Vol. 70, Nr. 6, 2005—JOURNAL OF FOOD SCIENCE C413Published on Web 7/14/2005

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Purification of Amylase from HoneySSSSSIBELIBELIBELIBELIBEL B B B B BABACANABACANABACANABACANABACAN ANDANDANDANDAND A A A A ARTHURRTHURRTHURRTHURRTHUR G. R G. R G. R G. R G. RANDANDANDANDAND

ABSTRAABSTRAABSTRAABSTRAABSTRACTCTCTCTCT: : : : : The major amylase in honey was concentrThe major amylase in honey was concentrThe major amylase in honey was concentrThe major amylase in honey was concentrThe major amylase in honey was concentrated bated bated bated bated by ultry ultry ultry ultry ultrafiltrafiltrafiltrafiltrafiltration, isolated bation, isolated bation, isolated bation, isolated bation, isolated by ultry ultry ultry ultry ultracentracentracentracentracentrifugation andifugation andifugation andifugation andifugation andgel filtrgel filtrgel filtrgel filtrgel filtration, and puration, and puration, and puration, and puration, and purified bified bified bified bified by ion-exy ion-exy ion-exy ion-exy ion-exchange chrchange chrchange chrchange chrchange chromatogromatogromatogromatogromatographyaphyaphyaphyaphy. . . . . The amylase activity was in the floThe amylase activity was in the floThe amylase activity was in the floThe amylase activity was in the floThe amylase activity was in the flow-thrw-thrw-thrw-thrw-throughoughoughoughoughfraction of the anion-exchange column, suggesting a high isoelectric point (>7.4) for the enzyme. The enzymefraction of the anion-exchange column, suggesting a high isoelectric point (>7.4) for the enzyme. The enzymefraction of the anion-exchange column, suggesting a high isoelectric point (>7.4) for the enzyme. The enzymefraction of the anion-exchange column, suggesting a high isoelectric point (>7.4) for the enzyme. The enzymefraction of the anion-exchange column, suggesting a high isoelectric point (>7.4) for the enzyme. The enzymefraction from the anion-exchange chromatography was loaded onto a cation-exchange column, and the amylasefraction from the anion-exchange chromatography was loaded onto a cation-exchange column, and the amylasefraction from the anion-exchange chromatography was loaded onto a cation-exchange column, and the amylasefraction from the anion-exchange chromatography was loaded onto a cation-exchange column, and the amylasefraction from the anion-exchange chromatography was loaded onto a cation-exchange column, and the amylaseactivity was eluted as a single band at 50 mactivity was eluted as a single band at 50 mactivity was eluted as a single band at 50 mactivity was eluted as a single band at 50 mactivity was eluted as a single band at 50 mMMMMM NaCl. The purification factor after this step was 531-fold. The NaCl. The purification factor after this step was 531-fold. The NaCl. The purification factor after this step was 531-fold. The NaCl. The purification factor after this step was 531-fold. The NaCl. The purification factor after this step was 531-fold. Thepurpurpurpurpurified enzyme was an ified enzyme was an ified enzyme was an ified enzyme was an ified enzyme was an �����-amylase-amylase-amylase-amylase-amylase, as deter, as deter, as deter, as deter, as determined bmined bmined bmined bmined by thin-layy thin-layy thin-layy thin-layy thin-layer chrer chrer chrer chrer chromatogromatogromatogromatogromatographyaphyaphyaphyaphy, with a molecular w, with a molecular w, with a molecular w, with a molecular w, with a molecular weight ofeight ofeight ofeight ofeight of57000 D57000 D57000 D57000 D57000 Da accora accora accora accora according to sodium dodecyl sulfate-polyacrding to sodium dodecyl sulfate-polyacrding to sodium dodecyl sulfate-polyacrding to sodium dodecyl sulfate-polyacrding to sodium dodecyl sulfate-polyacrylamide gel electrylamide gel electrylamide gel electrylamide gel electrylamide gel electrophorophorophorophorophoresis (SDS-Pesis (SDS-Pesis (SDS-Pesis (SDS-Pesis (SDS-PAAAAAGE). GE). GE). GE). GE). The rThe rThe rThe rThe resultsesultsesultsesultsesultssupported the concept that amylase in honey had a high degree of similarity with bee amylase.supported the concept that amylase in honey had a high degree of similarity with bee amylase.supported the concept that amylase in honey had a high degree of similarity with bee amylase.supported the concept that amylase in honey had a high degree of similarity with bee amylase.supported the concept that amylase in honey had a high degree of similarity with bee amylase.

KKKKKeyworeyworeyworeyworeywords: honeyds: honeyds: honeyds: honeyds: honey, amylase, amylase, amylase, amylase, amylase, diastase, diastase, diastase, diastase, diastase, pur, pur, pur, pur, purificationificationificationificationification

Introduction

The presence of enzymes in honey has been known for manyyears. The major honey enzymes have been reported as inver-

tase, glucose oxidase, and a mixture of �- and �-amylases, alsoknown as diastase (White 1978). Other enzymes, such as catalaseand acid phosphatase, have occasionally been reported in honey,but have not received the same analytical attention as the aboveenzymes.

Invertase, actually an �-glucosidase added to honey by bees,catalyzes hydrolysis of sucrose in nectar to its monosaccharides(Sporns 1992). Invertase was reported to be the major proteinpresent in the hypopharyngeal gland of the forager bee, reachingabout 50% of the total protein of the gland, whereas amylase andglucose oxidase were each estimated to account for 2% to 3% ofthe total protein (Ohashi and others 1999). Because sucrose crys-tallizes much more easily than the hydrolyzed product, the hydrol-ysis facilitates conversion of nectar to honey by allowing for thepreparation of the required supersaturated sugar solution(Sporns 1992). Invertase has been considered responsible formost of the chemical changes that take place during the conver-sion of nectar to honey. The enzyme has been purified and somecharacteristics have been established (White and Kushnir 1967a;Huber and Mathison 1976; Cho 1994).

Glucose oxidase would be another important enzyme added tohoney by the bees. It has also been partially purified and studiedin detail (Schepartz and Subers 1964). This enzyme was consideredmainly responsible for the antibiotic effect of honey through theproduction of hydrogen peroxide and gluconic acid (Sporns 1992).Hydrogen peroxide has been identified as the “inhibine,” whichhas long been regarded as a mysterious compound in honey(Schade and others 1958; White and others 1963). White (1978) re-ported that gluconic acid, in equilibrium with gluconolactone, wasthe principal acid and the reason behind low pH values of about 4for honey.

The amylase component of honey has received attention overthe years due to use as a freshness indicator (White 1975). However,it has been argued that because of the large degree of variation in

amylase activities found in untreated raw honeys, determinationof this enzyme should not be considered a valid criterion to mea-sure the freshness of honey (Oddo and others 1990). Various expla-nations for the low enzymatic activity of certain honeys have beenproposed, such as poor processing of nectar by the bees duringabundant nectar flow or seasonal activity of the pharyngeal glands(White 1992). Even international standards, such as Codex Alimen-tarius, include honeys with “a low natural content of enzymes” forwhich different limits are accepted.

The origin of amylase in honey has been attributed to the sali-vary secretions of bees, or to its presence in pollen or nectar (Oddoand others 1990). Today, the most widely accepted theory attributesthe origin of amylase in honey to salivary secretions of bees. Thisconclusion was based on the presence of amylase in honey pro-duced by sugar-fed bees (Stadelmeier and Bergner 1986) and onsimilarities between honey amylase and bee amylase (Rinaudoand others 1973). Recently, �-amylase from forager-bee hypopha-ryngeal glands was purified through ion-exchange chromatography(Ohashi and others 1999). The molecular weight of the bee �-amy-lase was estimated at 57 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The au-thors also reported the N-terminal amino acid sequence of thepurified amylase.

There have been a few attempts in the literature to separate andpurify the amylase of honey. Schepartz and Subers (1966) used ion-exchange cellulose chromatography, which provided 2 principalfractions, but could not characterize the pooled fractions becauseof instability. White and Kushnir (1967a) carried out Sephadex gelfiltration of dialyzed honey concentrates. They were able to eluteprotein peaks for the amylase and �-glucosidase components, al-though without baseline separation, suggesting molecular weightsof about 24000 and 51000, respectively. However, the authors alsomentioned that these values were only approximations becausethey observed interaction of the proteins with the column. Bergnerand Diemair (1975) separated honey proteins into 5 peaks onSephadex G-200, 1 of which showed amylase activity. Based on thedetermination of elution coefficients, they suggested that 3 of the5 components, including the amylase peak, originated from thebees whereas the other 2 were from plants. An extensive review ofthe literature has revealed no clear procedure to purify and studythe amylase component of honey. Therefore, the primary goal ofthis study was to concentrate, isolate, and purify honey amylase.

MS 20050049 Submitted 1/23/05, Revised 3/4/05, Accepted 4/15/05. The au-thors are with Food Science Research Center, Univ. of Rhode Island, 530Liberty Lane, West Kingston, RI 02892. Direct inquiries to author Rand (E-mail: enzyme@adelphia.net).

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Purification of Amylase from Honey

Materials and Methods

MaterialsMaterialsMaterialsMaterialsMaterialsTris (hydroxymethyl aminomethane), Sephadex G-100, and pure

�-amylase type I-B from sweet potato were purchased from SigmaChemical Co. (St. Louis, Mo., U.S.A.). Pure �-amylase from porcinepancreas was purchased from Worthington Biochemical Co. (Free-hold, N.J., U.S.A.). Sodium acetate trihydrate, glycine, sodium chlo-ride, butanol, sulfuric acid, and methanol were purchased fromFisher Scientific (Fair Lawn, N.J., U.S.A.). Maltose was purchasedfrom J.T. Baker, Inc. (Philipsburg, N.J., U.S.A.). Unmodified waxymaize starch (Novation 2600TM) was provided by the Natl. Starchand Chemical Co. (Bridgewater, N.J., U.S.A.). Raw honey blend wasdonated by Sioux Honey Assoc. (Sioux City, Iowa, U.S.A.). Pro-cessed honey was purchased from a local supermarket.

Amylase assayAmylase assayAmylase assayAmylase assayAmylase assayThe activity of amylase in honey was measured spectrophoto-

metrically by the procedure of Sigma Diagnostics (1999). This pro-cedure was based on the progressive hydrolysis of the �(1-4)-glu-cosidic bonds in 4,6-ethylidene-p-nitrophenyl-�-D-malto-heptaoside, which served as the substrate for the amylase. OneSigma unit of amylase activity was given as a millimole 4,6-eth-ylidene-p-nitrophenyl-�-D-maltoheptaoside degraded per min at37 °C. The Sigma assay was used for activity recovery determina-tions within purification steps.

Protein determinationProtein determinationProtein determinationProtein determinationProtein determinationProtein concentration during column chromatography and the

progress of purification was monitored by measuring absorbanceat 280 nm. Protein concentration was also determined by the coo-massie blue dye binding method of Bradford (1976) using a Bio-Rad protein assay kit (Bio-Rad Labs, Hercules, Calif, U.S.A.) andbovine serum albumin as the standard. Buffers that were identicalto those containing protein samples were used as blanks.

Purification of amylase in honeyPurification of amylase in honeyPurification of amylase in honeyPurification of amylase in honeyPurification of amylase in honeyStep 1: Concentration of honey proteins.Step 1: Concentration of honey proteins.Step 1: Concentration of honey proteins.Step 1: Concentration of honey proteins.Step 1: Concentration of honey proteins. Honey proteins were

concentrated and sugars were eliminated by ultrafiltration with aHollow Fiber Cartridge Adapter System Model DH4 (Amicon Co.,Lexington, Mass., U.S.A.). An aliquot of honey sample (300 g) wasdissolved in 0.01 M Tris-chloride buffer, pH = 7.4, to give a totalvolume of 380 mL. The ultrafiltration was conducted in a recyclebatch operation at room temperature with an inlet pressure of 11psi. The hollow-fiber cartridge was a Diaflo PM30 with a nominalmolecular weight cut-off of 30000 Da. The operation was carried outuntil a retentate of 70 mL was collected, which took about 1 h. Therecycled material was retained on ice at all times during the oper-ation. When completed, the hollow fiber cartridge was rinsed withdeionized water and then cleaned with 1% detergent (Liqui-Nox)from Alconox, Inc. (New York, N.Y., U.S.A.). The cartridge was storedin 0.2% sodium azide solution at 4 °C until next use.

Step 2: Ultracentrifugation.Step 2: Ultracentrifugation.Step 2: Ultracentrifugation.Step 2: Ultracentrifugation.Step 2: Ultracentrifugation. An aliquot (7 mL) of concentratedhoney proteins from the 1st step was centrifuged (100000 × g for 40min) in a Beckman L60 Ultracentrifuge from Beckman Instru-ments, Inc. (Columbia, Md., U.S.A.). A dark pellet at the bottom ofthe tube and a bright yellow pellet at the side of the tube were dis-carded. The process was repeated 3 times and the supernatant wasused for the next step.

SSSSStep 3: Gtep 3: Gtep 3: Gtep 3: Gtep 3: Gel-filtrel-filtrel-filtrel-filtrel-filtration chration chration chration chration chromatogromatogromatogromatogromatographyaphyaphyaphyaphy..... The supernatant fromthe ultracentrifugation step was applied to a Sephadex G-100 gel-filtration column (2.4 × 37.0 cm). The system also consisted of anISCO fraction collector and Model 400 volumeter from Instrumen-

tation Specialties Co. (Lincoln, Nebr., U.S.A.). The pump was aMasterflex L/S peristaltic pump (Model 7523-30) from Cole-ParmerIns. Co. (Vernon Hills, Ill., U.S.A.). Elution was carried out at roomtemperature with 0.01 M Tris-chloride buffer, pH 7.4, and at a flowrate of 1.5 mL/min. The collected fractions (2.5 mL) were transferredto ice. The amylase-containing fractions were pooled and used forthe next step in the purification scheme.

Step 4: Concentration of gel filtration peaks.Step 4: Concentration of gel filtration peaks.Step 4: Concentration of gel filtration peaks.Step 4: Concentration of gel filtration peaks.Step 4: Concentration of gel filtration peaks. The protein in thepooled amylase fractions (121 mL) from the gel filtration step wasconcentrated using a Virtis Genesis 25SL freeze-drier from The VirtisCo, Inc. (Gardiner, N.Y., U.S.A.). The drying process was carried outat a plate temperature of –4 °C for 15 h. The freeze-dried samplewas reconstituted in 12 mL deionized water and dialyzed against0.01 M Tris-chloride buffer, pH 7.4, overnight at 4 °C. The dialysisunit was a slide-a-lyzer® dialysis cassette with molecular weight cut-off of 10000 Da from Pierce (Rockford, Ill., U.S.A.).

SSSSStep 5: Anion-extep 5: Anion-extep 5: Anion-extep 5: Anion-extep 5: Anion-exchange chrchange chrchange chrchange chrchange chromatogromatogromatogromatogromatographyaphyaphyaphyaphy..... The anion-exchangechromatography was performed by the procedure of Ohashi andothers (1999) using a HiTrapTM Q sepharose high-performancestrong ion exchanger and a AKTA Fast Protein ChromatographySystem (FPLC) from Amersham Pharmacia Biotech, Inc. (Piscat-away, N.J., U.S.A.) at about 4 °C in a cold room. The proteins wereeluted from the column using a linear NaCl gradient (0 to 1.0 M) in0.01 M Tris-chloride buffer. Fractions (1 mL) were collected. Initially,the entire chromatogram was screened for amylase activity by com-bining 5 consecutive fractions (5 mL) and performing the Sigmaamylase assay. Then, the amount of amylase activity was deter-mined for each 1-mL fraction within the identified area and report-ed in the chromatogram.

SSSSStep 6: Ctep 6: Ctep 6: Ctep 6: Ctep 6: Cation-exation-exation-exation-exation-exchange chrchange chrchange chrchange chrchange chromatogromatogromatogromatogromatographyaphyaphyaphyaphy..... The amylase frac-tions from step 4 were combined and applied to a HiTrapTM SPsepharose high-performance strong ion-exchanger column. Themethod was identical to the method described in step 5.

ElectrophoresisElectrophoresisElectrophoresisElectrophoresisElectrophoresisElectrophoresis in the presence of sodium dodecyl sulfate

(SDS) was performed with 10% running gels and 4% stacking gelsas described by Laemmli (1970). Samples for electrophoresis weretreated at 100 °C for 2.5 min in the buffer containing 62.5 mMTris-chloride (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoeth-anol, and 0.0004% bromphenol blue. The running gel buffer was1.5 M Tris (pH 8.8) and the stacking gel buffer was 0.5 M Tris (pH6.8). Electrophoresis was carried out in a tank buffer of 0.25 M Tris(pH 8.3) containing 0.192 M glycine and 0.1% SDS at constantcurrent (40 mA per gel) for 35 min using a EC 120 Mini Vertical GelSystem from E-C Apparatus Co. (Holbrook, N.Y., U.S.A.) and a Bio-Rad Power Pac 300 power supply from Bio-Rad Laboratories. Pro-teins on SDS-polyacrylamide gels were stained in 0.025% coo-massie brilliant blue overnight and destained 1st in 40%methanol-7% acetic acid solution, and then in 7% acetic acid-5%methanol solution. Protein molecular weight standards used inelectrophoresis consisted of myosin (205000), �-galactosidase(116000), phosphorylase b (97000), fructose-6-phosphate kinase(84000), albumin (66000), glutamic dehydrogenase (55000), oval-bumin (45000), glyceraldehyde-3-phosphate dehydrogenase(36000), carbonic anhydrase (29000), trypsinogen (24000), trypsininhibitor (20000), alpha-lactalbumin (14200), and aprotinin(6500) obtained from Sigma Chemical Co. The molecular weightsof the bands within honey samples were determined from theplots of logarithm of the molecular weights of the standardsagainst the distance of migration (in millimeters) from the top ofthe running gel. The amylase-containing fractions from the ion-exchange chromatography steps had to be concentrated through

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freeze-drying before electrophoresis due to the low amount ofprotein present.

Thin-layThin-layThin-layThin-layThin-layer chrer chrer chrer chrer chromatogromatogromatogromatogromatography (aphy (aphy (aphy (aphy (TLTLTLTLTLC) analysis ofC) analysis ofC) analysis ofC) analysis ofC) analysis ofenzymatic degradation productsenzymatic degradation productsenzymatic degradation productsenzymatic degradation productsenzymatic degradation products

TLC was performed to analyze reaction products of the purifiedamylase. Aliquots (200 �L) of pure �-amylase from sweet potato(0.04 Sigma amylase units/mL), pure porcine �-amylase (about 0.2Sigma units/mL), and purified amylase from honey (0.04 Sigmaunits/mL) were incubated with 340 �L of 2% unmodified waxymaize starch, 420 �L of 0.2 M sodium acetate buffer, pH 5.3, and 40�L of 0.5 M sodium chloride for 1 h at 40 °C. Then, a 10-�L aliquotfrom each reaction mixture was loaded on a precoated K6 silica gel60A plate (20 × 20 cm with layer thickness of 250 �m), which waspurchased from Whatman Inc. (Clifton, N.J., U.S.A.). As controls,2% maltose and 2% unmodified waxy maize starch were also in-cluded. The hydolysates were developed with butanol, ethanol,and water (5:3:2). Sugars were detected by spraying the plates withsulfuric acid-methanol (1:3) solution and heating the plates in anoven at 100 °C for 30 min (Puki and others 1996).

Results and Discussion

Purification of amylase from honeyPurification of amylase from honeyPurification of amylase from honeyPurification of amylase from honeyPurification of amylase from honeyPreliminary work on purification involved a number of experi-

ments with Sephadex G-100 gel chromatography. First, an aliquot(4 mL) of processed honey (0.73 g/mL) was loaded onto a gel-filtra-tion column (2.4 cm × 13 cm) after minor dilution in 0.01 M Tris-chloride buffer. The resulting chromatogram (Figure 1a) gave 2absorbance peaks, the 1st of which was in the flow-through volumeand consisted of 85% of the total protein as determined by the Bio-Rad assay and contained 96% of the amylase activity. Although theabsorbance of the 2nd peak at 280 nm was very high, it did not con-sist of much protein. The presence of nonprotein, ultraviolet-ab-sorbing materials in honey, which could originate from nectar orpollen, have been reported (White and Kushnir 1967b). This obser-vation suggested that the purification of amylase from honey wouldinvolve separating the enzyme from the plant materials existing inhoney as well as the rest of the honey proteins, which were very low(<0.2%) in honey (Schepartz and Subers 1964; White and Rudyj1978). Thus, it was more appropriate to use A280 absorbance unitsin the specific activity calculations for monitoring progress in thepurification steps. This approach reflected not only the removal ofthe other proteins in honey during purification steps, but also theelimination of the plant materials, which was an important consid-eration in this study.

Another experiment involved utilization of an ultrafiltration stepwith a molecular weight cut-off of 30000 to remove the low-molec-ular-weight compounds, including sugars, while concentrating thehigh-molecular-weight compounds in honey. The result of thisstudy (Figure 1b, solid line) showed that while the height of theinitial peak in the gel-filtration chromatogram increased, theheight and the area of the 2nd peak in the chromatogram de-creased. In other words, the ultrafiltration step eliminated some ofthe UV-absorbing low-molecular-weight compounds present inhoney while concentrating the high-molecular-weight compounds,including proteins. As a complementary step, ultrafiltration wascoupled with ultracentrifugation to remove additional UV-absorb-ing high-molecular-weight compounds, which resulted in honeyamylase concentration, as shown by the decreased height of the 1stpeak in the gel-filtration chromatogram (Figure 1b, dashed line)without any loss in activity.

After the completion of the preliminary studies, a purification

scheme was developed. The steps were devised from experimen-tation on pilot lots at each stage with a market-purchased honey.The final procedure was then applied to 2 complete batches with araw honey blend, which gave essentially the same results. Thequantities reported in Table 1 are from 1 of these batches. The rawhoney blend was 1st passed through cheesecloth to remove anyparticulate impurities. The purification started with the ultrafiltra-tion step and resulted in the collection of 70 mL of retentate with78.9% of the initial amylase activity (Table 1). An aliquot of thisretentate was then ultra-centrifuged to remove 86% of the high-molecular-weight compounds that had absorbance at 280 nm whilekeeping amylase activity intact. The supernatant from this step wasloaded onto a Sephadex G-100 column. The resulting chromato-gram, Figure 2, was very similar to the one obtained in the prelimi-nary studies (Figure 1b). The amylase activity in each of the gel fil-tration fractions was determined by the Sigma-amylase assay,which revealed 2 peaks. The 1st of the 2 peaks came in the flow-through fraction with most of the other proteins in honey while the2nd peak appeared to elute separately. The reason why part of theamylase was excluded from the gel while the rest was retardedcould be due to interactions between plant materials still remainingin the preparation and the proteins of honey. In an earlier study

Figure 1—Preliminary purification of honey amylase onSephadex G-100 before and after ultrafiltration and ultra-centrifugation. Column (2.4 × 13.0 cm) eluted with 0.01 MTris-Cl buffer, pH 7.4; 4 mL of sample was loaded and 1.5-mL fractions were collected with a flow rate of 1.5 mL/min at room temperature.

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Purification of Amylase from Honey

(White and Kushnir 1967a), evidence was obtained of interactionbetween honey invertase and plant proteins, resulting in multipleforms that behaved differently in ion-exchange chromatographyand gel electrophoresis. After the gel-filtration step, all fractionswith the isolated amylase activity were pooled and concentratedthrough freeze-drying. The next step in the purification protocolwas anion-exchange chromatography. As shown in Figure 3, themajor amylase peak came through in the flow-through volume,indicating that the enzyme was not sufficiently negatively chargedat the chromatography pH (Tris-chloride buffer; pH = 7.4 at roomtemperature). This indicated a high isoelectric point for this en-zyme. There was also another area in the chromatogram, whichshowed some amylase activity (<7%) that was eluted between 58

mM and 75 mM NaCl concentration. Because this area had a verylow enzyme activity and did not consist of a well-defined proteinpeak, further characterization could not be performed. The anion-exchange chromatography resulted in 227-fold purification for themajor amylase in honey with 20% enzyme recovery. As a final step,the fractions with amylase activity were combined and added to acation exchange column under the same conditions (Figure 4). Thehoney amylase was eluted from the column at about 50 mM saltconcentration. This also suggested a high isoelectric pH for thisenzyme (> pH 7.4). Although a higher purification (531-fold) wasachieved with the cation-exchange chromatography step, the activ-ity recovery was very low (3.9%). A summary of the purification forthe honey amylase is presented in Table 2.

Aliquots from selected purification steps were subjected to SDS-PAGE (Figure 5). The electrophoresis pattern showed at least 4 pro-tein bands between the molecular weights of 84000 and 45000 Da

Table 1—Concentration of honey proteins and elimination of sugars by ultrafiltration with a hollow-fiber cartridgeb

Volume (mL) Total protein (mg) Total A280 units Total activitya (units) Activity recovery (%)

Starting sampleb 380 140.0 7,223 58.5 100.0Retentate 70 135.6 5,299 46.2 78.9Filtrate 310 0 2,015 0 —aOne unit of activity is defined as 1 millimole 4,6-ethylidene-p-nitrophenyl-�-D-maltoheptaoside degraded per 1 min.bAfter filtration through cheesecloth.

Table 2—Summary of the purification of amylase from honey

Purification Volume Total Total amylase Specific activity Purification Yieldstep (mL) A280 activity (units)a (units/A280) (-fold) (%)

Hollow-fiber retentate 7 530 4.41 0.0083 1.0 100.0UC supernatant 7 72.2 4.55 0.063 7.6 103.2Gel filtration 121 22.1 3.70 0.167 20.1 84Concentration 12 24.0 3.15 0.131 15.8 71.4Anion exchange 24 0.47 0.89 1.89 227.7 20.2Cation exchange 22 0.039 0.17 4.41 531.3 3.9aOne unit of activity is defined as 1 millimole 4,6-ethylidene-p-nitrophenyl-�-D-maltoheptaoside degraded per 1 min.

Figure 2—Gel-filtration chromatography of concentratedhoney amylase on Sephadex G-100 after ultracentrifuga-tion. Column (2.4 × 37.0 cm) eluted with 0.01 M Tris-Clbuffer, pH 7.4; 7 mL of sample was loaded and 2.5 mLfractions were collected with a flow rate of 1.5 mL/min atroom temperature.

Figure 3—Anion-exchange chromatography of the com-bined honey amylase peaks from Sephadex G-100 in 0.01M Tris-Cl buffer, pH = 7.4, with a linear NaCl gradient (0 to1 M). Fractions (1 mL) were collected at a flow rate of 0.5mL/min at 4 °C with an AKTA Purifier (Fast Protein Chro-matography System [FPLC]) system.

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for the starting honey sample. The concentrated and isolated en-zyme, after the ultracentrifugation step and gel-filtration had thesame major bands, which indicated that the contribution of the 1st3 purification steps, ultrafiltration, ultracentrifugation, and gel-fil-tration, was mainly to remove some non-protein ultraviolet-ab-sorbing materials from honey. The bands obtained with the amy-lase fractions from the cation-exchange step showed mainly themajor band (57 kDa) and faint traces of 2 smaller component

bands. This indicated that ion-exchange chromatography purifiedthe main amylase component to near homogeneity.

TLC analysis was performed to analyze the reaction products ofthe purified amylase from honey and determine the type (� or �) ofthis amylase. Commercially available pure �- and �-amylase werealso evaluated for comparison. Figure 6 shows that while pure �-amylase produced only maltose from waxy maize starch, pure �-amylase and honey amylase produced a number of intermediateproducts. This would be characteristic of �-amylases, which splitthe interior �-1,4-glycosidic bonds in a random manner (Kulp 1975).Thus, the purified amylase of honey was an �-amylase.

Evaluation of the purification results for honey amylase with ionexchange chromatography and with SDS-PAGE electrophoresis indi-cates a similarity to that of bee �-amylase, as reported by Ohashi andothers (1999). These authors purified �-amylase from forager-beehypopharyngeal glands through ion-exchange chromatography. Theamylase activity was in the flow-through fraction with the anion-ex-change column in both studies. In addition, amylase activity wasdetected as a single band at 45 mM NaCl with the bee amylase andat 50 mM NaCl with the honey amylase following elution from a cat-ion-exchange column. The molecular weight of both amylases wasestimated at 57 kDa as determined by SDS-PAGE. Solid confirmationof the origin of the honey amylase would be possible by determiningthe amino-acid sequence of the purified �-amylase from honey andcomparing it to that of the bee �-amylase.

Conclusions

In this study, a step-by-step procedure was developed to concen-trate, isolate, and purify the major amylase in honey. This was ac-

complished with a combination of ultrafiltration, ultracentrifuga-

Figure 5—Sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) pattern of honey proteins at puri-fication steps after staining with coomassie blue. Lanes:1 = molecular weight standards; 2 = starting honey sample;3 = after ultrafiltration and ultracentrifugation; 4 = aftergel filtration; 5 = after anion-exchange chromatography;6 = after cation-exchange chromatography.

Figure 6—Thin-layer chromatogram of the hydrolyzingproducts of waxy maize starch by pure �-amylase, �-amy-lase, and purified amylase from honey after incubation atpH 5.3 and 40 °C for 1 h. The reaction mixture was devel-oped by n-butanol:ethanol:water (5:3:2) and the sugarswere detected by sulfuric acid:methanol (1:3) solution andheating at 100 °C. Lanes: 1 = maltose; 2 = waxy maize; 3= pure �-amylase from sweet potato; 4 = pure porcine �-amylase; 5 = purified amylase from honey.

Figure 4—Cation-exchange chromatography of the com-bined amylase peaks from anion-exchange chromatogra-phy in 0.01 M Tris-Cl buffer, pH = 7.4, with a linear NaClgradient (0 to 1 M). Fractions (1 mL) were collected at aflow rate of 0.5 mL/min at 4 °C with an AKTA Purifier (FastProtein Chromatography System [FPLC]) system.

C418 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 6, 2005 URLs and E-mail addresses are active links at www.ift.org

C: Food Chemistry & Toxicology

Purification of Amylase from Honey

tion, and ion-exchange chromatography. The purified enzyme wasan �-amylase with a molecular weight of 57000 Da. The data fromthis research showed that the honey amylase had physical proper-ties similar to the bee enzyme.

AcknowledgmentsThis study was supported by the Rhode Island Agricultural Exper-iment Station and by a contract from the Natl. Honey Board.

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