INTRODUCTION

Amylolytic enzymes, such as -amylase (EC) and debranching pullulanase (pullulan 6-glucanohydrolase; EC), are industrially important for the liquefaction of starch and in saccharification processes (Norman, 1982). We have found and characterized some unique debranching enzymes, such as an alkaline pullulanase (Ara et al., 1992), an alkali-resistant neopullulanase (Igarashi et al., 1992), and an alkaline isoamylase (EC) (Ara et al., 1994), in strains of alkalophilic Bacillus, and these enzymes can be used as effective additives in dishwashing and laundry detergents under alkaline conditions. Amylopullulanases (APase)¹ (Saha et al., 1989) or type II pullulanases (Spreinat and Antranikian, 1990) have frequently been found in cultures of thermophiles (Coleman, 1993). Recently, we also found and characterized the first known alkaline APase (the product of the apuA gene) in cultures of alkalophilic Bacillus sp. KSM-1378 (Ara et al., 1995b). This APase is unique in that it efficiently hydrolyzes the -1,6 linkages of pullulan, as well as the -1,4 linkages of amylose, amylopectin, and glycogen at alkaline pH values, whereas other APases, the type that have frequently been found in cultures of some thermophiles, are all active at acid or neutral pH.

There are many reports that demonstrate unequivocally that a single active site is involved in the -1,4- and -1,6-hydrolyzing activities of several APases, such as the enzymes from Thermoactinomycetes (Sakano et al., 1982), Thermus sp. (Nakamura et al., 1989), Clostridium thermohydrosulfuricum (Melasniemi, 1988; Saha et al., 1988), Pyrococcus furiosus, and Thermococcus litoralis (Brown and Kelly, 1993). For example, it has been shown that the specific conversion of an aspartic acid residue and a glutamic acid residue individually to their amide forms in the APase from Thermoanaerobacter ethanolicus 39E (formerly C. thermohydrosulfuricum 39E; Lee et al. (1993a)) caused the loss of both the -amylase and the pullulanase activities, providing proof of the presence of a single active site in the enzyme (Mathupala et al., 1993). However, in the case of the alkaline APase from Bacillus sp. KSM-1378, all the kinetic data that we have obtained (Ara et al., 1995a, 1995b) can only be explained by assuming that the dual catalytic activity of this enzyme is associated with different active sites. In fact, we found that the 210-kDa APase of this organism could be cleaved by papain to generate a 114-kDa amylose-hydrolyzing polypeptide and a 102-kDa pullulan-hydrolyzing polypeptide (Ara et al., 1996). This report describes the molecular cloning of the apuA gene for the 210-kDa APase from Bacillus sp. KSM-1378 and the determination of its nucleotide sequence. Furthermore, the deduced amino acid sequence of the product of the apuA gene from this organism is compared with those of other acid and neutral APases, with emphasis on their respective catatytic properties and molecular structures.

EXPERIMENTAL PROCEDURES

The source of the enzyme examined in this study was Bacillus sp. KSM-1378, which had previously been isolated from a soil sample in our laboratory. It was grown in a liquid medium composed of 1.0% (w/v) pullulan (Hayashibara, Karushiki, Japan), 0.1% yeast extract (Difco), 0.2% tryptose (Difco), 0.03% K₂HPO₄, 0.02% MgSO₄·7H₂O, 0.1% (NH₄)₂SO₄, 0.02% CaCl₂·2H₂O, 0.001% FeSO₄·7H₂O, 0.0001% MnCl₂·4H₂O, and 1.0% Na₂CO₃ (pH 10). The solutions of trace metals and Na₂CO₃ were autoclaved separately. Escherichia coli HB101 (FhsdS20 recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 supE44 leuB6 thi-1) and pBR322 were used for cloning experiments. E. coli was grown with shaking in LB broth (Difco) at 37 °C. When appropriate, ampicillin and tetracycline were added at final concentrations of 100 and 15 µg/ml, respectively.

Genomic DNA from Bacillus sp. KSM-1378 was prepared as described by Saito and Miura (1963), and plasmid DNA was isolated by the alkaline extraction procedure of Birnboim and Doly (1979). E. coli HB101 and Bacillus subtilis ISW1214 (leuA8 metB5 hsrM1) cells were transformed with plasmids by the methods of Hanahan (1983) and Chang and Cohen (1979), respectively. Transformed B. subtilis cells were grown at 30 °C for 60 h with shaking in LB broth supplemented with tetracycline (15 µg/ml).

Genomic DNAs after digestion with restriction enzymes and electrophoresis were allowed to hybridize as described by Southern (1975). The probes, which were labeled with digoxigenin-dUTP, were prepared, and patterns of hybridization with the probes were examined with a digoxigenin DNA Labeling and Detection kit (Boehringer Mannheim).

Primer DNAs were designed for the amplification of appropriate regions between specific sites in the genomic DNA. The primer sequences used were as follows: primer A, 5-CGTATTTACCGATACCTGCAG-3; primer B, 5-CGTAACGAATCTTGCTCTAGA-3; primer C, 5-ATTTTTGATAATGCTCTAGA-3; primer D, 5-CCGGAACTGAGAATCAAAGAATTC-3; primer E, 5-TCCCTCATGGCGATTTCCGAATTC-3; primer F, 5-GTGGATGGTAATGAAATTCTAGA-3; primer G, 5-GCGCAAACGATTTCACATTTACAT-3; and primer H, 5-ATGCAATTCTGCCCCAAGCTTTC-3. They were prepared with a DNA synthesizer (model 392A, Applied Biosystems) and were purified with a DNA Refining System (model Dnastec-1000, Astec). PCR (Saiki et al., 1985) was performed in a DNA thermal cycler (model 480, Perkin-Elmer), using each primer (0.2 µg) plus genomic DNA (1.0 µg) from Bacillus sp. KSM-1378. The reaction conditions were as follows: 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min for 20 cycles. The reaction mixture contained 200 µM dNTPs, 25 mM KCl, 5 mM (NH₄)₂SO₄, 2 mM MgSO₄, 2.5 units of Pwo DNA polymerase, and 10 mM Tris-HCl buffer (pH 8.85) (Boehringer Mannheim) in a reaction volume of 100 µl. Products of PCR were purified with a Geneclean II kit (Bio 101 Inc.), and they were used for sequencing or for subcloning.

Sequencing was performed by the dideoxy chain termination method of Smith et al. (1986), using fluorescent terminators and an automated DNA sequencer (model 373A, Applied Biosystems). Both strands of the DNA were sequenced, and computer analysis was performed using a GENETYX program (SDC Software Development, Tokyo, Japan). When necessary, DNA fragments that had been amplified by PCR were sequenced directly after purification with a Geneclean II kit.

-Amylase and pullulanase activities were measured at 40 °C in a 1.0-ml reaction mixture that contained 0.5 ml of a 1.0% (w/v) solution of amylose (degree of polymerization, 17; Hayashibara) or pullulan (M_r 64,800, 95.2% pure, Hayashibara) in 80 mM glycine/NaCl/NaOH buffer (pH 9.0) and 0.1 ml of a suitably diluted solution of enzyme, as described elsewhere (Ara et al., 1995b). The reducing sugar formed was quantified by the dinitrosalicylic acid procedure (Miller et al., 1960). One unit of enzymatic activity was defined as the amount of protein that produced 1 µmol of reducing sugar as glucose per min under the conditions of the assay. Protein was quantitated by the method of Bradford (1976), with a protein assay kit (Bio-Rad) and bovine plasma albumin as the standard.

APase from Bacillus sp. KSM-1378 was purified such that it yielded a single band of protein after both nondenaturing PAGE and SDS-PAGE by chromatography on DEAE-cellulose, affinity chromatography on Sepharose 6B--cyclodextrin, and gel filtration on Sephacryl S-200, as reported previously (Ara et al., 1995b). Specific activities of the purified APase were 47 units/mg protein for amylose and 84 units/mg protein for pullulan under the standard conditions of the assays.

The purified intact APase (0.5 mg) was partially digested with papain (5 milliunits; Sigma) at 25 °C for 10 min in 2 ml of 10 mM Tris-HCl buffer (pH 8.0) to generate the two fragments, designated the 114-kDa amylose-hydrolyzing polypeptide and the 102-kDa pullulan-hydrolyzing polypeptide (Ara et al., 1996). The polypeptide fragments were each purified by HPLC (model LC-7A, Shimadzu, Kyoto, Japan) on a column of TSK DEAE-5PW (7.5-mm inner diameter × 7.5 cm; Tosoh, Tokyo, Japan), which had been equilibrated with the same buffer plus 250 mM NaCl. The active fragments were eluted from the column at a flow rate of 1.0 ml/min with a linear gradient of 250 to 350 mM NaCl in the equilibration buffer. The two purified fragments were homogeneous as judged by nondenaturing PAGE and SDS-PAGE. They displayed similar enzymatic properties, such as temperature optima, pH optima and substrate specificity, to the respective properties of the intact APase, as described elsewhere (Ara et al., 1996).

Pullulanase activity encoded by pBP101 was expressed in transformed E. coli cells that were cultured in LB broth at 37 °C for 2 days. Harvested cells were sonicated in 10 mM Tris-HCl buffer (pH 8.0) on ice, and the crude enzyme was prepared by treating the cell-free extract, namely, the centrifuged sonicate, with ammonium sulfate and pelleting the protein that precipitated between 35 and 80% saturation. The pellet was dissolved in 10 mM Tris-HCl buffer (pH 8.0), and the solution was dialyzed against the same buffer. The retentate was then applied to a column of DEAE-Toyopearl 650S (1.5-cm inner diameter × 65 cm; Tosoh) that had been equilibrated with the same buffer. The column was washed with 200 ml of the equilibration buffer, and proteins were eluted in 1.5-ml fractions at a flow rate of 15 ml/h with a 1.0-liter gradient of 0-1.0 M NaCl in the equilibration buffer. The active fractions were combined and concentrated by ultrafiltration on a PM-10 membrane (10-kDa cut-off; Amicon). The concentrated sample was applied to a column of Sephacryl S-200 (1.5-cm inner diameter × 96 cm; Pharmacia Biotech Inc.) that had been equilibrated with 10 mM Tris-HCl buffer (pH 8.0) plus 0.1 M NaCl. Elution was performed with the same buffer, and 1.5-ml fractions were collected at a flow rate of 7.0 ml/h. Pullulanase activity in some of the fractions was checked by staining for both protein and activity after nondenaturing PAGE. The active fractions were combined and dialyzed against a large volume of 10 mM Tris-HCl buffer (pH 8.0) and then used as the final preparation of recombinant enzyme (0.4 mg of protein). The purified enzyme was used for studies of the enzymatic properties of pullulanase, such as optimum pH, optimum temperature, and substrate specificity.

Nondenaturing PAGE was performed by the method of Davis (1964), and SDS-PAGE was performed essentially as described by Laemmli (1970) on slab gels (90 × 90 mm, 2.0-mm thickness). Concentrations of acrylamide were 9.0% (w/v) for nondenaturing gels and 7.5% (w/v) for SDS-PAGE. Activity staining of amylase in slab gels was performed essentially as described by Taniguchi et al. (1982), with agar sheets containing starch azure (Sigma) as replica plates. The slab gel after nondenaturing PAGE was laid on the replica sheet and was left for several hours at room temperature. The bands of protein that were associated with amylase activity were seen as clear zones on the replica sheet, on a dark blue background. Activity staining for pullulanase was performed as described by Ara et al. (1992), using red pullulan-agar sheets as replica plates.

Molecular masses were estimated by SDS-PAGE (7.5% (w/v) acrylamide) with high range molecular mass standards (Bio-Rad), which included rabbit skeletal muscle myosin (200 kDa), -galactosidase from E. coli (116.3 kDa), rabbit muscle phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), and hen egg white ovalbumin (45 kDa). Proteins were stained with Coomassie Brilliant Blue dye.

Each polysaccharide was dissolved in 1 ml of 40 mM Tris/HCl buffer (pH 8.0) at a final concentration of 0.5% (w/v). An aliquot of the enzyme preparation was added to the reaction mixture, which was then incubated at 30 °C for an appropriate time. At intervals, 10-µl aliquots were withdrawn and subjected to chromatography on thin-layer plates of silica gel 60 (10 × 20 cm; Merck) in a solvent system composed of butanol, pyridine, and water (6:4:3, v/v/v). Chromatograms were developed by spraying with aniline/diphenylamine reagent, as reported elsewhere (Ara et al., 1995b).

Amino- and carboxyl-terminal sequences of proteins were re-examined in this study. To determine the amino-terminal sequence of APase, the purified preparation of enzyme described above was further purified by nondenaturing PAGE (10% (w/v) acrylamide, 0.5-mm thickness). The gel was then immersed and equilibrated in transfer buffer (10 mM CAPS plus 20% (v/v) methanol, pH 11) for 10 min. A polyvinylidene difluoride membrane (ProBlott, Applied Biosystems) was wetted with methanol and then equilibrated in the transfer buffer. Electrotransfer of proteins to the membrane was performed for 50 min at 50 V in a Trans-Blot Cell (Bio-Rad). The electroblotted membrane was rinsed in methanol and stained for protein in a 0.1% (w/v) solution of Coomassie Brilliant Blue R-250 in 50% (v/v) methanol for 1 min, and then it was destained in 50% (v/v) methanol. The amino acid sequence of the protein band was directly determined in an amino acid sequencer (model 470A, Applied Biosystems) that was connected to an on-line PTH-derivative analyzer. Internal carboxyl-terminal sequences of proteins were determined after degradation with a lysyl endopeptidase in a protein sequencer (model PPSQ-10, Shimadzu) equipped with a carboxyl-terminal fragment fractionator (model CTFF-1, Shimadzu) in accordance with the manufacturer's recommendations.

The preparation of purified APase (or fragments after cleavage by papain) was diluted to 0.1 mg protein/ml with a 10 mM solution of Tris-HCl buffer (pH 8.0) that contained 60% (v/v) glycerol, and the solution was spread onto a freshly cleaved piece of mica with a spray gun (model SP-B, Olympus, Tokyo, Japan). The mica was dried quickly under a vacuum of 10-5 Torr and then rotary shadowed at an angle of 3-10 ° with platinum/carbon evaporated from an electron beam gun in a freeze-etching apparatus (model JFD-9010, JEOL, Tokyo, Japan) as described by Tyler and Branton (1980). After coating with a supporting film of carbon, the carbon film with the platinum replica was taken from the apparatus, mounted on a 200-mesh copper grid, and then examined with a transmission electron microscope (model JEM-1010, JEOL). The 114-kDa amylose- and the 102-kDa pullulan-hydrolyzing polypeptides, both of which were generated by limited proteolysis of intact APase with papain, were similarly examined under the electron microscope.

RESULTS AND DISCUSSION

A gene library for Bacillus sp. KSM-1378 was constructed by ligating PstI fragments of the genomic DNA into the PstI site of pBR322. After transformation of E. coli cells with the ligation mixture, an attempt was made to identify amylase- and pullulanase-positive transformants by the formation of halos on starch-azure and red pullulan agar plates, respectively. However, no transformants with both amylase and pullulanase activities nor any amylase-positive transformants were obtained under the cloning conditions that employed the E. coli pBR322 system.

A pullulanase-positive clone, designated pBP101 (10.6 kbp), was found to encode an active pullulanase on a 6.2-kbp PstI-PstI insert (fragment A), which had various other restriction sites, as shown in Fig. 1. Subcloning showed that a 4.2-kbp PstI-BamHI region of the insert contained the region essential for the pullulanase activity. Sequencing analysis indicated that neither a putative Shine-Dalgarno sequence nor a signal sequence was present in the 4.2-kbp PstI-BamHI region. In a separate experiment with pWE15 and E. coli NM554 (Gigapack II Plus Packaging Extract kit, Stratagene), a fragment containing the 6.2-kbp PstI-PstI region with the same restriction sites was found to have been inserted in the BamHI site of the cosmid, which was designated pWEK155. The sequence of pWEK155 included an additional 735-bp fragment in the flanking region upstream of the 5-terminal PstI site of the first insert (Fig. 1).

The 2.8-kbp fragment between the EcoRV sites in the 735-bp flanking region and in the 6.2-kbp PstI-PstI region (probe 1, see Fig. 2) was found to hybridize with a 2.8-kbp EcoRV fragment of the genomic DNA from Bacillus sp. KSM-1378. In addition, the deduced amino acid sequence of the first 0.5-kbp PstI-HindIII region of fragment A, TVPLALVSGEVLSDKL, was identical to the amino-terminal sequence of a 102-kDa pullulan-hydrolyzing polypeptide generated by limited proteolysis of the intact APase with papain (see Fig. 3). It appeared, therefore, that the 735-bp fragment was part of an unidentified gene, located upstream of the gene for pullulanase but not necessarily required for the expression of the active pullulanase.

The pBP101-encoded pullulanase was expressed in E. coli cells and was purified almost to homogeneity (see ``Experimental Procedures''). The purified enzyme had a molecular mass of 105 or 103 kDa, as judged by SDS-PAGE or by HPLC on a column of TSK gel G3000 SWXL (Ara et al., 1995b), respectively. The specific activity for pullulan of the enzyme was 217 units/mg protein. The optimum pH and temperature for the reaction were around pH 9.5 and 50 °C, respectively, and these values were almost identical to those for the pullulanase activity of APase secreted by Bacillus sp. KSM-1378. The recombinant enzyme efficiently hydrolyzed pullulan to generate maltotriose as the major end product, but it did not hydrolyze amylose at all, as judged by thin-layer chromatography (data not shown). It appeared, therefore, that the gene for the alkaline amylase domain of APase might possibly be located upstream of the gene for the alkaline pullulanase in pBP101.

The sequencing strategy and restriction map of the entire apuA gene, which was used to determine the sequence, are summarized in Fig. 2. Sequences of primers (A through F) used in the PCR experiments are given under ``Experimental Procedures.'' First, to assist in the choice of restriction enzymes for inverse PCR (Triglia et al., 1988; Ochman et al., 1988), we performed Southern hybridization analysis. The initial 730-bp PstI-XbaI region (probe 2) of the 4.2-kbp PstI-BamHI region was labeled for use as the probe. The genomic DNA from Bacillus sp. KSM-1378 was first digested with XbaI, and the resultant digest was subjected to agarose gel electrophoresis, and then the DNA bands were transferred onto a nylon filter and allowed to hybridize with digoxigenin-labeled probe 2. A 1.5-kbp XbaI fragment hybridized with the probe, and the XbaI site was located 0.8 kilobases upstream of the 5-terminal PstI site. The Bacillus sp. KSM-1378 genomic DNA (0.9 µg) was digested with XbaI and ligated under conditions that favored the formation of monomeric circles (Collins and Weissman, 1984) by T4 DNA ligase. The first inverse PCR was conducted to amplify a 0.8-kbp DNA fragment (fragment B), using the self-circularized molecules as template and primers A and B. The primers had been synthesized on the basis of the results of sequencing of the 4.2-kbp PstI-BamHI fragment. The sequence of the amplified 0.8-kbp DNA fragment included a sequence that encoded the deduced amino acid sequence DRYSGQEYQANEEGQVT, which was identical to the sequence of the internal carboxyl-terminal region of a 114-kDa amylose-hydrolyzing polypeptide that was generated by limited hydrolysis of intact APase with papain (see Fig. 3). However, neither the regulatory region nor the structural gene for intact APase was present in the 0.8-kbp fragment. Therefore, we performed Southern hybridization analysis of the genomic DNA to clone the flanking region of the XbaI site.

Southern hybridization analysis revealed that an EcoRI site was found 1.2 kilobases upstream of the 5-terminal XbaI site. Therefore, inverse PCR experiments were conducted to amplify a 1.2-kbp DNA fragment (fragment C) using EcoRI-self-circularized DNA molecules as template and suitably synthesized primers C and D, and then to amplify a 1.1-kbp DNA fragment (fragment D) using XbaI-self-circularized DNA molecules as template and appropriate primers E and F, so that we could detect the sequence that encoded the regulatory and amino-terminal regions of intact APase. The resultant fragment D was found to contain a putative regulatory region and a sequence that corresponded to the amino acid sequence ETGDKRIEFSYERP, which was identical to the amino-terminal sequence common to the intact APase from Bacillus sp. KSM-1378 and to a 114-kDa amylose-hydrolyzing polypeptide generated by limited digestion of the intact enzyme with papain (see Fig. 3).

The nucleotide sequence of apuA, extending from the 5-terminal XbaI site (nucleotide 1) to the 3 terminus at nucleotide 6,142, was determined and is shown in Fig. 3. Starting from an ATG initiation codon at nucleotide 145, there is a very long ORF of 5,814 bp that terminates in a TAA stop codon at nucleotide 5,959. Upstream from this ORF, there is a putative ribosome-binding site (Shine-Dalgarno sequence) with the sequence 5-AAAGGGG-3, followed after 8 bases by a potential ATG initiation codon. The sequence would have a free energy (G) of 11.9 kcal/mol (49.8 kJ/mol), as calculated by the method of Tinoco et al. (1973), when bound to the 3 end of the 16 S rRNA from B. subtilis. The sequence from nucleotides 35 to 65 resembled the consensus sequence of sigma A-type vegetative promoters (sigA or ^A) of B. subtilis (Moran et al., 1982; Haldenwang, 1995). It consisted of 5-TTTACA-3 as the potential 35 region and 5-TTAAAT-3 as the potential 10 region, separated by 19 nucleotides. A long inverted repeat sequence was found downstream of the stop codon of the ORF (from nucleotides 5,963 to 6,013). The G value of this sequence for a stem-loop structure was calculated to be 31.1 kcal/mol (130.1 kJ/mol), which would be sufficient for termination of transcription.

The ORF in the nucleotide sequence encoded 1,938 amino acid residues, as shown under the nucleotide sequence in Fig. 3. The amino acid sequence deduced from the apuA gene contains a short hydrophilic region from amino acids 1 to 9, followed by a hydrophobic region that extends from amino acids 10 to 32. The hydrophilic region is slightly basic because of the presence of two lysine and two arginine residues. The hydrophilic-hydrophobic sequence is similar to signal peptides of Bacillus (Murphy et al., 1984; Mezes and Lampen, 1985). A deduced sequence that was identical to the amino-terminal 14 amino acid residues of the APase secreted by Bacillus sp. KSM-1378 was found at amino acids 33-46. The residues Ala³⁰-Ala³¹-Ala³² in the hydrophobic region might be the recognition site of a signal peptidase (Perlman and Halvorson, 1983; Simonen and Palva, 1993). If this putative signal peptide were cleaved on the carboxyl-terminal side of Ala³², the molecular mass of the extracellular mature APase (from Glu³³ to Leu¹⁹³⁸) would be 211,450 Da, a value close to the 210 kDa determined by SDS-PAGE of the APase that was purified from the culture broth of Bacillus sp. KSM-1378 (Ara et al., 1995b).

As can unequivocally be seen in Fig. 3, the entire amino acid sequence of the product of apuA gene of Bacillus sp. KSM-1378 consists of the amylase domain in the amino-terminal half and the pullulanase domain in the carboxyl-terminal half. A long sequence with 35 amino acid residues, NTL(V)RIHYQ(E)RTD(N)N(A)S(D)YEN(G)L(W)GLWL(N)WG(E)DVA(E)A(S)PSE (D)N(G)WPS(N)G, is repeated between the two catalytic domains, with one copy extending from amino acids 834 to 868 and another from amino acids 944 to 978. The amino acid residues in parentheses are those found in the second sequence. The deduced amino acid sequences were found to exhibit strong homology to two repeated sequences, ⁸⁴⁶NNIRIH YKREDNVYKNYGAWLWNDVASPSANWPVG⁸⁸⁰ and ⁹⁵⁶NTVRIHYTREAVDYDDFGIWNWGDVASPSDGWPTG⁹⁹⁰, that were found in the carboxyl-terminal region of an alkaline amylase from a strain of Micrococcus (Kimura and Horikoshi, 1990). It exhibited no significant similarity to other sequences in the GenBank/SWISS-PROT/NBRF data bases. In the carboxyl-terminal region of this APase, three repeats of PGDGDGDGNTPP were also found, without interruptions, at amino acids 1,821-1,832, 1,833-1,844, and 1,845-1,856, and eight repeats of PGXG were found between amino acids 1,821 and 1,896, where X represents any of a number of amino acid residues. These short repeated sequences contain many -helix-breaking proline and glycine residues, but we have no idea at present why and how, in terms of structure, these repeated sequences are located in the carboxyl-terminal region of this enzyme. Similar sequences have been reported in the internal amino acid sequences in a metaloproteinase precursor from Bacillus polymyxa (YGDGDGDGSTFI; Takekawa et al., 1991), a glucoamylase from Clostridium (PWGDGQGDDNTGG; Ohnishi et al., 1992), and a chitinase from Streptomyces (PGTGGGSADLPP; NBRF accession number S32039[GenBank]), but these sequences are not repeated. There are many reports of the carboxyl-terminal repeats of amino acid residues, as in the surface layer proteins of Acetogenium kivui (Peters et al., 1989) and Thermus thermophilus (Faraldo et al., 1992), in surface layer-like sequences of an endoxylanase from Thermoanaerobacterium saccharolyticum (Lee et al., 1993b) and an alkaline cellulase from a strain of alkalophilic Bacillus (Ozaki et al., 1995), and in amylopullulanases from Thermoanaerobacterium thermosulfurigenes (Matuschek et al., 1994) and a strain of alkalophilic Bacillus (Lee et al., 1994). However, the repeated amino acid sequences in our enzyme also exhibited no similarity to these reported sequences.

The deduced amino acid sequence of the apuA product included several regions similar to sequences in other amylolytic enzymes reported to date (Matsuura et al., 1984; Takata et al., 1992; Holm et al., 1990; Svensson, 1994). For instance, the amino acid sequence of the amylase domain exhibited 27.3 (amino acids 332-702), 21.5 (amino acids 461-642), and 23.7% (amino acids 386-500) identity with those of -amylases from Clostridium thermosulfurogenes EM1 (Bahl et al., 1991), Bacillus stearothermophilus (Nakajima et al., 1985), and B. subtilis NA64 (Yamazaki et al., 1983), respectively. The amylase domain (amino acids 1-1,110) had the highest similarity (53.2%) to the -amylase from Micrococcus sp. 207 (Kimura and Horikoshi, 1990). The amino acid sequence of the pullulanase domain exhibited 34.0 (amino acids 1,317-1,637) and 19.7% (amino acids 1,181-1,799) identity to those of pullulanases from B. stearothermophilus TRS128 (Kuriki et al., 1990) and Klebsiella aerogenes W70 (Katsuragi et al., 1987), respectively.

The deduced amino acid sequence contained two sets of four highly conserved regions, designated I, II, III, and IV, one set being located between amino acids 462 and 645 in the amylase domain and the other set being between amino acids 1,396 and 1,581 in the pullulanase domain. The amino acid sequences of these four conserved regions were compared with those of representative bacterial -amylases and pullulanases, as shown in Fig. 4. The catalytic residues of the -amylase domain (and of the pullulanase domain) of APase from Bacillus sp. KSM-1378 were tentatively identified to be Glu⁵⁷⁹ (Glu¹⁴⁹³) in conserved region III, Asp⁵⁵⁰ (Asp¹⁴⁶⁴) in conserved region II, and Asp⁶⁴⁵ (Asp¹⁵⁸¹) in conserved region IV (Matsuura et al., 1984; Buisson et al., 1987). The last His residue in region I, the third Arg residue in region II and the fifth His residue in region IV were also conserved in all the aligned sequences of the amylolytic enzymes, including those of amylopullulanases from C. thermohydrosulfuricum DSM 3783 (Melasniemi et al., 1990), T. ethanolicus 39E (Mathupala et al., 1993), and Bacillus sp. XAL601 (Lee et al., 1994) and also from T. saccharolyticum B6A-RI (data not shown; Ramesh et al., 1994), all of which have a single active site for the dual -1,4 and -1,6 activities.

The entire apuA gene was constructed on the basis of the nucleotide sequences of PCR-amplified fragments, as shown in Fig. 5. First, we amplified the 3.6-kbp DNA fragment E, which included the regulatory region and the amylase domain, using primers G and H and the genomic DNA from Bacillus sp. KSM-1378 as template (see Fig. 2). The amplified fragment was then inserted in the SmaI site of vector plasmid pUC18 (2.7 kbp), and the resultant recombinant plasmid was designated pUCAMY (6.3 kbp). Colonies of E. coli HB101 cells that harbored pUCAMY formed clear halos on agar plates prepared with LB broth and starch azure. The 5-terminal PstI-BamHI segment (4.2 kbp) of the 6.2-kbp PstI-PstI fragment A (pBP101), which contained the region that encoded the pullulanase domain, was also inserted in the PstI-BamHI site of pHY300PLK (Ishiwa and Shibahara-Sone, 1986) and the resultant recombinant plasmid was designated pHYPUL (9.1 kbp). pHYPUL was then expressed in E. coli cells and formed halos around the colonies of transformants on red pullulan agar plates. After these two recombinant plasmids had been digested with appropriate restriction enzymes and partially sequenced, pUCAMY and pHYPUL were both digested with PstI and connected by T4 DNA ligase to construct the entire apuA gene in a plasmid designated pAP101 (13.7 kbp). During this procedure, the ampicillin resistance of pHYPUL was lost because of the presence of a PstI site on the recombinant plasmid. The sequence of the constructed apuA gene in pAP101 was determined, and it was entirely identical to the sequence shown in Fig. 3.

The recombinant plasmid pAP101 was introduced into B. subtilis ISW1214 cells, and one of the transformants obtained was grown at 30 °C for 60 h with shaking in LB broth supplemented with tetracycline (15 µg/ml). The product of the apuA gene was expressed extracellularly at a level of 60 units/liter, in terms of the alkaline pullulanase activity. The product of apuA in the culture supernatant was purified by chromatography on a column of DEAE-cellulose and then by HPLC on a column of TSK G3000 SWXL. The -1,4 and -1,6 hydrolytic activities were found to copurify through the two chromatography steps. The partially purified preparation of enzyme had pH optima of around pH 8-9 for the amylase activity and at pH 9.5 for the pullulanase activity, values close to the pH optima for the respective enzymatic activities of the APase purified from cultures of Bacillus sp. KSM-1378 (Ara et al., 1995b). The molecular mass of the purified enzyme was estimated to be approximately 200-210 kDa by SDS-PAGE, a value close to the 210 kDa determined for the native enzyme produced by Bacillus sp. KSM-1378 (Fig. 6A). The purified enzyme hydrolyzed soluble starch (from potato), amylopectin (from corn), amylose (degree of polymerization, 17), and pullulan (from Aureobasidium pullulans) at a relative rate of 100:58:31:46, a value similar to the relative rate (100:52:35:61) for the APase activity of Bacillus sp. KSM-1378. In addition, the band of the purified enzyme protein, detected by staining after nondenaturing PAGE, coincided fairly well with the band visualized by activity staining on a starch-azure agar sheet or on a red pullulan agar sheet, indicating that the single apuA protein encoded by pAP101 had both -amylase and pullulanase activities (Fig. 6B). There were additional protein bands, showing amylase activity and moving faster than the main APase band, which might be generated by proteolytic enzyme(s) contaminated in the enzyme preparations.

The 210-kDa APase from Bacillus sp. KSM-1378 and its 114-kDa amylose- and 102-kDa pullulan-hydrolyzing fragments, generated by partial digestion with papain, were visualized after rotary shadowing by the method of Tyler and Branton (1980). Samples dried from a solution in glycerol were rotary shadowed with platinum/carbon at a low angle, and the resultant specimens, after coating with a supporting film of carbon, were observed by transmission electron microscopy, as shown in Fig. 7. The intact APase molecules were seen as ``castanet-like'' or ``bent dumbbell-like'' shapes with a diameter of approximately 25 nm (Fig. 7, A and B). Two globular (ovoid) heads of different sizes were clearly seen, and they were joined by a thin, short linker region. When the intact molecule was cleaved by limited proteolysis with papain, the amylose- and pullulan-hydrolyzing polypeptides were observed as a mixture of globular molecules of different sizes under the electron microscope (Fig. 7C). The joint region might correspond to the linker sequence between the amylase and the pullulanase domains, which were deduced from the nucleotide sequence (see Fig. 3). The bent structure, with two heads, of the intact APase might be related to the presence of this linker sequence because many -turn-forming proline residues are concentrated in the linker region.

The alkaline APase from Bacillus sp. KSM-1378 has unique features, having -1,4 and -1,6 activity at different active sites and an unusual but noncontradictory molecular structure. The production of this enzyme is induced by pullulan but by neither amylose nor soluble starch.² Because the pullulanase domain lies in the carboxyl-terminal half, we postulate that this APase might be the product of gene fusion caused by recombination of a gene for a pullulanase with a gene for an amylase. It is possible that a foreign gene for an amylase gene was introduced by chance, in frame, between the regulatory region and the structural gene of the original pullulanase in Bacillus sp. KSM-1378. Similar events might have been responsible for formation of genes for other bifunctional enzymes, such as the enzyme with endo- and exoglucanase activities from Caldocellum saccharolyticum (Saul et al., 1990) and the enzyme with xylanase and (1,3-1,4)-glucanase activities from Ruminococcus flavefaciens (Flint et al., 1993).