further fractionated by ion exchange chromatography on Pharmacia Mono Q anion exchange resin at pH 8.5 in 50 mA·! TRIS buffer using a O.O-O.llv! NaCI gradient at a flow rate of I mIl min. One-milliliter fractions were collected. Hydroxylapatite Chromatography: Affinity-Unbound Enzymes Eluate containing nonbinding activity was concentrated with an Amicon Centriprep 10 filtration concentrator, dialyzed in 10 mM potassium phosphate, pH 6.0, containing 0.1 mM CaCI2, then purified by chromatography on a hydroxylapatite column with a bed volume of 7.5 ml, at a flow rate of I mil min. Proteins were eluted in two steps with 45 and 300 mM potassium phosphate. a-Amylases from the second hydroxylapatite peak were concentrated and dialyzed against chromatofocusing start buffer for 24 hr. Chromatofocusing Fractions from anion exchange chromatography and from the second hydroxylapatite peak were further purified by chromatofocusing on a Pharmacia Mono P column. Samples were introduced in 25 mM Bis-Tris, pH 6.5, containing 0.1 mM CaCI2. Then they were eluted with Polybuffer 74 that was diluted I: I0 and pH adjusted to 3.5, containing 0.1 mM CaCI2. The flow rate was I mil min. One-milliliter fractions were collected. Elution pH, approximately equivalent to the pI, was monitored with a flow-through pH electrode or by measuring the pH of individual fractions with a Beckman Expandomatic pH meter equipped with a combination electrode. Enzyme Activity Amylase activity was measured using the dinitrosalicylic acid (DNSA) assay (Bernfeld 1951). Depending on activity, 10-100 I.d of enzyme was added to 0.5 ml of 2% (w/v) soluble potato starch (Sigma S-2004) in extraction buffer. The reaction mixture was incubated at 25° C. Aliquots (50 I.d) of reaction mixture were taken after an appropriate reaction time and added to 1.0 ml of DNSA reagent. Assay solutions were placed in boiling water for 5 min. Water (4 ml) was added to the assay solution; the sample was mixed by vortexing; and the solutions were 384 CEREAL CHEMISTRY equilibrated to 25° C. Absorbance was measured at 540 nm and compared to values obtained from a maltose standard curve. One unit of activity was equivalent to I }.Lmol of maltose released per minute. (Robyt and Whelan [1972] have shown that this method is not accurate for determining absolute activity because absorbance varies with the chain length of the oligosaccharides being analyzed. In spite of this limitation, we found the method to be satisfactory for estimating the relative activitv values of different enzy~e fractions o~ different prepar~tions; the convenience of the method for our purposes outweighed the acknowledged lack of absolute accuracy.) Protein Analysis Protein was determined by the Bradford (1976) method using fatty acid-free bovine serum albumin as a standard. Samples for analysis (I ml or less) were mixed with 2 ml of acetone, held overnight at 4°C, then centrifuged 2 min at 15,000 X g in an Eppendorf micro centrifuge (model 5415). The pellet was redissolved in 100 }.LI of 50% sucrose, and duplicate 25-}.LI aliquots were analyzed. Effect of pH on Enzyme Activity Effects of pH were determined by assaying amylase activity in 2% starch solutions prepared in 50 mM buffers with pH ranging from 3.0 to 8.0. Buffers used were glycine-HCI (pH 3.0), Naacetate (pH 3.6-5.5), Bis-Tris-HCI (pH 5.5-7.0), and N-(2hydroxyethyl)piperazine-N'-(a-ethanesulfonic acid)-NaO H (p H 7.0-8.0). All substrate solutions contained I mM CaCI2. Reactions were run for 30 min at 25° C. Effect of Calcium on Enzyme Activity Major peaks from anion exchange (affinity-bound fractions) were evaluated for calcium dependence by dialyzing against 10 ml"! ethylendiamine tetraacetic acid (EDTA) at pH 5.2 for up to 138 hr. Aliquots of chromatofocusing fractions from the affinity-unbound peak were incubated directly with I mM or 10 mM ethylene glycol-bis(f3-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) in 50 mM Na-acetate, pH 4.5, for 85 min at 25° C. Reaction rates were then measured in 2% (w Iv) starch in 50 mM Na-acetate, pH 4.5, containing no added CaCI2• Effect of Heat on Enzyme Activity Enzyme samples were heated at 70°C for 10 or 15 min in 50 mM acetate buffer (pH 4.5) containing I mM CaCI2, and activity was measured to determine loss of activity due to heating. Analysis of Reaction Products Crude extracts, affinity chromatography fractions, and individual fractions from chromatofocusing were incubated with 4% (w/v) soluble starch in 20 mM acetate buffer at pH 4.5. For chromatofocusing fractions, 200 }.LI of enzyme from each I-ml fraction was incubated with 10 ml of starch solution. At intervals between 0.5 and 144 hr, an aliquot was removed from the reaction mixture and mixed with two volumes of absolute ethanol to stop enzyme action and to precipitate unreacted starch and large oligosaccharides. The supernatant was filtered through a 0.2-}.Lm filter. The composition of the reaction mixtures was determined by high-performance liquid chromatography (HPLC), using a Spectra-Physics SP8100 high-performance liquid chromatograph equipped with a Dionex PAD-2 pulsed amperometric detector and a Dionex Carbopak PAl carbohydrate column. Oligosaccharide components were separated by gradient elution at 40°C; mobile phases were 100 mM NaOH (solution A) and 100 mM NaOH plus 500 mM sodium acetate (solution B); the flow rate was I mil min. Maltooligosaccharides with degree of polymerization (DP) I to DP7 from Sigma Chemical Company were used as external standards for quantitation. The acetone-precipitated proteins from the crude extract were tested with other substrates to detect activity of other carbohydrate-degrading enzymes. Maltase activity was detected by incubating 200 }.LI of extract with 225 mg of maltose in IO ml of acetate buffer for 120 hr. Limit dextrinase activity was detected Fig. 2. Anion exchange chromatography of affinity-bound a-amylases. 50