THE JOURNAL OF BIOLOGICAL CHEMISTRY (0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 31, Issue of November 5, pp. 22108-22114,1992 Printed in U. S. A .

Catalytic Properties of the Cloned Amylase from Bacillus licheniformis*

(Received for publication, April 6, 1992)

In-Cheol Kim, Jae-Ho Cha, Jung-Ryul Kim, So-Young Jang, Byung-Cheol Seo, Tae-Kyou Cheong, Dae Si1 Lee$, Yang Do Choi6, and Kwan-Hwa Parkll From the Research Center for New Biomaterials in Agriculture and Department of Food Science and Technology, §Department of Agricultural Chemistrv. Seoul National Uniuersitv. Suwon 441-744 and the $.Genetic Engineering Research Institute,

“ I I,

Taeyon 305-601, Korea

A gene encoding a new amylolytic enzyme of Bacillus licheniformis (BLMA) has been cloned, and we char- acterized the enzyme expressed in Escherichia coli. The genomic DNA of B. licheniformis was double- digested with EcoRI and BamHI and ligated the pBR322. The transformed E. coli was selected by its amylolytic activity, which carries the recombinant plasmid pIJ322 containing a 3.5-kilobase fragment of B. licheniformis DNA. The purified enzyme encoded by pIJ322 was capable of hydrolyzing pullulan and cyclodextrin as well as starch. It was active over a pH range of 6-8 and its optimum temperature was 50 “C. The molecular weight of the enzyme was 64,000, and the isoelectric point was 5.4. It degraded soluble starch by cleaving maltose units preferentially but did not attack a-1,6-linkage. The enzyme also hydrolyzed pul- lulan to panose units exclusively. In the presence of glucose, however, it transferred the panosyl moiety to glucose with the formation of a-1,6-linkage. The spec- ificity of transferring activity is evident from the re- sult of the maltosyl-transferring reaction which pro- duces isopanose from maltotriose and glucose. The mo- lecular structure of the enzyme deduced from the nucleotide sequence of the clone maintains limited sim- ilarity in the conserved regions to the other amylolytic enzymes.

a-Amylase is one of the best known enzymes which hydro- lyzes a-1,4-linkage of starch producing a mixture of oligosac- charides. In recent years, evidence has mounted in support of the unusual action of amylases which exhibit transferring as well as hydrolyzing activity. New amylases have also been discovered from various microorganisms including maltose forming-, pullulan’ and/or cyclodextrin hydrolyzing-, and glu- cose-transferring amylases. a-Amylases from Thermoactino- myces vulgaris R-47 (1,2) and Bacillus stearothermophilus K P 1064 (3) degrade soluble starch, yielding maltose and glucose

* This research was supported by a grant from the Research Center for New Biomaterials in Agriculture, Seoul National University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X67133.

7 To whom correspondence should be addressed. Tel.: 82-331-292- 1943; Fax: 82-331-293-4789.

Structures of the oligosaccharides are defined pullulan, (-4Glcpcul-6Glcpnl-4Glcpnl-),; panose, Glcpal-6Glcp~l-4Glc; iso- panose, Glcpnl-4Glcpnl-6Glc; maltotriose, Glcpnl-4Glcpnl-4Glc.

as major products. They also hydrolyze cyclodextrins. These unusual enzymes convert pullulan to panose. A new type of pullulanase which produces panose from pullulan was also found in B. stearothermophilus (4, 5), and a gene for the enzyme was cloned and expressed in Bacillus subtilis. Suzuki et al. (6) isolated extracellular a-amylase I1 from Bacillus thermoamyloliquefaciem KP1071, which split a-1,6 bonds in amylopectin as well. This enzyme hydrolyzed a- and @-cy- clodextrins, and pullulan as well. David et al. (7) cloned a gene encoding the amylolytic enzyme of Bacillus megateriurn. Based on the action pattern of the enzyme, it has been proposed that this enzyme hydrolyzed pullulan as well as starch. Interestingly, it also exhibits glucose transferring ac- tivity with the formation of a-1,4-linkage. With the discovery of these new amylases, the scheme of the transferring activi- ties on the starch metabolism in the microorganisms was to be elucidated and the production of maltooligosaccharides in large quantities became easier.

We have cloned a gene encoding a new type of amylase, BLMA,2 from Bacillus licheniforrnis. The enzyme had the ability to hydrolyze pullulan and cyclodextrin as well as starch. It degraded soluble starch by cleaving maltose units preferentially. The usual thermostable amylase, BLTA, of B. licheniformis produces mostly maltohexaose and maltohep- taose from starch but cannot hydrolyze pullulan and cyclo- dextrins (13). BLMA also showed the catalytic properties of a new type of a-amylase which exhibited transferring activity. In the present work the molecular cloning of the gene for BLMA, catalytic properties, and the reaction pattern of the purified enzyme from E. coli are described.

EXPERIMENTAL PROCEDURES

Materials and Bacterial Strains-Maltooligosaccharides, CY-, p - , y- cyclodextrin, and panose were purchased from Sigma. Isopanose was a gift from Dr. Y. Sakano, Tokyo Noko University, Japan. B. lich- eniformis ATCC 27811 was obtained from the American Type Culture Collection (ATCC) and used as the donor strain for the BLMA gene. E. coli HB 101 was used as a host strain for recombinant DNA.

Cloning and Characterization of BLMA Gene-B. licheniformis chromosomal DNA was isolated by the spool method (8, 9). Isolated DNA was partially cleaved with EcoRI and BamHI and ligated into EcoRI and BamHI double-digested pBR322. The ampicillin-resistant colonies were toothpicked onto a solid LB plate containing 1% soluble starch and 50 mg/liter of ampicillin. Five ml of LB medium containing 3 mg of o-cycloserine and 0.6% agar was overlaid to each plate. After overnight incubation a t 37 “C, amylase-producing colonies were de- tected by the addition of 2.5% Lugol’s iodine solution. Positive

The abbreviations used are: BLMA, maltogenic amylase of B. licheniformis; BLTA, thermostable a-amylase of B. licheniformis; HPLC, high performance liquid chromatography; SDS-PAGE, SO- dium dodecyl sulfate-polyacrylamide gel electrophoresis; TLC, thin layer chromatography; bp, base pair.

22108

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Maltogenic Amylase of B. licheniformis 22109

colonies resulted in a clear zone on the agar medium. From these colonies plasmid DNA was isolated by the rapid alkaline extraction method of Sambrook et al. (8). Manipulation and analysis of DNA was also carried out by the procedure of Sambrook et al. (8).

Purification of BLMA from E. coli-Transformed cells containing the BLMA gene of B. licheniformis were cultured in 2.5 liters of LB medium containing 50 mg/liter of ampicillin a t 37 "C for 12-13 h in a jar fermenter and harvested by centrifugation a t 10,000 X g. The cells were resuspended in 30 ml of 50 mM Tris-HC1 buffer (pH 7.5) and sonicated for 5 min in an ice bath. The cell debris was removed by centrifugation a t 15,000 X g, and the supernatant was fractionated with solid ammonium sulfate a t 4 "C. The precipitate a t 70% satu- ration of ammonium sulfate was collected by centrifugation and dissolved in 30 ml of 50 mM Tris buffer (pH 7.5) containing 1% of streptomycin sulfate. After centrifugation the supernatant was di- alyzed at 4 "C against the same buffer without streptomycin. The dialysate was applied to Sephadex G-100 (3 X 80-cm) and fractionated with 50 mM Tris-HC1 buffer (pH 7.5). The collected active fractions were concentrated through PM-10 membrane (Amicon Co.) under a nitrogen atmosphere. The concentrate was applied on a DEAE- cellulose column (3 X 30-cm) equilibrated with 50 mM Tris buffer (pH 7.5). The column was washed with 250 ml of the same buffer (30 ml/h), and then the enzyme was eluted with a linear gradient of potassium chloride from 0 to 0.8 M in the same buffer. The active fractions were eluted a t 0.4 M NaCl, and the pooled fraction was purified further by a fast protein liquid chromatography (FPLC) system (Pharmacia LKB Biotechnology AB) under the following conditions: column, Mono Q HR 5/5 anion exchanger; buffer A, 20 mM Tris-HC1 (pH 7.0); buffer B, 0.5 M NaCl in buffer A; 0-100% gradient in 40 min; flow rate, 1.0 ml/min. Active fractions eluted at 0.4 M NaCl were collected and characterized.

Determination of Molecular Weight and Isoelectric Point-Molec- ular weight of purified BLMA was determined by SDS-PAGE using 4% stacking and 12% resolving gels of 1-mm thickness as described by Laemmli (10). The relative molecular weight of the purified enzyme was estimated by comparing its relative mobility with those of the following reference proteins: /3-galactosidase ( M , 116,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhy- drase ( M , 30,000), and a-lactalbumin ( M , 15,000). The isoelectric point of the enzyme was estimated using Phast Gel IEF 4-6.5 by Phast System (Pharmacia). The isoelectric point of the enzyme was estimated by comparing the relative migration distance of the band.

Enzyme Assay-Hydrolytic activity of BLMA was assayed as de- scribed by Suzuki et al. (6) with minor modifications. The mixture containing 0.5 ml of 1% soluble starch, 0.25 ml of 0.04 M Tris-malate buffer (pH 6.8), and 5 mM of EDTA was prewarmed a t 50 "C for 5 min. The enzyme solution of 0.25 ml was added to the prewarmed solution and incubated at 50 "C for 30 min. The reaction was termi- nated by adding 3 ml of dinitrosalicylate solution (11). After boiling for 5 min in a water bath, absorbance was measured a t 550 nm. One unit of the enzyme was defined as the amount of the enzyme giving a n increase of 1.0 absorbance for 30 min under the described condi- tions.

Action Pattern of BLMA-To determine the hydrolytic action mode, 20 p1 each of the purified enzyme from the Bacillus donor strain and from the E. coli transformant was incubated a t 50 "C with 0.5 ml of 1% starch, pullulan, or cyclodextrin solution and 0.25 ml of 0.04 M maleate buffer (pH 6.8) containing 5 mM EDTA for 12 h. For the transferring activity determination, 1% solution of maltose, mal- totriose, maltotetraose, maltopentaose, or maltohexaose was digested with BLMA. For the panosyl transferring activity, 1% pullulan so- lution was incubated in the presence of 9-, 18-, 27-, 36-, 45-, 54-, and 63% glucose under the same conditions as above. The reaction mix- ture was analyzed by TLC, paper electrophoresis, and/or HPLC.

Thin Layer Chromatography and Paper Electrophoresis-TLC was carried out on the Kiesel Gel-60 plate (Merck Co., Ltd.) with a solvent system of isopropyl alcohol/ethylacetate/water (3:1:1, v/v/v). After 5 h of development, the TLC plate was dried and visualized by spraying with 50% sulfuric acid in methanol and heating a t 110 "C. Paper electrophoresis was carried out on Whatman paper No.1 (20 X 20- cm) in 0.05 M borate buffer (pH 10.0) a t 500 V, 2 mA/cm for 1 h (12). After electrophoresis the paper was dried and sprayed with silver nitrate solution (0.1 ml of saturated silver nitrate + 20 ml of acetone). Then 4.5% pentaerythritol ethanolic solution in 0.5 M NaOH was sprayed.

High Performance Liquid Chromatography-The chromatographic analysis of the hydrolysates was carried out under the following conditions: column, Machery & Nagel Nucleosil 10NH2 (4.6 X 300

mm); detector, Pye Unicam PU4023 refractive index detector; solvent, acetonitrile/water (65-7035-30, v/v); flow rate, 1.0 ml/min; sample loop, 20 pl. Before injection, an equal volume of cold acetonitrile was added to the hydrolysate, mixed, and centrifuged. The supernatant was filtered through a membrane filter (0.45 pm), and the filtrate was applied to the injection port.

"C NMR Spectroscopy-The trisaccharide product from the pul- lulan hydrolysate by BLMA was purified by TLC. I3C NMR spectra of the purified trisaccharide and standard panose in D20 were re- corded a t 75 MHz (Bruker), in which the signal of internal standard (CHsOD) was at 6 50.4.

RESULTS

Isolation of a Gene for BLMA-To isolate a gene encoding amylase from B. licheniformis, chromosomal DNA was par- tially double-digested with EcoRI and BamHI, ligated into pBR322, and transformed into E. coli HB101. About 600 ampicillin-resistant colonies were screened for amylase gene, and one positive colony was selected by its amylolytic activity. Plasmid DNA, pIJ322, was isolated from the amylase-positive colony, and the insert size in the recombinant plasmid was 3.5 kbp. I t was confirmed by retransformation of E. coli HBlOl with pIJ322. The control transformant carrying pBR322 did not show any amylolytic activity.

To analyze the genomic structure of the gene encoding BLMA in B. licheniformis, genomic Southern blotting analy- sis was carried out. There were one EcoRI fragment of 6 kbp and two BurnHI fragments of 2.5 and 2.3 kbp which were hybridized to pIJ322 (Fig. IA). This result was consistent with the presence of the internal BamHI site on the pIJ322 insert. As shown in the partial restriction endonuclease map of pIJ322, the insert has a single restriction endonuclease site for BamHI, ClaI, and SalI, and two for Hind111 (Fig. 1B). Comparing the intensity of the plasmid pIJ322 insert recon- structed with the equivalent amount of a single copy gene, the intensity of bands suggest that there is only a single copy of BLMA gene in B. licheniformis (data not shown). A simple genomic restriction fragment pattern also supports a unique gene structure.

Structural Analysis of the BLMA Gene-To analyze the

A

2 0

1.6 1 9

1.3

0 98 0 . ~ 3

0.56

4.0

5.8 4.3

2 3

1.2

B

E w R I

sa l I

FIG. 1. Southern Blot analysis of the genomic DNA of B. licheniformis. Panel A, genomic DNA was digested with the restric- tion enzyme indicated, separated by 0.5% agarose gel electrophoresis, and was transferred onto a nitrocellulose filter. I t was hybridyzed with pIJ322 which was labeled by nick translation with [a-"PIdATP. Lane A, EcoRI and HinIII-digested phage DNA. Lanes B and C: BamHI- and EcoRI-digested genomic DNA, respectively. Lane D, BamHI and EcoRI double-digested genomic DNA. Lane E, BamHI and EcoRI double-digested pIJ322. Lane F, EcoRI-digested pBR322. Panel B, partial restriction enzyme map of recombinant plasmid pIJ322. Open region is vector DNA pBR322 and closed region is the insert containing the BLMA gene.

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22110 Maltogenic Amylase of B. licheniformis

structure of the BLMA gene, nucleotide sequencing was car- ried out (Fig. 2) There was one open reading frame encom- passing 1740 nucleotides encoding a protein of 580 amino acids. The molecular size of the deduced polypeptide was calculated to be 66,931 which is consistent with the apparent molecular weight of BLMA, 64,000, estimated by SDS-PAGE (Fig. 3A). The insert of the clone contains about 291 bp and 1.2 kbp of 5‘- and 3”flanking sequences, respectively. Puta- tive promoter elements, TTAACA (-35 element) and TGA- TAAT (-10 element), are present around -93 and -54 from the translational initiation site, respectively. A Shine-Dal- garno sequence, AGGGGG, was also noticed around -8. These results are quite consistent with the functional analysis of the pIJ322 by serial deletion (13).

Purification and Characterization of BLMA-To study the reaction mechanism of BLMA, the enzyme produced by trans- formed E. coli was purified to an apparent homogeneity by using fast protein liquid chromatography (14). The purified fraction did not contain any detectable amount of contami- nating proteins as judged by SDS-PAGE and electrofocusing

.226 -151

-76 -1

75 25

150 50

225 75

300 100

375 125

450 150

525 175

6M) 200 675 225

750 250

9:: 900 300

975 325

IO50 350

1125 375

I200 400

1275 425

1350 4 5 0

1425 475

1500 500 I575 525

1650 550

1725 575

1800 578

1875 1950 2025 2104 2175 2180

FIG. 2. Nucleotide and deduced amino acid sequence of BLMA gene.-The nucleotide sequence was numbered from the first nucleotide for translation initiation. A putative promoter (-35 and -10 region) and a probable Shine-Dalgarno sequence are underlined. The inverted repeat sequence upstream and downstream of coding region are shown as shaded. Four blocks of conserved sequences in amylases are shown in boxes.

A B

M B $“=SF” c pH 4.0

11 6,000- 67,000~ 43,000+ 30,000*

15,000-

- c p l 5.4

c pH 6.5

FIG. 3. Electrophoresis of purified BLMA. Panel A, SDS- PAGE was carried out by Phast System using Phast Gel SDS-PAGE 12.5% and was stained with Coomassie Blue. Lane M, molecular weight marker; lane B, purified BLMA. Panel B, electrofocusing was carried out with Phast Gel IEF 4-6.5. Separated proteins were de- tected by silver staining.

(Fig. 3). The molecular weight of BLMA was determined to be about 64,000 by SDS-PAGE, and the isoelectric point of the enzyme was found to be around pH 5.4. The optimum pH of hydrolyzing activity was pH 6 for pullulan, pH 7 for starch, but around pH 7-9 for cyclodextrin (13).

Hydrolytic Activity of BLMA-To test the substrate speci- ficity of BLMA, several different substrates were subjected to hydrolysis by BLMA. Substrate specificity was compared with that of BLTA. Soluble starch, pullulan, and cyclodextrin were readily hydrolyzed by BLMA, while BLTA hydrolyzed only soluble starch. To examine the hydrolysis pattern of BLMA, the reaction product was analyzed by TLC at various time points of digestion as shown in Fig. 4. The major product a t various stages of hydrolysis of soluble starch was apparently maltose (Fig. 4A). As the reaction proceeded, the quantity of maltose was increased. When P-cyclodextrin was used as the substrate, the enzyme could liberate a series of low molecular weight oligosaccharides, in which the main product was malt- ose (Fig. 4B). In case of pullulan, which contains one a-1,6- and two a-1,4-glycosidic linkages alternately, the major reac- tion product migrates at the position between maltotriose and maltotetraose (Fig. 4C). Compared with the panose standard, however, this product could be panose.

The result of TLC analysis for pullulan hydrolysis was consistent with that of HPLC analysis (Fig. 5). The major hydrolysis product from pullulan showed the same retention time as panose in HPLC (Fig. 5B). Panose and isopanose, however, were not distinctively resolved on TLC and HPLC under these conditions. Paper electrophoresis was carried out to identify this major product. Fig. 6 clearly shows that the hydrolysis product from pullulan was panose rather than isopanose.

The hydrolysis product of pullulan by BLMA was analyzed further by 13C NMR after isolation of the major product by TLC. The spectrum of the isolate was shown to be identical to that of the authentic panose (data not shown). These results made it sure that the trisaccharide produced by BLMA was clearly panose.

These results altogether suggest that BLMA hydrolyzes every other a-1,li-glycosidic linkage to produce maltose from soluble starch. It also hydrolyzes the a-lP-linkage of pullulan with the production of panose, which the usual @-amylase cannot. It, however, could not hydrolyze the a-l,ri-linkage right next to the a-1,6-linkage (Fig. 10).

Transferring Activity of BLMA-To examine the transfer-

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Maltogenic Amylase of B. licheniformis 22111

RICTKYl TIY.o((xR)

C Gl h

02 4 '5 Gl * G2

O3 e $ 03 M ) .'.!I b * @ #4 8 * m 05 Ge

05 DB

o w 0 0 s O ? 2 3 4 L l 2 o p l N s

REACTION TIME (mwm

FIG. 4. Thin layer chromatogram of the hydrolysis products of various substrates by BLMA. Soluble starch (panel A ) , p- cyclodextrin (panel R ) , and pullulan (panel C) were incubated with the purified enzyme, and the reaction products were taken a t various time points and analyzed by TLC using isopropyl alcohol/ethylace- tate/water (3:1:1, v/v/v) as solvent. G I , G2, G3, G4, G5, and G6 are standards of glucose, maltose, maltotriose, maltotetraose, maltopen- taose, and maltohexaose, respectively.

A B

i

. . . , .

FIG. 6. Paper electrophoresis to identify the panose and isopanose. I P , isopanose standard; P, panose standard H, hydrolysis product of pullulan by BLMA.

t

8 16 24 32 40 min

FIG. 7. High performance liquid chromatogram of the pul- lulan hydrolysis products by BLMA in the presence of 20% glucose. Position of standard glucose ( I ) , maltose ( 2 ) , maltotriose (3 ) , maltotetraose ( 4 ) , and maltopentaose (5) are indicated by arrows. P, panose; R, Glcpnl-6Glcpal-4Glcpnl-4Glc added as internal ref- erence; U, unknown branched tetrasaccharide assumed to be Glcpcul- 6Glcpcul-4Glcpcul-6GIc. The HPLC analysis was carried out under the following conditions: column, Machery & Nagel NucleosillONH2 (4.6 X 300 mm); detector, Pye Unicam PU4023 refractive index detector; solvent, acetonitrile/water (7525, v/v); flow rate, 1.0 ml/ min; sample loop, 20 PI.

10 20 30 40 min

10 20 30 40 min

Retention time

FIG. 5 . High performance liquid chromatographic analysis of the hydrolysis product of pullulan with BLMA. Panel A, standard solutions of glucose ( I ) , maltose (Z), maltotriose (3), panose (4), maltotetraose (S), maltopentaose (6), maltohexaose (7) . Panel H, reaction product of pullulan + BLMA. The HPLC analysis was carried out under the following conditions: column, Machery & Nagel Nucleosil 10NH2 (4.6 X 300 mm); detector, Pye Unicam PU4023 refractive index detector; solvent, acetonitrile/water (65:35, v/v); flow rate, 1.0 ml/min; sample loop, 20 PI.

ring activity of the enz$me, pullulan was incubated with BLMA in the presence of a high concentration of glucose, and the reaction products were analyzed by HPLC as shown in Fig. 7. The reaction products were identified as panose, glu-

cose, maltose, and unknown oligosaccharide which were de- tected between maltotetraose and maltopentaose. Retention time of the unknown oligosaccharide suggests that it might be a branched tetrasaccharide. It, however, was eluted later than one of the branched maltotetraoses, Glcpal-6Glcpal- 4Glcpa1-4Glc, which was added to the reaction product as an internal reference. This unknown branched tetrasaccharide was thought to be Glcpal-6Glcpal-4Glcpal-6Glc, which was made by the transferase activity of BLMA.

Specificity of transferring activity of the enzyme was dem- onstrated further by digesting maltooligosaccharide. Malto- triose was degraded by BLMA in the presence of glucose to form a product with the migration distance corresponding to that of isopanose as shown in Fig. 8. This result suggests that the bond formed by transferring activity of BLMA is a-1,6- linkage between the reducing end of maltose and C-6 of

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22112 Maltogenic Amylase of B. licheniformis

acceptor glucose. These results strongly argue that the un- known tetrasaccharide generated from pullulan in the pres- ence of glucose is Glcpal-6Glcpal-4Glcpal-6Glc (Fig. 7).

The effect of glucose concentration on the formation of branched tetrasaccharide was tested by digesting pullulan in various concentrations of glucose (Fig. 9). Formation of the branched tetrasaccharide was maximum at the concentration of 10-27% glucose, while at the concent,ration of 36% glucose the formation of the branched tetrasaccharide was dramati- cally decreased. The branched tetrasaccharide was not formed over 45% glucose in the reaction mixture. The concentration of the branched tetrasaccharide increased with incubation time (14). These results indicated that in the presence of

G3 P T IP

FIG. 8. Paper electrophoresis to identify panose and isopa- nose. G 3 , maltotriose; P, panose; T, maltotriose + glucose + RLMA; II' , isopanose. The fast migrating spot in lane T was identified as glucose.

20 40 glucose tonc (. )

FIG. 9. Effects of glucose concentration on the transferring activity of BLMA. Open circles denote panose and closed circles denote the branched oligotetrasaccharide concentration in the reac- tion mixture.

glucose pullulan was degraded by BLMA via transfer of the panosyl moieties to C-6 of glucose. In this process, glucose served as an acceptor for panose, and, therefore, the concen- tration of panose was kept relatively low during the coupling reactions, for the formation of branched tetrasaccharide. BLMA, however, did not catalyze the formation of branched tetrasaccharide from the mixture of panose and glucose, sug- gesting the coupling of hydrolysis and transferring reactions (14).

DISCUSSION

This study reports a new type of amylase. The a-amylase produced by B. licheniformis, BLTA, has been known to be extremely thermostable (13). The enzyme hydrolyzes starch into mostly maltohexaose and maltoheptaose but does not degrade pullulan, a- and P-cyclodextrin. However, a new amylase produced by E. licheniformis reported in this paper, BLMA, was not thermostable. The molecular weight of BLMA ( M , 64,000) differs from that of BLTA (Mr 55,200). In contrast to BLTA, BLMA hydrolyzes soluble starch, a-, and p-cyclodextrin to maltose.

Campbell (15) and Bliesmer and Hartman (16) have re- ported that two different amylase genes might exist in Bacillus sp. because two different a-amylases were produced a t differ- ent culture temperatures. BLMA could be produced by one of the two amylase genes which was barely expressed in B. licheniformis. BLMA was not detectable in the culture broth of the original B. licheniformis strain but barely detected in cell lysate. A fairly high level of enzyme activity, however, was noticed in E. coli transformed with the BLMA gene, which might result from regulated gene expression (13).

When the deduced amino acid sequence of BLMA was compared with those of various amylases reported, limited similarities a t four prominently conserved regions were no- ticed (Table I). A relatively high similarity was noticed at conserved region I which is considered to be the calcium- binding domain of amylase (16, 17). The various spacings between these conserved domains could reflect the chemical nature of the reaction catalyzing and thus product specificity of the enzyme.

Even though the substrate specificity of BLMA from the clone seems to be similar to that of a-amylase I1 from B. thermoamyloliquefaciens KP1071 (6), a-amylase from T. vul- garis R-49 (1, 2), maltogenic a-amylase from B. megaterium

TABLE I ComDarison of the deduced amino acid seauences of various amvlases

~

Amino acid sequencesa

Region 1 Region 2 Region 3 Region 4 Refs.

Consensus sqn DAVINH GFRLDAAKH EVID FVDNHD 23 RLMA 237 - - - F - - 242 318 -vwMwQM-L 326 349 -1WH 352 410 LL-S- - 415 This work Neopullulanase 240 - - -F- - 245 324 -W---V-NE332 357 -1WH360 419LLGS" 424 16 Pullulanase 281 - V - Y - - 286 348 ---F-LMGI 356 381 -GW- 384 464Y-ES" 469 17 CGTase 135 -FAD-- 140 225 - I -V- -V- - 233 268-YHQ271 293-1"" 298 24 BLTA 129 - V - I - - 134 256 - - - - - - V - - 264 29ODYWQ294 352 """357 25

Sequence similarities ( % ) h

Region 1 Region 2 Region 3 Region 4

nt aa nt aa nt aa nt aa

BLMA 100 100 100 100 100 100 100 100 Neopullulanase 88.8 100 25.9 11.1 91.6 100 77.7 83.3 Pullulanase 66.6 66.6 40.7 22.2 58.3 50.0 44.4 50.0 CGTase 50.0 50.0 44.4 22.2 50.0 25.5 66.6 50.0 BLTA 66.6 66.6 29.6 22.6 66.6 25.5 77.7 50.0

"Amino acid sequences different from consensus sequence are shown. Amino acid sequence similarities (aa) and nucleotide sequence similarities (nt) to BLMA at conserved region.

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Maltogenic Amylase of B. licheniformis

TABLE I1 ComDarison of the ohvsicochemical DroDerties of various amvlases

22113

~~~~

Enzyme Substrate specificity

Substrate Major product Origin M, Transfering

activities Refs.

BLMA B. licheniformis 64,000 Starch Maltose Formation of This work Pullulan Panose a-1,6-linkage

BLTA

Pullulanase

Isopullu- lanase

Neopullu- lanase

BMA

T. vulgaris a-amylase

Cyclodextrin

Pullulan Cyclodextrin

Pullulan Cyclodextrin

Pullulan Cyclodextrin

Pullulan Cyclodextrin

Pullulan Cyclodextrin

Pullulan Cvclodextrin

B. licheniformis 55,000 Starch

I(. pneumoniae 66,000 Starch

A. niger 62,000 Starch

B. stearothermophilus 62,000 Starch

B. megaterium 55,000 Starch

T. vulgaris 71,000 Starch

Maltose Maltopentaose - -

13

-

- - 26 Panose - - - 27 Isopanose

Maltose - 4 Panose ?b

Oligomer Formation of I Panose a-l,4-linkage Maltose Maltose - 1 Panose ?

-

~

-, no products. ?, not determined.

,,2% 0-0 maltose

% branched tetrasaccharide 11

FIG. 10. Proposed action pattern of BLMA, coupling of hy- drolysis, and transferring activity of BLMA generate. Open circles and slashed circles denote non-reducing glucopyranosyl resi- dues and reducing glucose residues, respectively. Horizontal lines and vertical lines connecting circles indicate a-1,4- and a-1,6-glucosidic linkages, respectively. The structure of branched tetrasaccharides I and I1 are Glcpal-6Glcpal-4Glcpal-6Glc and Glcpal-4Glcpal- GGlcpnl-4Glc, respectively.

(6, 20), pullulanase from Klebsiella aerogenes (21), isopullu- lanase from Aspergillus niger (22), and neopullulanase from B. stearothermophilus (4, 5, 19), the chemical nature of the reaction catalyzed by BLMA is different from theirs. BLMA can attack typically every other endo a-1,4-linkages but not the a-l,6-linkage. It, however, cannot hydrolyze the a-1,4- linkage next to the a-l,g-linkage, which results in the release of panose exclusively from pullulan rather than isopanose. Pullulanase, however, produces isopanose rather than panose. Neopullulanase from B. stearothermophilus could hydrolyze a-1,6-linkage as well as a-1,l-linkage and, therefore, releases a significant amount of glucose and maltose in addition to panose (4, 5 ) . The substrate specificities of various unusual

amylases are summarized in Table 11. I t is interesting to note that BLMA has transferring activity

in addition to hydrolyzing activity. The branched tetrasac- charide (Glcpal-6Glcpal-4Glcpal-6Glc) havinga new a-1,6- linkage is formed as BLMA degrades pullulan in the presence of glucose. Hydrolysis specificity of a-amylase from B. mega- terium and T. vulgaris seems to be quite similar to that of BLMA. David et al. (7), however, pointed out that the hy- drolysis reaction of a-amylase from E . megaterium in the presence of glucose is gradually replaced by the coupling reaction with the formation of Glcpal-6Glcpal-4Glcpal- 4Glc which was formed by panosyl transfer from pullulan to the C-4 position of the glucose molecule rather than the C-6 position. 'H and 13C NMR studies confirmed that it did catalyze formation of a-1,4-linkage in the transfer reactions. In contrast, BLMA catalyzes the formation of a-1,6-linkage (Table 11).

The transferring reaction by BLMA in the presence of maltotriose and glucose gave isopanose in which a-l,g-linkage was formed by the maltosyl transfer. Incubation of maltotriose and maltose with BLMA also results in the formation of branched maltotetraose in which two maltose units are linked by a-1,6-linkage (14). While pullulan was incubated with BLMA in the presence of glucose, branched maltotetraose was produced. The retention time of the branched maltote- traose in HPLC differed from those of maltotetraose and Glcpal-6Glcpal-4Glcpa!1-4Glc, indicating the existence of a-1,6-linkage in the tetraose. The retention time of the un- known products on the HPLC column was between linear maltotetraose and maltopentaose suggesting the existence of maltotetraose with a-l,g-linkage which did not exist in the reaction mixture. From the results, the action pattern of BLMA can be summarized as in Fig. 10.

Combinations of donors such as maltose or panose and acceptor molecules such as glucose or maltose in the presence of BLMA will give rise to a variety of branched oligosaccha- rides. Such a novel transferring activity of BLMA could be utilized to synthesize various kinds of branched oligosaccha- rides.

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H ParkI C Kim, J H Cha, J R Kim, S Y Jang, B C Seo, T K Cheong, D S Lee, Y D Choi and K

Catalytic properties of the cloned amylase from Bacillus licheniformis.

1992, 267:22108-22114.J. Biol. Chem. 

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