enzymatic properties of a novel liquefying α-amylase from ...mm tris-hcl (ph 7.5) plus 2 mm cacl 2....

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/98/$04.0010

Sept. 1998, p. 3282–3289 Vol. 64, No. 9

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Enzymatic Properties of a Novel Liquefying a-Amylase from anAlkaliphilic Bacillus Isolate and Entire Nucleotide and

Amino Acid SequencesKAZUAKI IGARASHI, YUJI HATADA, HIROSHI HAGIHARA, KATSUHISA SAEKI, MIKIO TAKAIWA,

TAKAAKI UEMURA, KATSUTOSHI ARA, KATSUYA OZAKI, SHUJI KAWAI,TOHRU KOBAYASHI, AND SUSUMU ITO*

Tochigi Research Laboratories of Kao Corporation, Ichikai, Haga, Tochigi 321-3497, Japan

Received 16 March 1998/Accepted 30 June 1998

A novel liquefying a-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378.The specific activity of purified LAMY was approximately 5,000 U mg of protein21, a value two- to fivefoldgreater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzymehad a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced fromsodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparentisoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield malto-triose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction.Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The struc-tural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 aminoacids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellularmature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases,such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearother-mophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad werefound in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent withthe tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and whichinclude the well-known (a/b)8 barrel motif in domain A.

a-Amylase (1,4-a-D-glucan glucanohydrolase [EC 3.2.1.1])and pullulanase (pullulan 6-glucanohydrolase [EC 3.2.1.41])are amylolytic enzymes of industrial importance, particularly inthe food and detergent industries. We have found and charac-terized some unique debranching enzymes, such as a high-alkaline pullulanase (2), an alkali-resistant neopullulanase(16), and an alkaline isoamylase (3), from cultures of alkali-philic Bacillus strains, and these enzymes can be used as effec-tive additives in dishwashing and laundry detergents underalkaline conditions, especially when used in combination witha-amylase. We have also found the first known alkaline amy-lopullulanase from alkaliphilic Bacillus sp. strain KSM-1378(4), which is very unique in that it efficiently hydrolyzes thea-1,6 linkages of pullulan, as well as the a-1,4 linkages ofvarious carbohydrates at different active sites (1, 13).

Liquefying a-amylases, particularly the Bacillus licheniformisenzyme (BLA) (35), are used widely in technical applicationfields, such as in bread making, production of glucose andfructose syrup and fuel ethanol from starch materials, andtextile treatment. The demand for a-amylase for use in laundryand automatic dishwashing detergents has also been growingfor several years (42). However, most of the Bacillus liquefyingamylases, such as the enzymes from Bacillus amyloliquefaciens(BAA) and Bacillus stearothermophilus (BSA) (28), includingBLA (35), have pH optima of between 5 and 7.5 (44). Theseneutrophilic enzymes are essentially not good for use in deter-gents, because the working pH range between 8 and 11 is

relevant to washing in detergents (17). Since Horikoshi (15)first reported an alkaline amylase from alkaliphilic Bacillus sp.strain A-40-2, many alkaline amylases have been found incultures of, for example, Bacillus sp. strain NRRL B-3881 (31),Bacillus sp. strain H-167 (14), Bacillus alcalothermophilus A3-8(7), and Bacillus sp. strain GM8901 (21). The alkaline amylasesfrom these alkaliphilic Bacillus strains reported to date are allof the saccharifying type, except for the enzymes from Bacillussp. strain 707 (22, 41) and B. licheniformis TCRDC-B13 (5).However, very limited or no information about enzymaticproperties of these two liquefying amylases is available. In thispaper, we report the isolation of a novel liquefying a-amylase(LAMY) from cultures of the alkaline amylopullulanase pro-ducer Bacillus sp. strain KSM-1378 (13). This enzyme is highlyactive at alkaline pH compared with those of other liquefyinga-amylases reported to date. Furthermore, analysis of the genefor this a-amylase (amyK) indicates that LAMY exhibits lowamino acid identity to the reported liquefying a-amylases.

MATERIALS AND METHODS

Organism and culture conditions. Bacillus sp. strain KSM-1378, a relative ofBacillus firmus, was used (4), which had previously been isolated from a soilsample collected in Tochigi City, Tochigi, Japan. The optimum temperature andpH for growth of this organism were around 30°C and pH 10, respectively. Theorganism was found to produce an alkaline a-amylase on an alkaline agar platecomposed 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% KH2PO4, 0.1% MgSO4 z 7H2O,0.1% CaCl2 z 2H2O, 0.001% FeSO4 z 7H2O, 0.0001% MnCl2 z 4H2O, 1.0%Na2CO3 (separately autoclaved), and 1.0% agar (pH 10).

The organism was propagated at 30°C for 2 days in 50-ml aliquots of analkaline medium placed in 500-ml flasks, with shaking on a reciprocal shaker (125strokes/min; Iwashiya, Tokyo, Japan). The medium contained 1% (wt/vol) solu-ble starch (Wako Pure Chemical), 0.2% tryptone, 0.1% yeast extract, 0.2%KH2PO4, 0.1% MgSO4 z 7H2O, 0.1% CaCl2 z 2H2O, 0.001% FeSO4 z 7H2O,

* Corresponding author. Mailing address: Tochigi Research Labo-ratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi321-3497, Japan. Phone: 81 (285) 68-7304. Fax: 81 (285) 68-7305.E-mail: [email protected].

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0.0001% MnCl2 z 4H2O, and 1.0% Na2CO3 (separately autoclaved). The final pHof the complete medium was about pH 10. After removal of cells by centrifuga-tion (12,000 3 g, 15 min) at 4°C, the supernatant (pH 8.6 to 8.8) was used as thestarting material for purification of the enzyme.

Purification of the enzyme. Enzyme purification was done at a temperaturebelow 4°C. The centrifugal supernatant of the culture broth was treated withammonium sulfate, and the fraction that precipitated at 60% saturation wascollected. The precipitates formed were dissolved in a small volume of 10 mMTris-HCl (pH 7.5) plus 2 mM CaCl2, and the solution was dialyzed twice over thecourse of 16 h against 50 vol of the same buffer. The retentate was then appliedto a column of DEAE-Toyopearl 650M (10 by 15 cm; Tosoh, Tokyo, Japan) thathad been equilibrated with 10 mM Tris-HCl plus 2 mM CaCl2 (pH 7.5). Thecolumn was washed with the equilibration buffer, and the nonadsorbed activefractions were combined and concentrated by ultrafiltration (PM-10; 10,000-Mrcutoff; Amicon, Danvers, Mass.). The concentrate was put on a column ofCM-Toyopearl 650S (2.5 by 50 cm; Tosoh) that had been equilibrated with 10mM Tris-HCl (pH 7.5) plus 2 mM CaCl2. The column was initially washed with300 ml of the equilibration buffer, and proteins were eluted with 2.0-liter lineargradient of 0 to 0.5 M NaCl in the same buffer, at a flow rate of 120 ml h21.Fractions of 15 ml were collected from the start of the gradient. The activefractions were combined and concentrated by ultrafiltration on a PM-10 mem-brane. The concentrate was dialyzed overnight against 10 mM Tris-HCl (pH 7.5)plus 2 mM CaCl2. The resulting retentate was used exclusively for furtherexperiments as the final preparation of purified enzyme. For comparison, we alsopurified a commercially available, thermostable BLA (Termamyl; Novo Nordisk,Bagsvaerd, Denmark) to homogeneity by the method described above.

Enzyme assay. a-Amylase activity was routinely measured at 50°C in a 1-mlreaction mixture that contained 0.5 ml of a 1.0% (wt/vol) solution of solublestarch (from potato; Sigma) in 50 mM Tris-HCl buffer (pH 8.5) and 0.1 ml of asuitably diluted solution of enzyme. The reducing sugar formed was measured bythe dinitrosalicylic acid procedure (26). One unit of enzymatic activity wasdefined as the amount of protein that produced 1 mmol of reducing sugar asglucose per min under the conditions of the assay. Maltooligosaccharides in theG3 to G7 range and in the G8 to G15 range were purchased from HayashibaraBiochemical (Kurashiki, Japan) and Funakoshi (Tokyo, Japan), respectively.Other polysaccharides used as substrates were the products of Sigma. Proteinwas determined with a protein assay kit (Bio-Rad, Richmond, Calif.) with bovineserum albumin as the standard protein. Protein in column effluents was alsoroutinely monitored by measuring the A280.

Electrophoretic analysis. Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) was done essentially as described by Laemmli (23) withslab gels (10% [wt/vol] acrylamide, 70 by 50 mm, 2.0-mm thickness), and sampleswere stained for protein with Coomassie brilliant blue R-250. Activity staining ofamylase in slab gels was done essentially as described previously (13), with agarsheets containing starch azure (Sigma) as replica plates. The slab gel afterSDS-PAGE was laid on the replica sheet and left for several hours at roomtemperature. The bands of protein that were associated with amylase activitywere seen as clear zones on a dark blue background on the replica sheet.

Molecular masses were estimated by SDS-PAGE (10% [wt/vol] acrylamidegel) with low-range molecular mass standards (Bio-Rad), which included phos-phorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonicanhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa).

Isoelectrofocusing of proteins was done with a Multiphore II gel electrofocus-ing system with a PAG-Plate and a broad pI calibration kit (Pharmacia FineChemica AB, Uppsala, Sweden), which included amyloglucosidase (pI 3.50),methyl red (pI 3.75), soybean trypsin inhibitor (pI 4.55), b-lactoglobulin A (pI5.20), bovine carbonic anhydrase b (pI 5.85), human carbonic anhydrase b (pI6.55), horse myoglobin-acidic band (pI 6.85), horse myoglobin-basic band (pI7.35), lentil lectin-acidic band (pI 8.15), lentil lectin-middle band (pI 8.45), lentillectin-basic band (pI 8.65), and trypsinogen (pI 9.30).

Chromatographic analysis of the products of hydrolysis of carbohydrates. Thehydrolysis 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 ofbutanol-pyridine-water (6:4:3 or 9:2:2 [vol/vol], the spots were visualized byspraying with diphenylamine-aniline reagent (12) and then baking at 90°C for 30min. The products were quantified by high-performance liquid chromatography(HPLC). The purified enzyme was incubated at 30°C with substrate in 10 mMpotassium phosphate buffer (pH 8.0). Samples were removed at intervals and

heated immediately in boiling water for 5 min to terminate the reaction, and theproducts 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 ata flow rate of 1.4 ml min21. Each product was quantified by using a data analysissoftware, 805 Data Station (Waters), with authentic maltooligosaccharides.

NMR spectroscopy. Spectra were recorded on a JNM A-500 nuclear magneticresonance (NMR) spectrometer (JEOL, Tokyo, Japan) operated at 20°C and at500 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 selectiveirradiation. Chemical shifts were measured relative to the calibrated resonanceof internal sodium 3-(trimethylsilyl)-1-propane sulfonate (Merck). The substrateused was p-nitrophenyl a-D-maltooctaoside (pNP-G8; Calbiochem, La Jolla,Calif.)

Sequencing of amino-terminal regions of protein. The enzyme sample wasblotted on a polyvinylidene difluoride membrane (Prosorb; Perkin-Elmer, FosterCity, Calif.), which had been wetted with methanol. The amino-terminal se-quence of the protein was determined directly by a protein sequencer (model476A; Perkin-Elmer).

Isolation of DNA and transformation. Genomic DNA from Bacillus sp. strainKSM-1378 was prepared as described by Saito and Miura (34) and plasmid DNAwas isolated by the alkaline extraction procedure of Birnboim and Doly (6).Escherichia coli HB101 (F2 hsdS20 recA13 ara14 proA2 lacY1 galK2 rpsL20 xyl-5mtl-1 supE44 leuB6 thi-1) cells were transformed with plasmids by the methodsof Hanahan (11). Transformed E. coli cells were grown at 37°C for one day onLuria-Bertani (LB) agar plates supplemented with 0.4% (wt/vol) starch azureand ampicillin (100 mg ml21).

Southern hybridization. Genomic DNAs after digestion with restriction en-zymes and the subsequent electrophoresis were subjected to Southern hybrid-ization (37). Patterns of hybridization of the digested DNAs, which were labeledwith digoxigenin-dUTP with probes, were examined with a digoxigenin DNAlabeling detection kit (Boehringer Mannheim, Mannheim, Germany).

Amplification and sequencing of DNA. Primer DNAs were designed for theamplification of appropriate regions between specific sites in the genomic DNA.The primer sequences used were as follows: primer A, 59-TNGAYGCNGTNAARCAYATHAA-39; primer B, 59-CGNCANTGNAARCANCTRTTRGTRCT-39; primer C, 59-AGCCAATCTCTCGTATAGCTGTA-39; primer D, 59-GTACAAAAACACCCTATACATG-39; primer E, 59-AATGGWACWATGATGCAKTA-39; primer F, 59-CATTTGGCAAATGCCATTCAAA-39; primer G, 59-A

FIG. 1. SDS-PAGE of LAMY purified from cultures of Bacillus sp. strainKSM-1378. The purified enzyme (40 mg) was visualized by activity staining (laneA) and Coomassie brilliant blue staining for protein (lane B). Lane M, molecularmass markers (calibration in kilodaltons). The arrows indicate the positions ofthe purified enzyme.

TABLE 1. Purification of LAMY produced by Bacillus sp. strain KSM-1378

Purification step Total amt ofprotein (mg)Total activity

(U)Sp act(U/mg)

Yield(%)

Purification(fold)

Culture filtrate 353.6 292,401 827.0 100 1.060% Ammonium sulfate precipitation 68.8 399,572 3,811.9 137 4.6DEAE-Toyopearl, unadsorbed 55.4 218,067 3,935.3 75 4.8CM-Toyopearl chromatography 20.4 102,039 5,009.3 35 6.1

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AAATTGATCCACTTCTGCAG-39; primer H, 59-CAGCGCGTGATAATATAAATTTGAAT-39; and primer I, 59-AAGCTTCCAATTTATATTGGGTGTAT-39. 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 mg) plus genomic DNA (1.0 mg) fromBacillus sp. strain KSM-1378 (94°C for 1 min, 55°C for 1 min, and 72°C for 2 minfor 30 cycles). The reaction mixture contained 200 mM deoxynucleotide triphos-phates, 25 mM KCl, 5 mM (NH4)2SO4, 2.5 U of Pwo DNA polymerase (Boehr-inger Mannheim) and 10 mM Tris-HCl buffer (pH 8.85) in a reaction volume of100 ml. 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 Smithet al. (36), by using fluorescent terminators and an automated DNA sequencer(model 373A; Perkin-Elmer). Both strands of the DNA were sequenced, andcomputer analysis was done with a GENETYX program (SDC Software Devel-opment, Tokyo, Japan). Amino acid sequence alignments were done with aGENETYX MAlign program (SDC Software Development).

Nucleotide sequence accession number. The nucleotide sequence data re-ported in this paper have been submitted to the DDBJ, EMBL, and GenBankdatabases under accession no. AB008763.

RESULTS AND DISCUSSION

Purification and physicochemical properties of LAMY. Ahighly purified preparation of LAMY was obtained by a simplepurification procedure with high yield (35%), as summarized inTable 1. Approximately 6.1-fold purification to a specific ac-tivity of 5,009 U mg of protein21 was obtained for the a-amy-

lase activity when measured at 50°C and at pH 8.5 in 50 mMTris-HCl buffer. The protein was homogeneous, as judged bySDS-PAGE, and the band of protein coincided fairly well withthe band that was visualized by activity staining, as shown inFig. 1. The molecular mass of the purified LAMY was deter-mined to be approximately 53 kDa by SDS-PAGE. The iso-electric point was estimated to be around pH 9. The N-termi-nal amino acid sequence was HHNGTNGTMMQYFEW.BLA from a commercial product was also purified to homo-geneity. The molecular mass and the specific activity of thereference amylase were approximately 53 kDa and 1,600 U/mgof protein, respectively, when measured under our standardassay conditions.

Effects of pH and temperature on activity and stability. Theranges of pH at which LAMY was active and stable weredetermined with soluble starch as the substrate. As shown inFig. 2A, the maximum activity was observed around pH 8.0 to8.5 when measured in various buffers at 50 mM. More than50% of the maximum activity was detectable between pH 6 andpH 9. In this pH range, the specific activity of LAMY is two- tofivefold greater than that of BLA. When Britton-Robinsonbuffers (50 mM phosphoric acid-acetic acid-boric acid; with pHadjusted with NaOH) at different pH values were used, the pHoptima were around pH 7.5 to 8.0 for LAMY and pH 5.0 to 6.0for BLA. To determine the pH stability of LAMY, the enzymewas preincubated at 40°C for 30 min in 10 mM Britton-Rob-inson buffer and assayed at 50°C in 50 mM Tris-HCl buffer at

FIG. 2. Effect of pH on the activity and stability of LAMY. (A) The pHactivity curves of purified LAMY and BLA (each at 0.2 U ml21) are shown bysolid and dotted lines, respectively. The buffers used (50 mM each) were asfollows: glycine-HCl, pH 3.0 to 3.5 (Œ); acetate, pH 4.0 to 6.0 (■); Tris-HCl, pH6.5 to 8.5 (F); glycine-NaOH, pH 9.0 to 10.5 (h); glycine-NaCl-NaOH, pH 8.0to 10.5 (E); and carbonate, pH 10.0 to 11.0 (‚). The values are shown aspercentages of the maximum specific activity of LAMY observed at pH 8.0 to 8.5,which is taken as 100%. (B) To assess the pH stability of LAMY, the enzyme (2.0U ml21) was preincubated at the indicated pH in 10 mM Britton-Robinsonbuffer and at 40°C for 30 min, and then samples (0.1 ml) were used for themeasurements of the residual activity under the standard conditions of theenzymatic assay. The values are shown as percentages of the original activity,which is taken as 100%.

FIG. 3. Effect of temperature on the activity and stability of LAMY. (A) Thetemperature activity curves of purified LAMY (F) and BLA (E) (each at 0.18 Uml21) are shown. The reactions were done at the indicated temperatures for 10min and at pH 8.5 in 50 mM Tris-HCl buffer. The values are shown as percent-ages of the specific activity of LAMY observed at 55°C, which is taken as 100%.(B) For determination of the thermostability of LAMY, the enzyme (1.8 U ml21)was heated at the indicated temperatures in the presence (at 50°C [h], 60°C [‚],and 70°C [E]) or absence (at 50°C [■] and 60°C [Œ]) of 0.1 mM Ca21 ions in 50mM Tris-HCl buffer (pH 8.5). Samples (0.1 ml) were used for the determinationof the residual activity under the standard conditions of the assay.

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pH 8.5. LAMY was stable over a range between pH 6 and pH10 (Fig. 2B).

LAMY activity was measured at various temperatures at pH8.5 in 50 mM Tris-HCl buffer. The optimum temperature foractivity of LAMY was around at 55°C (Fig. 3A), while that ofBLA was around at 80°C. At 80°C, the specific activities ofBLA and LAMY were almost equal, and they were approxi-mately 25% of the maximum activity of LAMY observed at55°C. The time course of the thermal inactivation of LAMYwas monitored at various temperatures and at pH 8.5 in 20 mM

Tris-HCl buffer (Fig. 3B). In the absence of CaCl2, the enzymeretained its full activity after 60 min of incubation at 45°C, butonly 32 and 3% of the original activity remained after 60 minof incubation at 50 and 60°C, respectively. However, in thepresence of 0.1 mM CaCl2, nearly 100 and 65% of the originalactivity remained at 50 and 60°C, respectively. The enzymaticactivity was abolished after being heated at 70°C for 60 min,even in the presence of 0.1 mM Ca21 ions. In contrast, BLAwas quite stable with incubation at 70°C, at least up to 60 min,regardless of whether CaCl2 was present or not.

Effects of metal ions and chemical reagents. The purifiedenzyme was incubated with various cations (1 mM) at 40°C for30 min, and the residual activities were assayed at 50°C in 50mM Tris-HCl buffer. Ni21, Cd21, Zn21, and Hg21 ionsstrongly inhibited the enzymatic activity by 82, 91, 100, and100%, respectively. LAMY activity was also inhibited almostcompletely by iodoacetate (0.5 mM), but not by the thiol in-hibitors p-chloromercuribenzoate (0.5 mM) and N-ethylmale-imide (1 mM), as shown in Table 2. The following chemicalreagents were without effect on LAMY activity under our assayconditions: citrate (10 mM), diethyl pyrocarbonate (2 mM),and di-isopropyl fluorophosphate (1 mM). When the enzymewas preincubated with 10 mM EDTA or EGTA, the enzymeactivity decreased to 10 or 9% of the initial activity, respec-tively. It has been reported that saccharifying amylases fromBacillus sp. strain A-40-2 (15), Bacillus sp. strain NRRL B-3881(31), and Bacillus alcalothermophilus A3-8 (7) are all stable inresponse to EDTA treatment, whereas the liquefying enzymesfrom Bacillus strains require Ca21 ions for expression of en-zyme activity (44). LAMY activity was completely abolished bya low concentration of N-bromosuccinimide (0.1 mM), which

FIG. 4. Patterns of hydrolysis of soluble starch and maltooligosaccharides by LAMY. (A) Products from soluble starch (0.2% [wt/vol]). The reaction (0.2 U ml21)was done at 30°C and at pH 8.5 in 50 mM Tris-HCl buffer. Samples were taken at the indicated intervals and boiled for 5 min to terminate the reaction. The productsformed were analyzed by TLC with butanol-pyridine-water (6:3:4 [vol/vol]) as the solvent system. Std denotes authentic maltooligosaccharides. (B) Maltooligosaccha-rides in the G2 to G15 range (0.2% [wt/vol]), shown in lanes 2 through 15, respectively were hydrolyzed for 15 min by LAMY (0.2 U ml21) under the standard conditionsof the assay. The products formed were boiled for 5 min, and then they were analyzed by TLC with butanol-pyridine-water (9:2:2 [vol/vol]) as the solvent system. (C)Cleaved-bond distribution in the hydrolysis of maltooligosaccharides G7 and G8 by LAMY. All reactions were performed at 40°C and at pH 8.5 in 50 mM Tris-HClbuffer. The products formed were boiled for 5 min, and then they were quantified by HPLC, as described in Materials and Methods. Solid symbols (F) representnonreducing ends of the substrates. The numerals indicate the cleavage frequency of the bond. Catalytic efficiency for each substrate is expressed as k0/Km.

TABLE 2. Effects of chemical reagents on the activity of LAMY

AdditiveReagent

concn(mM)

Residualactivity (%)a

None 100N-Bromosuccinimideb 0.1 0Iodoacetatec 0.5 2N-Ethylmaleimide 1 107p-Chloromercuribenzoatec 0.5 80Citrate 10 98EDTA 10 10EGTA 10 9

a Enzymatic activity was measured after the enzyme had been treated witheach inhibitor for 30 min at the indicated temperature and pH in the appropriatebuffer. Usually, the enzyme was treated with inhibitor at 30°C and at pH 8.5 in50 mM Tris-HCl buffer, and an aliquot (0.1 ml) was used for assay of the residualactivity in the standard buffer. The values shown are the percentages of theactivity without additives, which is taken as 100%.

b Preincubated for 15 min at 4°C and at pH 5.0 in 10 mM acetate buffer.c Preincubated for 15 min at 40°C and at pH 5.5 in 10 mM acetate buffer.



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oxidizes tryptophan residues. Tryptophan residues have beensuggested to be involved in substrate binding and/or catalysisfor an a-amylase (25), a pullulanase (2), and cellulases (20, 45).LAMY was resistant to incubation at 40°C for 1 h with varioussurfactants, such as SDS, polyoxyethylene alkyl ether, sodiuma-sulfonated fatty acid ester, linear-alkylbenzene sulfonate,and alkyl glucoside (each added at 0.1% [wt/vol]) (data notshown).

Substrate specificity. The purified LAMY was examined forits ability to hydrolyze various carbohydrates under the stan-dard conditions of the assay. It hydrolyzed soluble starch, amy-lopectin (from potato), amylose (from potato; degree of poly-merization, 17-mers), glycogen (from oyster), and dextrin(from corn) at a relative rate of 100:114:37:83:7. No reactionwas observed on dextran, pullulan, and a-, b-, and g-cyclodex-trins. When soluble starch was used as the substrate, the hy-drolysis ratio (amount of reducing sugar formed as glucose[initial amount of total reducing sugars as glucose]21 3 100)after completion of the reaction (a 20-h digestion) was approx-imately 48%.

Purified LAMY is unique in that at the early stage of hy-drolysis of soluble starch, the enzyme produced G5 to G7 andlarger sizes in quantities much greater than G2 and G3, asanalyzed by TLC (Fig. 4A). G1 and G4 were formed at thisstage in trace amounts. On further incubation, G3 and G5increased, while G6 to G8 decreased. When analyzed quanti-tatively by high-performance liquid chromatography (HPLC),the typical molar ratios of products at equilibrium (after 20 h)were as follows: G7, 0.38 mM; G6, 2.02 mM; G5, 2.16 mM; G4,0.51 mM; G3, 3.05 mM; G2, 2.05 mM; and G1, 0.59 mM. The

action patterns of the enzyme on amylose, amylopectin, andglycogen were very similar to that on soluble starch. Therefore,LAMY is classified as liquefying-type amylase, because largemaltooligosaccharides in the G4 to G6 range were generatedfrom starch (18, 40, 44). The main products of starch hydrolysisby saccharifying-type enzymes are G1, G2, and G3 (44).

Under the standard conditions of the reaction, LAMY couldnot act on maltooligosaccharides in the G2 to G6 range, butcould hydrolyze the substrates in the G7 to G15 (or larger)range to generate G3, G5, and G6 as the major end products,as shown in Fig. 4B. When excess LAMY was used, only G6,but not G1 through G5, was cleaved slightly to yield G1 andG5. The Km and Vmax values were 6.37 mM and 16.6 V min

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for G7, 2.18 mM and 58.5 V min21 for G8, and 0.89 mM and136.1 V min21 for G17, respectively. The frequency distribu-tion of bond cleavage of maltooligosaccharides was analyzedby HPLC with p-nitrophenyl (PNP)-labeled and nonlabeledmaltooligosaccharides as substrates. LAMY hydrolyzedpNP-G8 to generate G5, G6, PNP-G3, and PNP-G2 as themajor end products, indicating that it cleaves the a-glucosidiclinkages at the 5th and 6th positions from the nonreducing endof the substrate. As shown in Fig. 4C, G7 was hydrolyzed toyield G2 almost exclusively (99% of the total products), and G8was hydrolyzed to yield both G3 (61%) and G2 (36%) as theend products. The catalytic efficiencies were 3.33 3 107 for G8and 3.65 3 106 min21 M21 for G7. These results indicate thatlarger maltooligosaccharides are better substrates for LAMY.

The anomeric form of the products was examined by 1H-NMR spectrometry and polarimetry. Figure 5A shows a 1H-NMR spectrum of the anomeric region of pNP-G8 in deuter-ated 10 mM phosphate buffer (p2H 7.4). In a spectrumrecorded 40 min after addition of LAMY (72 mg/ml), ana-anomeric proton signal of the hydrolyzed product appearedat 5.21 ppm (doublet, J 5 3.5 Hz), as shown in Fig. 5B. Inaddition, optical rotation of the reaction solution containing 1mM amylose (degree of polymerization, 17-mers) was de-creased sharply after completion of the reaction (data notshown). These results indicate that the product(s) from thesubstrate has an a-configuration, and, hence, LAMY is classi-fied as an a-amylase.

Cloning of the gene for LAMY. To clone and sequenceamyK, we first designed primers A and B from two commonregions, DAVKHIK and DVTFVDNHD, respectively, ofamino acid sequences of typical liquefying a-amylases, such asBLA (46), BAA (39), and BSA (29). The PCR was conductedto amplify a 0.3-kb fragment (fragment A) with the Bacillus sp.strain KSM-1378 DNA as the template and primers A and B.To assist in the choice of restriction enzymes for inverse PCR,we performed Southern hybridization analysis. The genomicDNA from Bacillus sp. strain KSM-1378 was digested withsome endonucleases, and the resultant digests were subjectedto agarose gel electrophoresis, and then the DNA bands weretransferred onto a nylon membrane and allowed to hybridizewith digoxigenin-labeled primer C, which was a 59-terminalcomplementary sequence of fragment A. A 1.0-kb XbaI frag-ment hybridized with the probe, and the XbaI site was located1.0 kb upstream or downstream of the XbaI site in fragment A.The Bacillus sp. strain KSM-1378 genomic DNA (0.5 mg) wasdigested with XbaI and ligated under conditions that favoredthe generation of monomeric circles by T4 DNA ligase. Thefirst inverse PCR was conducted to amplify a 0.7-kb DNAfragment (fragment B), with the self-circulated molecules asthe template and primers C and D. The primers had beensynthesized on the basis of the results of sequencing of frag-ment A. The sequence of fragment B included a sequence thatencoded the deduced amino acid sequence, which was identi-

FIG. 5. 1H-NMR spectra of the anomeric proton regions of pNP-G8 and itsproducts of hydrolysis by LAMY. (A) Before addition of enzyme. (B) Fortyminutes after addition of enzyme (72 mg ml21). The enzymatic reaction wasperformed at 20°C and at p2H 7.4. The arrow in the upper panel indicates anewly appearing doublet signal. Signals around at 5.4 ppm and at 5.82 ppm(doublet) are due to protons at C-2 to C-8 and C-1, respectively, of the G8 moietyof the substrate. The detailed experimental procedure is described in Materialsand Methods.



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cal to the C-terminal amino acid sequence of typical liquefyinga-amylases, and also included a putative stop codon, TAA.

We designed and synthesized primer E, which was deducedfrom the N-terminal amino acid sequence of the purifiedLAMY from culture broth of Bacillus sp. strain KSM-1378.

The PCR experiment was performed to amplify a 0.7-kb frag-ment (fragment C), with the genomic DNA from Bacillus sp.strain KSM-1378 as the template and primers C and E. South-ern hybridization analysis showed that a HindIII site was found0.5 kb upstream of the N-terminal sequence in the amplified

FIG. 6. Amino acid sequence alignment of LAMY, strain 707 enzyme, BAA, BSA, and BLA. Each numbering starts after the respective signal peptide. The fourconserved regions, I, II, III, and IV (30), are boxed. Domains A and B are indicated by dotted boxes and shading, respectively. The remaining C-terminal sequencecorresponds to domain C. Underlined sequences correspond to the secondary structure elements a1 to a8 and b1 to b8 of the BLA (a/b)8 barrel (domain A) accordingto Machius et al. (24). BLA residues involved in the catalytic site (.), calcium binding site (E), and chloride binding site (F) are indicated above the sequences.Sequence accession numbers (Swiss-Prot database) were as follows: strain 707 enzyme, P19571; BAA, P00692; BSA, P06279; and BLA, P06278.



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fragment. Therefore, inverse PCR experiments were con-ducted to amplify a 0.8-kb DNA fragment (fragment D) byusing HindIII self-circularized DNA molecules as the templateand suitably synthesized primers F and G. The resulting frag-ment, D, contained a putative regulatory region and a se-quence that corresponded to the amino acid sequence HHNGTNGTMMQYFEW, which was identical to the N-terminalsequence of LAMY produced by Bacillus sp. strain KSM-1378.Using the genomic DNA from Bacillus sp. strain KSM-1378 asthe template and primers H and I, we amplified a 1.8-kbfragment (fragment E) by PCR, which would contain the entireamyK gene. The 1.8-kb fragment E was inserted into the SmaIsite of pUC19. The resulting plasmid was introduced into E.coli HB101. When the recombinant E. coli strain was grown onthe LB agar plate containing starch azure and ampicillin at37°C for 1 day, clear halos were found to form around thecolonies. This indicates that an amylase gene on fragment Ewas actually expressed in E. coli (data not shown).

The entire nucleotide sequence of amyK. The 1.8-kb frag-ment E containing the amylase gene was sequenced. The frag-ment contains a single open reading frame (ORF), which be-gins with an ATG codon at nucleotide 1 and ends with a TAAcodon at nucleotide 1545 in the 1,786-bp nucleotide sequencedetermined. Upstream from this ORF, the putative ribosome-binding sequence AGGAGA is found, separated 11 bp fromthe initiation codon ATG. The sequence at nucleotides be-tween 220 and 250 resembles the consensus sequence of thesigma A (sigA, or sA)-type promoter of Bacillus subtilis (27).This sequence consists of TTGACT as the potential 235 re-gion and TAAATT as the potential 210 region, separated by19 bp (data not shown).

Amino acid sequence analysis. The ORF in the nucleotidesequence encoded 516 amino acid residues, as shown in Fig. 6.The amino acid sequence deduced from amyK contains a short,relatively basic hydrophilic region, from amino acid 231 toamino acid 226, followed by a hydrophobic region that extendsfrom amino acid 227 to amino acid 21. This hydrophilic-hydrophobic sequence resembles signal peptides of Bacillus(32). A deduced sequence that was identical to the N-terminal15 amino acid residues of LAMY secreted by Bacillus sp. strainKSM-1378, HHNGTNGTMMQYFEW, was found at aminoacids 1 to 15. The residues AQA in the hydrophobic regionmight be the recognition site of a signal peptidase. If thisputative signal peptide were cleaved on the C-terminal side ofAla21, the molecular mass of the extracellular mature enzyme(from 1 to 485) would be 55,391 Da, a value close to the 53 kDadetermined by SDS-PAGE of LAMY that was purified fromthe culture broth of Bacillus sp. strain KSM-1378.

When suitably aligned, the deduced amino sequence ofLAMY exhibits only 66.7, 68.6, and 68.9% identity to those ofBAA, BSA, and BLA, respectively (Fig. 6). The deducedamino acid sequence of G6-producing a-amylase from alkali-philic Bacillus sp. strain 707 (41) has the highest identity to thatof LAMY (83.5%), although the physicochemical and enzy-matic properties of the strain 707 enzyme have not yet beendescribed. The sequence of LAMY shows almost no homologyto those of the saccharifying a-amylases reported to date (datanot shown).

The deduced amino acid sequence of LAMY was comparedwith that of BLA that contains three domains, A, B, and C, asfirst reported by Buisson et al. (8) for porcine pancreatica-amylase. Essentially, LAMY has the core of the (a/b)8 barreldomain; the structure elements are underlined and labeled bya1 to a8 for a-helices and b1 to b8 for b-sheets in Fig. 6. Theamino acid sequences of domains A, B, and C in LAMY andBLA showed 68.0, 72.5, and 67.8% identity, respectively. Four

conserved regions (I through IV) which are necessary for thecatalytic activity of a-amylase (30, 33) are well conserved inLAMY as Asp102 to His107, Gly232 to His240, Glu266 toLys269, and Phe328 to Asp333 for regions I, II, III, and IV,respectively. They are believed to form the active center, thesubstrate binding site, and the calcium binding site. The threeBLA residues (Asn104, Asp200, and His235) involved in cal-cium binding are conserved in LAMY as Asn106, Asp205, andHis240. The BLA residues for chloride binding, Arg229 andAsn326, correspond to Arg234 and Asn331 in LAMY. Likeother liquefying a-amylases, LAMY has a distinct internalsequence, a B domain loop (Arg171 to Tyr200), which is be-lieved to play an important role in liquefaction of starch (30).Thus, the overall structure of LAMY may be basically similarto that of BLA, but their catalytic properties are significantlydifferent. For instance, BLA is a rather acidic enzyme, whereasLAMY is highly active at the semialkaline pH side (Fig. 2A).Furthermore, BLA is stable and active at high temperature andstable at least after being heated at 70°C for 60 min, and theoptimum temperature for its activity is observed at around80°C (Fig. 3A). In contrast, LAMY is rapidly inactivated at60°C (Fig. 3B).

Declerck et al. (9, 10) and Joyet et al. (19) have indepen-dently reported hyperthermostable mutants of BLA; two sub-stitutions in the amino acid sequence, His133Ile (orHis133Tyr) and Ala209Val (or Ala209Ile), can together in-crease in the half-life of BLA at 90°C up to 10-fold. It is veryinteresting that the original amino acid sequence of LAMYconserves the corresponding amino acid residues at aminoacids 135 (Tyr) and 214 (Ile), respectively. Suzuki et al. (38)compared the deduced amino acid sequences of BAA andBLA and demonstrated that the thermostability of BAA wasdrastically improved by deletion of Arg176 and Gly177. Thethermostability of LAMY can undoubtedly be improved bysite-directed mutagenesis, since the amino acid sequences ofLAMY, strain 707 enzyme, BAA, and BSA all conserve thecorresponding Arg and Gly residues at the respective aminoacid positions (Fig. 6). Recently, the substitution of bothSer187Asp and His133Tyr in the amino acid sequence of BLAwas found to increase the specific activity of the enzyme three-fold (43). The high specific activity of LAMY, compared withthat of BLA, may be related to its original amino acid sequencethat conserves Tyr and Asp at amino acids 135 and 192, re-spectively.

We have already improved the thermostability of LAMY bysite-directed mutagenesis and are now analyzing the acquiredthermostability of mutant proteins by computer-aided struc-ture modeling. The results will shortly be published elsewhere.

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enzymatic properties of a novel liquefying α-amylase from ...mm tris-hcl (ph 7.5) plus 2 mm cacl 2....

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