Journal of Cereal Science 9 (1989) 17-33

Measurement of p-Amylase in Cereal Flours andCommercial Enzyme Preparations

BARRY V. McCLEARY and RACHEL CODD

Biological and Chemical Research Institute, Department of Agriculture and Fisheries,P.M.B. 10, Rydalmere, 2116, Australia

Received 18 July 1988

A procedure previously developed for the assay of cereal-flour p·amylase has beenimproved and standardised. The improved procedure uses the substrate p-nitrophenylmaltopentaose (PNPG5) in the presence of near saturating levels of IX-glucosidase.PNPG5 is rapidly hydrolysed by p-amylase but less readily by cereal et-amylases. Thesubstrate is hydrolysed by p-amylase to maltose and p-nitrophenyl maltotriose(pNPG3). With the levels of et-glucosidase used in the substrate mixture, PNPG3 israpidly cleaved to glucose and p-nitrophenol, whereas PNPG5 is resistant tohydrolysis by the et-glucosidase. The assay procedure has been standardised forseveral p-amylases and the exact degree of interference by cereal et-amylasesdetermined. The procedure can be readily applied to the selective measurement of p­amylase activity in cereal and malted cereal-flours.

Introduction

J3-Amylase plays a central role in the complete degradation of starch to metabolisableor fermentable sugars during the germination or malting of cereal grains. However,interest in this activity has been hampered by the lack of a specific assay procedure.

In ungerminated cereal grains, ~-amylase is the major amylolytic activity present.Thus, non-specific reducing sugar assays with starch as substrate, can be employed, tomeasure this activityl.2. In malted cereals, non-specific reducing-sugar assays can be usedto measure ~-amylase only if a-amylase is selectively inhibited. Ammonium oxalate hasbeen shown to be useful for this purpose3

• The level of ~-amylase in malted barley issufficient to ensure almost complete degradation of starch to maltose [with lesserquantities of glucose, maltotriose and higher degree of polymerisation (D.P.) dextrins]during the mashing step in the brewing process. However, with the use of less malt andmore adjuncts in the mash, it is important to have accurate measurements of theamounts of a-amylase and p-amylase in the malt. Materials used as alternative sourcesof fermentable sugars (adjuncts) include unmalted cereal grains (which may be a goodsource of ~-, but not a-amylase) and starches or heat-treated grains such as parboiledrice (which are essentially devoid of starch-degrading enzymes). .

A simple and specific assay for ~-amylase could also be of considerable value to plantbreeders and plant physiologists as well as to industrial operations involved in theproduction, preparation or use of ~-amylase. ~-Amylase currently finds considerable

0733-5210/89/010017+ 17 $03.00/0 © 1989 Academic Press Limited

18 B. V. McCLEARY AND R. CODD

application, together with starch debranching enzymes (pullulanase and isoamylase), inthe production of high maltose syrups.

The observation by Mathewson and Seabourn4 that Pantrak® reagent can be used toassay ~-amylase selectively in the presence of cereal a-amylase, was a major advance inthe development of a useful and specific assay for ~-amylase. Pantrak® reagent(Calbiochem Behring, La Jolla, CA), when reconstituted, contains 0·85 mM p­nitrophenyl a-maltopentaoside (PNPG5), 0·65 mM p-nitrophenyl a-maltohexaoside(PNPG6) and approximately 800 units/litre of microbial a-glucosidase. This reagentwas developed for the quantitative determination of a-amylase in human serum andurine. The substrate cannot be used to measure cereal a-amylases because these enzymesrequire (I -+ 4)-o:-linked maltosaccharides with a chain length of seven to nine D-glucosylresiduess .6 • PNPG5 and PNPG6 are too short effectively to satisfy the substratesub-site binding requirements of these amylases.

In the present communication we have extended the work of Mathewson andSeabourn4 by addressing a range of questions either not answered, or incompletelyanswered by them, such as: (a) what is the optimal maltosaccharide chain-length toensure maximum selectivity for ~- over a-amylase; (b) what is the optimal level of yeasta-glucosidase to ensure maximum sensitivity of the assay; (c) can the assay procedurebe used to assay ~-amylases from various sources; and (d) is the relationship betweenreducing sugar activity on starch substrate and activity on the PNP-maltosaccharidesubstrate constant for a range of ~-amylases.

The present method has also been applied to the measurement of both soluble andtotal ~-amylase in cereal grains with the formulation of appropriate extraction bufferswhich ensure effective extraction and maximum stability of the extracted enzyme.

Experimental

Materials

Para-nitrophenyl maltose, malta-triose, -tetraose, -pentaose and -hexaose and p-nitrophenyl ex­glucopyranoside were obtained from Boehringer Mannheim Australia Pty. Ltd. Yeast rx­glucosidase (BC 3.2.1 .20) was from Genzyme Biochemicals Ltd, Maidstone, U.K. Bacillus cereus~-amylase (BC 3 .2. 1. 2) was from Amano Pharmaceuticals 2-7, I-Chome, Nishiki Naka-ku,Nagoya, and wheat flour p-amylase was from Hankyu Kyoei Bussan Co. Ltd, 1-10, 7-Chome,Tenjinbashi, Osaka, Japan. Crystalline sweet-potato p-amylase was obtained from SigmaChemical Company, St Louis, Missouri, U.S.A. Soybean p-amylase was extracted from soy-beanflour with 0'1 M acetate buffer (pH 5'0), precipitated with ammonium sulphate (50 g/IOO ml),dialysed against 20 mM sodium acetate buffer (pH 5'0) in 1 M ammonium sulphate. The enzymewas applied to a column (2'5 x 12 em) of Phenyl Sepharose Fast Flow and eluted with a lineargradient of ammonium sulphate (1'0 -+ 0'0 M) in 20 mM sodium acetate (pH 5'0; total volume600 mI). Contamination of recovered p-amylase with ex-amylase was < 0-001 %. A sample ofhighly purified malted barley rx-amylase I was a kind gift from Dr A. W. MacGregor, GrainResearch Laboratory, Canadian Grain Commission, Winnipeg, Canada.

Extraction of p-amylase

Extraction buffer (Buffer A). The standard buffer (Buffer A) used in this work (unless otherwisestated) for the extraction of l3-amylase from flour, for enzyme dilution, and in assay mixtures

MEASUREMENT OF p-AMYLASE 19

contained Na maleate (100 mM), diNa EDTA(l mM), bovine serum albumin (BSA, 1 mg/ml) andNaNa (3 mM), adjusted to pH 6'2. This buffer extracts 'soluble' p-amylase from cereal flours.Total p-amylase (soluble plus insoluble) was extracted using the above buffer mixture to whichcysteine (20 mM) was added (with pH adjustment to 6,2) immediately before use (Buffer B).

Extraction procedure. Wheat, barley malt or other cereal grains were milled to pass a 0'5 mmscreen. Samples of the resulting flour (0'5 g) were accurately weighed into 12 ml capacitycentrifuge tubes and either Buffer A or B (5 ml) added to each. The tubes were capped, mixed toensure complete suspension and allowed to extract (with occasional mixing) over a period of 2 hat room temperature. The suspensions were then filtered through Whatman OF jA glass-fibre filterpaper or, alternatively, the tubes were centrifuged at 1000 g for 10 min. An aliquot (0'2 ml) of thefiltrate (or the supernatant) was diluted to 10·0 ml in Buffer A, and then an aliquot (0,2 ml) of thissolution was further diluted to 2·0 ml in Buffer A. The enzyme extracts were incubated at 40 °C forapproximately 10 min before assay. Assays were performed within 2 h by the standard PNPG5assay procedure.

Effect of time ofextraction with buffer and time ofstorage ofenzyme after extraction on p-amylaseactivity. Extractions were performed by the procedure as described above except that in someexperiments extraction times up to 24 h were employed and in other experiments, the filteredenzyme extract was stored at 22 °C for up to 48 h before assay.

Assay of fJ-amylase

(a) PNPG5 standard assay. Pre-equilibrated and suitably diluted enzyme preparation or flourextract (0,2 ml) in Buffer A (pH 6'2) was incubated with 0·2 ml of pre-equilibrated substratemixture (PNPG5, 5 mM; a-glucosidase, 20 D, in distilled water) at 40 °c for 10 min: The reactionwas terminated and colour developed by 1% (wjv) Trizma base (3-0 ml, pH > 10, SigmaChemical Co.) and the absorbance at 410 nm measured (Em" of p-nitrophenol of pH> 10 is17'8). One unit of enzyme is defined as the amount of enzyme which releases I j.lmole of p­nitrophenoljmin under defined assay conditions.

Calculation of activity

Malt flour p-amylase (D/g flour) =

AAm total vol. in cell 1 extraction d'l .. x x - x X 1 utlOnIncubation time aliquot assayed Em" volume

AAm = A (reaction)-A (blank)For example, with the following conditions: incubation time = 10 min, total volume in cell = 3·4ml, aliquot assayed = 0·2 ml, Em'" of p-nitrophenol in 1% Trizma base = 17'8, extractionvolume = 10 mljg flour, dilution = 500, the calculated activity would be given by:

. AA H 1umtsjg flour =~ x - x--x 10 x 500

10 0·2 17·8= AAm x 447·5

(b) PNPG5 sequential assay. Enzyme preparation (0,2 ml) in Buffer A (pH 6'2) was incubated with0'2 ml of pre-equilibrated PNPG5 (5 mM; in distilled water) at 40 °C for 10 min. The reaction wasterminated by immersing the reaction tubes in boiling water for 1 min. The tubes were equilibratedto 40 °C and 0·1 ml of a-glucosidase (20 U) was added to each with incubation at 40 °C for 10 min.The reaction was terminated and colour developed by the addition of 1 % (wjv) Trizma base(2,9 ml) and the absorbance at 410 nrn measured. p-Arnylase activity was calculated as for thePNPG5 standard assay.

20 B. V. McCLEARY AND R. CODD

(c) Nelson-Somogyi method? Enzyme preparation (0,2 ml) in Buffer A was incubated with 0'5 mlof soluble starch solution (10 mg/ml) in Buffer A at 40°C for 0,5,10, 15 and 20 min. The reactionwas terminated by the addition of Nelson-Somogyi reagent 0 (0'5 ml) and colour developed aspreviously describedB. One unit of enzyme activity is defined as the amount of enzyme whichreleases I I!mole of maltose reducing sugar equivalents per minute at 40°C and pH 6·2.

(d) Bernfeld method. Assays were performed as described2 with soluble starch as substrate atpH 4'8.

Preparation of soluble starch substrate

Soluble starch (I g, May and Baker Ltd, Dagenham, U.K.) was slurried in 10 ml of cold BufferA (not containing BSA) and this was added to 80 ml of boiling and vigorously stirred Buffer A(devoid of BSA). Stirring was continued for 5 min and then the solution was cooled to roomtemperature, and adjusted to 100 ml.

Assays for other enzymes

ex-Glucosidase and ex-amylase were assayed as previously described9• The activity of Aspergillusniger ex-amylase on PNPG5 was determined with the standard ~-amylase assay procedure atpH 6·2 (unless otherwise stated).

pH-Activity of p-amylases

Enzyme preparation (0,2 ml) was incubated with 0'2 ml of substrate-enzyme mixture containing5 mM PNPG5 and ex-glucosidase (l00 Vlml) prepared in Na maleate (50 mM) plus Na phosphate(50 mM) buffered at pH 5·0 to 8,0, at 40 DC for 10 min. The reaction was terminated by the additionof Trizma base (3 ml, 1%, w/v) and the absorbance at 410 nm measured, With soluble starch(10 mg/ml) as substrate, the same buffer mixtures were employed, and reaction was monitoredwith the Nelson-Somogyi reducing sugar procedure?,B.

Determination of Km and Vmax

Wheat-flour ~-amylase (0'2 ml) in Buffer A was incubated with 0·2 ml of substrate-enzymemixture containing ex-glucosidase (100 Vlml) and PNPG5 (0-20 mM) for 0, 3, 6 and 10 min at40°C. The reaction was terminated by the addition of Trizma base (3 ml, I %, wIv).

Effect of concentration of (X-glucosidase in the assay

The assay was performed in duplicate under standard conditions using wheat-flour l3-amylase inBuffer A. The substrate mixture contained PNPG5 (5 mM) with a-glucosidase (0-200 UIml) indistilled water.

Stability of substrate mixture

AUquots (5 ml) of the standard substrate mixture, containing PNPG5 (5 mM) plus ex-glucosidase(l00 U/ml) in stoppered vials, were stored at 4 and 25°C for up to 6 days. At various times,aliquots were removed and both the blank absorbance values and the values obtained onincubating the substrate with 10·7 mU (on PNPG5) of wheat ~-amylase for 10 min at 40°C weredetennined in duplicate. Parallel experiments were performed with Pantrak reagent (Calbiochem

MEASUREMENT OF ~·AMYLASE 21

Behring) according to the procedure of Mathewson and Seabourn4 • Samples of the PNPG5ja­glucosidase reagent were also stored at - 20°C for up to 6 months and lyophilizedsubstrate-enzyme mix was stored at 37°C for up to 4 weeks. The lyophilized substrate wasreconstituted in distilled water and used in the standard assay procedure.

Linearity of PNPG5 p-amylase assay procedures

Wheat-flour B-amylase (0'2 ml) at 0-30 mD/0'2 ml on starch substrate (Nelson/SomogyF) inBuffer A was incubated in duplicate with 0'2 ml of substrate using both the combined and thesequential assay procedures. Linearity of the reaction with time was determined by incubatingwheat-flour B-amylase (0'2 ml, 13'4 mD on PNPG5) with 0·2 ml of standard PNPG5 substratemixture for 0 to 10 min. Reaction was terminated by Trizma base (3 ml, 1%, w/v) and theabsorbance at 410 nm measured.

Action of a-amylase on PNPG5 substrate mixture

Assays were performed using the standard format except that highly purified malted wheat-floura-amylase, malted barley-flour a-amylase or malted barley-flour a-amylase po (0'2 ml, 0-320 mU)was used either alone, or in admixture with wheat-flour B-amylase. In other experiments, purifiedA. niger a-amylase (0-16 mU per assay on BPNPG7 at pH 5'2) was employed and assays wereperformed in Buffer A (pH 6'2). The a-amylases used in these studies were standardised on eitherblocked p-nitrophenyl maltoheptaoside (BPNPG7) or soluble starch (10 mg/m1) in malate buffer(pH 5'2)9.

Purification of malted-wheat-flour and malted-barley-flour a-amylasell

Commercial malted barley (Weeah), or malted wheat (Kite, 4 days at 25°C) was dried and thenmilled to pass a 0'5 mm screen. A sample of each flour (l00 g) was suspended in 500 ml of sodiumacetate buffer (50 mM, pH 5,0) containing 2 mM CaCI2, and stirred at room temperature for 2 h.The slurries were centrifuged (3000 g, 20 min) and the supernatants treated with ammoniumsulphate (50 g/IOO ml) and stored at 4 °C for 4 h. The pellet recovered on centrifugation (3000 g,20 min) was dissolved in 100 ml of 50 mM sodium acetate buffer (pH 5'0) containing 2 mMCaCl2 and dialysed against 4 I of the same buffer for 20 h at 4°C. The preparation was thencentrifuged (12000 g, 20 min) and applied to a column (2'5 x 10 em) of B-cyclodextrin linked toepoxy-activated Sepharose 4B. The column was eluted with sodium acetate buffer (350 ml, 50 mM,pH 5'0) containing 2 mM CaCI2 • a-Amylase was then eluted by washing with the same buffercontaining B-cyclodextrin (10 mM). The recovered enzyme was dialysed exhaustively againstseveral changes of acetate buffer containing 2 mM CaCl2 and then treated with ammoniumsulphate (5 g/IO ml) and sodium azide (0,02 %) and stored at 4°C.

Hydrolysis of PNPG3, PNPG4 and PNPG5 by wheat-fiour p-amylase and pure maltedbarley ~-amylase

Aliquots (l·0 ml) of PNP-maltosaccharide (20 mM) in 5 mM maleate buffer (pH 6,2) wereincubated with wheat-flour B-amylase (500 mD, 0·1 ml) at 40°C. Reaction was stopped after 0,30and 90 min by incubating individual tubes at 100°C for 2 min. Solutions were diluted 10-fold,filtered (Millipore 0·45 micron durapore filter) and analysed by high performance liquidchromatography (HPLC) with a Waters® Sugar Analyser fitted with a Sugar Pak I column. Theeluant was HPLC grade water containing 20 ppm of Ca EDTA run at 90°C and 0'5 mljmin.Elution patterns were monitored by refractive index changes. The action of both ri- and l3-amylaseon PNP-maltosaccharides was also followed by thin layer chromatography (TLC) on Merck

22 B. V. McCLEARY AND R. CODD

Hohbo~o~ab-o-V-N02p-nitrophenyl moltopenlooside (PNPG5)- I

II ;a-AmyloseIt

HcJ=lAoH·Hobbab~N02maltose e-nilrophenyl maltolrioside (PNPG3)

II ",-Glucosidaseft

}-o, • HO-Q-N02HO~OH

glucose p-nilrophenol (PNP/f -

ITrizmo baset

Reaction stopped andyellow colour developed

FIGURE 1. Schematic representation of the reactions involved in the measurementof ~-amylase using p-nitrophenyl maltopentaose in the presence of excess quantities

of a-glucosidase.

(a) (b)

v '"UI

t:l ~a. C'"

z ::;: vUI a. t:l

Jja.

N zt:l a.a.za.

~, , , , , ,0 15 30 0 15 30

(e) (d)

'"UI

IC) ~CD "0a. ::;:

'" z roUI a. t:l~ a.

u> z0 ro a.::;: ~ t:l

a. a.z za. a.

I I I I I ,

o 15 30 0 15 30

FIGURE 2. HPLC of the products of hydrolysis ofPNPG4 and PNPG5 with wheat­flour f3-amylase. Reactions were terminated at 30 and 90 min. (a) PNPG4, 30 min; (b)

PNPG4, 90 min; (c) PNPG5, 30 min; (d) PNPG5, 90 min.

MEASUREMENT OF ~-AMYLASE 23

'PNPG

'PNPG2·PNPG3'PNPG4

• PNPG!>

'G

'G4

cBA6030

P PG6

30o 60 0 30 60 0~ -..JI 1-1 ---J1 LI ..J

PNPG4 PNPG5

FIGURE 3. TLC of the products of hydrolysis of PNPG4, PNPG5 and PNPG6 bymalted barley a-amylase (total). Experimental conditions are described in the text.PNP-maltosaccharide was incubated with a-amylase and aliquots taken at 0, 30 and60 min for chromatography. (a) PNPG, PNPG3 and PNPG5; (b) PNPG2, PNPG4

and PNPG6; (c) glucose-maltotetraase.

DC-Alufolien Kieselgel 60 (0'2 mm) prepared plates. PNP-maltosaccharide (20 mM, 1 rot) wasincubated with wheat-flour ~-amylase (2'0 U, 0·1 ml) or pure malted barley a-amylase (8'4 U,0'14 ml) at 40°C and the reaction in individual tubes terminated after 0,5,10,20,60 and 120 minby incubating the tubes at 100°C for 2 min. Aliquots (5 or 10 Jll) of the reaction mixtures wereanalysed by TLC. Plates were developed once with 7: I :2 n-propanol-ethanol-water. Spots weredetected by spraying with 5% sulphuric acid in ethanol and heating to 110°C.

Results and Discussion

The assay procedure described in this paper is a modification of a method described byMathewson and Seabourn4 . The major advantages of the current procedure are that adefined maltosaccharide substrate (rather than a mixture) is employed, the level of addeda-glucosidase is near-saturating (ensuring maximum linearity and sensitivity), the assay

24 B. V. McCLEARY AND R. CODD

(al (bl1·2

6-

1·0 §y~E

<:0·8 p.0

~~ ___0~(\.I

/>~o--u

0·6c:0-e0

"'.0

/a//« 004

0·2 ~/

16 20 4 8 12 16 20

.a-Amylase (mU/assaylFIGURE 4. Effect of PNPGS and a.glucosidase concentrations on the PNPGS ~­

amylase assay procedure. (a) PNPGS concentration in substrate; D, 1 mM; .... ,2·5mM; 0,5 mM; 6" 10 mM; (b) a-glucosidase concentration in substrate; D. 25 D/ml;

.... ,50 Ujml; 0,100 D/ml; 6" 150 D/ml; .,200 UjmI.

has been carefully calibrated and standardised against alternative methods, and thedegree of interference by other enzymes has been established. The principle of the assayprocedure is shown in Fig. 1. The substrate mixture comprises PNPG5 and an a­glucosidase which has little action on PNPGS, but rapidly cleaves PNPG3. Onincubation of this mixture with p-amylase, as maltose is removed by the p-amylases, thea-glucosidase gives essentially instantaneous removal of the remaining glucosyl residuesfrom PNPG3, releasing free p-nitrophenol. The reaction is terminated and colourdeveloped by adding Trizma base to adjust the pH to > 10.

PNP-maltosaccharides vary in their rates of cleavage by p-amylase. Substratescontaining less than four D-glucosyl residues are hydrolysed slowly. PNPG3 ishydrolysed at about 10 % the rate for PNPG4 (data not shown), which is hydrolysed atabout 80 % the rate for PNPGS (Fig. 2). PNPGS and PNPG6 are hydrolysed at similarinitial rates by p-amylases. Hydrolysis of these PNP-maltosaccharides by a-amylases isalso dependent on the D-gluco-oligosaccharide chain length. The optimal degree ofpolymerisation of the a-I,4-linked maltosaccharides for cereal a-amylase action is 9.However, nitrophenyl-maltasaccharides of d.p. 7 are readily cleaved9• The relativesusceptibility of PNPG4, PNPG5 and PNPG6 to hydrolysis by high levels of highlypurified malt·flour a-amylase (total) is shown in Fig. 3. PNPG4 is hydrolysed mainly toPNPG2 and maltose, and PNPG5 is hydrolysed mainly to PNPG3 and maltose. Thesaccharide mixture produced on incubation with PNPG6 is much more complex,indicating a high degree of transglucosylation is occurring under the assay conditionsemployed. PNPG6 is cleaved at approximately four times the rate for PNPGS (byHPLC; data not shown) which is cleaved at about the same rate as for PNPG4. AtpH S'2, PNPG5 is cleaved at only 3 % the rate for starch (10 mg/ml at pH 5'2).

MEASUREMENT OF ~-AMYLASE 25

1,4-

E 0·8-e:

o1 I I I

5 10 15 20 25 30

,a-Amylose, mU/ossoy (storch substrate)

FIGURE 5. Standard curves relating activity of wheat-flour ~-amylase on starch toabsorbance increase on incubation with PNPG5 under standard assay conditions in

either a combined C.) or a sequential CJ..) assay format.

2·0

/

/

o I 2 3 6

Length of incubation at room temperature (days)

FIGURE 6. Stability ofPNPG5jtx-glucosidase and Pantrak® reagent mixtures for theassay of ~-amylase after storage at 22°C for up to 6 days. 0, blank absorbance valuefor modified Pantrak® reagent; /:::" blank for PNPG5 reagent; .... , reaction value onincubation of 10·7 mU of wheat-flour ~-amylase with PNPG5 reagent under standard

assay conditions; x, reaction-blank for PNPG5 reagent.

26 B. V. McCLEARY AND R. CODD

TABLE I. Reproducibility of the PNPG5 assay for the measurement of barley and malt flourp-amylase"

Sample Day 1 Day 2 Day 3 Day 4

A 0'5054B 0·7282C 0·2725D 0'7689

S.E.M. b = 0·0368C.V. (%) =: 6·4

Absorbance (410 nm) without cysteine0·5248 0-4510 0·4592 0·4884 0·4741 0-44330,7281 0·6969 0·7202 0·7467 0·7438 0·73150·2806 0·2789 0·2703 0·3232 0·3140 0·39800·7978 0·7604 0'7599 0·8567 0·8369 0·7833

0·45400·71470·38110·7833

A 0'6851B 0·7594C 0·6435o 0·8484

S.E.M. b = 0·0412C.V. (%) = 5·7

0'68480·73600·63540·8525

Absorbance (410 nm) with cysteine0·6139 0·6181 0·6632 0·65890-7008 0·7116 0·7488 0'73700·6050 0·5945 0·6577 0·63080·8150 0'7825 0,8236 0'8918

0·69520·77300·75170,8527

0·70390·76170·74750·8245

" Duplicate analyses of single extracts made on four separate days.b Based on model assuming fixed samples and random days and duplicates.

These experiments demonstrate that the preferred substrate for the selectivemeasurement of l3-amylase in the presence of cereal (X-amylase is PNPG5.

With starch as substrate, the optimal pH for activity of wheat-flour ~-amylase is6-0-6'5, whereas the value for B. cereus ~-amylase is 7·0. With the PNPG5jc<-glucosidasereagent mixture, the pH optimum for both enzymes is in the range 6·0-6·5. The slightlydifferent optima and shapes of the pH activity curves when starch or PNPG5j(X­glucosidase are used as substrate is considered to be due to the pH activity properties ofthe a-glucosidase in the PNPG5 substrate mixture. For all further work, independentof the l3-amylase being assayed, or the substrate being employed, a pH of 6·2 was used.This value is similar to that used by Snider (pH 6-6)1 and Mathewson and Seabourn(pH 6'0)4 for cereal (X-amylases, but higher than the value employed by Meyer et al.(pH 5'2)12.

The effect of PNPG5 concentration on the activity value for wheat-flour ~-amylase isshown in Fig. 4(a). The Km value was 0·32 mM. At the substrate concentration used inthe final assay mixture (2,5 mM), the rate ofsubstrate hydrolysis was approximately 95 %that of the theoretical maximum. To determine the concentration of a-glucosidaserequired to optimise the assay, a substrate concentration of 2·5 mM (final assay mixture)and a concentration of a-glucosidase of0-200 U jml (final assay mixture) were employed[Fig. 4(b)]. The level of a-glucosidase required to optimise the assay is greater than200 U jml in the substrate mixture. At an a-glucosidase concentration of 100 Ujml, thereaction proceeded at greater than 85 % of the maximal rate, and for reasons ofeconomy, this was the level of a-glucosidase employed in the standard substratemixture.

The rate of release of free p-nitrophenol on incubation of an aliquot (0'2 ml) of

MEASUREMENT OF f3-AMYLASE

TABLE II. Ratio of activity· of affinity-purified IX-amylases on various substrates

27

Relative activities for different IX-amylaseSubstrate andpH of assay

BPNPG7, pH 5,2Starchd , pH 5,2PNPG5, pH 6·2

Whole wheat b

10075'42'5

Whole maltb

10071-6

3·0

Malt IX-amylase"

10070·20-4

• All determinations were performed in duplicate on two separate days.b Purified by affinity chromatography on Cyclodextrin-Sepharose 4B," Highly purified, supplied by Dr A. MacGregor.'I Using the Nelson/Somogyi reducing sugar assay procedure,

standard substrate solution [5 mM PNPG5 plus a-glucosidase (100 D/ml)] with suitablydiluted p-amylase (0'2 ml, 13'4 mD), is not linear with incubation time. There is an initiallag-phase over a period of about 4 min before the reaction becomes linear. This lag isprobably due to both the restricted ability of the a-glucosidase to remove D-glucosylresidues from PNPG3 (released on p-amylase cleavage of PNPG5) and to the lowconcentrations of PNPG3 in the reaction mixture in the initial stages of incubation. Infact, the rate of release by a-glucosidase of the terminal D-glucosyl residue from PNPG3is 7·5 times less than the rate of release of the terminal residue from PNPG2. To checkthis proposal, the standard PNPG5 assay procedure (combined procedure) wascompared to an assay in which p-amylase was incubated with PNPG5 and the reactionterminated (by heating) before treatment of the resultant mixture with a-glucosidase.The results of these experiments are shown in Fig. 5. With the sequential assay, theabsorbance values obtained with a given level of p-amylase were about 15 % higher thanthose obtained with the combined assay, confirming that the rate limiting step in thetime-course incubation is the cleavage of released-PNPG3 to glucose plus PNP.Although the sequential assay is technically more precise, the combined assay isconsiderably more convenient, and in our opinion is the method of choice.

A major consideration in the development of any assay procedure is to identifyconditions under which the component being assayed for and the assay reagent mixturehave the maximum degree of stability. In Fig. 6 the relative stabilities of the currentreagent mixture and that described by Mathewson and Seabourn4 based on theCalbiochem Behring Pantrak® reagent are shown. The reconstituted substrate mixtureswere stored at room temperature ( "" 22°C) and after various time intervals the amountof free PNP in aliquots (0'2 ml) of the mixtures were determined (standard reactionblanks) by adding 0·2 ml of water and 3-0 ml of Trizma base (l %, wIv). The modifiedPantrak reagent deteriorated rapidly, being completely unusable after about 8 h. Incontrast, the current reagent was quite stable for periods of up to 3 days at roomtemperature, and even after storage for 6 days the blank absorbance values hadincreased to only 0·4 but the reagent mixture was still usable. On subtraction of blankabsorbance values from those obtained on incubation with 10·7 mD of p-amylase (understandard assay conditions), the absorbance values were very similar to those obtained

28 B. V. McCLEARY AND R. CODD

a -Amylose (mU/ossay on BPNPG7 ot pH 5·2)

o 100 150 200'·2

~ 1-(1

'0<t

~.

~.

~.

~.§ o·e ...........15 .........5 .."'.0

.3l(') 0·6""

~Z0..

, I

..................0,.. .....

a

'0 0'4

50 100 150 200

a - Amylase (mU/ossay on storch at llH 5'2)

FIGURE 7. Action of highly purified wheat-flour a-amylase on PNPG5 substratemixture, and the effect of this enzyme on the assay of ~-amylase using the samesubstrate mixture. 0, a-amylase (0-320 mD on BPNPG7/assay); ., ex-amylase

(0-320 mD/assay) plus ~-amylase (13-7 mUjassay).

with freshly prepared substrate mixture. Both substrate mixtures were stable for at least6 months when stored at 4 °C as lyophilised powder or at - 20°C after reconstitution.

~-Amylase is quite stable in crude cereal-flour extracts which contain high levels ofother protein. However, when highly diluted, i.e. to a level suitable for assay, the enzymeis unstable. This property of the enzyme is well documented in literature13

• Consistentwith published data, we found that stability is consideraply increased by adding cysteine(20 mM), mercaptoethanol (l mM) or dithiothreitol (l mM) to the buffer, but by far thebest stabiliser was BSA at 1 mg/ml. In the presence of this concentration of BSA, therewas essentially no loss in activity on storage of the diluted enzyme at 22°C for 2 h. Verysimilar results were obtained for wheat-flour, barley, sweet potato, soybean and B.cereus ~-amylases.

The reproducibility of the extraction and assay procedure for barley-flour and maltedbarley-flour soluble and total ~-amylase content is shown in Table 1. Four flour sampleswere extracted in duplicate with Buffer A or Buffer B on four different days and theextracts assayed in duplicate. Blank absorbance values did not vary significantly betweensamples.

In the current assay format, PNPG5 was selected as the substrate of choice becauseit is readily hydrolysed by ~-amylase, it is much more resistant to cleavage by yeast ct.­glucosidase than is PNPG4, and it is approximately four times more resistant tohydrolysis by cereal !X-amylases than is PNPG6. However, since the assay procedure

MEASUREMENT OF !3-AMYLASE 29

Nelson-Somogyi units = BPNPG7 units x 0'73.

does not specifically measure f3-amylase, it was essential to determine the degree ofinterference by a-amylase so that this could be allowed for in accurately calculating thelevel of ~-amylase in a given flour sample. To do this, cereal-flour a-amylases werepurified by a specific affinity-chromatography procedure employing ~-cyclodextrin

linked through a 'carbon-spacer' to Sepharose 4B11 • On application of a partly-purifiedflour-extract of a malted cereal to the chromatography column, ~-amylase elutedunbound and a-amylase was eluted with a solution of ~-cyclodextrin.The recovered a­amylase was devoid of ~-amylase.The relative activity of highly purified malted-wheat(total), malted-barley (total) and malted barley fraction I a-amylases on PNPG5 (atpH 6,2) and on blocked p-nitrophenyl maltoheptaoside (BPNPG7) and starch (at pH5,2) is shown in Table II. With the malted-wheat and malted-barley enzymes, the ratioof activity on PNPG5 (pH 6'2) and BPNPG7 (pH 5'2) is approximately 1: 40. Thus forevery 100 units of activity of a-amylase (as assayed on BPNPG7 at pH 5,2) there areonly ~ 2·5 units of activity on PNPG5 at pH 6·2. Consequently, where cereal a-amylaseand ~-amylase occur in admixture (i.e. in a malt-flour sample), the exact ~-amylase

activity can be determined by assaying the extract on PNPG5 (pH 6'2) to give apparent~-amylase activity, and then substracting from this one fortieth of the activity of thepreparation on BPNPG7 at pH 5·2 (a-amylase activity). As previously shown, (seeMcCleary and Sheehan9

, Fig. 6), ~-amylase has no action on BPNPG7, nor does itaffect the values for a-amylase obtained using this substrate/assay procedure.Thus, ~-Amylase (U/ g flour) = Activity on PNPG5 (pH 6'2) - [Activity on BPNPG7(pH 5·2)]/40.

From Table IIit is evident that the ratio of activity of the three highly purified cereala-amylase preparations on starch (Nelson-Somogyi units) compared to BPNPG7 (bothat pH 5'2) is:

This contrasts with a conversion factor of3-0 which we reported previously9 for a wheat­flour a-amylase purified by affinity chromatography on Glycogen AH-Sepharose 4B14.The reason for this difference was that the a-amylase previously employed wasapparently not completely free of ~-amylase. This would not affect the activity onBPNPG7, but would lead to inflated values for the Nelson-Somogyi units. In an attemptto ensure that the a-amylase preparations used here were free of p-amylase, the enzymeswere rechromatographed on ~-cyclodextrin-epoxy-activated Sepharose 4B11 three times.Rechromatography did not change the ratio of activity on PNPG5 (pH 6,2) andBPNPG7 (pH 5,2). The Nelson-Somogyi/BPNPG7 ratio value (3'2: 1) previouslyreportedO for a heat-stable Bacillus subtiUs a-amylase was reconfirmed.

The highly purified barley-malt ~-amylase I showed the same ratio of activity onBPNPG7 and starch as did the other enzymes, but it had a considerably lower relativeactivity on PNPG5 at pH 6·2. The reason for this is not clear.

The effect of wheat-flour a-amylase on the hydrolysis of PNPG5 by ~-amylase isshown in Fig. 7. When present in large excess, a-amylase gives increased absorbancevalues. However, the effect is linear with a-amylase concentration, thus the ~-amylase

values can be readily corrected using the above equation.Another enzyme which could potentially hydrolyse PNPG5 and thus reduce the

specificity of the assay for ~-amylase is a-glucosidase present in the barley or malt flour.

30 B. V. McCLEARY AND R. CODD

1·2r----------------,-----------11--.--/

1·0 j- .----.~'--- ........

E 0·8 :;-,fC===::::::==:::===~=::~I::-:"lt-~ , °---11-0

i"[:_-------O--l/--O! 0.4 yO

0'----:-----!;-2---:!3:----+-1 J-+---l

Extraction time (h)

FIGURE 8. Effect of time of extraction and presence or absence of added cysteineon the amount of f3-amylase extracted from barley and malt flours. Barley flour A;D, no cysteine; ., plus cysteine. Barley-flour B; 0, no cysteine; e, plus cysteine.

Malt-flour A; f:,., no cysteine; A, plus cysteine.

To determine the importance of this enzyme, the activity of enzymes in malt- and barley­flour extracts on PNPG5 in the presence and absence of yeast maltase (l00 Ujml) wasdetermined. Itwas found that in the absence of yeast maltase, the release of PNP fromPNPG5 was reduced by '" 4000-fold. Further, the level of ex-glucosidase in malt flourextracts (assayed on p-nitrophenyl-ex-D-glucopyranoside) was approximately 2000 timesless than the level of J3-amylase in the same extracts. Itcan thus be concluded that theeffect of the endogenous malt-flour iX-glucosidase on the specificity of the PNPG5 ~­

amylase assay is negligible.13-Amylase occurs in cereal-flours in both a 'soluble' and an 'insoluble' form. It is

believed that insolubility is due to cfosslinking of the sulphydryl groups of the enzymeto sulphydryl groups in other insoluble proteins13 . Hydrogen sulphide, cysteine andinactivated papain, through their ability to act as reducing agents, can split disulphidebonds and thus solubilise the 'insoluble' 13-amylase. The ability of cysteine to catalysethis solubilisation is shown in Fig. 8. For maximal extraction of 13-amylase, an extractiontime of 4 h is required. However, since more than 95 % of the activity is extracted after2 h, this time of extraction was routinely employed. Continued extraction with Buffer A(containing no cysteine) gave no apparent extraction of the insoluble ~-amylase, eventhough this fraction represented 30-50 % of total ~-amylase. Buffers containing 20 mMcysteine (Buffer B) were very effective in extracting both the soluble and the insolublecomponents of total f)-amylase. In contrast to the situation with barley flour, the 13­amylase in malted barley flour appears to be essentially all in the soluble form. Addition

MEASUREMENT OF B-AMYLASE 31

f-_sl_e_ep_+_M_a_lti_ng ~1 Kilning

250

50

0. 500

·0<l....- 4003,g.::':::J

300tri"(.!)a..Za..c: 2000c:a

"-5<t 100

~

6200 ""

~r-:

150 [?Za../,-J-',ooj

/XX_X

a.......-""2;::0~-4b.0----,6b.0---:Bb.0--;-!,IO:;::-0 --'*120,,------;-;;;!1400

Time from initiation of steep (h)

FIGURE 9. Changes in activity of a-amylase (x) and soluble (e, 0) and total (.,,6,) ~-amylase on malting and kilning of the barley variety Weeah.•, e, activitiesas measured on PNPG5 substrate mixture; ,6" 0, corrected ~-amylase activity values

allowing for the action of a-amylase on PNPG5.

of cysteine (20 mM) to the extraction buffer gave no significant increase in extractable~-amylase.

The PNPG5 ~-amylase assay procedure as described in this paper can be used tomeasure accurately the level of ~-amylase in a range of cereal products. The applicationof the method to the measurement of ~-amylase in barley grains removed during themalting process is shown in Fig. 9. Total extractable p-amylase increases about 70 %during malting, and there is a dramatic increase in ex-amylase activity after about 60 hfrom initiation of the steep. The corrected (actual) ~-amylase levels are shown by theopen circles (soluble) and triangles (total). These values were calculated as describedearlier in this paper. There is little difference between the' apparent' and' actual' values,and thus activities obtained using the PNPG5 assay procedure are an accurate measureof ~-amylasecontent. With the particular barley variety (Weeah) used in this study, 25 %of the ~-amylase occurred in the insoluble form. However, as malting proceeded,essentially all the ~-amylase was converted to a soluble form. This was also confirmedwith a range of other commerically available and experimentally produced malts, and isconsistent with literature reports15 •

The PNPG5 p-amylase assay procedure is directly applicable to the measurement ofa range of p-amylases other than the cereal-flour enzymes just described. The ratios ofactivity of ~-amylases from wheat-flour, soybean-flour, sweet potato and B. cereus onPNPG5 and starch substrates (Nelson-Somogyi and Bernfe1d assays) are shown inTable III. The Bernfeld assay procedure2 is performed at pH 4'8, which is below theoptimal pH for activity of ~-amylase. This explains the higher PNPG5/Bernfeldactivity ratios (Table III) compared to the PNPG5/Nelson-Somogyi activity ratios. It

32 B. V. McCLEARY AND R. COOD

TABLE III

Ratios of activities'

Source of p-amylasePNPG5/

Nelson-Somogyi PNPG5/Bernfeld Nelson-Somogyi/Bernfeld

Wheat-flourSoybeanSweet potatoBacillus cereus

1·251·031'010'77

3'292·642·33],92

2·602·562·302·49

• All determinations were performed in duplicate on two separate days.

is interesting to note that the rates of cleavage of maltose from PNPG5 and from starch(Nelson/Somogyi assay?) are very similar. This supports a multi-chain mode of actionof the enzyme on starch rather than a multiple attack action pattern as proposed byBailey and French16

•

PNPG5 is readily attacked by an (X-amylase purified (devoid of amyloglucosidase anda-glucosidase) from a commercial A. niger preparation. This enzyme, unlike cereal (X­amylases, can readily cleave malto-oligosaccharides of degree of polymerisation 4 and5 due to its less demanding substrate sub-site binding requirements. In fact, for thisenzyme, the ratio of activity on PNPG5 compared to starch was 0·69 (at pH 5,2), a valuesimilar to that obtained for ~-amylase preparations ('" 1,0, Table III). Consequently,the PNPG5/a-glucosidase assay mixture can theoretically be used to measure fungal (X­amylase, as suggested by Mathewson and Seabourn4

• However, fungal a-amylase,preparations contain significant and varying quantities of amyloglucosidase andtransglucosidase (an (X-glucosidase with a different substrate specificity to the enzymeused in the current assay mixture1?), both of which readily act on PNPG5. Thus, for themeasurement of fungal a-amylase we recommend the use of an end~blocked p­nitrophenyl maltosaccharide (e.g. BPNPG78 ,18). The presence of varying amounts ofamyloglucosidase and transglucosidase in fungal (X-amylase preparations could explainthe differences in slope values reported by Mathewson and Seabourn4 (refer to Fig. 5)with different commercial fungal ct-amylase preparations.

The authors thank Ms H. Sheehan and Mrs A. Casey for valuable technical assistance in the initialstages of this work, which was performed at Biocon Biochemicals, Carrigaline, Co. Cork, Ireland.Statistical analyses were performed by Mr J. Evans and we thank Ms M. Daniels for assistancein the preparation of this manuscript. This work was supported in part by a grant from theAustralian Wheat Research Council.

References

I. Snider, S. R. Proc. Arner. Soc. Brew. Chern. (1940) 49-62.2. Bemfeld, P. Methods Enzymol. 1 (1955) 149-159.3. Delcour, J. A. and Verschaeve, S. G. J. Insi. Brew. 93 (1987) 296-301.4. Mathewson, P. R. and Seabourn, B. W. J. Agric. Food Chern. 31 (1983) 1322-1326.

MEASUREMENT OF p-AMYLASE 33

5. Svanborg, K. and Myrback, K. Ark. Kerni 6 (1953) 113-121.6. MacGregor, E. A. and MacGregor, A. W. Carbohydr. Res. 142 (1985) 223-236.7. SOlI\ogyi, M. J. Bioi. Chern. 195 (1952) 19-23.8. McCleary, B. V. and Glennie-Holmes, M. J. Inst. Brew. 91 (1985) 285-295.9. McCleary, B. V. and Sheehan, H. J. Cereal Sci. 6 (1987) 237-251.

10. MacGregor, A. W. Cereal Chern. 55 (1978) 754-765.11. Weselake, R. J. and Hill, R. D. Carbohydr. Res. 108 (1982) 153-161.12. Meyer, K. H., Spahr, P. F. and Fischer, E. H. Helv. Chim. Acta 36 (1953) 1924-1936.13. Robyt, J. R. and Whelan, W. J. in 'Starch and its Derivatives', 4th edn, (J. A. Radley, ed.), Chapman and

Hall, London, (1968), pp. 477-497.14. Tkachuk, R. FEBS Lett. 52 (1975) 6CHi8.15. Pollock, J. R. A. and Pool, A. A. J. Inst. Brew. 64 (1958) 151-156.16. Bailey, J. M. and French, D. J. Bioi. Chern. 226 (1957) 1-14.17. McCleary, B. V., Gibson, T. S., Sheehan, H., Casey, A., Horgan, L. and O'Flaherty, J. Carbohydr. Res.

(in press).18. Sheehan, H. and McCleary, B. V. Biotechnol. Techn. (1988) (in press).

CER9