Vol. 174, No. 11JOURNAL OF BACrERIOLOGY, June 1992, p. 3439-34440021-9193/92/113439-06$02.00/0Copyright C) 1992, American Society for Microbiology

Haloalkaliphilic Maltotriose-Forming ox-Amylase from theArchaebacterium Natronococcus sp. Strain Ah-36

TETSUO KOBAYASHI,,* HARUHIKO KANAI,2 TAKAYA HAYASHI,' TERUHIKO AKIBA,1RYOICHI AKABOSHI,2 AND KOKI HORIKOSHI1

Laboratory of Microbiology, The RIKEN Institute, 2-1 Wako, Saitama 351-01,1 and Department ofApplied Chemistry,Toyo University, 2100 Nakanodai, Kujirai, Kawagoe, Saitama 350,2 Japan

Received 21 October 1991/Accepted 12 March 1992

A haloalkaliphilic archaebacterium, Natronococcus sp. strain Ah-36, produced extracellularly a maltotriose-forming amylase. The amylase was purified to homogeneity by ethanol precipitation, hydroxylapatitechromatography, hydrophobic chromatography, and gel filtration. The molecular weight of the enzyme was

estimated to be 74,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibitedmaximal activity at pH 8.7 and 55°C in the presence of 2.5 M NaCl. The activity was irreversibly lost at lowionic strength. KCI, RbCl, and CsCl could partially substitute for NaCl at higher concentrations. The amylasewas stable in the range of pH 6.0 to 8.6 and up to 50°C in the presence of 2.5 M NaCl. Stabilization of theenzyme by soluble starch was observed in all cases. The enzyme activity was inhibited by the addition of 1 mMZnCI2 or 1 mM N-bromosuccinimide. The amylase hydrolyzed soluble starch, amylose, amylopectin, and,more slowly, glycogen to produce maltotriose with small amounts of maltose and glucose of an a-configuration.Malto-oligosaccharides ranging from maltotetraose to maltoheptaose were also hydrolyzed; however, malto-triose and maltose were not hydrolyzed even with a prolonged reaction time. Transferase activity was detectedby using maltotetraose or maltopentaose as a substrate. The amylase hydrolyzed 'y-cyclodextrin. a-Cyclodex-trin and ,B-cyclodextrin, however, were not hydrolyzed, although these compounds acted as competitiveinhibitors to the amylase activity. Amino acid analysis showed that the amylase was characteristically enrichedin glutamic acid or glutamine and in glycine.

Amylases that specifically produce malto-oligosaccharidesfrom starch have been reported. These include malto-hexaose-forming amylases from Bacillus sp. strain H-167 (2),Bacillus circulans (15, 19), and Klebsiella pneumoniae (Aero-bacter aerogenes) (4); a maltopentaose-forming amylasefrom Bacillus licheniformis (7, 14); maltotetraose-formingamylases from Pseudomonas stutzeri (13) and Bacillus sp.strain MG-4 (18); and maltotriose-forming amylases fromStreptomyces griseus NA-468 (21), Bacillus subtilis (16), andMicrobacterium impeniale (17).

In spite of many publications on amylases, all purifiedamylases but one are from eubacteria or eukaryotes, thesingle exception being an amylase from a hyperthermophilicsulfur-metabolizing archaebacterium, Pyrococcus woesei(5). Purification of amylases from other archaebacteria hasnot yet been reported. A great deal of information oneukaryotic and eubacterial amylases is currently available;research on an archaebacterial amylase, therefore, may yieldfundamental information about extracellular enzymes of thearchaebacteria.

Strains of the genus Natronococcus are haloalkaliphilicarchaebacteria. They require an alkaline pH as well as a highsalt concentration for growth (20). Enzymes from extremelyhalophilic bacteria are excellent materials for understandingthe nature of halophily. These enzymes function underextremely high salt conditions, and they often lose theiractivities at low ionic strength. Only a small number ofhalobacterial enzymes have been purified to homogeneitybecause conventional procedures for purification are unsuit-able in high-salt conditions. The only amylase from anextreme halophilic bacterium so far reported is a Halobac-

* Corresponding author.

terium enzyme which has not, as yet, been purified. Thisamylase was revealed by examination of a crude preparationto be halotolerant rather than halophilic (1).

This article deals with the purification and characteriza-tion of a haloalkaliphilic maltotriose-forming amylase from ahaloalkaliphilic archaebacterium, Natronococcus sp. strainAh-36.

MATERIALS AND METHODS

Bacterial strain and medium. Strain Ah-36 was isolated asan amylase producer from a soil sample from the shores of aKenyan soda lake, Lake Magadii. The taxonomy of thestrain was investigated by described methods (20). Thisorganism was a haloalkaliphilic archaebacterium and be-longed to the genus Natronococcus (this study).The standard medium used for cultivation of Natronococ-

cus sp. strain Ah-36 contained (per liter) 5.0 g of yeastextract (Difco), 5.0 g of Casamino Acids (Difco), 5.0 g ofpotato starch, 1.0 g of KCl, 1.0 g of NH4Cl, 1.0 g of KH2PO4,5.0 g of Na2CO3, 200 g of NaCl, 0.17 g of CaCl2. 2H20, 1.8mg of MnCl2. 4H20, 4.5 mg of Na2B407. 10H20, 0.22 mgof ZnSO4 7H20, 0.55 mg of CuCl2 2H20, 0.03 mg ofNa2MoO4 2H20, 0.03 mg of VOS04 2H20, and 0.01 mgof CoSO4 7H20 (pH 9.0).Amylase purification. The temperature was kept at 4°C

throughout the purification unless otherwise noted. A 2-day-old culture of Ah-36 (75 ml) was inoculated into 1.5 liters ofthe standard medium in a 5-liter Erlenmeyer flask andcultivated aerobically for 3 days at 37°C on a rotary shakeroperating at 350 rpm with a stroke of 7.5 cm. The culture wascentrifuged twice at 4,500 x g for 30 min to remove the cells.One volume of cold ethanol (-20°C) was gradually added tothe supernatant. The precipitate was collected by centrifu-

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3440 KOBAYASHI ET AL.

gation at 4,500 x g for 10 min and dissolved in 150 ml of abuffer containing 50 mM Tris-HCl (pH 8.0) and 2.5 M NaCI(buffer A). After removal of insoluble matter by centrifuga-tion at 4,500 x g for 10 min, the solution was dialyzedagainst buffer A overnight.The hydroxylapatite column chromatography was carried

out at 30°C to avoid crystallization of salts in the column.The enzyme solution was loaded onto a hydroxylapatite(Seikagaku Kogyo Co. Ltd.) column (1.5 by 20 cm) equili-brated with buffer A. The column was washed with 1.0 literof 50 mM sodium phosphate buffer (pH 8.0) containing 2.5 MNaCl. The enzyme was eluted with a 320-ml linear gradientof 50 to 400mM sodium phosphate buffer (pH 8.0) containing2.5 M NaCl at a flow rate of 0.8 ml/min. The eluted fractionswere immediately chilled on ice. The active fractions werepooled and dialyzed against buffer A overnight at 4°C.The hydrophobic chromatography was carried out at 9°C.

The enzyme solution containing 40% saturated (NH4)2SO4was loaded onto a column (1.5 by 20 cm) of butyl Sepharose4B (Pharmacia AB) equilibrated with buffer A containing40% saturated (NH4)2SO4 (buffer B). The column waswashed with 200 ml of the same buffer and then developedwith a negative linear gradient of (NH4)2SO4 from 40%saturation to 0% saturation at a flow rate of 0.7 ml/min. Theactive fractions were dialyzed against buffer A overnight.The enzyme solution was brought to 40% saturation of(NH4)2SO4 and loaded onto the same column. The columnwas washed with 200 ml of buffer B and eluted with anegative linear gradient of (NH4)2SO4 from 40% saturationto 20% saturation at 0.7 ml/min. The active fractions weredialyzed against buffer A overnight at 4°C.The enzyme solution was concentrated to 0.4 ml by using

Ficoll 400 powder and then a Centricon-10 microconcentra-tor (Amicon Co.) and loaded onto a column (1.5 by 40 cm) ofSephacryl S-200 (Pharmacia AB) equilibrated with buffer A.The enzyme was eluted at a flow rate of 0.07 ml/min, and theactive fractions were collected. The gel filtration step wasrepeated under the same conditions.

Gel electrophoresis. Polyacrylamide gel electrophoresis(PAGE) was carried out by using a premade gel system (TEFCo.) according to the supplier's instructions. Phosphorylaseb (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonicanhydrase (30 kDa), and trypsin inhibitor (20 kDa) were usedas molecular mass markers. Protein bands were visualizedby staining with 0.2% Coomassie brilliant blue R-250.

Active staining of the amylase in a native polyacrylamidegel was performed by incubating the gel in a buffer contain-ing 50 mM Tris-HCI (pH 9.0), 2.5 M NaCl, and 1% solublestarch at 50°C for 3 h. The amylase activity was visualized bystaining the gel in a solution containing 0.05% I2 and 0.5%KI.Amylase assay. The enzyme solution of 50 ,Ll was mixed

with 0.45 ml of 0.5% soluble starch in a buffer containing 50mM Tris-HCI (pH 9.0) and 2.5 M NaCl (buffer R) andincubated at 50°C for 30 min. The amount of reducing sugarliberated was determined by Nelson's adaptation of themethod of Somogyi (8). One unit of amylase activity wasdefined as the amount of enzyme which releases 1 ,umol ofreducing sugar equivalent to glucose per min.

Protein determination. Protein concentration was deter-mined by using a BCA protein assay kit (Pierce ChemicalCo.). Bovine serum albumin was used as the standard.TLC. The reaction products from several substrates pro-

duced by the amylase were desalted by using AG 501-X8(D)resin (Bio-Rad Laboratories) and subjected to thin-layerchromatography (TLC) with a precoated silica gel plate

(Kieselgel 60 F254; E. Merck). The products on the TLCplate, developed by multiple ascents with a solvent systemof n-butyl alcohol-methyl alcohol-water (4:2:1 [vol/vol/vol])were detected by the method described by Pastuska (10).

Identification of the anomeric form of product. The mu-tarotation of products from soluble starch with the amylasewas determined by the method of Robyt and French (12) byusing a digital polarimeter (Perkin-Elmer 241MC).Amino acid analysis. The amylase was hydrolyzed in

vacuo with 4 N methanesulfonic acid at 106°C for 22 h.Amino acid analysis was carried out with a Shimadzu LC-9Aamino acid analyzer.

Materials. Soluble starch was purchased from KantoChemical Co. Inc. Short-chain amylose (degree of polymer-ization [DP], 17), amylopectin, oyster glycogen, and pullulanwere the products of Nacalai Tesque Inc. Cyclodextrins andmalto-oligosaccharides were obtained from Nihon ShokuhinKako Co. The other chemicals used were of reagent grade.

RESULTS

Characterization of strain Ah-36. Strain Ah-36 was anaerobic, nonmotile, coccoid-shaped bacterium (1 to 2 ,m indiameter) occurring in irregular clusters, pairs, and singlecells. Cells did not lyse in distilled water. The temperaturerange for growth extended from 20 to 55°C, with an optimaltemperature of 40 to 45°C. Growth of strain Ah-36 occurredwithin a pH range of 8.0 to 10.0 (with an optimal pH of 9.0)and with a concentration of NaCl of between 8 and 30%(with an optimal concentration of 15 to 20%). Strain Ah-36hydrolyzed starch but did not liquefy gelatin. Reactions foroxidase and catalase production and for nitrate reductionwere shown to be positive. Growth inhibition was notobserved in the presence of ampicillin, chloramphenicol,polymyxin B, or streptomycin. The cells were sensitive toanisomycin, bacitracin, erythromycin, novobiocin, and tet-racycline. Strain Ah-36 had C20-C20 and C25-C20 diether corelipids. The molar ratio of G+C was calculated to be 63.5mol%. These results indicated that strain Ah-36 was ahaloalkaliphilic archaebacterium and belonged to the genusNatronococcus.

Production of amylase. Strain Ah-36 was aerobically culti-vated in 1.5 liters of the standard medium in a 5.0-literErlenmeyer flask at 37°C. The maximum cell concentrationwas achieved after 70 h of incubation. Amylase activity wasdetected at 35 h (0.01 U/ml) and reached a maximum level at90 h (0.12 U/ml). The level was retained for the following 110h. The activity was detected in the culture broth but not inthe cells. No activity was detected in the absence of starch.The addition of 0.1% glucose to the medium completelyinhibited the production.

Purification of the amylase. Since the amylase was irre-versibly inactivated at low ionic strength, the NaCl concen-tration was kept at 2.5 M throughout the purification. Theamylase activity was eluted between 250 and 300 mMsodium phosphate buffer from the hydroxylapatite columnand between 27 and 22% saturated (NH4)2SO4 from the butylSepharose 4B column. The enzyme was finally purified tohomogeneity by being passed twice through a SephacrylS-200 gel filtration column (2,000-fold purification and 10%recovery). The single and symmetrical absorption peak ofprotein was parallel to the peak of the amylase activity. Thefinal preparation had a specific activity of 160 U/mg ofprotein and gave a single protein band on sodium dodecylsulfate (SDS)-PAGE and native PAGE (Fig. 1). The amylaseactivity on the native polyacrylamide gel was visualized by

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HALOALKALIPHILIC AMYLASE FROM A NATRONOCOCCUS SP. 3441

94 _g

67 _

43 _____

1000"

20.1

M 1 2 3FIG. 1. SDS-PAGE (left, lane 1) and native PAGE (right, lanes 2

and 3) of the purified amylase. The amylase was visualized byCoomassie brilliant blue staining (lane 2) and by activity staining(lane 3). The enzyme (4 ,ug) was applied to each lane. The minimumamounts of the enzyme required for detection were 50 ng and 2 ng byCoomassie brilliant blue staining and activity staining, respectively.Lane M, molecular mass markers (in kilodaltons).

active staining as well as by Coomassie brilliant blue stain-ing. The mobility of the active amylase band coincided withthat of the single protein band stained by Coomassie brilliantblue.

Molecular mass. The molecular mass of the purified amy-lase was estimated to be 74,000 Da by SDS-PAGE (Fig. 1).The molecular mass estimated by the gel filtration purifica-tion step was 76,000 Da. These results indicated that theamylase had its activity in a monomeric form.

Effect of pH. The activity of the amylase was assayed inbuffers of various pH values in the presence of 2.5 M NaCl.The results are shown in Fig. 2. The amylase exhibited asharp pH activity profile with an optimum pH value of 8.7.To examine pH stability of the amylase, the enzyme wasincubated in buffers of various pH values for 30 min at 50°Cand the remaining activity was determined (Fig. 2). Theamylase was stable in a pH range of 6.0 to 8.6. The activitysharply declined in acidic conditions (44% inactivation at pH5.0 and 80% inactivation at pH 4.2), but in an alkaline rangethe enzyme was relatively stable, with only 30% loss ofactivity at pH 10.5. In the presence of 0.5% soluble starch,the enzyme was more stable: 100% activity remained be-tween pH 6.0 and 10.0, with 20% activity still remaining evenat pH 3.5.

Effect of temperature. The activity of the amylase wasmeasured at pH 9 for 30 min at various temperatures (Fig. 3).The optimum temperature was 55°C. To examine the ther-mostability, the amylase was incubated in a buffer containing50 mM Tris-HCl (pH 8.0) and 2.5 M NaCl for 30 min atvarious temperatures and the remaining activity was mea-sured. As shown in Fig. 3, 50% activity was observed at55°C. Soluble starch stabilized the enzyme; 100% activityremained at 55°C in the presence of 0.5% soluble starch. Thethermal stability was not enhanced by Ca2+ ion (data notshown).

Effect of salt concentrations. A high-ionic-strength require-ment for activity is a characteristic feature of halophilicenzymes. The amylase activity was determined at various

100

;O.*4-

4-

P50

02 4 6 8 10 12

pHFIG. 2. Effect of pH on the amylase activity and stability. Closed

circles represent the activity at each pH value. For determining thestability, the amylase was incubated for 30 min at 50'C in buffers ofvarious pH values containing 2.5 M NaCl in the presence (triangles)or absence (open circles) of 0.5% soluble starch. The remainingactivity was measured in buffer R containing 0.5% soluble starch asdescribed in Materials and Methods. Citrate-phosphate buffer (0.1M) was used at pH values between 2.5 and 7.0, Tris-HCl (0.1 M) wasused at pH values between 7.0 and 9.0, and glycine-NaCl-NaOH(0.1 M) was used at pH values between 8.5 and 11.0.

NaCl concentrations. The amylase showed maximum activ-ity in the presence of 2.5 M NaCl (Fig. 4). No activity wasdetected below 1.0 M NaCl; however, 20% activity could bedetected even at 5 M NaCl. KCl, RbCl, and CsCl weresubstitutable for NaCl, although higher concentrations wererequired for maximum activity. Ninety-two percent maxi-mum activity was observed at 4.0 M KCl, 35% was observedat 4.5 M RbCl, and 28% was observed at 5.0 M CsCl. Noactivity was detected with LiCl or NH4C1.

100

e 80. -

* 600

.> 40

20

020 30 40 50 60

Temperature (°C)70 80

FIG. 3. Effect of temperature on the amylase activity (dottedline) and stability (solid lines). For determining the thermostability,the amylase was incubated at each temperature for 30 min in thepresence (squares) or absence (triangles) of 0.5% soluble starch andthe remaining activity was then measured.

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3442 KOBAYASHI ET AL.

100

~80

>~ 60

>~ 40

~20

00 1 2 3 4 5

Salt concentration (NFIG. 4. Effect of monovalent cations on the am3

The activity was measured in the reaction buffer contconcentrations of NaCl (closed circles), KCI (tri(squares), and CsCl (open circles). KCI was insolubletions above 4 M.

The amylase was incubated at 50°C and pH 9various NaCl concentrations, and the remainingthen measured under the standard condition. TIshown in Fig. 5. The amylase was most stable alunder this condition. The amylase was inactpletely by incubation at 1.3 M NaCl, and 80% oiactivity was inactivated at 1.7 M NaCl. On thethis amylase was relatively stable at higher NaCtions, with 70% activity remaining at 4.4 M. Inof 0.5% soluble starch, no loss of activity was cconcentration between 2.1 and 3.6 M NaCI. The

100

tkm.

U- 50

0

0 1 2 3Salt concentration (M)

FIG. 5. Effect of NaCl and KCl on the amylaseamylase activity was measured after incubation at 5001various concentrations of NaCl (closed circles and sc(open circles and squares) in the absence (circles(squares) of 0.5% soluble starch.

GI

G2

G4 §sE||2

G5G6G77 .

,W 1N 11E_WM 1 2 3 4 5 6 7 8 9 10 11 M

FIG. 6. Action patterns of the amylase on various substrates.6 7 The reaction products from soluble starch (lane 1), amylose (DP, 17)

i) (lane 2), amylopectin (lane 3), glycogen (lane 4), and -y-cyclodextrin(lane 5) were analyzed by TLC. Lanes 6 to 11 are the products from

ylase activity. malto-oligosaccharides ranging from maltose to maltoheptaose (leftaining various to right). Lane M represents a standard mixture of malto-oligosac-angles), RbCl charides ranging from glucose (Gl) to maltoheptaose (G7).at concentra-

act30mivt wat at a low NaCl concentration was irreversible; i.e., no activ-for 30. mm a ity was recovered by increasing the NaCl concentration. The

enzyme was also stabilized in the presence of higher con-e results are centrations of KCI.

t 2.5 MNaCl The effect of various anions (as sodium salts) on thetivated com- amylase activity was determined. The amylase preferredf the enzyme anions in the following order: citrate>chloride>acetate. The

Kother hand, relative activity for each anion was 130, 100, and 80%,'I concentra- respectively. In all of these cases, the amylase activitythe presence reached its maximum at 2.5 M sodium ion even when sodium)bserved at a citrate was used (0.83 M sodium citrate). No activity wasinactivation detected when NaF, NaBr, or NaCIO4 was used.

Effect of metal ions and chemical reagents. The amylaseactivity was assayed in the presence of various metal ions (aschloride salts) at 1 mM or in the presence of chemicalreagents by using the purified amylase which had beendialyzed against a buffer containing 10 mM Tris-HCI (pH8.0), 2.5 M NaCl, and 1 mM EDTA. The enzyme did not loseany activity by this dialysis. None of the metal ions stimu-lated enzyme activity. Zn2+ completely inhibited enzymeactivity. Hg2" ion, which inactivates most amylases at 0.1mM, did not have a strong inhibitory effect on this amylase;85% activity remained at 1 mM and 25% remained even at 10mM. Ca2" and Ni2+ ions showed 33% inhibition. Theenzyme lost 80 and 74% activity in the presence of 0.1% SDSand 1 M urea, respectively. lodoacetic acid and phenylmeth-ylsulfonyl fluoride did not have an inhibitory effect at 1 mM,while N-bromosuccinimide showed 73% inhibition of theenzyme activity, suggesting the involvement of tryptophanin the catalytic action.Mode of action of the amylase. The amylase hydrolyzed

soluble starch, amylose, amylopectin, and glycogen to mal-4 5 totriose and small amounts of maltose and glucose (Fig. 6).

The enzyme did not hydrolyze pullulan, ao-cyclodextrin, or

stability. The 3-cyclodextrin. -y-Cyclodextrin, however, was hydrolyzed,C for 30 min at indicating that the amylase action was endolytic. Table 1quares) or KCl shows Km and ko values of the enzyme for various sub-

or presence strates. The amylase hydrolyzed amylose most rapidly,whereas glycogen was slowly hydrolyzed. The enzyme had

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HALOALKALIPHILIC AMYLASE FROM A NATRONOCOCCUS SP. 3443

TABLE 1. Km and ko values for various substrates

Substrates Km (mM)a ko (s-1)b

Soluble starch 25 250Amylose (DP, 17) 19 610Amylopectin 49 370Oyster glycogen 42 48y-Cyclodextrin 6.8 70

a The Km values are shown as glucose equivalents.b The ko values are obtained as Vmax/eo, where eo was the molar concen-

tration of the enzyme (1.9 pmol).

the smallest Km value for -y-cyclodextrin, although the kovalue was very low.

Figure 6 also shows the action of this amylase on variousmalto-oligosaccharides ranging from maltose to maltohep-taose. Maltoheptaose and maltohexaose were mainly hydro-lyzed to maltotriose; maltopentaose was mainly hydrolyzedto maltotriose and maltose; and maltotetraose was mainlyhydrolyzed to maltotriose and glucose. Production of smallamounts of maltose and/or glucose was observed in all cases.Maltotriose and maltose were not hydrolyzed even with aprolonged reaction time. When maltotetraose was used as asubstrate, maltopentaose and maltohexaose were formed.Maltohexaose was also detected when maltopentaose wasused. These results indicate that the amylase has transferaseactivity.The anomeric form of product produced by the amylase

was examined with a polarimeter. The results indicated thatthe product had an ao-configuration.

Competitive inhibition by a- and 13-cyclodextrins. A Line-weaver-Burk plot of the amylase in the presence of 0.25%a-cyclodextrin or 0.025% ,B-cyclodextrin indicated that theinhibition was competitive, with Ki values of 1.63 and 0.41mM (9.8 and 2.9 mM as glucose equivalent) for a- and,-cyclodextrins, respectively.Amino acid composition. The amino acid composition of

the amylase was compared with those of nonhalophilicamylases from B. subtilis (22) and P. woesei (5) (Table 2).The most prominent features of the Natronococcus amylasewere remarkably high contents of glutamic acid or glutamineand of glycine. The content of dicarboxylic amino acids ortheir amides was about 30% of the total amino acid residues.On the other hand, the basic amino acid content was low,particularly that of lysine. The amylase was also character-istic in its low serine and threonine content.

DISCUSSION

In this article we described the purification of Natrono-coccus amylase and its properties. This amylase was the firstamylase purified from halophilic archaebacteria. This mal-totriose-forming amylase required a high salt concentrationof 2.5 M NaCl for its enzyme activity and stability. Further-more, the amylase activity was irreversibly and completelyinactivated by exposure to low ionic strength. Conventionalion-exchange chromatography was not available underhigher saline conditions. Therefore, we used hydroxylapatitechromatography and butyl Sepharose 4B chromatographyfor purification.There have been only a few reports on extracellular

enzymes of halophilic archaebacteria. Until now, Halobac-tenium amylase was the only known amylase of extremehalophiles. Good and Hartman reported the properties of theHalobactenum amylase as determined by using a crude

TABLE 2. Comparison of the amino acid composition ofa-amylase from Natronococcus sp. strain Ah-36 with those of

oa-amylases from nonhalophilic bacteria

Amino acid composition (mol%) of a-amylase from:Amino acid Natronococcus sp. B. subtilia P. woesei

strain Ah-36

Asx 14.7 14.9 10.8Thr 4.2 6.9 7.7Ser 2.9 8.9 5.1Glx 14.7 9.6 8.1Pro 7.0 2.9 4.3Gly 13.3 7.4 8.4Ala 8.8 7.8 6.9Cys 0.5 0.2 NDbVal 7.1 4.4 5.5Met 1.7 1.8 2.0Ile 2.0 5.6 4.0Leu 7.0 6.5 6.7Tyr 3.5 4.2 5.7Phe 2.7 4.2 4.8His 3.6 2.7 3.4Lys 0.8 4.7 5.7Trp 0.9 2.2 NDArg 4.2 4.2 4.6

a The amino acid composition of the mature amylase of B. subtilis wascalculated from the amino acid sequence deduced from the DNA sequence.

b ND, not determined.

preparation (1). The amylase was found to be halotolerantrather than halophilic, although 33% activity was maintainedin 4 M NaCl. The production of a halophilic amylase in amoderately halophilic Micrococcus species was reported byOnishi (9). The amylase exhibited maximal activity in 1.4 to2 M NaCl or KCl, and no difference between the effects ofthe two salts was shown. In the case of Halobacteriumprotease, the effects of NaCl and KCl were the same (3).Natronobacterium protease showed a marked preference forNaCl over KCl (23). In this study, the optimum concentra-tions of NaCl and KCl for the Natronococcus amylase were2.5 and 4 M, respectively. The maximal activity of theNatronococcus amylase in KCl was 90% of that in 2.5 MNaCl. Thus, this amylase preferred NaCl over KCl. Theseobservations may reflect a higher concentration of NaCl thanof KCl outside the cell.

Interestingly, increased ion radii of alkali metal ionsresulted in a requirement for higher concentrations formaximal activities: Na+ at 2.5 M, K+ at 4.0 M, Rb+ at 4.5M, and Cs+ at 5.0 M. When sodium citrate was used, themaximal activity was detected at 2.5 M Na+ (0.83 M sodiumcitrate), a concentration which coincided with the optimumconcentration of NaCl or sodium acetate. These observa-tions suggested that the optimum salt concentration for theamylase activity was determined only by cation concentra-tion. A possible explanation of the effect of alkali metal ionson the Natronococcus amylase is that the amylase requiresthe binding of a certain amount of cations for its activity andstability. The affinity of the cations to the enzyme maydecrease with increased ion radii or with decreased positive-charge density.An extremely high content of acidic amino acids is one of

the general characteristics of halophilic proteins (6). TheNatronococcus amylase exhibited a remarkably high contentof glutamic acid or glutamine in comparison to those exhib-ited by mesophilic amylases, while the content of asparticacid or asparagine exhibited was similar to that of the

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3444 KOBAYASHI ET AL.

mesophilic enzymes. The lysine content was extremely low,resulting in the low content of basic amino acids. Therefore,it is highly possible that the amylase is, like other halophilicproteins, extremely acidic. Rao and Argos reported that agreat increase of acidic charges was found at the surface ofa halophilic 2Fe-2S ferredoxin when it was compared withthe Spirulina platensis ferredoxin, the three-dimensionalarchitecture of which had been determined by X-ray crys-tallography (11). They suggested that halophilic proteins cancompete very effectively with cytoplasmic salts for water bymeans of their external carboxyl groups and thus obtainoptimal hydration. Water is a hidden substrate of the amy-lase reaction. Therefore, in the case of the Natronococcusamylase, the catalytic center must compete with salts forwater. Inhibition of the enzyme activity at a higher NaClconcentration may be caused by this competition.The amylase isolated from Natronococcus sp. strain

Ah-36 hydrolyzed starch, amylose, amylopectin, glycogen,and -y-cyclodextrin to maltotriose of a-configuration as themain product. Therefore this enzyme is an a-amylase whichcleaves a-1,4 linkages endolytically. There are three knownamylases that specifically produce maltotriose from starch,which were found in S. griseus NA-468 (21), B. subtilis (16),and M. imperiale (17). The Streptomyces amylase hydro-lyzes starch with maltotriose units from its nonreducing endsby means of an exo mechanism. The other amylases hydro-lyze starch by an endo mechanism producing maltotriosewith small amounts of maltose and glucose. The action ofNatronococcus amylase is also endolytic. In addition, thisamylase possesses transglycosylating activity.

ACKNOWLEDGMENTSWe thank W. D. Grant and T. Hamamoto for providing us with

the soil samples from Lake Magadii. We are also grateful for manyhelpful and creative discussions with T. Kudo. We thank K. J.Sutherland for reading the manuscript and for useful discussions.

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