JOURNAL
OF
Vol. 134, No. 2
BACrERIOLOGY, May 1978, p. 389-393
0021-9193/78/0134-0389$02.00/0 Printed in U.S.A.
Copyright i 1978 American Society for Microbiology
a-Amylase from Five Strains of Bacillus amyloliquefaciens: Evidence for Identical Primary Structures PETER T. BORGIAt* AND L. LEON CAMPBELLtt Department of Microbiology, University of Illinois, Urbana, Illinois 61820
Received for publication 11 November 1977
The a-amylases from five strains of Bacillus amyloliquefaciens were compared to determine whether differences in primary structure are responsible for variations in catalytic properties previously reported among the enzymes. Amino acid analysis established virtually identical compositions for the proteins. Reaction with dimethylaminonaphthylene sulfonylchloride indicated the amino-terminal amino acid of each amylase to be valine. Carboxyl termini of the enzymes have been determined by digestion with carboxypeptidase A. The resulting kinetic data indicate tyrosine as the carboxyl terminus and leucine as the penultimate residue for all five proteins. Isoelectric focusing of the enzymes yielded isoelectric points in the pH range of 5.09 to 5.18. Tryptic digests of the enzymes chromatographed on a cation-exchange column showed identical elution patterns. It is concluded that the primary structure of the amylase from the five strains is identical or exhibits only conservative substitutions. Numerous reports concerning the a-amylases from several strains of Bacillus subtilis have appeared in the literature. On the basis of DNA base compositions, inter- and intrastrain transduction, physiological-biochemical properties, and DNA hybridization, Welker and Campbell demonstrated that many highly amylolytic strains used in previous studies were not B. subtilis but strains of B. amyloliquefaciens (13). Accordingly, it was found that the a-amylase of genuine strains of B. subtilis differs in immunological and electrophoretic properties from that of B. amyloliquefaciens (11). Authentic strains of B. amyloliquefaciens are also highly proteolytic and are responsible for the commercial production of subtilisins BPN' and Novo. Welker and Campbell (12) have described a method for crystallizing the a-amylase from five strains of B. amyloliquefaciens. The enzymes were shown to be identical with respect to temperature and pH optima, electrophoretic mobility in polyacrylamide gels, UV absorption spectrum, and immunological properties. The enzymes were demonstrated to differ with respect to activity on various substrates, pH and temperature stability, Km, and energy of activation. These findings prompted us to propose that such differences may be a reflection of slight alterations in the primary structure or in the confor-
mational state of the proteins. This report is concerned with attempts to determine whether differences in primary structure among the enzymes exist. MATERIALS AND METHODS
Organisms and enzyme production. The strains of B. amyloliquefaciens used were described by Welker and Campbell (13). The organisms were grown, enzyme purified, and assayed according to the procedures of Welker and Campbell (12). Amino acid analysis. Protein was hydrolyzed at 110°C with constantly boiling 6 N HCI in evacuated and sealed vials for 24, 48, and 72 h. The hydrolysates were analyzed on a Beckman 120B amino acid analyzer. Cysteine was determined as cysteic acid by the method of Moore (7). Tryptophan was determined spectrophotometrically according to Edelhoch (4). Peptide analysis. Five milligrams of each enzyme was dissolved in 1 ml of 5 M guanidine-HCI (Mann, ultrapure) and heated at 90°C for 10 min. The denatured protein was dialyzed for 24 h against 4 liters of distilled water. The precipitated protein suspension was brought to 5 ml, the pH was adjusted to pH 8.00, and the suspension was transferred to a Metroohm pH-stat at 37°C. Digestion was initiated by the addition of 10 Id of 1% (wt/vol) tolylsulfonyl phenylalanyl chloromethyl ketone-trypsin (Worthington) in 0.1 M CaCl2. The pH of the mixture was maintained by automatic addition of 0.01 N NaOH. After 2 h, another 10Al of trypsin solution was added, and the reaction was allowed to continue for 10 more h. The digestion and School of address: Present Microbiology t Department mixture was rotary evaporated, and the residue was of Medicine, Southern Illinois University, Carbondale, IL dissolved in 1 ml of 0.2 M pyridine-acetate, pH 3.10. 62901. Cation-exchange chromatography was carried out tt Present address: Provost, University of Delaware, Newon a column (0.9 by 20 cm) of Aminex A-5 (Bio-Rad) ark, DE 19711. 389
390
BORGIA AND CAMPBELL
in an automatic system similar to those described by Benson et al. (3). Peptides were eluted with a 500-ml linear gradient from 0.2 M pyridine-acetate, pH 3.10, to 2.0 M pyridine-acetate, pH 5.00. After completion of the gradient, limiting buffer was continued for an additional 200 ml. Buffer and ninhydrine flow rates were 30 ml/h. Effluent was monitored at 570 nm in a Gilford 300 recording spectrophotometer. Amino-terminal analyses. Enzyme was dansylated by the procedure of Gros and Labouesse (5). The dansyl protein was hydrolyzed at 1100C for 18 h in 6 N HCI, and the dansyl amino acids were separated on cellulose MN 300 thin layers (Brinkmann) by the technique of Arnott and Ward (1). Carboxyl-terminal analyses. About 35 mg of heat-denatured protein was dissolved in 8 ml of 0.2 M N-ethylmorpholine-acetate, pH 8.4-0.056 M sodium dodecyl sulfate containing 0.125 ,umol of norleucine per ml. Digestion was initiated by the addition of 1 mg of carboxypeptidase A (Worthington) in 50A of buffer. Samples of 1 ml were removed at intervals up to 180 min and added to 1.0 ml of glacial acetic acid. Precipitated protein was removed by centrifugation, the precipitate was washed twice with 1 N acetic acid, and the supernatants were combined and lyophilized. The residues were used for amino acid analysis. Protein concentration was determined by hydrolyzing an undigested portion of amylase solution with 6 N HCI for 24 h followed by amino acid analysis. Isoelectric focusing. One-milligram amounts of amylase were focused at 40C on a 110-ml electrofocusing column in an Ampholine gradient (LKB) of pH 4 to 6. An initial power of 2.4 W was applied to the column, and focusing was continued for 40 h. The cathode was at the bottom of the column. The pH of 1-ml fractions was read at 40C with a Corning 112 pH meter standardized between pH 4.00 and 7.00 ± 0.01. Peptide mapping. Tryptic digests were fingerprinted on sheets (35 by 46 cm) of Whatman 3MM paper. Electrophoresis was carried out in 0.6 M pyridine-acetate (pH 6.6) at 1,000 V for 90 min. Descending chromatography in the second dimension was performed in pyridine-butanol-acetic acid-water (40:60:-
J. BACTERIOL. 12:48) for 16 h. Peptide maps were stained with ninhydrine or potassium chloroplatinate according to Bennett (2).
RESULTS Isoelectric focusing. Although Welker and Campbell (12) reported no differences in electrophoretic mobility among the enzymes, we decided to subject the enzymes to the more sensitive technique of isoelectric focusing. Figure 1 shows the results of focusing of strain P enzyme. The enzyme was separable into two components of pl 5.19 and 5.03. Each of the other proteins except strain F enzyme also separated into two components (Table 1). No significant differences in pI among the enzymes were evident. The enzymes are known to form dimers in the presence of zinc ions, presumably by salt linkage (9). Therefore, experiments were conducted to determine whether the two components observed were monomers and dimers. Preparations in which 10' M Zn2+ was present were still separable into two components. Enzyme that had been dialyzed against 2 x 10' M ethylenediaminetetraacetic acid before focusing showed a single component (Fig. 2), which corresponded in isoelectric point to that of the trailing peak. It therefore is likely that the trailing component represents the monomer. The pI of the dimer was higher since acidic groups are bound by zinc ions. Complete conversion to dimers may not be possible due to the removal of zinc ions in the electrical field. Amino acid analysis. Table 2 shows the amino acid analyses of the five enzymes presented as molar ratios on the basis of five methionine residues per molecule of protein. The number of methionine residues per molecule was determined by staining of tryptic peptide maps
-
250
-2001 150E t X~~~~~~
-4inn
c
FIG. 1. Isoelectric-focusing profile of strain P amylase. Sample was focused in an LKB 110-mi column in a pH 4 to 6 Ampholine gradient. One milliliter fractions were collected.
B. AMYLOLIQUEFACIENS a-AMYLASE
VOL. 134, 1978
391
with potassium chloroplatinate. Recovery of to- essentially identical (data not presented), but tal nitrogen in the five analyses ranged from sufficient ambiguity existed to warrant further 99.41 to 101.9%. The amino acid compositions of analysis by a more sensitive technique. The trypthe enzymes were identical within experimental tic peptides from each enzyme were chromatoerror and agreed with the analysis reported by graphed on Aminex A-5 cation-exchange resins. Junge et al. (6) for strain T amylase. The molec- Figure 4 shows the elution patterns of strain N ular weights of the five enzymes calculated from (upper) and strain SB (lower) amylase. The the analyses were: strain P, 49,627; strain N, profiles, except for a few slight quantitative dif49,134; strain SB, 49,610; strain T, 48,109; and ferences, were identical. This reflects the high strain F, 49,297. degree of sequence homology between the enCarboxyl and amino termini. The amino zymes. Identical chromatograms were obtained ternini of all the enzymes were found to be for strain P, T, and F enzymes. These data valine. This agrees with the results of Sugae and Honda (10) for strain N amylase determined by TABLE 2. Amino acid compositions of B. the dinitrofluorobenzene method. amyloliquefaciens a-amylase Figure 3 shows the results of carboxypeptidase Integer value from strain:a digestion of strain F amylase. The data indicate Amino acid that tryosine is the carboxyl terminus and leuP F N T SB cine is the penultimate residue. Interpretation of 27 26 27 25 26 the kinetics is made difficult since more than Lys .. 11 11 10 11 one residue of both tyrosine and leucine was His .10 20 19 19 19 19 present in the carboxyl-terminal region. How- Arg ........ 57 56 57 56 58 Asp . ever, since the rate of release of leucine becomes Thrrb 24 24 25 26 26 .. greater than that of tyrosine after about 15 min, Serb ........ 27 29 28 29 27 a sequence of (Tyr, Leu) "Ser"-Leu-Tyr-COOH Glu ........ 47 46 46 45 47 is proposed. Identical results have been obtained Pro ........ 17 17 17 17 18 for the T, SB, N, and P proteins. 40 40 41 39 Gly ........ 40 31 31 30 31 Peptide analysis. The two-dimensional pa- Ala .30 27 27 27 27 per peptide maps from the five enzymes were Val ........ 27 TABLE 1. Summary of isoelectric-focusing data pI Strain
N SB T F
P
Monomer
Dimer
5.11 5.09 5.19 5.19 5.19
4.91 4.95 5.03 5.03
5 Met ........ Ile .18 Leu ........ 25 Tyr ........ 26 18 Phe ........
5
5 5 18 18 25 25 26 26 18 18 12 13 TrpC .14 a Data expressed to nearest integer on the basis of five methionine residues/molecule of protein. b Determined by extrapolation to zero hydrolysis
18 25 26 18 14
5 18 25 26 18 13
time. c Determined,spectrophotometrically.
FIG. 2. Isoelectric-focusing profile of strain P amylase that was first dialyzed against 2 x 1O-4 M ethylenediaminetetraacetic acid.
392
BORGIA AND CAMPBELL
J. BACTERIOL.
z M
0
o-
K
TYR LEU SER
0.
MINUTES FIG. 3. Kinetics of release of amino acids from strain F amylase by carboxypeptidase A.
0.86.I-JO
1
0.41 _
LU 0.21 z
o 1.0 0 U,
lo0.8
. .~ ~ ~ ~ ~ ~ ~ ~ ,2
0.6 OA-
0.2 120
240
360
480
ML. EFFLUENT
FIG. 4. Elution profile of tryptic peptides from strain N (upper) and SB (lower) amylase. Peptides were chromatographed on a column (0.9 by 20 cm) of Aminex A-5 and were eluted with a linear 500-ml gradient from 0.2 Mpyridine-acetate, pH 3.10, to 2.0 Mpyridine-acetate, pH 5.0.
strongly indicate that the primary structures of the a-amylases from the five strains are identical or exhibit only conservative substitutions. Approximately 65 peptides have been resolved by this technique. The number of tryptic peptides is greater than that calculated from the lysine-plus-arginine content for the proteins (45 to 47) due presumably to incomplete hydrolysis of trypsin-sensitive bonds or to some nontryptic cleavages. This finding is not unusual when sensitive detection methods are used for peptide analysis (3). DISCUSSION The results presented in this paper strongly indicate that the a-amylases from the five strains
of B. amyloliquefaciens are identical in primary structure or exhibit only conservative modifications. These findings strengthen the conclusions of Welker and Campbell (12, 13) that the five strains represent a species distinct from B. subtilis, since the enzymes differ in electrophoretic mobility and immunological properties from B. subtilis a-amylase. A similar situation exists concerning the alkaline proteases produced by these organisms. Differences in electrophoretic mobility between subtilisin BPN' (or Novo) and sub-
tilisin Carlsberg have been shown to be the result of 83 substitutions in the amino acid seof the BPN' enzyme (8). Subsequently, B. subtilis BPN' (13) and B. subtilis Novo (this laboratory, unpublished data) have been shown
quence
B. AMYLOLIQUEFACIENS a-AMYLASE
VOL. 134, 1978
to be strains of B. amyloliquefaciens. The amino acid composition reported for B. amyloliquefaciens enzymes closely resembles that reported by Junge et al. (6) for "B. subtilis" (B. amyloliquefaciens strain T) a-amylase. The amino acid composition of enzyme from genuine strains of B. subtilis differs from that reported here (14). Additionadly, B. subtilis enzyme-does not cross-react with antiserum prepared against B. amyloliquefaciens aLamylase, and B. sutilis enzyme is of the saccharifying type whereas that of B. amyloliquefaciens is a liquefying enzyme (11). In conclusion, no evidence for differences in the primary structure of the five enzymes could be found. The differences in catalytic properties reported by Welker and Campbell (11) may be due to slight alterations in conformation among the proteins. We have found that the mo!ecular weights of some of the enzymes during sedimentation equilibrium are concentration dependent (unpublished data), whereas others are not. These properties may be a reflection of conformational differences. Since the enzymes are calcium and zinc metalloproteins (9), differences in the trace metal content of the proteins may be involved. LITERATURE CITD 1. Arnott, M. S., and D. N. Ward. 1967. Separation of dansyl amino acids in a single analysis. Anal. Biochem.
21:50-56. 2. Bennett, J. C. 1967. Paper chromatography and electro-
393
phoesis; special procedure for peptide maps. Methods Enzymol. 1,:330-339. 3. Benson,,J. V., R. T. Jopes, J. Cormick, and J. A. Patte,rson. 1i36. Accelerated automatic chromatographlc analysla)of peptides on a spherical resin. Anal. Biochem. 16:91-106. 4. Edelhoch, H. 1067.§$;pectroscopic determination of tryptophan and tyrosifi residues in proteins. Biochemistry 6:194&49545. Gros, C., and B. Labouesse. 1969. Study of /he dansylatioa of amnno acids, peptides and proteins. Euw. J. Biochem. 1:4j3-470. 6. Junge, J. M., 'R. A. Stc4n, H. Neurath, and E. H. Fischer. 1958. The amino acid composition of a-amylae fmom Bacillus subtilis. J. Biol. Chem. 234:556-561. 7. Moore, S. 1963. On the determination of cysteine as cysteic acid. J. Biol. them. 238:235-237. 8. Smith, E. L., F. S. Markland, C. B. Kasper, R. J. Delange, K. Landon, and W. H. Evans. 1966. The complete amino acid sequence of two types of subtilisin, BPN' and Carlaberg. J. Biol. Chem. 241:5974-5976. 9. Stein, E. A., and E. H. Fischer. 1960. Bacillus subtilis a-amylase, a zinc-protein complex. Biochim. Biophys. Acta 39:287-296. 10. Sugae, K., and Y. Honda. 1960. Studies on bacterial amylase. III. On the amino terminal peptide of bacterial amylase. J. Biochem. 47:307-314. 11. Welker, N. E., and L. L. Campbell. 1967. Comparison of the a-amylase of BaciUus subtilis and Bacillus amyloliquefaciens. J. Bacteriol. 94:1131-1135. 12. Welker, N. E., and L L. Campbell. 1967. Crystallization and properties of a-amylase from five strains of Bacillus amyloliquefaciens. Biochemistry 6:3681-3689. 13. Welker, N. E., and L. I. Campbell. 1967. Unrelatedness of Bacilus amyloliquefaciens and Bacilus subtilis. J. Bacteriol. 94:1124-1130. 14. Yamane, K., K. Yamaguchi, and B. Maruo. 1973. Purification and properties of a cross reacting material related to a-amylase and biochemical comparison with the parent a-amylase. Biochim. Biophys. Acta 295:323-340.
OF
Vol. 134, No. 2
BACrERIOLOGY, May 1978, p. 389-393
0021-9193/78/0134-0389$02.00/0 Printed in U.S.A.
Copyright i 1978 American Society for Microbiology
a-Amylase from Five Strains of Bacillus amyloliquefaciens: Evidence for Identical Primary Structures PETER T. BORGIAt* AND L. LEON CAMPBELLtt Department of Microbiology, University of Illinois, Urbana, Illinois 61820
Received for publication 11 November 1977
The a-amylases from five strains of Bacillus amyloliquefaciens were compared to determine whether differences in primary structure are responsible for variations in catalytic properties previously reported among the enzymes. Amino acid analysis established virtually identical compositions for the proteins. Reaction with dimethylaminonaphthylene sulfonylchloride indicated the amino-terminal amino acid of each amylase to be valine. Carboxyl termini of the enzymes have been determined by digestion with carboxypeptidase A. The resulting kinetic data indicate tyrosine as the carboxyl terminus and leucine as the penultimate residue for all five proteins. Isoelectric focusing of the enzymes yielded isoelectric points in the pH range of 5.09 to 5.18. Tryptic digests of the enzymes chromatographed on a cation-exchange column showed identical elution patterns. It is concluded that the primary structure of the amylase from the five strains is identical or exhibits only conservative substitutions. Numerous reports concerning the a-amylases from several strains of Bacillus subtilis have appeared in the literature. On the basis of DNA base compositions, inter- and intrastrain transduction, physiological-biochemical properties, and DNA hybridization, Welker and Campbell demonstrated that many highly amylolytic strains used in previous studies were not B. subtilis but strains of B. amyloliquefaciens (13). Accordingly, it was found that the a-amylase of genuine strains of B. subtilis differs in immunological and electrophoretic properties from that of B. amyloliquefaciens (11). Authentic strains of B. amyloliquefaciens are also highly proteolytic and are responsible for the commercial production of subtilisins BPN' and Novo. Welker and Campbell (12) have described a method for crystallizing the a-amylase from five strains of B. amyloliquefaciens. The enzymes were shown to be identical with respect to temperature and pH optima, electrophoretic mobility in polyacrylamide gels, UV absorption spectrum, and immunological properties. The enzymes were demonstrated to differ with respect to activity on various substrates, pH and temperature stability, Km, and energy of activation. These findings prompted us to propose that such differences may be a reflection of slight alterations in the primary structure or in the confor-
mational state of the proteins. This report is concerned with attempts to determine whether differences in primary structure among the enzymes exist. MATERIALS AND METHODS
Organisms and enzyme production. The strains of B. amyloliquefaciens used were described by Welker and Campbell (13). The organisms were grown, enzyme purified, and assayed according to the procedures of Welker and Campbell (12). Amino acid analysis. Protein was hydrolyzed at 110°C with constantly boiling 6 N HCI in evacuated and sealed vials for 24, 48, and 72 h. The hydrolysates were analyzed on a Beckman 120B amino acid analyzer. Cysteine was determined as cysteic acid by the method of Moore (7). Tryptophan was determined spectrophotometrically according to Edelhoch (4). Peptide analysis. Five milligrams of each enzyme was dissolved in 1 ml of 5 M guanidine-HCI (Mann, ultrapure) and heated at 90°C for 10 min. The denatured protein was dialyzed for 24 h against 4 liters of distilled water. The precipitated protein suspension was brought to 5 ml, the pH was adjusted to pH 8.00, and the suspension was transferred to a Metroohm pH-stat at 37°C. Digestion was initiated by the addition of 10 Id of 1% (wt/vol) tolylsulfonyl phenylalanyl chloromethyl ketone-trypsin (Worthington) in 0.1 M CaCl2. The pH of the mixture was maintained by automatic addition of 0.01 N NaOH. After 2 h, another 10Al of trypsin solution was added, and the reaction was allowed to continue for 10 more h. The digestion and School of address: Present Microbiology t Department mixture was rotary evaporated, and the residue was of Medicine, Southern Illinois University, Carbondale, IL dissolved in 1 ml of 0.2 M pyridine-acetate, pH 3.10. 62901. Cation-exchange chromatography was carried out tt Present address: Provost, University of Delaware, Newon a column (0.9 by 20 cm) of Aminex A-5 (Bio-Rad) ark, DE 19711. 389
390
BORGIA AND CAMPBELL
in an automatic system similar to those described by Benson et al. (3). Peptides were eluted with a 500-ml linear gradient from 0.2 M pyridine-acetate, pH 3.10, to 2.0 M pyridine-acetate, pH 5.00. After completion of the gradient, limiting buffer was continued for an additional 200 ml. Buffer and ninhydrine flow rates were 30 ml/h. Effluent was monitored at 570 nm in a Gilford 300 recording spectrophotometer. Amino-terminal analyses. Enzyme was dansylated by the procedure of Gros and Labouesse (5). The dansyl protein was hydrolyzed at 1100C for 18 h in 6 N HCI, and the dansyl amino acids were separated on cellulose MN 300 thin layers (Brinkmann) by the technique of Arnott and Ward (1). Carboxyl-terminal analyses. About 35 mg of heat-denatured protein was dissolved in 8 ml of 0.2 M N-ethylmorpholine-acetate, pH 8.4-0.056 M sodium dodecyl sulfate containing 0.125 ,umol of norleucine per ml. Digestion was initiated by the addition of 1 mg of carboxypeptidase A (Worthington) in 50A of buffer. Samples of 1 ml were removed at intervals up to 180 min and added to 1.0 ml of glacial acetic acid. Precipitated protein was removed by centrifugation, the precipitate was washed twice with 1 N acetic acid, and the supernatants were combined and lyophilized. The residues were used for amino acid analysis. Protein concentration was determined by hydrolyzing an undigested portion of amylase solution with 6 N HCI for 24 h followed by amino acid analysis. Isoelectric focusing. One-milligram amounts of amylase were focused at 40C on a 110-ml electrofocusing column in an Ampholine gradient (LKB) of pH 4 to 6. An initial power of 2.4 W was applied to the column, and focusing was continued for 40 h. The cathode was at the bottom of the column. The pH of 1-ml fractions was read at 40C with a Corning 112 pH meter standardized between pH 4.00 and 7.00 ± 0.01. Peptide mapping. Tryptic digests were fingerprinted on sheets (35 by 46 cm) of Whatman 3MM paper. Electrophoresis was carried out in 0.6 M pyridine-acetate (pH 6.6) at 1,000 V for 90 min. Descending chromatography in the second dimension was performed in pyridine-butanol-acetic acid-water (40:60:-
J. BACTERIOL. 12:48) for 16 h. Peptide maps were stained with ninhydrine or potassium chloroplatinate according to Bennett (2).
RESULTS Isoelectric focusing. Although Welker and Campbell (12) reported no differences in electrophoretic mobility among the enzymes, we decided to subject the enzymes to the more sensitive technique of isoelectric focusing. Figure 1 shows the results of focusing of strain P enzyme. The enzyme was separable into two components of pl 5.19 and 5.03. Each of the other proteins except strain F enzyme also separated into two components (Table 1). No significant differences in pI among the enzymes were evident. The enzymes are known to form dimers in the presence of zinc ions, presumably by salt linkage (9). Therefore, experiments were conducted to determine whether the two components observed were monomers and dimers. Preparations in which 10' M Zn2+ was present were still separable into two components. Enzyme that had been dialyzed against 2 x 10' M ethylenediaminetetraacetic acid before focusing showed a single component (Fig. 2), which corresponded in isoelectric point to that of the trailing peak. It therefore is likely that the trailing component represents the monomer. The pI of the dimer was higher since acidic groups are bound by zinc ions. Complete conversion to dimers may not be possible due to the removal of zinc ions in the electrical field. Amino acid analysis. Table 2 shows the amino acid analyses of the five enzymes presented as molar ratios on the basis of five methionine residues per molecule of protein. The number of methionine residues per molecule was determined by staining of tryptic peptide maps
-
250
-2001 150E t X~~~~~~
-4inn
c
FIG. 1. Isoelectric-focusing profile of strain P amylase. Sample was focused in an LKB 110-mi column in a pH 4 to 6 Ampholine gradient. One milliliter fractions were collected.
B. AMYLOLIQUEFACIENS a-AMYLASE
VOL. 134, 1978
391
with potassium chloroplatinate. Recovery of to- essentially identical (data not presented), but tal nitrogen in the five analyses ranged from sufficient ambiguity existed to warrant further 99.41 to 101.9%. The amino acid compositions of analysis by a more sensitive technique. The trypthe enzymes were identical within experimental tic peptides from each enzyme were chromatoerror and agreed with the analysis reported by graphed on Aminex A-5 cation-exchange resins. Junge et al. (6) for strain T amylase. The molec- Figure 4 shows the elution patterns of strain N ular weights of the five enzymes calculated from (upper) and strain SB (lower) amylase. The the analyses were: strain P, 49,627; strain N, profiles, except for a few slight quantitative dif49,134; strain SB, 49,610; strain T, 48,109; and ferences, were identical. This reflects the high strain F, 49,297. degree of sequence homology between the enCarboxyl and amino termini. The amino zymes. Identical chromatograms were obtained ternini of all the enzymes were found to be for strain P, T, and F enzymes. These data valine. This agrees with the results of Sugae and Honda (10) for strain N amylase determined by TABLE 2. Amino acid compositions of B. the dinitrofluorobenzene method. amyloliquefaciens a-amylase Figure 3 shows the results of carboxypeptidase Integer value from strain:a digestion of strain F amylase. The data indicate Amino acid that tryosine is the carboxyl terminus and leuP F N T SB cine is the penultimate residue. Interpretation of 27 26 27 25 26 the kinetics is made difficult since more than Lys .. 11 11 10 11 one residue of both tyrosine and leucine was His .10 20 19 19 19 19 present in the carboxyl-terminal region. How- Arg ........ 57 56 57 56 58 Asp . ever, since the rate of release of leucine becomes Thrrb 24 24 25 26 26 .. greater than that of tyrosine after about 15 min, Serb ........ 27 29 28 29 27 a sequence of (Tyr, Leu) "Ser"-Leu-Tyr-COOH Glu ........ 47 46 46 45 47 is proposed. Identical results have been obtained Pro ........ 17 17 17 17 18 for the T, SB, N, and P proteins. 40 40 41 39 Gly ........ 40 31 31 30 31 Peptide analysis. The two-dimensional pa- Ala .30 27 27 27 27 per peptide maps from the five enzymes were Val ........ 27 TABLE 1. Summary of isoelectric-focusing data pI Strain
N SB T F
P
Monomer
Dimer
5.11 5.09 5.19 5.19 5.19
4.91 4.95 5.03 5.03
5 Met ........ Ile .18 Leu ........ 25 Tyr ........ 26 18 Phe ........
5
5 5 18 18 25 25 26 26 18 18 12 13 TrpC .14 a Data expressed to nearest integer on the basis of five methionine residues/molecule of protein. b Determined by extrapolation to zero hydrolysis
18 25 26 18 14
5 18 25 26 18 13
time. c Determined,spectrophotometrically.
FIG. 2. Isoelectric-focusing profile of strain P amylase that was first dialyzed against 2 x 1O-4 M ethylenediaminetetraacetic acid.
392
BORGIA AND CAMPBELL
J. BACTERIOL.
z M
0
o-
K
TYR LEU SER
0.
MINUTES FIG. 3. Kinetics of release of amino acids from strain F amylase by carboxypeptidase A.
0.86.I-JO
1
0.41 _
LU 0.21 z
o 1.0 0 U,
lo0.8
. .~ ~ ~ ~ ~ ~ ~ ~ ,2
0.6 OA-
0.2 120
240
360
480
ML. EFFLUENT
FIG. 4. Elution profile of tryptic peptides from strain N (upper) and SB (lower) amylase. Peptides were chromatographed on a column (0.9 by 20 cm) of Aminex A-5 and were eluted with a linear 500-ml gradient from 0.2 Mpyridine-acetate, pH 3.10, to 2.0 Mpyridine-acetate, pH 5.0.
strongly indicate that the primary structures of the a-amylases from the five strains are identical or exhibit only conservative substitutions. Approximately 65 peptides have been resolved by this technique. The number of tryptic peptides is greater than that calculated from the lysine-plus-arginine content for the proteins (45 to 47) due presumably to incomplete hydrolysis of trypsin-sensitive bonds or to some nontryptic cleavages. This finding is not unusual when sensitive detection methods are used for peptide analysis (3). DISCUSSION The results presented in this paper strongly indicate that the a-amylases from the five strains
of B. amyloliquefaciens are identical in primary structure or exhibit only conservative modifications. These findings strengthen the conclusions of Welker and Campbell (12, 13) that the five strains represent a species distinct from B. subtilis, since the enzymes differ in electrophoretic mobility and immunological properties from B. subtilis a-amylase. A similar situation exists concerning the alkaline proteases produced by these organisms. Differences in electrophoretic mobility between subtilisin BPN' (or Novo) and sub-
tilisin Carlsberg have been shown to be the result of 83 substitutions in the amino acid seof the BPN' enzyme (8). Subsequently, B. subtilis BPN' (13) and B. subtilis Novo (this laboratory, unpublished data) have been shown
quence
B. AMYLOLIQUEFACIENS a-AMYLASE
VOL. 134, 1978
to be strains of B. amyloliquefaciens. The amino acid composition reported for B. amyloliquefaciens enzymes closely resembles that reported by Junge et al. (6) for "B. subtilis" (B. amyloliquefaciens strain T) a-amylase. The amino acid composition of enzyme from genuine strains of B. subtilis differs from that reported here (14). Additionadly, B. subtilis enzyme-does not cross-react with antiserum prepared against B. amyloliquefaciens aLamylase, and B. sutilis enzyme is of the saccharifying type whereas that of B. amyloliquefaciens is a liquefying enzyme (11). In conclusion, no evidence for differences in the primary structure of the five enzymes could be found. The differences in catalytic properties reported by Welker and Campbell (11) may be due to slight alterations in conformation among the proteins. We have found that the mo!ecular weights of some of the enzymes during sedimentation equilibrium are concentration dependent (unpublished data), whereas others are not. These properties may be a reflection of conformational differences. Since the enzymes are calcium and zinc metalloproteins (9), differences in the trace metal content of the proteins may be involved. LITERATURE CITD 1. Arnott, M. S., and D. N. Ward. 1967. Separation of dansyl amino acids in a single analysis. Anal. Biochem.
21:50-56. 2. Bennett, J. C. 1967. Paper chromatography and electro-
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phoesis; special procedure for peptide maps. Methods Enzymol. 1,:330-339. 3. Benson,,J. V., R. T. Jopes, J. Cormick, and J. A. Patte,rson. 1i36. Accelerated automatic chromatographlc analysla)of peptides on a spherical resin. Anal. Biochem. 16:91-106. 4. Edelhoch, H. 1067.§$;pectroscopic determination of tryptophan and tyrosifi residues in proteins. Biochemistry 6:194&49545. Gros, C., and B. Labouesse. 1969. Study of /he dansylatioa of amnno acids, peptides and proteins. Euw. J. Biochem. 1:4j3-470. 6. Junge, J. M., 'R. A. Stc4n, H. Neurath, and E. H. Fischer. 1958. The amino acid composition of a-amylae fmom Bacillus subtilis. J. Biol. Chem. 234:556-561. 7. Moore, S. 1963. On the determination of cysteine as cysteic acid. J. Biol. them. 238:235-237. 8. Smith, E. L., F. S. Markland, C. B. Kasper, R. J. Delange, K. Landon, and W. H. Evans. 1966. The complete amino acid sequence of two types of subtilisin, BPN' and Carlaberg. J. Biol. Chem. 241:5974-5976. 9. Stein, E. A., and E. H. Fischer. 1960. Bacillus subtilis a-amylase, a zinc-protein complex. Biochim. Biophys. Acta 39:287-296. 10. Sugae, K., and Y. Honda. 1960. Studies on bacterial amylase. III. On the amino terminal peptide of bacterial amylase. J. Biochem. 47:307-314. 11. Welker, N. E., and L. L. Campbell. 1967. Comparison of the a-amylase of BaciUus subtilis and Bacillus amyloliquefaciens. J. Bacteriol. 94:1131-1135. 12. Welker, N. E., and L L. Campbell. 1967. Crystallization and properties of a-amylase from five strains of Bacillus amyloliquefaciens. Biochemistry 6:3681-3689. 13. Welker, N. E., and L. I. Campbell. 1967. Unrelatedness of Bacilus amyloliquefaciens and Bacilus subtilis. J. Bacteriol. 94:1124-1130. 14. Yamane, K., K. Yamaguchi, and B. Maruo. 1973. Purification and properties of a cross reacting material related to a-amylase and biochemical comparison with the parent a-amylase. Biochim. Biophys. Acta 295:323-340.
