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Protein Expression and Purification 18, 111–114 (2000)doi:10.1006/prep.1999.1172, available online at http://www.idealibrary.com on

Purification of Wheat Germ Amylase by Precipitation

Aparna Sharma, Shweta Sharma, and M. N. Gupta1

Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India

Received September 11, 1998, and in revised form October 1, 1999

a-Amylase from various sources was found to bindalginate in free solution. The alginate–enzyme com-plex could be precipitated with Ca21. The enzyme ac-tivity could be recovered by dissolving the precipitatein 1 M maltose and precipitating alginate alone byaddition of Ca21. Based upon these observations,a-amylase from wheat germ was purified with 68-foldpurification and 72% recovery. The molecular weightestimated by SDS–PAGE was 18 kDa. The method alsoworked equally well with a-amylase for the whole

heat seed. The latter enzyme could be purified 54-old with 70% activity recovery. The molecular weightf this second enzyme was estimated to be 45 kDa byDS–PAGE. © 2000 Academic Press

One way of reducing the cost of enzyme/protein pro-duction is to develop purification protocols with fewersteps (1). An important trend has been to introduceaffinity interactions at early stages of purification. Twosuch approaches, which have emerged, are expanded-bed affinity adsorption (2) and affinity precipitation (3).Affinity precipitation combines the power of resolutionof affinity approach with the convenience and numer-ous advantages of precipitation (3–6). This techniquebasically consists of precipitating the target enzymeactivity with a polymer or polymer bioconjugate whichhas affinity for the target enzyme/protein. The precip-itation is carried out by exploiting the reversibly solu-ble insoluble nature of the polymer. Some successfulapproaches of the technique have been described in therecent years (6–8). In our laboratory, we have beenfocusing on developing purification protocols for en-zymes/proteins which exclusively use precipitation(8,9). Precipitation as a unit process has several advan-tages (10). In this paper, we describe the purification ofwheat germ a-amylase from crude extracts by two suc-cessive precipitation steps, viz. ammonium sulfate pre-

1 To whom correspondence should be addressed. Fax: 91-11-

6581073. E-mail: mngupta@chemistry.iitd.ernet.in.

1046-5928/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

cipitation followed by an affinity precipitation withalginate.

MATERIALS AND METHODS

Sodium alginate (Cat. No. A-2158, composed pre-dominantly of mannuronic acid residues), wheat germpowder, and porcine pancreatic a-amylase (EC 3.2.1.1,a-D-glucan-glucanohydrolase) were purchased fromSigma (St. Louis, MO). Starchzyme was purchasedfrom Jaysons Agritech Pvt.Ltd., Mysore, India, andwas an industrial preparation of the enzyme from As-pergillus oryzae. BAN 240L (Bacillus subtilis) weregenerously supplied by NOVO Nordisk A/S (Bangalore,India). All other chemicals used were of analyticalgrade.

Estimation of Enzyme Activity

Activity of a-amylase was estimated using starch asa substrate. One enzyme unit (U) liberates 1 mmol ofreducing sugar (calculated as maltose) per minute at25°C and pH 5.6 under the specified conditions fromsoluble starch (11).

Estimation of Protein

Protein was estimated according to the dye-bindingmethod of Bradford (12) using bovine serum albuminas a standard.

Preparation of Alginate Solution

Alginate (2% w/v) was dissolved in water the pH ofwhich has been adjusted to pH 5.6. The solution wasstored at 4°C for further use and diluted with appro-priate buffer for further use.

Extraction of Wheat Germ Amylase from Wheat GermPowder

Ten grams of wheat germ powder was stirred with 10ml of chilled acetone in cold for 2 h. The dry powder so

obtained was used for extraction of amylase activity.

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112 SHARMA, SHARMA, AND GUPTA

The extraction was done with 20 ml of 50 mM acetatebuffer, pH 5.6, with constant stirring in cold for 4 h.The extract was centrifuged at 12,000g for 15 min at°C and the clear supernatant thus obtained was useds crude wheat germ amylase preparation.

inding of Amylase with Alginate Solution

One milliliter of BAN 240L (9.2 U) or starchzyme9.2 U) or wheat germ amylase (9.5 U) in 50 mMcetate buffer, pH 5.6, and 1 ml of a-amylase from

porcine pancreas (8.3 U) in 20 mM Tris buffer, pH 6.9,were added separately to 0.5 ml of alginate solution(2% w/v). The final volume of the solution was made to1.6 ml with 50 mM acetate buffer, pH 5.6. After 1 h ofincubation with continuous shaking in a water bath at25°C, the enzyme-bound alginate was precipitated byadding 0.1 ml of 1 M CaCl2 solution (final concentra-tion of CaCl2 in the solution was 60 mM). This amountof calcium ions was found to precipitate alginate nearlyquantitatively (98%) (13). After 15 min of incubation at25°C, the precipitate was centrifuged at 8000g for 15min at 25°C. The precipitate was washed with 1 ml of50 mM acetate buffer, pH 5.6, containing 60 mM CaCl2

twice till no enzyme activity was detected in the wash-ings. The difference between the total enzyme activitybefore the addition of alginate and the total activity ofthe supernatant (and washings) represented theamount of the enzyme bound. The enzyme bound algi-nate was dissolved in 1 ml of 50 mM acetate buffer, pH5.6, containing 1 M maltose and kept at 4°C for 4 h14). Enzyme was then recovered by precipitating thelginate with 0.1 ml of 1 M CaCl2. This procedure was

repeated twice in order to recover all the bound enzymeactivity. The activity in the supernatant was deter-mined after extensive dialysis of maltose.

Polyacrylamide Gel Electrophoresis

SDS–polyacrylamide gel with 12% gel was per-formed according to Hames (15) using BioRad MiniProtean II electrophoresis unit.

TABLE 1

Binding of a-Amylase from Four Different Sourcesto Alginate Solution

Amylase from different sourcesPercentage bound activity on

alginate

Porcine pancreas 85.7BAN 240L 86Starchzyme 74Wheat germ amylase 96

Note. The binding of amylase to alginate solution was done as

described under Materials and Methods.

RESULTS AND DISCUSSION

Recently, it has been reported that alginate beadsshow adequate and selective affinity for a-amylasefrom various sources (13). In this paper, we show thatthis affinity could be exploited by purifying a-amylasesby affinity precipitation. Table 1 shows the binding ofalginate to a-amylase from four different sources.While the enzyme preparations from pig pancreas (an-imal), A. oryzae (fungal), and B. subtilis (bacterial)were commercial preparations, wheat amylase prepa-ration was the crude extract obtained from wheat germ(plant) as described under Materials and Methods.While the alginate bound to amylases from differentsources at a significant level, maximum binding wasobserved in case of wheat amylase (Table 1). Furtherwork was carried out with wheat germ a-amylase.

The separation of the alginate–a-amylase complex

TABLE 2

Effect of Polymer Concentration at the Time ofPrecipitation on the Binding and Recovery

of the Wheat Germ Amylase

Polymerconcentration (%)

Activitybound (%)

Recovery with 1 Mmaltose at 4°C (%)

0.2 56 12.50.4 65 19.00.6 96 74.00.8 93 68.01.0 87 50.0

Note. Wheat germ amylase (containing 9.5 U) was added to 1 ml ofvarious alginate solutions. The binding and recovery of enzyme wascarried out as described under Materials and Methods.

TABLE 3

Recovery of Wheat Germ Amylase

Sugars/Chemicals % Activity recovered

0.5 M maltose 25°C (1 h) 210.5 M maltose 4°C (1 h) 260.5 M maltose 4°C (4 h) 391.0 M maltose 4°C (4 h) 751.0 M mannose 4°C (4 h) 01.0 M glucose 4°C (4 h) 121.0 M galactose 4°C (4 h) 01.0 M trehalose 4°C (4 h) 01.0 M sucrose 4°C (4 h) 00.1 M borate 4°C (4 h) 191.0 M NaCl 25°C (1 h) 0

Note. The wheat amylase was bound to the alginate solution asdescribed under Materials and Methods. 9.5 units of enzyme activitywas added to the alginate solution and nearly complete binding (99%in terms of activity loaded) was observed. Bound enzyme activity wasrecovered as described under Materials and Methods but using the

sugar/chemical during the incubation as specified below.

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113WHEAT GERM AMYLASE PURIFICATION

was based upon precipitation of the polymer and thepolymer–enzyme complex with Ca21 (16). It was foundhat presence of 60 mM CaCl2 does not inhibit the

a-amylase activity (data not shown).The effect of polymer concentration at the time of pre-

cipitation on the binding and recovery of the amylaseactivity and the results are shown in Table 2. It wasexpected that as the polymer concentration was in-creased, more enzyme bound to polymer. However, as thepolymer concentration was increased beyond 0.6%, ex-tent of binding leveled off and in fact decreased at 1%.Presumably, at the higher concentration, enzyme hasproblem in accessing binding sites on the polymer sincetoo many polymer chains “crowd out” the enzyme. It hasbeen reported earlier that polymer concentration at thetime of precipitation influences subsequently enzyme re-covery in precipitation (17). In this case, we found thatthere is a common optimum polymer concentration formaximum binding as well as maximum recovery.

Table 3 shows that incubation of the alginate–en-zyme precipitate with 1 M maltose at 4°C for 4 h gavethe best recovery of the enzyme activity. The data alsosuggested that the affinity process is involved sinceother sugars like mannose and other chemicals did not

FIG. 1. SDS–PAGE of purified a-amylase from wheat germ. LaneM, marker proteins; lane 1, purified wheat a-amylase; lane 2, crude

heat germ a-amylase. Same amount of protein (25 mg) was appliedin lanes 1 and 2. The gel was stained with Coomassie brilliant blueR-250 for 15 min and then destained in 40% methanol and 10% acetic

TAB

Purification of Wheat Germ Amylase

Steps Activity (U) Protein (mg)

Crude extract 9.6 720Ammonium

sulfateprecipitation 9 392

Elution 1 7 7.9Elution 2 1.1 21

Note. Elution 1: Bound activity was eluted by incubating the enzym1 M maltose at 4°C for 4 h. The activity was determined after extensiprecipitate was carried out in a manner identical to elution 1 proceduin the readings in duplicates was less than 65%.

acid.

result in significant recovery of the enzyme activity.Greater recovery of enzyme activity with 1 M maltoseompared to that obtained with 1 M glucose is in agree-

ment with the known behavior of the sugars with amy-lases (18). The above results prompted us to attemptpurification of a-amylase from wheat germ crude ex-tract. Table 4 shows that an initial step of ammoniumsulfate precipitation followed by affinity precipitationwith alginate gave about 68-fold purification with 72%activity recovery. Further 8% activity could be recov-ered but this fraction is considerably less pure. This issimilar to the results reported with other systems likepectinase (19). The enzyme showed a single band onSDS–PAGE which corresponded to a molecular weightof 18 kDa (Fig. 1). The identical protocol was alsoapplied for isolation and purification of a-amylase fromwhole wheat. The purification details are given in Ta-ble 5. The SDS–PAGE analysis of the purified enzymeshows the molecular weight of 45 kDa which agreeswell with the literature (18). The SDS–PAGE (Fig. 2)also shows a faint band around 18 kDa which confirmsthe relatively low amount of the wheat germ enzyme inthe preparation.

FIG. 2. SDS–PAGE of purified a-amylase from whole wheat seed.Lane M, marker proteins; lane 1, purified wheat a-amylase; lane 2,crude whole wheat a-amylase. The same amount of protein (25 mg)was applied in lanes 1 and 2. The gel was stained with Coomassiebrilliant blue R-250 for 15 min and then destained in 40% methanol

4

Precipitation with Alginate Solution

Specific activity(U/mg)

Yield(%)

Purificationfactor

13 100.0 1.0

23 95 1.7886 72 6852 10 4

und alginate with 1.0 ml of 50 mM acetate buffer, pH 5.6, containingdialysis of maltose. Elution 2: Second elution of the activity from theAll the experiments were performed in duplicate and the difference

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114 SHARMA, SHARMA, AND GUPTA

a-Amylase is an enzyme which is widely used forstarch hydrolysis (20). The numerous applications ofthe enzyme in various industries are also well docu-mented (21). The simple approach described here maybe useful for purifying amylases from other sources aswell. As the only unit process used in the above puri-fication protocol is precipitation, it should be possibleto scale up the purification. Alginate is an inexpensivepolymer of marine origin. It is nontoxic and is used infood processing industries as well (16). Hence a-amy-lase purified by the simple process can be used in foodprocessing industries as well.

ACKNOWLEDGMENTS

This work was supported by The Council of Scientific and Indus-trial Research (India). This work was also partially supported byfunds from the Department of Biotechnology (Government of India)and Department of Science and Technology (Government of India).

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TAB

Purification of Wheat a-Amylase from Whole W

Steps Activity (U) Protein (mg)

Crude extract 10 780Ammonium

sulfateprecipitation 9 290

Elution 1 7 10Elution 2 1 70

Note. Purification and isolation of a-amylase was done as for whea: Bound activity was eluted by incubating the enzyme bound alginat 4°C for 4 h. The activity was determined after extensive dialysis oas carried out in a manner identical to elution 1 procedure. All t

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15. Hames, B. D. (1986) An introduction to polyacrylamide gel elec-trophoresis in “Gel Electrophoresis of Protein: A Practical Ap-proach” (Hames, B. D., and Rickwood, D., Eds.), pp. 1–86, IRLPress, Oxford.

16. Smidsrod, O., and Skjak-Braek, G. (1990) Alginate as immobili-zation matrix for cells. Trends. Biotechnol. 8, 71–78.

17. Kumar, A., Agarwal, R., Batra, R., and Gupta, M. N. (1994)Effect of polymer concentration on recovery of the target proteinsin precipitation methods. Biotechnol. Tech. 8, 651–654.

18. Thoma, J. A., Spradlin, J. E., and Dygert, S. (1971) “The En-zyme” (Boyer, P. D., Ed.) Vol. 5, pp. 115–189, Academic Press,New York.

19. Gupta, M. N., Guogiang, D., and Mattiasson, B. (1993) Purifica-tion of endopolygalacturonase by affinity precipitation using al-ginate. Biotechnol. Appl. Biochem. 18, 321–327.

20. Chen, J. P., Sun, Y. M., and Chu, D. H. (1998) Immobilization ofalpha-amylase to a composite temperature-sensitive membranefor starch hydrolysis. Biotechnol. Prog. 473–478.

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5

t Seed by Precipitation with Alginate Solution

Specific activity(U/mg)

Yield(%)

Purificationfactor

13 100.0 1.0

31 90 2.4700 70 5414 10 1.1

erm a-amylase (as described under Materials and Methods. Elutionith 1.0 ml of 50 mM acetate buffer, pH 5.6, containing 1 M maltose

altose. Elution 2: Second elution of the activity from the precipitatexperiments were performed in duplicate and the difference in the

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91–93, Macmillian, London.