Organism and culture conditions. Bacillus
sp. strain KSM-1378, a relative of Bacillus firmus
, was used (4
), which had previously been isolated from a soil sample collected in Tochigi City, Tochigi, Japan. The optimum temperature and pH for growth of this organism were around 30°C and pH 10, respectively. The organism was found to produce an alkaline α-amylase on an alkaline agar plate composed of 1% (wt/vol) soluble starch (Wako Pure Chemical, Osaka, Japan), 0.4% starch azure (Sigma, St. Louis, Mo.), 0.2% tryptone (Difco Laboratories, Detroit, Mich.), 0.1% yeast extract (Difco), 0.2% KH2
, 0.1% MgSO4
O, 0.1% CaCl2
O, 0.001% FeSO4
O, 0.0001% MnCl2
O, 1.0% Na2
(separately autoclaved), and 1.0% agar (pH 10).
The organism was propagated at 30°C for 2 days in 50-ml aliquots of an alkaline medium placed in 500-ml flasks, with shaking on a reciprocal shaker (125 strokes/min; Iwashiya, Tokyo, Japan). The medium contained 1% (wt/vol) soluble starch (Wako Pure Chemical), 0.2% tryptone, 0.1% yeast extract, 0.2% KH2PO4, 0.1% MgSO4 · 7H2O, 0.1% CaCl2 · 2H2O, 0.001% FeSO4 · 7H2O, 0.0001% MnCl2 · 4H2O, and 1.0% Na2CO3 (separately autoclaved). The final pH of the complete medium was about pH 10. After removal of cells by centrifugation (12,000 × g, 15 min) at 4°C, the supernatant (pH 8.6 to 8.8) was used as the starting material for purification of the enzyme.
Purification of the enzyme.
Enzyme purification was done at a temperature below 4°C. The centrifugal supernatant of the culture broth was treated with ammonium sulfate, and the fraction that precipitated at 60% saturation was collected. The precipitates formed were dissolved in a small volume of 10 mM Tris-HCl (pH 7.5) plus 2 mM CaCl2, and the solution was dialyzed twice over the course of 16 h against 50 vol of the same buffer. The retentate was then applied to a column of DEAE-Toyopearl 650M (10 by 15 cm; Tosoh, Tokyo, Japan) that had been equilibrated with 10 mM Tris-HCl plus 2 mM CaCl2 (pH 7.5). The column was washed with the equilibration buffer, and the nonadsorbed active fractions were combined and concentrated by ultrafiltration (PM-10; 10,000-Mr cutoff; Amicon, Danvers, Mass.). The concentrate was put on a column of CM-Toyopearl 650S (2.5 by 50 cm; Tosoh) that had been equilibrated with 10 mM Tris-HCl (pH 7.5) plus 2 mM CaCl2. The column was initially washed with 300 ml of the equilibration buffer, and proteins were eluted with 2.0-liter linear gradient of 0 to 0.5 M NaCl in the same buffer, at a flow rate of 120 ml h−1. Fractions of 15 ml were collected from the start of the gradient. The active fractions were combined and concentrated by ultrafiltration on a PM-10 membrane. The concentrate was dialyzed overnight against 10 mM Tris-HCl (pH 7.5) plus 2 mM CaCl2. The resulting retentate was used exclusively for further experiments as the final preparation of purified enzyme. For comparison, we also purified a commercially available, thermostable BLA (Termamyl; Novo Nordisk, Bagsvaerd, Denmark) to homogeneity by the method described above.
α-Amylase activity was routinely measured at 50°C in a 1-ml reaction mixture that contained 0.5 ml of a 1.0% (wt/vol) solution of soluble starch (from potato; Sigma) in 50 mM Tris-HCl buffer (pH 8.5) and 0.1 ml of a suitably diluted solution of enzyme. The reducing sugar formed was measured by the dinitrosalicylic acid procedure (26
). 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. Maltooligosaccharides in the G3 to G7 range and in the G8 to G15 range were purchased from Hayashibara Biochemical (Kurashiki, Japan) and Funakoshi (Tokyo, Japan), respectively. Other polysaccharides used as substrates were the products of Sigma. Protein was determined with a protein assay kit (Bio-Rad, Richmond, Calif.) with bovine serum albumin as the standard protein. Protein in column effluents was also routinely monitored by measuring the A280
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done essentially as described by Laemmli (23
) with slab gels (10% [wt/vol] acrylamide, 70 by 50 mm, 2.0-mm thickness), and samples were stained for protein with Coomassie brilliant blue R-250. Activity staining of amylase in slab gels was done essentially as described previously (13
), with agar sheets containing starch azure (Sigma) as replica plates. The slab gel after SDS-PAGE was laid on the replica sheet and left for several hours at room temperature. The bands of protein that were associated with amylase activity were seen as clear zones on a dark blue background on the replica sheet.
Molecular masses were estimated by SDS-PAGE (10% [wt/vol] acrylamide gel) with low-range molecular mass standards (Bio-Rad), which included phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa).
Isoelectrofocusing of proteins was done with a Multiphore II gel electrofocusing system with a PAG-Plate and a broad pI calibration kit (Pharmacia Fine Chemica AB, Uppsala, Sweden), which included amyloglucosidase (pI 3.50), methyl red (pI 3.75), soybean trypsin inhibitor (pI 4.55), β-lactoglobulin A (pI 5.20), bovine carbonic anhydrase b (pI 5.85), human carbonic anhydrase b (pI 6.55), horse myoglobin-acidic band (pI 6.85), horse myoglobin-basic band (pI 7.35), lentil lectin-acidic band (pI 8.15), lentil lectin-middle band (pI 8.45), lentil lectin-basic band (pI 8.65), and trypsinogen (pI 9.30).
Chromatographic analysis of the products of hydrolysis of carbohydrates.
The hydrolysis products of LAMY were analyzed by thin-layer chromatography (TLC) with a precoated silica gel plate (Kieselgel 60 F254; E. Merck AG, Darmstadt, Germany). After development of the products in a solvent systems of butanol-pyridine-water (6:4:3 or 9:2:2 [vol/vol], the spots were visualized by spraying with diphenylamine-aniline reagent (12
) and then baking at 90°C for 30 min. The products were quantified by high-performance liquid chromatography (HPLC). The purified enzyme was incubated at 30°C with substrate in 10 mM potassium phosphate buffer (pH 8.0). Samples were removed at intervals and heated immediately in boiling water for 5 min to terminate the reaction, and the products in them were separated in a carbohydrate column (4.6 by 250 mm; Waters, Milford, Mass.) with acetonitrile-water (70:30 [vol/vol]) as an eluent at a flow rate of 1.4 ml min−1
. Each product was quantified by using a data analysis software, 805 Data Station (Waters), with authentic maltooligosaccharides.
Spectra were recorded on a JNM A-500 nuclear magnetic resonance (NMR) spectrometer (JEOL, Tokyo, Japan) operated at 20°C and at 500 MHz for protons in deuterated 10 mM sodium phosphate buffer (p2H 7.4). The spectral width, data point, and the number of accumulation were 6,500 Hz, 16K, and 256, respectively. The water resonance was suppressed by selective irradiation. Chemical shifts were measured relative to the calibrated resonance of internal sodium 3-(trimethylsilyl)-1-propane sulfonate (Merck). The substrate used was p-nitrophenyl α-d-maltooctaoside (pNP-G8; Calbiochem, La Jolla, Calif.)
Sequencing of amino-terminal regions of protein.
The enzyme sample was blotted on a polyvinylidene difluoride membrane (Prosorb; Perkin-Elmer, Foster City, Calif.), which had been wetted with methanol. The amino-terminal sequence of the protein was determined directly by a protein sequencer (model 476A; Perkin-Elmer).
Isolation of DNA and transformation.
Genomic DNA from Bacillus
sp. strain KSM-1378 was prepared as described by Saito and Miura (34
) and plasmid DNA was isolated by the alkaline extraction procedure of Birnboim and Doly (6
). Escherichia coli
HB101 (F− hsdS20 recA13 ara14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 supE44 leuB6 thi-1
) cells were transformed with plasmids by the methods of Hanahan (11
). Transformed E. coli
cells were grown at 37°C for one day on Luria-Bertani (LB) agar plates supplemented with 0.4% (wt/vol) starch azure and ampicillin (100 μg ml−1
Genomic DNAs after digestion with restriction enzymes and the subsequent electrophoresis were subjected to Southern hybridization (37
). Patterns of hybridization of the digested DNAs, which were labeled with digoxigenin-dUTP with probes, were examined with a digoxigenin DNA labeling detection kit (Boehringer Mannheim, Mannheim, Germany).
Amplification and sequencing of DNA.
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′-TNGAYGCNGTNAARCAYATHAA-3′; primer B, 5′-CGNCANTGNAARCANCTRTTRGTRCT-3′; primer C, 5′-AGCCAATCTCTCGTATAGCTGTA-3′; primer D, 5′-GTACAAAAACACCCTATACATG-3′; primer E, 5′-AATGGWACWATGATGCAKTA-3′; primer F, 5′-CATTTGGCAAATGCCATTCAAA-3′; primer G, 5′-AAAATTGATCCACTTCTGCAG-3′; primer H, 5′-CAGCGCGTGATAATATAAATTTGAAT-3′; and primer I, 5′-AAGCTTCCAATTTATATTGGGTGTAT-3′. They were prepared on a DNA synthesizer (model 392A; Perkin-Elmer) and were purified with a DNA refinement system (model Dnastec-1000; Astec, Fukuoka, Japan). PCR was performed with a DNA thermal cycler (model 480; Perkin-Elmer) with each primer (0.2 μg) plus genomic DNA (1.0 μg) from Bacillus sp. strain KSM-1378 (94°C for 1 min, 55°C for 1 min, and 72°C for 2 min for 30 cycles). The reaction mixture contained 200 μM deoxynucleotide triphosphates, 25 mM KCl, 5 mM (NH4)2SO4, 2.5 U of Pwo DNA polymerase (Boehringer Mannheim) and 10 mM Tris-HCl buffer (pH 8.85) in a reaction volume of 100 μl. Products of PCR were purified with a PCR product purification kit (Boehringer Mannheim), and they were used for sequencing or for subcloning.
Sequencing was performed by the dideoxy chain termination method of Smith et al. (36
), by using fluorescent terminators and an automated DNA sequencer (model 373A; Perkin-Elmer). Both strands of the DNA were sequenced, and computer analysis was done with a GENETYX program (SDC Software Development, Tokyo, Japan). Amino acid sequence alignments were done with a GENETYX MAlign program (SDC Software Development).
Nucleotide sequence accession number.
The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank databases under accession no. AB008763.