Agric. Biol. Chem., 42 (9), 1681-1688, 1978

Purification and Some Properties of

a Novel ƒ¿- Produced by a Strain of

Thermoactinomyces vulgaris•õ

Mizuho SHIMIZU, Mutsuo KANNO, Masaki TAMURA

and Mikio SUEKANE

Nakatani Memorial Laboratory, CPC Japan Ltd., Chiyoda-ku, Tokyo 102, Japan

Received February 8, 1978

An a-amylase[ƒ¿-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1.], found in the culture filtrate of a strain of Thermoactinomyces vulgaris, was purified by ammonium sulfate fractionation , and DEAF-cellulose and CM-cellulose chromatographies. The purified showed

a single band on disc gel electrophoresis. The optimum reaction pH and temperature were determined to be around pH 5.0 and 70°C. The isoelectric point was determined to be pH

5.2. The a-amylase was stabilized by Cal+. The a-amylase was found to hydrolyze pullulan to panose. Therefore, the hydrolytic

pattern of this enzyme is different from those of and .

The authors have screened thermophilic acti reciprocal shaker. Inoculation was conducted by nomycetes strains for a-amylase production transferring spores and mycelia from a slant culture with a wire loop. The medium composition was as and found that one actinomycetes strain iso follows; 3% soluble starch (Junsei Chemical Co.), lated from a soil sample at 50°C produced a 0.5% Bacto-Tryptone (Difco), 0.5% yeast extract new type of ƒ¿-amylase. The a-amylase had (Kyokuto Seiyaku), 0.05% CaCl2. 2H2O,0.05% MnC12 the ability to hydrolyze pullulan and showed 4H2O, 0.05% MgCl2.7H2O and 0.1 % KH2PO4. The

different properties from the Thermoactino pH of the medium was adjusted to 6.0. myces a-amylase reported by M. J. Kuo and Afaterials. DEAE-cellulose (DE 32) and CM- P. A. Hartman.1,2) Since no ƒ¿- have cellulose (CM-23) were purchased from Whatman Ltd.

been reported to hydrolyze the part of pullulan Isomaltose, maltotriose, pullulan, and the pullulanase of Klebsiella aerogenes3) were purchased from Hayashi consisting of maltotriose unit, this enzyme bara Biochemical Laboratories, Inc. Ampholine was seems to be a new type of a-amylase. The purchased from LKB Produkter Co. Panose, iso present report describes the procedure of puri panose, and the isopullulanase of Asp. niger4) were fication, some properties, and the pattern of kindly supplied by Dr. T. Kobayashi of Tokyo Noko

pullulan of this ƒ¿-amylase. University. Other chemicals were purchased from Iwai Kagaku Yakuhin Co.

Determination of a-amylase activity.') One milli MATERIALS AND METHODS liter of suitably diluted enzyme solution was incubated with 10 ml of 0.5% soluble starch (Merck) dissolved in Microorganism and culture. A thermophilic acti 0.03 M acetate buffer (pH 4.5) containing 0.03 M CaC12 nomycetes strain showing both ƒ¿-amylase and pullulan at 60°C for 10 min. Then 1 ml of the reaction mixture hydrolyzing activities was isolated from soil at 50°C. was pipetted into a 100 ml volumetric flask containing The microorganism was identified as Thermoactinomyces 50 ml of 0.02 N HCl. After the addition of 3 ml of vulgaris and was named strain R-47, which will be 0.05% iodine solution, the flask was filled up to 100 ml reported elsewhere. Flask culture of the isolated acti with distilled water. The optical density of the result- nomycetes strain was carried out for 2 days at 50°C in ing blue color was measured at 620 nm. One unit of 50 ml of medium in a 500 ml Sakaguchi flask on a a-amylase activity was the amount of enzyme cataly

•õ This paper was presented at the Annual Meeting zing a 20 % decrease in the optical density per min under of Agricultural Chemical Society of Japan, held in the assay conditions as calculated by the following Yokohama on April 1•`4, 1977. formula. 1682 M. SHIMIZU, M. KANNO, M. TAMURA and M. SUEKANE

Enzyme activity (units) acrylamide gel, by the method of Davis.i) After the electrophoresis, the gel was stained for proteins with 1% Amino Black 1OB in a 7% acetic acid solution and then destained electrophoretically with 7% acetic acid.

Determination of isoelectric point. Electrofocusing A linear relationship between the enzyme activity was carried out using an LKB Ampholine column at and the decrease in the optical density was found in 300-700 volts for 70 hr under a linear gradient of the range of 10%•`60% decrease in the optical density. glycerol from 0 to 60%. Ampholine of pH 3.5- 10 was used. After the run, the Ampholine solution was Determination of pullulan-hydrolyzing activity. A half milliliter of suitably diluted enzyme solution was drained from the column in 3 ml fractions. The pH and a-amylase and pullulan-hydrolyzing activities of incubated with 0.5 ml of 2% pullulan dissolved in 0 .1 M acetate buffer (pH 4.5) containing 0.03 M CaC12 at 40°C each fraction were determined. for 1 hr. Then the reaction was stopped by heating

the mixture in a boiling water bath for 5 min. The reducing sugar in the reaction mixture was determined RESULTS

by the Nelson-Somogyi method6) and calculated as Purification of T. vulgaris R-47 a-amylase equivalents. One unit of pullulan-hydrolyzing activity is the amount of enzyme which produces reduc Step 1. Concentration by ammonium sulfate

ing sugar equivalent to 1 i mole of glucose per min fractionation. Solid ammonium sulfate was under the assay conditions. added to the culture filtrate until 90% ammo

Determination of protein. The protein content was nium sulfate saturation was reached . Then

determined according to the Folin-Lowry method .7) the precipitate was separated by centrifugation Bovine serum albumin was used as the standard. and dissolved in a minimal amount of 0 .05M acetate buffer (pH 5.5) containing 5mm CaCl2 Hydrolyses of pullulan and starch. Two percent .

pullulan or soluble starch was dissolved in a 0.05M The solution was dialyzed against the same acetate buffer (pH 4.5) containing 5mm CaCl2 and in buffer at 4°C overnight, changing the buffer cubated with a given amount of T. vulgaris R-47 ƒ¿- twice. amylase at 60°C. The reducing sugar and total sugar Step 2. First DEAE-cellulose column chro in the hydrolyzed products were determined by the matography. The dialyzate was applied to Nelson-Somogyi method6) and the phenol-sulfuric acid method,') respectively. The degree of hydrolysis was a DEAE-cellulose column (2.6cm_??_ x 30cm)

expressed as the percent of the reducing sugar against equilibrated with 0.05 M acetate buffer (pH the total sugar. 5.5) containing 5mm CaC12 and eluted with

the same buffer. Paper chromatography. Samples were spotted on a Whatman No. 1 filter paper and this was developed The protein having a-amylase activity was

with a solvent system of butanol: pyridine: water not adsorbed to the column . The a-amylase (6: 4: 3 v/v) by the descending method for 24 hr. fractions were pooled and the ƒ¿-amylase was After drying, the sugars were detected by the silver concentrated by ammonium sulfate precipita nitrate dip method.') tion (90 % saturation). The ƒ¿-amylase solu For the purification of the trisaccharide from pullulan

hydrolyzed with T. vulgaris R-47 a-amylase, the hy tion was dialyzed against 0 .05 M acetate buffer drolyzed product (200 mg as dry substance) was linearly (pH 5.0) containing 5 mm CaC12. applied to a Whatman 3MM filter paper and the paper Step 3. CM-cellulose column chromato was developed with the same solvent system for 3 days. graphy. The dialyzed enzyme solution was After drying, guide strips were cut off from both sides applied to a CM-cellulose column (1 of the paper and the sugars were detected by the same .6 cm¢ X

method. Then the part of the paper corresponding 20 cm) equilibrated with 0 .05 M acetate buffer to the position of the trisaccharide indicated by the (pH 5.0) containing 5mm CaC12, The ƒ¿- guide strips was cut off. The trisaccharide was eluted amylase did not adsorb to the column and was from the paper with distilled water and concentrated eluted with the same buffer . The ƒ¿-amylase at 60°C under a vacuum. was recovered by ammonium sulfate precipi

Disc gel electrophoresis. Electrophoresis of the tation (90% saturation) and was then dis enzyme was carried out using a pH 8.8 - 9.0 poly solved in 0.05 M Tris-HC1 buffer (pH 7 .8) Purification and Properties of T. vulgaris ƒ¿-Amylase 1683

TABLE I. PURIFICATION OF R-47 ƒ¿-AMYLASE

containing 5mm CaC12. The enzyme solution saturation), dissolved in 0.05 M CaC12 and was dialyzed against the same buffer. dialyzed against the same buffer. A summary Step 4. Second DEAE-cellulose column of this purification procedure is shown in chromatography. The dialyzed enzyme solu Table I. tion was applied to a DEAE-cellulose column

(1.6cm_??_•~20cm) equilibrated with 0.05 M Tris- Disc gel electrophoresis HCl buffer (pH 7.8) containing 5 mm CaC12. The disc gel electrophoresis pattern of the

The enzyme was eluted from the column by purified ƒ¿-amylase is shown in Fig. 1. The linearly increasing the NaCl concentration in the buffer from 0 to 0.5 M. The a-amylase fractions were collected, and the a-amylase was precipitated with ammonium sulfate (90

FIG. 2. Pullulan-hydrolyzing and a-Amylase Ac tivities on Acrylamide Gel.

FIG. 1. Disc Electrophoresis Pattern of Purified The purified enzyme protein (50 ƒÊg) was subjected to

T. vulgaris R-47 ƒ¿-Amylase. polyacrylamide disc gel electrophoresis at pH 9.0. After electrophoresis, the gel was sliced into 2 mm- The purified enzyme protein (50 ƒÊg) was applied to wide strips and each strip was separately eluted with polyacrylamide gel and electrophoresis was carried 0.5 ml of 0.05 M acetate buffer (pH 5.0). Then, the out at a constant current of 5 mA per tube for 1 hr. a-amylase and pullulan-hydrolyzing activities of the After the electrophoresis, the gel was stained for eluted solution were determined.•›•\•› proteins with 1% Amido Black 10B in a 7% acetic acid solution and then destained electrophoretically with , a-amylase activity; •-0, pullulan-hydrolyzing activity. 7% acetic acid. 1684 M. SHIMIZU, M. KANNO, M. TAMURA and M. SUEKANE

purified enzyme showed a single band. The a-amylase and pullulan-hydrolyzing activities on the unstained gel were detected by slicing

the gel and determining the enzymatic ac tivities of each slice. The results are shown

in Fig. 2. Both the a-amylase and pullulan - hydrolyzing activities were seen to coincide with the protein band.

Properties of T. vulgaris R-47 ƒ¿-amylase 1) Optimum pH. The effect of the reac

tion pH on the a-amylase and pullulan

- hydrolyzing activities is shown in Fig. 3. The FIG. 4. Effect of Temperature. optimum pH for the a-amylase and pullulan- a-Amylase and pullulan-hydrolyzing activities were hydrolyzing activities was around pH 5 and estimated at pH 4.5 and various temperatures: 0--0, both activities showed similar relationships to a-amylase activity; •-0, pullulan-hydrolyzing ac the pH. tivity.

in the presence of 10 mM Ca2+. Without Cat+, however, it was gradually inactivated. The Ca 14 had a strong stabilizing effect on both the a-amylase and pullulan-hydrolyzing ac tivities. The rates of inactivation of the a- amylase and pullulan-hydrolyzing activities were similar under all conditions.

FIG. 3. Effect of pH. a-Amylase and pullulan-hydrolyzing activities were estimated at 60°C and various pH's using a 0.05 M acetate buffer containing 0.03 M CaC12: 0-0, a- amylase activity; 9--0, pullulan-hydrolyzing activity.

2) Optimum temperature. The effect of the reaction temperature on the a-amylase and FIG. 5. Heat-stability. pullulan-hydrolyzing activities is shown in Fig. 4. The optimum temperature for the a- The purified enzyme was incubated at pH 5 .0 and 60°C or 70°C in the presence of 10mM Ca2+ or with no Ca" . amylase and pullulan-hydrolyzing activities was The residual a-amylase and pullulan-hydrolyzing ac

70°C. The a-amylase activity showed higher tivities were determined after 15 , 30, 60 and 120 min: relative activity than the pullulan-hydrolyzing 0-0 a-amylase activity; •œ•\•œ pullulan-hydrolyzing activity at 80°C and 90°C. This was assumed activity. to be caused by a difference between the starch 1. 60°C, 10mM Ca2+; 2 . 70°C, 10mM Cal 1; 3. 60°C, no Ca2+; 4. 70°C, no Ca2+ and pullulan substrates in terms of the heat- . protection they afford to the enzyme. 4) Isoelectric point. The isoelectric point 3) Heat stability. As shown in Fig. 5, of both the a-amylase and pullulan -hydroly the enzyme was fairly stable at 60°C and pH 5.0 zing activities was pH 5.20+0 .15 as shown in Purification and Properties of T. vulgaris ƒ¿-Amylase 1685

Fig. 6. The elution patterns of protein , a- amylase and pullulan-hydrolyzing activities were consistent with each other. This shows the homogeneity of the enzyme.

FIG. 7. Hydrolysis of Pullulan.

FIG. 6. Isoelectric Point. Hydrolysis of pullulan (2%) was carried out for 20 hr at 40°C using 10 units of pullulanase, isopullulanase The purified enzyme protein (0.5 mg) was applied to and the T. vulgaris R-47 a-amylase per gram of an Ampholine column and electrofocusing was carried pullulan. The pH of the pullulan solution was 6.0 out at 300 - 700 volts for 70 hr. After the run the with the pullulanase and 4.5 with the isopullulanase Ampholine solution was collected in 3 ml fractions and R-47 a-amylase. The hydrolysis percent of the with a fraction collector. The pH, and the a-amylase hydrolyzates was determined by the Somogyi-Nelson

and pullulan-hydrolyzing activities of each fraction method6) and the phenol-sulfuric acid method8): were determined. •›•\•›, a-amylase activity; •œ•\•œ •œ•\•œ, pullulanase; •¢•\•¢, isopullulanase; •›•\•›, T. vulgaris R-47 ƒ¿-amylase. pullulan-hydrolyzing activity; pH; x---x, absorbance at 280 nm.

5) Hydrolysis of pullulan. Pullulan (2%) was hydrolyzed with pullulanase, isopullu

lanase and the T. vulgaris R-47 a-amylase. The progress of these hydrolyses is shown in

Fig. 7. The degrees of hydrolysis of the final

products were 31.3% with pullulanase, 32.8 with isopullulanase and 33.1% with T. vulgaris

R-47 ƒ¿-amylase. The sugar composition of the hydrolyzates (4 hr reaction) was analyzed by paper chromatography. The paper chro

matogram is shown in Fig. 8. The sugar com-

position of the hydrolyzate produced by the T. vulgaris R-47 ƒ¿-amylase was similar to that

in the isopullulanase hydrolyzate but different from that in the pullulanase hydrolyzate.

Pullulan (2%) was completely hydrolyzed using FIG. 8. A Paper Chromatogram of the Products of Pullulan Hydrolyzed by Pullulanase, T. vulgaris the T. vulgaris R-47 ƒ¿-amylase and the sugar R-47 a-Amylase and Isopullulanase. composition of this hydrolyzate was deter- Paper chromatography was carried out by the pro mined by paper chromatography. The results cedure described under MATERIALS AND METHODS: 1, glucose; 2, maltose; 3, maltotriose; 4, pullulanase are shown in Table II. The content of tri hydrolyzate; 5, T. vulgaris R-47 ƒ¿-amylase hydroly saccharides reached 96.5%. This saccharide zate; 6, isopullulanase hydrolyzate; 7, isopanose; was thought to be panose or isopanose, or 8, panose. 1686 M. SHIMIZU, M. KANNO, M. TAMURA and M. SUEKANE

TABLE II. COMPOSITION OF SACCHARIDES IN PULLULAN HYDROLYZATES

a mixture of both saccharides based upon the glucose and isomaltose. Therefore, the tri Rf value in paper chromatography and the saccharide was panose, and was not contami structure of pullulan. nated with isopanose. 6) Identification of the trisaccharide. The 7) Hydrolysis of starch. Two percent trisaccharide was purified by Whatman 3 MM soluble starch was hydrolyzed with 10 units paper chromatography. Then the purified tri of T. vulgaris R-47 a-amylase at 60°C and saccharide (2% solution) was incubated over- pH 4.5. The increase in the degree of hydroly night with 10 units of isopullulanase per gram sis and decrease in starch-iodine color reaction of substrate at 40°C and pH 4.5. The hydroly accompanying the hydrolysis are shown in zed products were analyzed by paper chromato Fig. 10. The blue color of the starch-iodine graphy as shown in Fig. 9. This indicates that reaction disappeared when the hydrolysis the trisaccharide was completely hydrolyzed to

FIG. 10. Hydrolysis of Soluble Starch .

Hydrolysis of soluble starch (2%) was carried out at 60°C and pH 4.5 using 10 a-amylase units of the FIG. 9. A Paper Chromatogram of the Products T. vulgaris R-47 a-amylase per gram of the starch from the Trisaccharide Hydrolyzed by Isopullulanase . . Hydrolysis percentage was determined by the Nelson- Paper chromatography was carried out as described Somogyi6) and the phenol-sulfuric acid method .') under MATERIALS AND METHODS: 1, glucose; 2 , Starch-iodine color reaction was determined by mixing maltose; 3, purified trisaccharide; 4, products from 0.03 ml of the hydrolyzates with 0 .5 ml of 0.5 N acetic the purified trisaccharide hydrolyzed by isopullu acid and 3 ml of 0.015 % 12 solution and measuring lanase; 5, isopanose; 6, isopanose treated with iso the blue color at 700 nm: 0-0 , hydrolysis percentage; pullulanase; 7, isomaltose. •œ•\•œ starch-iodine reaction.

TABLE Ill. COMPOSITION OF SACCHARIDES IN STARCH HYDROLYZATES Purification and Properties of T. vulgaris a-Amylase 1687

FIG. 11. Comparison of Hydrolysis of Starch and Pullulan. FIG. 12. Patterns of Enzymatic Hydrolysis of Hydrolysis of starch and pullulan (2%) was carried Pullulan. out at 60°C and pH 4.5 using 20 a-amylase units of 0, glucose; o, reducing end; -, ƒ¿-1,4 linkage; j, ƒ¿- the T. vulgaris R-47 a-amylase per gram of substrate. 1,6 linkage. Hydrolysis percentage of the hydrolyzates was deter- mined by the Somogyi-Nelson method6) and the phe The other type was an ƒ¿-amylase with an acidic nolsulfuric method8):•›•\•›, starch; •œ•\•œ, pullulan. pH optimum, and this ƒ¿-amylase had the

ability to hydrolyze pullulan. The hydrolyzed reached about 18•`20%. The mutarotation product was found to be mainly panose. of the hydrolyzed product was determined ac- The action pattern of this a-amylase on cording to the method described by H. Fuwa soluble starch was found to be similar to that and Z. Nikuni.ll) As a result, a decrease in of saccharifying a-amylases such as BSA the optical rotation was observed. Therefore, (Bacillus subtilis), Taka a-amylase (Asp. oryzae) the hydrolyzed products should have an a- and porcine pancreatic a-amylase. However, configuration. Figure 11 shows a comparison those a-amylases do not hydrolyze pullulan, of the hydrolysis of soluble starch and pullulan unlike this T. vulgaris R-47 ƒ¿-amylase. when equal amounts of T. vulgaris R-47 ƒ¿- Regarding pullulan-hydrolyzing , amylase were employed. The sugar com- three types of enzymes have been known, as

position of the final hydrolyzate from the solu shown in Fig. 12. This ƒ¿-amylase corresponds ble starch was determined by paper chromato to a fourth type of enzyme, which has not graphy and the results are shown in Table III. hitherto been reported. The main products were maltose (74.1 %) and Acknowledgements. We wish to thank Dr. Y. glucose (11.8%). These results indicate that Sakano and Dr. T. Kobayashi of Tokyo Noko Univer this enzyme is a saccharifying ƒ¿-amylase. sity for their kind gifts of panose, isopanose and iso

pullulanase.

DISCUSSION

REFERENCES In 1967, P. A. Hartman et al.2 reported the

purification of an ƒ¿-amylase from Thermo 1) M. J. Kuo and P. A. Hartman, J. Bacterial., 92, actinomyces vulgaris. The authors screened 723 (1966). 2) M. J. Kuo and P. A. Hartman, Can. J. Microbiol., thermophilic actinomycetes strains for ƒ¿-amy 13,1157 (1967). lase production and found that two types of 3) M. Abdullah and D. French, Arch. Biochem. a-amylase were produced by thermophilic ac Biophys., 137, 483 (1970).

tinomycetes strains. One type was a neutral 4) Y. Sakano, N. Masuda and T. Kobayashi, Agric. a-amylase similar to that reported by Hartman. Biol. Chem., 35, 971 (1971). 1688 M. SHIMIZU, M. KANNO, M. TAMURA and M. SUEICANE

5) J. Robyt, Ph. D. Dissertation, Iowa State Univer 86, 681 (1963). sity, 1962. 10) B. J. Davis, Ann. New York A cad. Sci., 121, Art. 2, 6) M. Somogyi, J. Biol. Chem., 195, 19 (1952). 321 (1964). 7) 0. H. Lowry, N. J. Rosebrough, A. L. Farr and 11) H. Fuwa and Z. Nikuni, Nippon Nogeikagaku R. J. Randall, ibid., 193, 265 (1951). Kaishi, 26,154 (1951). 8) M. Dubris, K. L. Gilles, J. K. Hamilton, P. A. 12) Y. Sakano, N. Masuda and T. Kobayashi, Agric. Rebers and F. Smith, Anal. Chem., 28, 350 (1956). Biol. Chem., 35, 971 (1971). 9) N. E. Welker and L. L. Campbell, J. Bacteriol.,