Agric. Biol Chem., 49 (4), 1091 -1097, 1985 1091

An Amylase Producing Maltotriose from Bacillus subtilis^Yoshiyuki Takasaki

Fermentation Research Institute, Agency of Industrial Science and Technology,Ministry of International Trade and Industry,1-1-3, Azuma, Yatabe-machi, Tsukuba-gun,

Ibaraki 305, JapanReceived September 12, 1984

An amylase which produces maltotriose from starch as the main product was found in theculture filtrate of a strain of Bacillus subtilis newly isolated from soil. The enzymewas purified toalmost complete homogeneity, as judged by disc electrophoresis in polyacrylamide gel, by means ofammoniumsulfate fractionation, DEAE-Sepharosecolumn chromatography and Sephadex gelfiltration.

The optimum pH and temperature of the enzyme were around 6~7 and 50°C, respectively.Metal ions such as Hg2+, Cu2+, Zn2+, Ni2+ and Fe2+ strongly inhibitied the enzyme activity. Themolecular weight was found to be about 25,000 by gel filtration. The yields of maltotriose fromshort-chain amylose (DP 17), amylopectin, soluble starch and glycogen were about 69, 56, 56 and40%, respectively.

Numerous studies on amylases producingglucose or maltose from starch have hithertobeen reported. However, there have onlybeen a few reports on amylases specificallyproducing maltooligosaccharides from starch.G6-producing amylase have been found inKlebsiella pneumoniaeX) and Bacillus cir-

culans,2'3) a G5-produeing amylase in Bacilluslicheniformis,4) a G4 and G5-producing amy-lase in Bacillus circulans5) and a G3-producingamylase in Streptomyces griseus NA-468.6)The author has isolated a microorganismproducing an amylase which specifically pro-duces maltotriose from starch. It seems thatthe enzyme differs in its action pattern onstarch from the G3-producing amylase of S.griseus NA-468.

This paper describes the purification andcharacteristics of the enzyme.

MATERIALS AND METHODS

Microorganism used. The microorganism used in thisstudy was isolated from soil. The taxonomical propertiesof the strain are presented in Table I. The strain wasidentified as a strain of Bacillus subtilis from such proper-ties as: it was gram-positive, spore-forming, growth-aerobic, catalase-positive, acetylmethylcarbinol-pro-ducing, starch-hydrolyzing, nitrate-reducing etc. The

stain was tentatively named Bacillus subtilis G3.

Cultivation method. The stain was cultivated in a me-dium (50ml, pH 8.0) containing 4% polypepton, 0.3%K2HPO4, 0.1% MgSO4-7H2Oand 2% soluble starch in a200ml Erlenmeyer flask. Cultivation was carried out for48hr at 30°C on a rotary shaker at 200rpm. Cell wereremoved by centrifugation and the resultant supernatantwas used as the material for purification.Assaying of the enzyme. The enzyme activity was mea-sured in a l ml reaction mixture containing 0.5ml of 2%soluble starch in 0.1 m phosphate buffer (pH 7.0) and theenzymesolution. The reaction was carried out for lOminat 40°C and the reducing sugar formed was determined bythe Somogyi & Nelson method.

One unit of the enzymewas defined as the enzyme

f Studies on Enzymatic Production of Oligosaccharides. Part III.This paper was presented at the Annual Meeting of the Agricultural Chemical Society of Japan in Sendai on April

1 -4, 1983 (Abstracts of Papers, p. 169).Remarks: G1? G2, G3, G4, G5, G6 etc. in this paper denote glucose, maltose, maltotriose, maltotetraose,

maltopentaose, maltohexaose, etc.

1092 Y. Takasaki

Table I. Taxonomical Properties of a Strain

Rod: 0.5 to 0.7 by 0.8 to 1.2 microns, occurring singly

or in pairs, non-motile, gram positive.Spores: Ellipsoidal, terminal, sporangia not swollen.Growth: Good growth under aerobic conditions, scant

if any growth under anaerobic conditions.G elatin stab : L iq uefaction .Agar colonies: Round and smooth growth, cream-

colored to yellow.Agar slants: Smooth, cream-colored to yellow.Nutrient agar slants: Growth abundant, smooth,

cream -co lored to yello w .Glucose-nutrient agar slants: Growth abundant,

smooth, cream colored to yellow.G lu co se-nitrate agar slants: G row th scan t.Soybean agar slants: Growth more abundant than on

agar slants.Tyrosine-agar slants: Good growth, smooth, yellow.Nutrient broth: Uniform and light turbidity with

sedim en t.NaC l broth: Good growth up to lO% NaCl.Milk: Slowly peptonized.Starch : H ydrolyzed .A cetylm ethy lcarbinol: P rod uced .C atalase: Produ ced.

R e d u c t i o n o f n i t r a t e : P o s i t i v e .C itrate: U tilized .Temperature for growth: Optimum growth temperature

is 38 ~46-C, maximum growth temperature is46 ~ 51-C .

Utilization of carbohydrates: Acid but not gas formedfrom D-glucose, D-fructose, D-mannose, D-galactose.D -xylose, L-arab ino se, m annito l, sucrose, m altosean d starch .

amountproducing reducing sugar corresponding to one/miol of glucose from soluble starch in one min under theassay conditions.

Determination of protein. Protein was determined ac-cording to Lowry et al.7) using bovine serum albumin

(Fraction V) as a standard protein, and also from theoptical density at 280nm.

Paper chromatography. Paper chromatography for de-tection and determination of sugar was carried out asdescribed in the previous paper.2)

Disc electrophoresis. The electrophoresis was carried outfor 40min at 3.5mA per gel at room temperature, andprotein was stained with Amido black.

Estimation of molecular weight. The molecular weight ofthe purified enzyme was estimated according to Andrew'smethod.8) A column (1.5x88cm) of Sephadex G-100

which was equilibrated with 2.5 x 10~3 m Tris buffer (pH

7.0) was used.

Materials. Four kinds of protein (cytochrome c,chymotrypsinogen, hen egg albumin and bovine serumalbumin) were obtained from Boehringer MannheimGmbH; pullulan, maltotriose and short chain amylose

(DP 17) were from Hayashibara Biochemical Labora-tories, Okayama, Japan; pullulanase (from Klebsiellapneumoniae) from Nagase Biochemicals Ltd., Japan;and potato starch, amylopectin (corn) and glycogen

(oyster) from Wako Chemical Ltd., Japan.

RESULTS

/. Purification of the enzymeThe G3-producing amylase of B. subtilis wasproduced extracellularly. After the maximumyield of the enzymewas obtained in the culturemedium, the broth was centrifuged to removethe cells. To 1 liter of the supernatant, am-moniumsulfate was added to 70%saturation.The precipitate was collected, dissolved indistilled water and dialyzed against 2.5x1(T3 M Tris buffer (pH 7.0).

DEAE-Sepharose column chromatography.The dialyzate (400ml) was applied on aDEAE-Sepharose column (2.5 x 31 cm)equilibrated with 2.5 x 10~3 m Tris buffer (pH7.0). After washing with the same buffer so-lution, the enzymewas eluted from the columnby linearly increasing the concentration of KC1in the buffer from 0 to 0.5m. The G3-producing amylase fraction was collected, con-centrated with a membrane filter and dialyzedagainst 2.5 x 10~3m Tris buffer (pH 7.0).Sephadex G-100 and G-150 column chroma-tography. The enzyme solution (10 ml) from theprevious stage was applied on a SephadexG-100 column (2.5 x92cm) equilibrated with2.5x 10~3m Tris buffer (pH 7.0) and elutedwith the same buffer. The G3-producing amy-lase fraction was collected, concentrated andthen further purified on a Sephadex G-150

column (1.5 x 90cm). A summary of the purifi-cation results is given in Table II.

Figure 1 shows the results of disc elec-trophoresis of the purified enzyme. The en-

zyme was found to be almost homogeneousondisc electrophoresis.Estimation of molecular weight. The molec-

Production of Maltotriose by an Amylase from B. subtilis 1093

Table II. Purification of the Enzymefrom B. subtHis

Speci ficFra cti on a nd P rote in A myla se a ctiv ity Yie ld

step (O D 280nm) (units) (units/ (% )O D 280nJ

M ateria l(A m . su lfate 3,230 620 0. 192 100

-80% )

D E A E -Seph arose 140 283 2 .02 45.6S e ph a d e x G - 1 0 0 3 4 . 3 1 6 6 4 . 84 2 6. 8S e p h a d e x G - 1 5 0 9 . 8 8 1 0 1 1 0 . 2 1 6 . 3

-start

Fig. 1. Disc Electrophoretic Pattern of the PurifiedEnzyme.

Theelectrophoresis wascarried out at a constant currentof 5mA per tube for 40min on polyacrylamide gel at pH

9.4.ular weight of the enzyme was estimated bythe Sephadex G-100 gel filtration method. Theresults are shown in Fig. 2. The molecularweight of the enzyme was estimated to beabout 25,000.

//. Enzymatic properties of the enzymeEffect of pH and temperature on the enzymeactivity. Figure 3 shows the optimum pHand pH stability of the enzyme. The optimumpH was 6~7 and the enzyme was stable inthe pH range of from about 6 to 8.

Figure 4 shows the optimum temperature

100-i

I80 \£ å  \

160- \4

c 3No- \

5^oLU

1 04 105Molecular weight

Fig. 2. Plots of Elution Volume against Logarithms ofMolecular Weight of Proteins on Sephadex G-100.Column, Sephadex G-100 (1.5 x 88cm); flow rate, 20ml/hr; fraction, 3 ml.1, cytochrome c; 2, chymotrypsinogen; 3, albumin (from

hen egg); 4, albumin (from bovine serum); å , the amylasefrom B. subtHis.

^60- ff \ \

3 A 5 6 7 8 9 rOPH

Fig. 3. Effect of pH on Activity and Stability of theEnzyme.

O-O, pH-activity curve (the reaction was carried outunder the standard assay conditions except for the re-action.pH); D-å¡, pH-stability curve (the mixture(0.4ml) containing 0.1 m buffer solution and 0.1 units ofthe enzyme was kept for 3 hr at 25°C. After adjustment ofthe pH to 6.0 by adding 0.4ml of 0.4m phosphate buffer(pH 6.0), the residual enzyme activity was measured.).

and heat stability of the enzyme. The optimumtemperature was about 50°C (30 min reaction).The enzyme was unstable above 45°C. How-ever, the presence of calcium ions protect-ed the enzyme from heat as shown in Fig. 4.Effect of metal ions. Table III shows the

effects of various metal ions on the enzymeactivity. Stimulation of the enzyme activitywas not observed. Metal ions such as Ag+,

Hg2+, Cu2+, Zn2+, Fe2+ etc. showed a stronginhibitory effect.

1094 Y. Takasaki

Action pattern of the enzyme. Figure 5 (A)shows a paper chromatogram illustrating theaction q{ the enzyme from B. subtilis on sol-uble starch, amylopectin, amylose, glycogen,pullulan and cyclodextrin. Maltotriose wasformed as the main product from starch, amy-lopectin, amylose and glycogen. The yields of

maltotriose were about 56% from soluble

starch, about 56% from amylopectin, about69% from amylose (DP 17) and about 40%from glycogen (Table IV). The yield of malto-triose from soluble starch remarkably increas-ed in the presence of pullulanase (Table V).

~100å  å * *><?I-^

S60- / \ I\

Ia°" V\\20- \\\

20 30 40 50 60

Temperature(°C)

Fig. 4. Effect of Temperature on Activity and Stabilityof the Enzyme.O-O, temperature-activity curve (the reaction was car-ried out for 30min under the standard assay conditionsexcept for the reaction temperature.); å¡-D, heat stabili-ty (the enzyme solution (0.13units) in 5x 10~2m Tris

buffer (pH 7.0) was heated for lOmin at various tempera-tures as indicated and the residual enzymeactivity wasmeasured.); å -å , calcium chloride (5x 10~3m) was

added in the above mixture for the heat treatment.

Table III. Effect of Metal Ions on theEnzyme Activity

The reaction mixture containing 0.05 m Tris buffer (pH7.0), 1% soluble starch, 5x 10"3m metal salt and 0.13

units of the enzyme in a total volume of 0.2ml.M eta l io n R ela tiv e a c tiv ity

(5 x 1(T 3 m ) (% )

N o n e 10 0

M g S O 4 10 2.4

M n C L 10 1. 6

C o C L 8 4.0

C aC l, 10 2.2

S rC l, 6 4.2

B a C l2 7 2.2

F e S O 4 2 8. 8

Z n S O 4 4.8

N iC l, 4 7 .3

C u S O 4 2.6

A g N O 3 17. 3

H g C l, 1. 6

å PiiHi

å å å  !à"à"à"à"t

å I

(A)

(B)(C)

Fig. 5. Paper Chromatograms of Hydrolysates of Several Substrates with the Enzyme.(A) substrate: starch, amylose, amylopectin, glycogen, a-cyclodextrin, /?-cyclodextrin, pullulan, standards.

(B) substrate, maltotriose (commercial); s, standards.(C) substrate, soluble starch; s, standards.

Production of Maltotriose by an Amylase from B. subtilis 1095

The enzyme did not attack pullulan, a-cyclo-dextrin or /J-cyclodextrin. Maltotriose was

the final product and was not hydrolyzed bythe enzyme as shown in Fig. 5(B).The time course of hydrolysis of starch bythe enzyme is shown in Figs. 5(C) and 6. Thereaction was carried out in 2%potato starch.The reducing sugar formed was determined bythe Somogyi-Nelson method and the sugarcomposition was analyzed by liquid chroma-tography. Typical results of liquid chromatog-raphy of the hydrolysate are shown in Fig. 7.The formation of reducing sugar and that ofmaltotriose in the hydrolysate (expressed as

the saccharification ratio) showed good coin-cidence at the early stage of the saccharifi-cation reaction. The iodine-staining power ofthe hydrolysate gradually decreased as thereaction progressed and finally almost com-

pletely disappeared.Table IV. Sugar Compositions of Hydrolysates

of Various Substrates Producedby the Enzyme

Each reaction mixture containing lO mg of substrate,0.1 ml of0.2m Tris buffer(pH 7.0), 1 x 10~2mCaCl2 andthe enzymein a total volume of 1.0ml was incubated for24hr at 50°C.

S u b stra teG lu c o se M a lto se M alto trio se O th e rs

(% ) ( % ) (% ) (% )

S ta rc h 4 . 8 2 5 . 8 5 6 . 1 1 3 . 3

A m y lo se(D P 1 7 )

4 . 2 2 3 . 4 6 8 . 7 3 . 7

A m y lo p e c tin 5 . 2 2 2 . 0 5 6 . 0 1 6 . 7G ly co g e n 2 . 1 8 . 4 3 9 . 7 4 9 . 7

§20- 80rt

å i |\ / Q5eS s? \ / S

3 o \ ^^ -03=

I5-20. >\ ^ui / "V -0.1 oå -~-*~

J J 1 ; 1 1 4£^*-'2 U 6 8 24

Reaction time(hr)

Fig. 6. Time Course of Hydrolysis of Starch by the

Enzyme.

The reaction mixture containing 80mgof potato starch,2xl(T2M Tris buffer (pH 6.0), 5x10~3m CaCl2 and

0.21units of the enzymein a total volume of 4ml wasincubated at 40°C.x-x , iodine reaction; O-O, content of maltotriose;A-A? sacchariflcation (as glucose).

m

m

å  _|.-mi r*-ff

Fig. 7. Chromatogram of the Hydrolysate of SolubleStarch on Liquid Chromatography.Column, Shodex NH-pack J-411; eluent, acetonitrile-

water (65 : 35); detector, Shodex RI, model SE-ll.1, glucose; 2, maltose; 3, maltotriose; 4, maltotetraose.

Table V. Effect of Pullulanase on Hydrolysis of Starch

Each reaction mixture containing 50mg of liquefied starch (DE 4.3), 0.1 ml of 0.2m Tris buffer (pH 7.0)and 0.31 units of the enzyme (with or without 0.15 units of pullulanase) in a total volume of 1.0ml wasincubated for 48hr at 50°C.One unit of pullulanase was defined as the amount of the enzyme producing reducing sugar corresponding

to one fimol of glucose from pullulan in one min under the assay conditions of 0.5% pullulan and 0.05mphosphate buffer (pH 7.0).

G lu c o s e M a lt o s e M a l t o t r i o s e M a lt o t e t r a o s e O t h e r s

( % ) ( % ) W o ) ( % ) ( % )

C o n tr o l 2 . 8 1 2 . 1 5 5 . 3 4 . 4 2 5 . 4

4 - P u l l u l a n a s e 5 . 1 1 0 . 5 7 3 . 3 3 . 8 7 . 3

1096 Y. Takasaki

60f o-a-^_^__o

50å 

rA0'

|30-

o%20- _ n-

g, -D-n n-a a510-

à"à" à" à" à"t |à"

2 A 6 8 10 12

DE ofsubstrateFig. 8. Effect of DE of Liquefied Starch on Yield ofMaltotriose.

The reaction mixtures containing of 50mgof liquefiedstarch of various DE, 0.02m Tris buffer (pH 7.0), l x10~2m CaCl2 and 0.31units of the enzyme in a total

volume of 1.0ml were incubated for 48hr at 50°C.O-O, maltotriose; å¡-D, maltose; $-#, glucose.

Figure 8 shows the effect of the DE ofliquefied starch on the yield of maltotriose.The liquefied starch having DE of 1.5 to 12.6used in this experiment was prepared by theprocedure described in the previous paper.9)The yield of maltotriose was slightly influencedby the DE of the substrate. A 55-60% yield ofmaltotriose was obtained with the use of a

substrate of a DE below 8.Mutarotation of the product. The mutaro-tation of the product from soluble starch withthe enzymefrom B. subtHis was examinedwitha polarimeter. Adecrease in the optical ro-tation was observed after cessation of thereaction. This indicates that the product hasthe a-configur-ation and the enzyme is a kind ofa-amylase.

DISCUSSION

An amylase which produces maltotriosefrom starch as the main product was found in

the culture filtrate of B. subtilis which wasisolated from soil and identified by the author.

The enzyme was purified to almost com-pletely homogeneity, as judged by disc elec-

trophoresis, by means of ammoniumsulfatefractionation, DEAE-Sepharosecolumn chro-matography and gel filtration with Sephadex

G-100 and G-150.

The enzyme produced maltotriose as themain and final product from various a-1,4-glucan. The yields of maltotriose from short-chain amylose (DP 17), amylopectin (corn),soluble starch and glycogen (oyster) wereabout 69, 56, 56 and 40%, respectively. Theenzyme produced small amounts of glucose

and maltose from the early stage of the hy-drolysis reaction. A typical sugar compositionof the end product from liquefied starch (DE4.3) was 2.8% G1? 12.1% G2, 55.3% G3, 4.4%G4 and 25.4% others. The yield of maltotriosewas about 73% from liquefied starch ofDE 4.3whenthe enzymereaction was carried out inthe presence of pullulanase. This high yield ofmaltotriose might be due to the longer chainlength of the liquefied starch compared withthe case of short chain amylose (DP 17). Theenzyme did not attack pullulan, a-cyclodextrinor /?-cyclodextrin.

Wako et al.6) found an amylase inStreptomyces griseus NA-468 which hy-

drolyzes starch with maltotriose units from itsnon-reducing ends by means of the exo-mechanism. The enzyme does not produceglucose and maltose from soluble starch.Therefore, the enzyme from S. griseus NA-468may be regarded as having a different actionpattern from that of the enzyme from B.sub tilis.

From the facts that the anomer form of theproduct with the enzyme from B. subtilis wasfound to be the a-form in the study on themutarotation of the product and that theiodine-staining power of the hydrolysate grad-ually decreased and finally disappeared withthe progress of the hydrolysis reaction, it couldbe concluded that the enzyme from B. subtilisis a kind of a-amylase exhibiting the endo-mechanism.

REFERENCES

1) K. Kainuma, K. Wako, S. Kobayashi, N. Nogamiand S. Suzuki, Biochim. Biophys. Acta, 410, 333(1975).

2) Y. Takasaki, Agric. Biol. Chem., 46,.1539 (1982).3) H. Taniguchi, M. Tei, Y. Maruyama and D.

Nakamura, Dempun Kagaku, 29, 107 (1982).

Production of Maltotriose by an Amylase from B. subtHis 1097

N. Saito, Arch. Biochem. Biophys., 155, 290 (1973).Y. Takasaki Agric. Biol. Chem., 47, 2193 (1983).K. Wako, S. Hashimoto, S. Kubomura, K. Yokota,K. Aikawa and J. Kanaeda, Dempun Kagaku, 26, 175

(1979).

7) O. H. Lowry,N.J. Rosebrough,A. L. FarrandR. J.Randall, /. Biol. Chem., 193, 265 (1951).

8) P. Andrews, Biochem. /., 91, 222 (1964).

9) Y. Takasaki, Agric. Biol. Chem., 40, 1515 (1976).