Apr 6, 1992 - In-Cheol Kim, Jae-Ho Cha, Jung-Ryul Kim, So-Young Jang, Byung-Cheol Seo, ... a-Amylase is one of the best known enzymes which hydro-.
THEJOURNALOF (0
Vol. 267, No. 31, Issue of November 5, pp. 22108-22114,1992 Printed in U.S.A .
BIOLOGICAL CHEMISTRY
1992 by The American Society for Biochemistry and Molecular Biology, Inc.
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, Taeyon 305-601, Korea “ I
I ,
A gene encoding a new amylolytic enzyme of Bacillus licheniformis (BLMA) has been cloned, and we characterized the enzyme expressed in Escherichia coli. The genomic DNA of B. licheniformiswas doubledigested with EcoRI and BamHI and ligatedthe pBR322. The transformed E. coli was selected by its amylolyticactivity,whichcarries 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 apH 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 pullulan 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 specificity of transferring activity is evident from the result of the maltosyl-transferring reaction which produces isopanose from maltotriose and glucose. The molecular structure of the enzyme deduced from the nucleotide sequence of the clone maintains limited similarity in the conserved regions to the other amylolytic enzymes.
a-Amylase is one of the best known enzymes which hydrolyzes a-1,4-linkage of starch producinga mixture of oligosaccharides. In recentyears, evidence has mounted in supportof the unusual action of amylases which exhibit transferring as well as hydrolyzing activity. New amylaseshave also been discovered from variousmicroorganismsincluding maltose forming-, pullulan’ and/or cyclodextrin hydrolyzing-, and glucose-transferring amylases. a-Amylases from Thermoactinomyces vulgaris R-47 (1,2) and Bacillus stearothermophilus K P 1064 (3) degrade soluble starch, yielding maltose and glucose
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 foundin B. stearothermophilus (4, 5), and a gene for the enzyme was cloned and expressed in Bacillus subtilis. Suzuki et al. (6) isolated extracellulara-amylase I1 from Bacillus thermoamyloliquefaciem KP1071, which split a-1,6 bonds in amylopectin as well. This enzyme hydrolyzed a- and @-cyclodextrins, and pullulan as well. David et al. (7) cloned a gene encoding theamylolytic enzyme of Bacillus megateriurn. Basedontheactionpattern of the enzyme, ithas been proposed that this enzymehydrolyzed pullulan as well as starch. Interestingly, it also exhibits glucose transferring activity with the formationof a-1,4-linkage. With the discovery of these new amylases, the scheme of the transferringactivities on the starch metabolism in the microorganisms was to be elucidated and the productionof maltooligosaccharides in large quantities became easier. We have cloned a gene encoding a new type of amylase, BLMA,2from Bacilluslicheniforrnis. The enzyme hadthe abilityto hydrolyze pullulanand 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 maltoheptaose from starch but cannot hydrolyze pullulan and cyclodextrins (13). BLMA also showed the catalytic properties of a new type of a-amylase which exhibited transferringactivity. 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. EXPERIMENTALPROCEDURES
Materials and Bacterial Strains-Maltooligosaccharides, CY-, p-, ycyclodextrin, and panose were purchased from Sigma. Isopanose was a gift from Dr. Y. Sakano, Tokyo Noko University, Japan. B. licheniformis ATCC27811 was obtained from the American Type Culture Collection (ATCC) and used as the donor strainfor the BLMA gene. E. coli HB 101 was used as a host strain for recombinant DNA. Cloning and Characterization of BLMA Gene-B. licheniformis * This research was supported by a grant from the Research Center chromosomal DNA was isolated by the spool method (8, 9). Isolated for New Biomaterials in Agriculture, Seoul National University. The DNA was partially cleaved with EcoRI and BamHI and ligated into costs of publication of thisarticle were defrayed inpart by the EcoRI and BamHIdouble-digested pBR322. The ampicillin-resistant payment of pagecharges. Thisarticlemusttherefore be hereby colonies were toothpicked ontoa solidLB plate containing1% soluble marked “aduertisement” in accordance with 18 U.S.C. Section 1734 starch and50 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 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted overnight incubation a t 37 “C, amylase-producing colonies were detected by theaddition of2.5% Lugol’s iodinesolution.Positive to the GenBankTM/EMBLDataBank with accession number(s) X67133. The abbreviations used are: BLMA, maltogenic amylase of B. 7 T o whom correspondence should be addressed. Tel.: 82-331-292licheniformis; BLTA,thermostablea-amylase of B. licheniformis; 1943; Fax: 82-331-293-4789. HPLC, high performance liquid chromatography; SDS-PAGE, SOStructures of the oligosaccharides are defined pullulan, dium dodecyl sulfate-polyacrylamide gel electrophoresis; TLC, thin (-4Glcpcul-6Glcpnl-4Glcpnl-),; panose, Glcpal-6Glcp~l-4Glc; isolayer chromatography; bp, base pair. panose, Glcpnl-4Glcpnl-6Glc; maltotriose, Glcpnl-4Glcpnl-4Glc.
22108
Maltogenic Amylase of B. licheniformis 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 supernatantwas fractionated with solid ammonium sulfate at 4 "C. The precipitate a t 70% saturation of ammonium sulfate was collected by centrifugationand dissolved in 30 ml of 50 mM Tris buffer (pH 7.5) containing 1% of streptomycin sulfate. After centrifugation the supernatant was dialyzed 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. Theconcentrate wasapplied on a DEAEcellulose column (3 X 30-cm) equilibrated with 50 mM Tris buffer (pH 7.5). The column was washed with 250 ml of the samebuffer (30 ml/h), and then the enzyme was elutedwitha 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 fastprotein liquid chromatography (FPLC) system(PharmaciaLKB Biotechnology AB) underthe 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-Molecular 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 relativemolecularweight of the purified enzyme was estimated by comparing its relative mobility with those ofthe following reference proteins: /3-galactosidase ( M , 116,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase ( 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 described 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 terminated 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 amountof the enzyme giving a n increase of 1.0 absorbance for 30 min under the described conditions. Action Pattern of BLMA-To determinethe hydrolytic action mode, 20 p1 each of the purified enzyme from the Bacillus donor strain andfrom 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, maltotriose, maltotetraose, maltopentaose, or maltohexaose was digested with BLMA. For the panosyl transferring activity, 1% pullulan solution was incubated in the presence of 9-, 18-, 27-, 36-, 45-, 54-, and 63% glucose under the same conditions as above. The reaction mixture was analyzed by TLC, paper electrophoresis, and/or HPLC. Thin Layer Chromatography and PaperElectrophoresis-TLC was carried outon the Kiesel Gel-60 plate (Merck Co., Ltd.) with asolvent 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 20cm) in 0.05 M borate buffer (pH 10.0) at 500 V, 2 mA/cm for 1h (12). After electrophoresis the paper was dried and sprayed with silver nitrate solution (0.1ml 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
22109
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 pullulan hydrolysate by BLMA was purified by TLC. I3C NMR spectra of the purified trisaccharide and standard panose in D20 were recorded a t 75 MHz (Bruker), inwhich 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 partially 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. It 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 analysis 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 partialrestriction 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 intensityof the plasmid pIJ322 insert reconstructed with the equivalent amount of a single copy gene, the intensityof bands suggest that thereis 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 BLMAGene-To analyze the B
A
EwRI
5.8 4.3 4.0
23 20 19
1.6 1.3
1.2
0 98 0 . ~ 3
0.56
sal I
FIG.1. Southern Blot analysis of the genomic DNA of B. licheniformis.Panel A, genomic DNA was digested with the restriction enzyme indicated, separated by 0.5% agarose gel electrophoresis, and was transferred onto a nitrocellulose filter. It 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, partialrestriction enzyme map of recombinant plasmid pIJ322. Open region is vector DNA pBR322 and closed region is the insert containing theBLMA gene.
22110
Maltogenic Amylase of B. licheniformis
structure of the BLMA gene, nucleotide sequencing was carried out (Fig. 2) There was one open reading frame encompassing 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. Putative promoter elements, TTAACA (-35 element) and TGATAAT (-10 element), are present around-93 and -54 from the translational initiationsite, respectively. A Shine-Dalgarno sequence, AGGGGG, was also noticed around -8. These results arequite consistent withthe functional analysisof the pIJ322 by serial deletion (13). Purification and Characterization of BLMA-To study the reaction mechanism of BLMA, the enzyme produced by transformed 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 contaminating proteins asjudged by SDS-PAGE andelectrofocusing .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 450
1425 475 1500
500
I575 525
1650 550
1725
575
1800 578
1875 1950
2025
2104
2175
2180
FIG.2. Nucleotideanddeducedaminoacidsequence 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
116,000-
67,000~ 43,000+ 30,000*
-
c p l 5.4
15,000-
c pH 6 . 5 FIG. 3. Electrophoresis of purified BLMA. Panel A, SDSPAGE 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 detected 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 pH5.4. The optimum pH of hydrolyzing activity was pH 6 for pullulan, pH 7 for starch, but around pH7-9 for cyclodextrin (13). Hydrolytic Activity of BLMA-To test the substratespecificity 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 at 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, theenzyme could liberate a seriesof low molecular weight oligosaccharides, in which the main product was maltose (Fig. 4B). In case of pullulan, which contains one a-1,6and two a-1,4-glycosidic linkages alternately, themajor reaction product migratesat theposition between maltotriose and maltotetraose (Fig. 4C). Compared withthe 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 spectrumof the isolate was shown to be identical to that of the authentic panose (data not shown). These results made it sure that thetrisaccharide produced by BLMA was clearly panose. These resultsaltogether suggest that BLMA hydrolyzes every other a-1,li-glycosidic linkage to produce maltose from soluble starch. Italso hydrolyzes the a-lP-linkageof pullulan with the production of panose, which the usual @-amylase cannot. It, however, could not hydrolyze the a-l,ri-linkage right next to thea-1,6-linkage (Fig. 10). Transferring Activity of BLMA-To examine the transfer-
Maltogenic Amylase of B. licheniformis
22111 .
. . ,
.
R I C T K Y l TIY.o((xR)
C Gl h 02
4
O3
e
'5
Gl
$
03
* .'.!I
M )
b
05
*
@ #4
8
G2
* m
FIG.6. Paper electrophoresis to identify the panose and isopanose. I P , isopanose standard; P, panose standardH, hydrolysis product of pullulan by BLMA.
t
05
Ge
DB
s
o w 0 0 O
?
2 3 4 Ll2oplNs REACTION TIME (mwm
FIG.4. Thin layer chromatogram of the hydrolysis products of various substrates by BLMA. Soluble starch (panel A ) , pcyclodextrin (panel R ) , and pullulan (panel C) were incubated with the purified enzyme, and the reaction productswere taken a t various time points and analyzed by TLC using isopropyl alcohol/ethylacetate/water (3:1:1, v/v/v) as solvent. G I , G2, G3, G4, G5, and G6 are standards of glucose, maltose, maltotriose, maltotetraose, maltopentaose, and maltohexaose, respectively. A
B
i
8
16
24
32
40 min
FIG.7. High performance liquid chromatogramof the pullulan 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 indicatedby arrows. P, panose; R, Glcpnl-6Glcpal-4Glcpnl-4Glc added as internal refbe Glcpculerence; U, unknown branched tetrasaccharide assumed to
6Glcpcul-4Glcpcul-6GIc. The HPLC analysis was carried out under the following conditions: column, Machery& Nagel NucleosillONH2 Unicam PU4023 refractiveindex (4.6 X 300 mm);detector,Pye 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 solutionsof 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,PyeUnicamPU4023 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 incubatedwith BLMA in the presence of a highconcentration 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 detected 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-6Glcpal4Glcpa1-4Glc, which was added to the reaction product as an internal reference. This unknown branched tetrasaccharide was thought tobe Glcpal-6Glcpal-4Glcpal-6Glc, which was made by the transferase activityof BLMA. Specificity of transferring activityof the enzyme was demonstrated further by digesting maltooligosaccharide. Maltotriose was degraded by BLMA in the presence of glucose to form a product with the migration distance corresponding to that of isopanose as shown inFig. 8. This resultsuggests that the bond formed by transferring activity of BLMA is a-1,6linkagebetween the reducing end of maltoseandC-6 of
of B. licheniformis
Amylase Maltogenic 22112 acceptor glucose. These results strongly argue that the unknown tetrasaccharide generated from pullulan in the presence 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 theconcent,ration of 36% glucose the formation of the branched tetrasaccharide was dramatically 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
IP
T
FIG.8. Paper electrophoresis to identify panose and isopanose. G 3 , maltotriose; P, panose; T, maltotriose + glucose + RLMA; II', isopanose. The fast migrating spot in lane T was identified as glucose.
20
40 (. )
tonc
glucose
FIG. 9. Effects of glucose concentrationon thetransferring activity of BLMA. Opencircles denote panose and closedcircles denote the branched oligotetrasaccharide concentration in the reaction 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 forpanose, and, therefore, theconcentration of panose was kept relatively low during the coupling reactions, for theformation of branchedtetrasaccharide. BLMA, however, did not catalyze the formation of branched tetrasaccharide from the mixture of panose and glucose, suggesting the coupling of hydrolysis and transferringreactions (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 notthermostable.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 reported thattwo different amylase genesmight existin Bacillus sp. because two different a-amylases were produced a t different culture temperatures.BLMA could be produced by one of B. the two amylase geneswhich was barelyexpressedin 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, wasnoticed in E. coli transformed with the BLMA gene, which might result from regulated gene expression (13). When the deduced amino acidsequence of BLMA was compared with those of various amylases reported, limited similarities a t four prominently conserved regions were noticed (Table I). A relatively high similarity was noticed at conserved region I which is considered to be the calciumbinding domain of amylase(16, 17). The variousspacings between these conserved domains could reflect the chemical nature of the reaction catalyzing and thus productspecificity 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. thermoamyloliquefaciensKP1071 (6), a-amylase from T.vulgaris R-49 (1, 2), maltogenic a-amylase from B. megaterium
TABLE I ComDarison of the deduced amino acid seauencesof various amvlases Amino acid sequencesa
~
Region 1
Consensus sqn RLMA Neopullulanase Pullulanase CGTase BLTA
DAVINH 237 - - F 240 - - - F - 281 - V - Y - 135 - F A D - 129 - V - I - -
-
Region 2
Region 4
EVID 349 -1WH 352 324 -W---V-NE332 357 -1WH360 348 ---F-LMGI 356 381 -GW- 384 225 -I-V--V-- 233 268-YHQ271 256 - - - - - - V - - 264 29ODYWQ294 Sequence similarities ( % ) h
- 242 245 286 140 134
Region 1
BLMA Neopullulanase Pullulanase CGTase BLTA
Region 3
GFRLDAAKH 318 -vwMwQM-L 326
Region 2
Refs.
410 LL-S- - 415 419LLGS" 424 464Y-ES" 469 293-1"" 298 352 """357
Region 3
nt
aa
ntaa
aa
100 88.8 66.6 50.0 66.6
100 100 66.6 50.0 66.6
100 25.9 40.7 44.4 29.6
100 11.1 22.2 22.2 22.6
nt
23
FVDNHD
This work 16 17 24 25 Region 4
nt
aa
100 91.6 58.3 50.0 66.6
100 100 50.0 25.5 25.5
"Amino acid sequences different from consensus sequence are shown. Amino acid sequence similarities (aa) and nucleotide sequence similarities (nt) to BLMA at conserved region.
100 77.7 44.4 66.6 77.7
100 83.3
50.0 50.0 50.0
22113
Maltogenic Amylaseof B. licheniformis TABLE I1 ComDarison of the ohvsicochemical DroDerties of various amvlases ~~~~
Substrate specificity
M,
Origin
Enzyme
Substrate
BLMA
B. licheniformis 64,000 a-1,6-linkage Panose
BLTA
B. licheniformis
55,000
Pullulanase
I(.pneumoniae
66,000
Isopullulanase
A. niger
62,000
Neopullulanase
B. stearothermophilus
62,000
BMA
B. megaterium
55,000
T. vulgaris
T. vulgaris
71,000
a-amylase
Major product
Starch Formation Maltose Pullulan Maltose Cyclodextrin Maltopentaose Starch Pullulan Cyclodextrin Starch Pullulan Panose Cyclodextrin Starch Pullulan Isopanose Cyclodextrin Maltose Starch Pullulan Panose Cyclodextrin ?b Oligomer Starch Pullulan Panose Cyclodextrin Maltose Maltose Starch Panose Pullulan Cvclodextrin ?
Transfering activities
of
Refs.
This work
-
13
-
26
-
27
-
4
Formation of a-l,4-linkage
I
-
1
~
-, no products. ?, not determined.
, 2 %
%
0-0
maltose
branched tetrasaccharide 11
FIG. 10. Proposed action pattern of BLMA, coupling of hydrolysis, and transferring activity of BLMA generate. Open circles and slashed circles denote non-reducing glucopyranosyl residues andreducing 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 andGlcpal-4GlcpalGGlcpnl-4Glc, respectively.
(6, 20), pullulanase from Klebsiella aerogenes (21), isopullulanase 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-1,4linkage next to the a-l,g-linkage, which results in therelease of panose exclusively from pullulan rather than isopanose. Pullulanase, however, produces isopanose rather thanpanose. Neopullulanase from B. stearothermophilus could hydrolyze a-1,6-linkage aswell 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 Table11. It is interesting to note that BLMA has transferring activity in addition to hydrolyzing activity. The branched tetrasaccharide (Glcpal-6Glcpal-4Glcpal-6Glc) havinga new a-1,6linkage is formed as BLMA degrades pullulan in thepresence of glucose. Hydrolysis specificity of a-amylase from B. megaterium and T. vulgaris seems to be quite similar to that of BLMA. David et al. (7), however, pointed out that the hydrolysis reaction of a-amylase from E . megaterium in the presence of glucose is graduallyreplacedby the coupling reactionwiththeformation of Glcpal-6Glcpal-4Glcpal4Glc 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 andglucose gave isopanose in which a-l,g-linkage was formed by the maltosyl transfer. Incubationof maltotriose and maltose with BLMA also results in the formation of branched maltotetraosein which two maltose units arelinked by a-1,6-linkage(14).Whilepullulan was incubated with BLMA in the presence of glucose, branched maltotetraose was produced. The retention time of the branched maltotetraose in HPLC differedfrom those of maltotetraoseand Glcpal-6Glcpal-4Glcpa!1-4Glc, indicating the existence of a-1,6-linkage in the tetraose. The retention time of the unknown products on the HPLC column was between linear maltotetraose and maltopentaosesuggesting the existence of maltotetraose with a-l,g-linkage which did not exist in the reactionmixture.Fromtheresults,theactionpattern 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 oligosaccharides. Such a novel transferring activity of BLMA could be utilized to synthesize various kinds of branched oligosaccharides.
Maltogenic Amylase of B. licheniformis
22114 REFERENCES
1. Shimizu, M., Kanno, M., Tamura,M., and Suekanne,M. (1978) Agric. Bid.
Chem., 42, 1681 2. Sakano. Y., Hiraiwa, S. I., Fukushima, J., and Kobayashi, T. (1982) Agric. Biol. Chem. 46,1121 3. Suzuki, Y., and Imai, T. (1985) A pl Microbiol. Biotechnol. 21, 20 4. Kuriki, T., Okada, S., and Imanafa,T. (1988) J. Bactenol. 170, 1554 5. Imanaka, T., and Kuriki,T. (1989) J . Bacteriol. 171, 369 - Y.,'Nagayama, T., Nakano, H., and Oischi, K. (1987) Starch 39, 6. Suzuki, "
XI1
7. David, M. H., Gdnther, H., and Roper (1987) Starch 39, 436 8. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 9. Dubnau, 0.. and Ahelson, R. D. (1971) J. Mol. Biol. 5 6 , 209 10. Laemmli, U. K. (1970) Nature 227,680-685 11. Miller, G. L. (1959) Anal. Chem. 31,426 12. Frahn, J. L., and Mills, J. A. (1959) A u t . J. Chern. 12,65 Seoul, 13. Kim, I. C. (1991) Ph.D. thesis,SeoulNationalUniversityPress, Korea
14. Kim, J. R. (1991) M. S. thesis, Seoul National University Press, Seoul, Korea 15. Campbell, L. L., Jr. (1955) Arch. Biochem. Biophys. 54, 154 16. Bliesmer, B. O., and Hartman, P. A. (1973) J. Bacteriol. 1 1 3 , 526 17. Kuriki, T., and Imanaka, T. (1989) J. Gen. Microbiol. 135,1521 18. Park. J. H. (1990) Ph.D. thesis, Seoul National University Press, Seoul, Korea 19. Suzuki, Y., and Imai, T. (1985) Appl. Microbiol. Biotechnol. 2 1 , 20 20. Hebeda, R. E., Styrlund, C. R., and Teague M. (1988) Starch 4 0 , 3 3 21. Bender, H., and Wallenfels, K. (1961) Biochm. 2. 3 3 4 , 7 9 22. Sakano, Y., Masuda, N., and Kobayashi, T. (1971) Agric. Biol. Chem. 35, 971 23. Rogers, J. C. (1985) Biochem. Biophys. Res. Commun. 128,470 24. Kaneko, T., Hamamoto, T., and Horokosbi, T. (1988) J. Gen Microbiol. 134,97 25. Yuuki, T., Monura, T., Tezuka,H., Tsuboi, A,, Yamagata, H., Tsukagoshi, N., and Udaka, S. (1985) J. Biochen, (Tokyo) 98, 1147 . 39,967 26. Ohha, R., and Ueda, S. (1975) Agric. B ~ o lChem. 27. Sakano, Y., Higuchi, M., and Kobayashi,T. (1972)Arch. Biochem. Biophys. 153,180
