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Modulation of Bacillus amylolytic enzymes and production of branched oligosaccharides
Enzymesfor CarbohydrateEngineering K.H. Park, J.F. Robyt and Y-D. Choi (Editors) 9 1996 Elsevier Science B.V. All rights reserved.
43
Modulation of Bacillus amylolytic enzymes and production of branched oligosaccharides Tae-Kyu Cheong, Tae-Jip Kim, Myo-Jeong Kim, Yang-Do Choi ~ In-Cheol Kim 2, Jung-Wan Kim3,and Kwan-Hwa Park. Department of Food Science and Technology, ~Department of Agricultural Chemistry and Research Center for New Bio-Materials in Agriculture, Seoul National University, Suwon, 441-744, Korea. 2Samyang Genex Research Institute, Taejeon, 305-348, Korea. 3Department of Biology, University of Inchon, Inchon, 402-749, Korea. Introduction
Many interesting and useful findings of new type amylases have been reported. They catalyze hydrolysis of a-l,4-glucosidic linkages not only in starch but also in other carbohydrates such as pullulan and cyclodextrin (Kim et al., 1992 ; Bender and Wallenfels, 1961; Sakano et al., 1971; Nakamura and Horikoshi, 1976). Debranching enzymes which hydrolyze a-l,6-glucosidic linkage were found in higher plants or microorganisms (David et al., 1987; Pazur and Ando, 1960). Some amylolytic enzymes exhibit glucose transferring activity as well as hydrolyzing activity (Kim et al., 1992 ; David et al., 1987; Matsumoto and Matsuda, 1983; Kaneko et al., 1987; Imanaka and Kuriki, 1989). Several microbial amylases which produce oligosaccharides with specific lengths or structures have been reported by numerous researchers (Robyt and Ackerman, 1971; Takasaki, 1985; Kainuma, 1988). Design of specific oligosaccharide synthesis became possible due to the discovery and modification of new amylases. Maltooligosaccharides mixture, maltotetraose syrup, and branched oligosaccharide mixture are used as substitutes for sucrose and other saccharides in the food industry due to their lower viscosity, less sweet taste, and smaller freezing point depression (Osaki et al., 1988). They also can be used to prevent crystallization of sucrose in foods and are useful in controlling microbial contamination as well as retrogradation of starchy foods because they have low water activity and high moisture-retaining capacity (Kweon et al., 1994; Komoto et al., 1993). Branched oligosaccharides are of benefit in in preventing dental caries (Glor et al., 1988) and effective for the growth of Bifidobacteria (Park et al., 1992) in human intestinal tract. They are hardly digested by human body. Therefore, the low calorie content of branched oligosaccharides has a great appeal to low calorie dieters. Due to these useful properties, the production of BOS mixtures has been increased by 50-100% per year during last few years. Generally, branched oligosaccharides are manufactured by a 2-step procedure that uses a-amylase, [~-amylase, and transglycosidase on starch solution (Takaku, 1988). The starch slurry is liquefied to a degree of
44
0
0
0
t~ 0 t~
c~
~r~
0 0
0 0 t~
0
0
~ ~o.~
~
,
~
~
0
0
'
o~
~
o b~
o
-
o.o
0~
o
~0
0 0 0
'
0 0 0
,
0
~
+
o ~
r~D
0 0 0
+
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~D
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o
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0 0 0
c~ o
O o O
0 0 0
~ o o~~o
o
~
~8
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t~O
o 00
0 0 0
o
o
~0
o.
0 0 0
i
"--'
0 0 0
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~
~
r~O
E-,
45 hydrolysis (DE) 6-10 by the action of a thermostable c~-amylase, then saccharified by t r e a t m e n t with soybean ~-amylase and transglycosidase of AspergiUus niger at 60~ for 72 hours. It is a time-consuming procedure. A series of amylolytic enzymes including a thermostable a-amylase (BLTA), a maltogenic amylase (BLMA), a CDase, and a CGTase were isolated from two Bacillus species,B, licheniformis and alkalophilic Bacills I-5 strain, in our laboratory. The genes responsible for them were cloned in E. coli and the physicochemical properties and the action patterns of these amylases on various carbohydrates were characterized. Recently, we developed a one-step procedure which simplified and shortened the reaction for the production of branched oligosaccharides. It applies BLTA and BLMA to starch solution simultaneously (Kim et al., 1994). The two amylases were prepared from an E. coli transformant harboring a recombinant plasmid which carried both of genes for the enzymes. The reaction was carried out for 24 hours and 45% of the product was branched oligosaccharides of various lengths. The amount of branched oligosaccharides in the mixture was comparable to that of the Alo (anomalously linked oligosaccharide) mixture in market (Takaku, 1988). However, it contained much less glucose than the Alo mixture (27% vs. 40%). Development of a continuous process of branched oligosaccharides production using immobilized BLMA and yeast cells would also improve the efficiency of the whole process and the quality of the product. Modification of the enzymes by recombinant DNA techniques would make application of the amylases to food industry more diverse and effective. Improving thermostability and inducing secretion of BLMA would be of significant benefit in applying the enzyme to the production of branched oligosaccharides. The predicted amino acid sequences indicated that they share homology with various amylolytic enzymes at four conserved active domains (Ihara et al., 1985; Ryoichi et al., 1986). Based on amino acid sequence comparison to that of Taka amylase, whose tertiary structure is known (Matsuura et al., 1984), BLTA and BLMA are likely to have the structure of 8 (~/a) barrels with intervening loops (MacGreger, 1993; Jespersen et al., 1993). Mutagenesis of the genes responsible for the enzymes is under investigation as an effort to improve the process for the production of oligosaccharides. Results and Discussion
Catalytic properties of starch degrading enzymes Physicochemical properties of the starch hydrolyzing enzymes isolated in the laboratory and those reported by others are summarized in Table 1. Substrate specificity of BLMA was compared with that of BLTA. Soluble starch, pullulan, and cyclodextrin were hydrolyzed by BLMA, while BLTA hydrolyzed soluble starch only. Optimum temperature of BLTA was 70~ and it was increased to 90~ in the presence of 5raM Ca +* (Kim, 1991). Optimum temperature of BLMA was 50~ (Kim et al., 1992). The activity of BLMA was inhibited by most of divalent cations such as Ca ++, Zn §247and Mn §247 CGTase produced a-, B-, ~-cyclodextrins at the ratio of 0.7 : 3.3 : 1.0
46 from starch in the mother strain. The ratio among various CD products was changed when the enzyme prepared from recombinant DNA in E. coli (see below) was used. CDase hydrolyzed cyclodextrin very rapidly into maltose. BLMA and CDase exhibited transferring activity in addition to hydrolyzing activity in the presence of excess glucose. A BOS mixture is prepared from liquefied starch using the hydrolyzing and the transferring activities of BLMA (Kim et al., 1994). The BOS mixture was analyzed by various methods including high performance liquid chromatography (HPLC) and found to contain various small branched sugars such as isomaltose, panose, and isopanose etc. Based on the results, a model of BLMA action mode was proposed as shown in Fig. 1. BLMA is likely to synthesize branched oligosaccharides in a complicated m a n n e r : it hydrolyzes liquefied starch further to glucose and maltose, and at the same time transfers the resulting molecules onto cleavage sites of other sugar moieties by forming c~-l,6-1inkages. The whole reaction is completed by repeating the coupled hydrolysis and transfer reactions, thereby creating a new population of branched oligosaccharide molecules.
Pullulan
4, Starch
s ~
/ t ~' "" " ~ l
%
i
Figure 1. A proposed model of BLMA action mode involved in the production of branched oligosaccharides from pulllulan or maltooligosaccharide. BLMA hydrolyze pullulan to panose and the resulting molecule is transferred to acceptor (in this case glucose) by forming a-l,6-glycosidic linkage. Maltooligosaccharide is hydrolyzed mainly to maltose, but also to glucose or maltotriose etc. They are then transferred to acceptor molecules such as glucose, maltose, or maltotriose. Resulting branched molecules are likely to be hydrolyzed further by BLMA.
47
Continuous production of the BOS mixture The procedure for continuous production of the BOS mixture and a highly concentrated BOS (high BOS) mixture is illustrated in Fig. 2. BLMA has an optimal temperature of 50~ and its thermostability could be enhanced by immobilization on CPC Silica. Decimal reduction time of immobilized BLMA at 55~ was 96.8 min, while that of free BLMA at the same temperature was only 20.5 min. Thirty percents (w/v) corn starch suspension in 50mM maleate-NaOH buffer (pH 6.8) was liquefied by thermostable c~-amylase (Termamyl ; Novo Nordisk, Denmark). The sample was heated in a boiling water bath for 10 min and the reaction was stopped by autoclaving the hydrolysate for 10 min when the DE value was about 20-25. Five millimoles of EDTA was added to the liquefied solution and its final pH was adjusted to 6.8 using 1N NaOH. For the synthesis of the BOS mixture, the liquefied starch was run through a column of immobilized BLMA at 45-50~ Residence time of the
Starch slurry
l
30% Soluble starch
Liquefaction
~-
alpha-Amylase 100~ 10min
Saccharification
--
45~
}_
~-
BLMA
15hrs
Filtration
BOS
i
Yeast fermentation ~-
I_.
Immobilized Yeast
27~ 2days Freeze drying
High B O S
Figure 2. mixtures.
Procedure
for
the
production
of the
BOS
and
high
BOS
48 liquefied starch solution in the column was 2.5 hours. It took 24 hours at 40~ to prepare a BOS mixture with similar composition when free BLMA was used. The reaction was stopped by boiling, filtered through a W h a t m a n paper No. 5, and then dried using a freeze-drier. The BOS mixture was analyzed by HPLC (Fig. 3) and its composition is shown in Table 2. It contained BOS over 60% including panose, branched DP4, and branched DP5 etc. The shoulder of the maltose peak indicated that isomaltose might be present in the mixture. Further analysis of the mixture using ion-chromatography showed that isomaltose and isopanose were also present in the mixture (data will be discussed elsewhere). The BOS mixture contained about 30% glucose and maltose. In order to get rid of sweet taste of the mixture, it was fermented by yeast. As the result of glucose and maltose fermentation by yeast, BOS would be concentrated to make a high BOS mixture. For the preparation of a high BOS mixture, the BOS mixture (30%; w/v) prepared as described above was fermented by Saccharomyces cerevisiae var. ellipsoideus that was immobilized on sodium-alginate matrix. Fermentation was carried out at 27-28~ for 2 days to remove glucose and maltose contained in the BOS mixture. Upon the completion of fermentation, the mixture was filtered and dried as described above. As the result of fermentation, almost all of glucose and maltose was removed and the BOS content increased to over 90% (Table 2).
o-o +
0
aD --"
N
Retention Time (min)
A
Retention Time (min)
B
Figure 3. High performance liquid chromatography analysis of the BOS (A) and high BOS (B) mixtures. Saccharide which is likely to constitute each peak is shown above the peaks. HPLC analysis was carried out as described previously (Kim et al., 1992).
49 Table 2 The compositions of the BOS and the high BOS mixtures Saccharide
BOS mixture
High BOS mixture
Glucose
10.2 %
1.3 %
Maltose
18.9 %a
_
Isomaltose
-
2.7 %
Maltotriose
6.8 %
7.2 %
Panose b
15.4 %
20.4 %
Branched tetraose
30.4 %
43.8 %
Branched pentaose
18.3 %
24.6 %
> Branched pentaose
trace
trace
Total a m o u n t of BOS
64 %
91.5 %
a : m a y contain isomaltose b : m a y contain isopanose
Application of the BOS mixture to bread as h u m e c t a n t for starchy foods was a t t e m p t e d . It retarded starch retrogradation and lowered w a t e r activity (manuscript submitted). The high BOS mixture would be of great benefit to apply it as h u m e c t a n t to food.
Comparison of amino acid sequences of various amylases The
genes
responsible
for
BLTA
or
BLMA
were
isolated
from
B.
licheniformis by shotgun cloning EcoRI or BamHI/EcoRI genomic DNA digests into an E. coli vector, pBR322. A CDase gene was isolated from an alkalophilic Bacillus strain I-5 using the same method. The positive clones were screened for their starch hydrolyzing phenotype in E. coli HB101 using iodine test. The starch hydrolyzing activity of BLMA or CDase was observed only when cell m e m b r a n e of the t r a n s f o r m a n t carrying one of the genes was disrupted by D-cycloserine. This indicated the two enzymes were cytoplasmic proteins. In order to isolate a CGTase gene from Bacillus I-5 strain, two oligonucleotides were synthesized based on homology among CGTase genes. An 1.1kb DNA fragment was amplified from I-5 genomic DNA and used as a probe to screen a genomic DNA library constructed using EMBL3 k phage DNA (Amersham, U.S.A.). The insert of a plaque with positive signal was subcloned into pUC18. The CGTase positive phenotype of the clone was confirmed by a phenolphthalein tested (Park et al., 1989) and HPLC analysis of the product produced by the enzyme p r e p a r e d from the clone. The restriction m a p s of pIJ322, pTA322, pCGTJ322, and pTJ3, which
50 contain the BLMA, BLTA, CGTase, and CDase gene, respectively, are shown in Fig. 4. Each gene was expressed from its own promoter and stably m a i n t a i n e d in E. coli. The gene products were characterized in detail and three of them, BLMA, BLTA, and CGTase, had properties t h a t were in good correlation with those found in the mother strains. The activity of CDase has not been detected in the mother strain, alkalophilic Bacillus I-5. To analyze the structure of the BLTA and BLMA genes, nucleotide sequencing was carried out. Nucleotide sequences of the two B. licheniformis amylases were determined by sequencing the plasmids
EcoRI
EcoRI
Pvull
Hindlll SlII
/ X
c.,
cloning
m"'
Pstl
Pvull
~
J J
Hlndlll Pvull
EcoRI
EcoRI
hmHI
BIImHI
EcoRI
Sail
oa,
BamHI
pBR322
BImHI
Smal
Hindlll
EcoRI
Figure 4. Restriction maps of pTA322, pIJ322, pTJ3, and pCGTJ322. pTA322 carries the BLTA gene on a 3.1kb insert; pIJ322, the BLMA gene on a 3.5kb insert; pTJ322, a CDase gene on a 3.2kb insert; pCGTJ322, a CGTase gene on a 4.8 kb insert. All of them are cloned on an E. coli vector, pBR322, and selected by resistance to ampicillin in E. coli.
51 carrying the genes according to Sanger's chain t e r m i n a t i o n method (Sanger et al., 1977) and using Sequenase kit purchased from U.S.B. Corp. The DNA f r a g m e n t s were sequenced in both strands. The BLTA gene coded for a 55 kD protein of 483 amino acids. The m a t u r e BLTA protein was proceeded by 29 amino acids which are likely to function as a signal sequence for secretion (Kim, 1991; Yuuki et al., 1986). The protein was localized in both periplasmic and cytoplasmic spaces. The BLMA gene was capable of encoding 584 amino acids from a promoter located 5' flanking region and the molecular weight of the gene product was predicted to be 66.5 kD. There was no signal sequence like sequence found at the amino t e r m i n a l of the gene. The protein was found only in cytoplasm of E. coli and the mother strain. The deduced amino acid sequences were aligned with those of various a m y l a s e s reported previously (Jespersen et al., 1993) and four highly conserved regions were found in Table 3. Conserved regions III and IV are considered to include active sites of amylases. The specificity of the anti-BLTA antibody was tested using Ouchterlony double immunodiffusion analysis (Jang et al., 1994). The antibody formed a precipitate with Taka-amylase, which is a thermostable amylase. However, neither pullulanase nor glucoamylase cross-reacted with the antibody. Termamyl (Novo Inc., Denmark) formed a precipitate with the antibody (data not shown). In a recent study, the antibody raised in the laboratory against another amylase of B. licheniformis, BLMA, did not cross-reacted with BLTA (Shim, 1994). Pullulanase, Termamyl, and glucoamylase are also Table 3 Comparison of amino acid sequences in the conserved domains Domains
I
II
III
IV
Reference
Enzymes
DAVINH GFRLDAAKH
EVIH
FVDNHD
BLTA
DVVINH GFRLDAVKH
EYWQ
FVDNHD Kim, 1991
BLMA
DAVFNH GWRLDVANE
EIWH
LLDSHD Kim et al., 1991
Neopullulanase
DAVFNH GWRLDVANE
EIWH
LLGSHD Kuriki & Imanaka, 1989
Pullulanase
DVVYNH GFRFDLMGI
EGWD
YVESHD Nakajima et al., 1985
CGTase
DFADNH GIRVDAVKH
EYHQ
FIDNHE Kaneko et al., 1988
ct-amylase a
DAVFNH GWRLDVANE
EIWH
LLDSHD Tonozuka et al., 1993
a: neopullulanse-type a-amyalse of T. vulgaris
52 known to have four active domains conserved among various amylases (Kim et al., 1992). The results obtained in this study suggest that the antigenic epitope determinant is located on a different portion of the protein than the conserved domains. Also, it is likely that the two amylases (BLTA and BLMA) of B. licheniformis should be easily separated from each other by immunoaffinity chromatography due to the specificity of the antibodies. AP BLTA BP DG GD IA KP BSMA BLMA CD NP OG PP TA
62 10 35 17 28 21 28 31 36 36 36 17 5 28
27 37 41 27 37 46 103 18 27 27 27 26 34 34
15 8
I105
1~7
9
:3
12
21 ' lO 53 I 27 101 151 151 15 , 19 ' 25
5
o
8
~4
20 5 3 6 12 9 9
' 72 1 261
156 48 50 60 61 60 "/6 75 6,3
g
20
t
[
i.4
~
27 2O
271
9
34 37 45
27 27 14 53 27
~'JP~ R ~
Sttarcls
9 i
BLTA BP DG GD IA KP BLMA BSMA CD NP OG PP TA
5 160 g 74 177 423 130 134 134 134 8 9 9
4
4
6
4
4
3 2 1
6 34 6
7
5 2 4
4 4 4
7 4
4
7 2
6
4
4
5
4
1 1 1 1 1 2 1
5 6 6 6 6 3 6
3 3 3 3 6 15 3
5 5
4 4
1
~0
4
9 g
1 4 4
9
4
5
5
6
4
8
2
To
G-t~minai domtms
Figure 5. Comparison of numbers of amino acid residues in loops between the 8 (~/c0 barrels in BLTA, BLMA, and other amylolytic enzymes. The presence of 8 (B/a) barrels in BLTA and BLMA was predicted by aligning the amino acid sequences to that of Taka-amylase. Numbers in the boxes represent the number of amino acid residues in loops between B-strand (El-E8) and c~-helix (HI-H8 and H) or vice versa. Abbreviations for the enzymes are : AP, c~-amylase-pullulanase from C. thermohydrosulfuricum; BA, BLTA ; BP, pullulanase from B. stearothermophilus; DG, dextran glucosidase; GD, gycogen debranching enzyme; IA, isoamylase; KP, pullulanase from K. pseudomonas; MA, BLMA; MB, maize branching enzyme; NP, neopullulanase; OG, oligo-1,6-glucosidase from B. cereus; PP, porcine pancreatic mamylase; TA, Taka amyalse A. The format of the figure was cited from Jespersen et al., 1993.
53
Mutagenesis of the BLTA and BLMA genes The c~-amyalse family are known to have catalytic domain consisted of a barrel of eight parallel ~-strands surrounded by eight helices (MacGreger, 1993; Jespersen et. al., 1993). Differences in specificities of starch metabolizing enzymes are in the numbers of subsites at active and catalytic sites. Structure of such (~/c0s-barrel was likely to be present in BLTA and BLMA based on circular dichroism a n a l y s i s , sequence comparison analysis, and prediction according to Chou and Fasman's method (Fig. 5). Enzymes specific for forming a-l,6-1inkage might resemble a-amylases at subsites of active site but differ from them in loops 4 and 5. The lysine-histidine residues of loop 4 (in conserved domain II) should be important for specificity of amylases, since they are absent from enzymes that hydrolyze or synthesize a-l,6-glucosidic bonds. Therefore, mutation in one or more residues might alter the ratio of a-l,4-bond hydrolysis to a-l,6-bond hydrolysis. Among the amylolytic enzymes, only a-amylase from Aspergillus oryzae (Taka-amylase A ; Matsuura et al., 1984) and from porcine pancreas (Buisson et al., 1987) have been investigated by X-ray crystallographic analysis. Three-dimensional structures of enzymes acting on a-l,6-glucosidic linkages are yet to be elucidated. Deletion mutation of the BLTA gene Spacing among the four conserved regions of various amylolytic enzymes is compared in Table 4. A large variation among the enzymes in the spacing between conserved regions I and II was found. Spacing between the conserved region I and II of BLTA was changed using Bal 31 exonuclease after linearizing the plasmid carrying the BLTA gene at the unique KpnI site occurring between the first and the second conserved domains (Fig. 6). The DNA fragments with various lengths of deletion Table 4 Comparison of the spacing between the conserved domains Domain N-terminal 1st-2nd-3rd-- 4th/C-terminal Enzyme'~'-----_~ BLTA 101 127 33 63 159 BSA a
101
129
34
62
189
BLMA
244
78
33
62
168
Neopullulanase
242
82
33
62
169
Pullulanase GG producing amylase G4 producing amyalse
281
67
33
83
194
107
133
92
112
80
95
a: c~-amylase of Bacillus stearothermophilus (Ihara et al., 1985)
54 were ligated and transformed into E. coli. Mutants were tested for their starch hydrolyzing activity on a starch agar plate by staining with iodine solution. Four of them showed less activity than wild type BLTA. These m u t a n t s were less stable at 75~ and had narrower pH range for stability than wild type BLTA. Also, the sizes of starch hydrolysis products were changed. The mutants produced glucose and maltose from starch, while wild type BLTA produced oligosaccharides of G l-G6. This draws a hypothesis : the size of the reaction product might be closely related to the spacing between the first and second conserved regions. The spacings between the conserved domains could reflect the chemical nature of the reaction and product specificity of an enzyme.
pUCNF18 RI
Kpnl
r--''
Hd RIm i Hd
i
l
"
Hindlll deletion
pUCNF18 A Hd RI
Kpnl
Hd
Ba131 deletion Hd
! pUCNF~I
n
i ~ pUCNF~.2 "
pUCNF~3
Figure 6. Construction of BLTA deletion mutants using Bal31 exonuclease. The 3.1kb EcoRi fragment of pTA322 harboring the BLTA gene was subcloned into pUC18 (pUNCF18). The KpnI site on the polylinker was removed by simply digesting pUCNF18 with HindIII and religating it. The resulting plasmid, pUCNF18AHd, was linearized at the unique KpnI site and treated with Ba131 to create deletion between the 1st and 2nd conserved domains, the open box represents the BLTA coding region; the line, the vector; the dark boxes, four conserved domains. The restriction sites shown are : RI, EcoRi; Hd, HindIII.
55
Figure. 7. TLC analysis of starch hydrolyzed by wild type BLTA or by the m u t a n t s (A1-A4).
Site directed mutagenesis of the BLMA gene The BLMA gene was mutated by synthetic oligonucleotide mediated site directed mutagenesis (Kunkel, 1985; Vieira and Messing, 1987). The resulting mutations were confirmed by DNA sequence analysis and activities of the mutants were tested by DNS method (Kim et. al., 1992). Three of the mutants are listed in Table 5. Glu-356 in the conserved domain III of BLMA was assumed to be an active site and was replaced with Asp by site directed mutagenesis. The m u t a n t did not exhibit the activity of wild type BLMA at all. The result indicated t h a t Glu-356 may constitute the active center of the Table 5 BLMA m u t a n t s created by site-directed mutagenesis Conserved Domain I II BLMA Wild type
DAVFNH
H250Q
.....
GWRLDVANE
III
EIWH
IX"
LLDSHD
Q
E356D D420G
- -G-
- -
55 enzyme as known for other enzymes (Kuriki et al., 1991). The mutant, H250Q, in which the histidine residue at the position 250 was substituted with glutamine retained only 10% cyclodextrin hydrolyzing activity of the wild type BLMA. The mutant, D420G, in which the aspartic acid residue at the position 420 was substituted with glycine showed only 5% cyclodextrin hydrolyzing activity of the wild type BLMA. The conserved histidine residue at 250 is known to be one of the substrate binding sites. Substitution of the residue with another basic amino acid caused significant loss of enzyme activity. Neopullulanase of B. stearothermophilus (Imanaka and Kuriki, 1989) carries out c~-l,6-glucosidic linkage hydrolysis and c~-l,4-transglycosylation reactions as well as the a-l,4-glucosidic linkage hydrolysis and a-l,6-transglycosylation reactions. BLMA shares significant homology (over 80%) with neoplullanase at the amino acid sequence level, especially around the four conserved regions. All conserved residues except one amino acid, aspartic acid at 420 (glycine in neoplullanase), are identical in these enzymes (Kuriki and Imanaka, 1989). The aspartic acid was substituted with glycine to test how the BLMA activity would be changed. In addition to decrease in activity, change of substrate specificity was observed in these mutants. The wild type BLMA hydrolyzed cyclodextrin best, then pullulan, and then soluble starch. The relative activity of the wild type BLMA on these substrate was 8 : 1 : 0.4. The ratio was changed to 10 : 1 : 1.4 and 27 : 1 : 1.1 by H250Q and D420G, respectively. The two m u t a n t s did not lose the transglycosylation activity of BLMA. More BOS was produced by the mutants (Table 6) when equal amount of pullulan hydrolyzing activity of the wild type BLMA or the m u t a n t s was added to 15% (w/v) liquefied starch solution. Especially, increase of branched DP4 molecules in the product was significant. This could be due to less efficient hydrolysis of oligosaccharides by the m u t a n t BLMA enzymes. More mutants have been created and they are under investigation in the laboratory.
Table 6 Production of BOS by the BLMA mutants Reaction 12 hours a time Enzyme BOS (%)
BDP4 (%)
36 hours a BOS (%)
BDP4 (%)
Wild Type
55.6
19.2
40.4
8.7
H250Q
54.1
19.2
56.2
17.3
D420G
56.9
25.3
58.8
25.9
a : the reaction was carried out using 15% (w/v) liquefied starch solution
57
Conclusions Four genes encoding various amylolytic enzymes were cloned from two strains. Two of them, the BLTA and BLMA genes were characterized in detail and their gene products were applied to the production of BOS. Immobilization of BLMA enabled continuous production of BOS at higher temperature than the process using free BLMA. Fermentation of the BOS mixture using immobilized yeast was useful to remove glucose and maltose, and the resulting high BOS mixture contained 91.5% BOS. DNA sequencing analysis of the BLTA and the BLMA genes suggested that the enzymes are likely to have the (~/~)s structure common to many amylolytic enzymes. Deletion mutation of the BLTA and BLMA enzymes indicated that the spacing between conserved domain I and II is important in determining the enzyme activity and the reaction product. Histidine at 250 and aspartic acid at 420 of BLMA were important for the hydrolysis activity of the enzyme. However, the transferase activity of the enzyme was not changed by the mutation introduced to the residues. The m u t a t e d enzymes showed preference to starch over pullulan as substrate. This would be of benefit for controlling the production of oligosaccharides.
Bacillus
References Bender, H., and K. Wallenfels. 1961. Untersuchungen an pullulan. II. Spezifisher abbau durch ein basterielles enzyme. Biochem. Z. 334 : 1913-1920 Buisson, G., E. Du~e, R. Haser, and F. Payan. 1987. Three dimensional structure of porcine pancreatic a-amylase at 2.9/k resolution, role of calcium in structure and activity. EMBO J. 6:3909-3916. David, M. H., H. Gunter, and H. Roper. 1987. 39:436-440
Catalytic properties of
Bacillus megaterium amylase. Starch
Glor, E. B., C. H. Miller, and D. F. Spandan. 1988. Degradation of starch and hydrolytic products by oral bacteria. J. Dent. Res. 67 : 75-81. Ihara, H., T. Sasaki, A. Tsuboi, H. Yamakata, N. Tsukagoshi, and S. Udaka. 1985. Complete nucleotide sequence of a thermophilic a-amyalse gene : homology between procaryotic and eukaryotic a-amyalses at the active site. J. Biochem. 9 8 : 9 5 - 1 0 3 . Imanaka,
T.
and
T.
Kuriki.
1989.
Pattern of action of Bacillus J. Bacteriol. 171 : 369-374.
stearothermophilus neopullulanase on pullulan.
Jang, S. Y., T. K. Cheong, W. Shim. J. W. Kim, and K. H. Park. 1994. Purification of Bacillus licheniformis thermostable c~-amylase by immunoaffinity chromatography. Korean Biochem. J. 27 : 38-41.
58 Jespersen, H. M., E. A. MacGregor, B. Henrissat, M. R. Sierks, and B. Svensson. 1993. Starch- and glycogen-debranching and branching enzymes : prediction of structural features of the catalytic (B/c0s barrel domain and evolutionary relationship to other amylolytic enzymes. J. Prot. Chem. 12 : 791-805 Kainuma, K. 1988. a-Amylases which produce specific oligosaccharides. In "Handbook of Amylases and Related Enzymes: Their Sources, Isolation Methods, Properties and Applications," The Amylase Research Society of Japan, Osaka, Japan (Ed.). pp 50-62, Pergamon Press, New York Kaneko, T., T. Hamamoto, and K. Horikoshi. 1988. Molecular cloning and nucleotide sequence of the cyclomaltodextrin glucanotransferase gene from the alkalophilic Bacillus sp. strain No. 38-2. J. Gen. Micro. 134 : 97-103. Kaneko, T., T. Kato, N. Nakamura, and K. Horikoshi. 1987. Spectrophotometric determination of cyclization activity of beta-cyclodextrin-forming cyclomaltodextrin glucanotransferase. J. Japan. Soci. Starch. Sci. 3 4 : 45-48. Kim, I. C. 1991. Molecular cloning of thermostable a-amylase and maltogenic amylase from Bacillus licheniformis and characterization of their enzymatic properties. PhD thesis, Seoul National University Kim, I. C., J. H. Cha, J. R. Kim, S. Y. Jang, B. C. Seo, T. K. Cheong, D. S. Lee, Y. D. Choi, and K. H. Park. 1992. Catalytic properties of the cloned amylase from Bacillus licheniformis. J. Biol. Chem. 267: 22108-22114. Kim, I. C., S. H. Yoo, S. J. Lee, B. H. Oh, J. W. Kim, and K. W. Park. 1994. Synthesis of branched oligosaccharides from starch by two amylases cloned from Bacillus licheniformis. Biosci. Biotech. Biochem. 58 : 416-418 Kunkel, T. A. 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection Proc. Nat. Acad. Sci. 82:488-492 Kuriki, T., H. Takata, S. Okata, and T. Imanaka. 1991. Analysis of the active center of Bacillus stearothermophilus neopullulanase. J. Bacteriol. 173:6147-6152. Kuriki, T., T. Imanaka. 1989. Nucleotide sequence of the neopullulanase gene from Bacillus stearothermophilus. J. Gen. Microbiol. 135 : 1521-1528. Kweon, M. R., C. S. Park, J. H. Auh, B. M. Cho, N. S. Yang, and K. H. Park. 1994. Phospholipid hydrolysate and antistaling amylase effects on retrogradation of starch in bread. J. Food. Sci. 59 : 3953-3958 MacGreger, E. A. 1993. Relationships between structure and activity in the a-amylase family of starch-metabolizing enzymes, Starch 45: 232-237.
59 Matssura, Y., M. Kusunoki, W. Harada, and M. Kakudo. 1984. Structure and possible catalytic residues of Taka-amylase. J. Biochem., 95 : 697-702. Matsumoto, A., and K. Matsuda. 1983. Role of branching enzyme in glycogen biosynthesis in Neurospora crassa. J. J a p a n Soc. Starch Sci., 30 : 212-222. Nakajima, R., T. Imanaka, and S. Aiba. 1985. Nucleotide sequence of the Bacillus stearothermophilus ~-amylase gene. J. Bacteriol., 163 : 401-406. Nakamura, N. and K. Horikoshi. 1976. Purification and properties of neutral cyclodextrin glucanotransferase of an alkalophilic Bacillus sp. Agri. Biol. Chem. 40 : 1785-1790. Osaki, S., Z. Yoshino, Y. Tsujisaka, H. Takaku. 1988. Manufacture of Oligosaccharides. In "Handbook of Amylases and Related Enzymes: Their Sources, Isolation Methods, Properties and Applications," The Amylase Research Society of Japan, Osaka, Japan (Ed.). pp 210-217, Pergamon Press, New York Park, C. S., K. H. Park and S. H. Kim. 1989. A rapid screening method for alkalophilic ~-cyclodextrin glucanotransferase using phenolphthalein methylorange containing solid medium. Agri. Biol. Chem. 53: 1167-1169. Park, J. H., J. Y. Yoo, O. H. Shin, H. K. Shin, S. J. Lee, and K. W. Park. 1992. Growth effect of branched oligosaccharides on principal intestinal bacteria. Kor. J. Appl. Microbiol. Biotechnol. 20 : 237-242. Pazur, J. H., and T. Ando. 1960. The hydrolysis oligosaccharide with a-D-(1,4) and a-D-(1,6) bonds amyloglucosidase J. Biol. Chem. 235 : 297-302.
of by
glycosyl fungal
Robyt, J. R. and R. J. Ackerman. 1971. Isolation, purification, and characterization of a maltotetraose-producing amylase from Pseudomonas stutzeri. Arch. Biochem. Biophys. Acta. 145 : 105-114. Ryoichi, N., T. Imanaka, and S. Aiba. 1986. Comparison of amino acid sequence of eleven different a-amylase. Appl. Microbiol. Biotechnol. 23 : 355-360 Sakano, Y., N. Masuda, and T. Kobayashi. 1971. Hydrolysis of pullulan by a novel enzyme from Aspergillus niger. Agric. Biol. Chem. 35:971-973 Sanger, F., S. Nicklen and A. R. Coulson. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74:5463-5467 Shim, W. 1994. Purification of Bacillus licheniformis maltogenic amylase by Immunoaffinity chromatography, MS thesis, Seoul National University.
60 Takaku, H. 1988. in "Handbook of Amylases and Related Enzymes: Their Sources, Isolation Methods, Properties and Applications," The Amylase Research Society of Japan, Osaka, Japan (Ed.), Pergamon Press, New York, 1988, pp.215-217. Takasaki, Y. 1985. An amylase producing maltotetraose and maltopentaose from Bacillus circulans. Agric. Biol. Chem. 47 : 2193-2199. Tonozuka, T., M Ohtsuka, S, Mogi, H, Sakai, T. Ohta, and Y. Sakano. 1993. A neopullulanase-type ~-amylase gene from Thermoactinomyces vulgaris R-47. Biosci. Biotech. Biochem. 57 : 395-401 Vieira, J. and J. Messing. 1987. Production of single-stranded plasmid DNA. Methods in Enzymology. 153 : 3-11. Yuki, T., Nomura, T., Tezuka, H., Tsuboi, A., Yamagata, H., Tsukagoshi, N., and Udaka, S. 1985. Complete nucleotide sequence of a gene coding for heat- and pH-stable a-amylase of Bacillus licheniformis : comparison of the amino acid sequences of three bacterial liquefying a-amylase deduced from the DNA sequences. J. Biochem. 98:1147-1156.
43
Modulation of Bacillus amylolytic enzymes and production of branched oligosaccharides Tae-Kyu Cheong, Tae-Jip Kim, Myo-Jeong Kim, Yang-Do Choi ~ In-Cheol Kim 2, Jung-Wan Kim3,and Kwan-Hwa Park. Department of Food Science and Technology, ~Department of Agricultural Chemistry and Research Center for New Bio-Materials in Agriculture, Seoul National University, Suwon, 441-744, Korea. 2Samyang Genex Research Institute, Taejeon, 305-348, Korea. 3Department of Biology, University of Inchon, Inchon, 402-749, Korea. Introduction
Many interesting and useful findings of new type amylases have been reported. They catalyze hydrolysis of a-l,4-glucosidic linkages not only in starch but also in other carbohydrates such as pullulan and cyclodextrin (Kim et al., 1992 ; Bender and Wallenfels, 1961; Sakano et al., 1971; Nakamura and Horikoshi, 1976). Debranching enzymes which hydrolyze a-l,6-glucosidic linkage were found in higher plants or microorganisms (David et al., 1987; Pazur and Ando, 1960). Some amylolytic enzymes exhibit glucose transferring activity as well as hydrolyzing activity (Kim et al., 1992 ; David et al., 1987; Matsumoto and Matsuda, 1983; Kaneko et al., 1987; Imanaka and Kuriki, 1989). Several microbial amylases which produce oligosaccharides with specific lengths or structures have been reported by numerous researchers (Robyt and Ackerman, 1971; Takasaki, 1985; Kainuma, 1988). Design of specific oligosaccharide synthesis became possible due to the discovery and modification of new amylases. Maltooligosaccharides mixture, maltotetraose syrup, and branched oligosaccharide mixture are used as substitutes for sucrose and other saccharides in the food industry due to their lower viscosity, less sweet taste, and smaller freezing point depression (Osaki et al., 1988). They also can be used to prevent crystallization of sucrose in foods and are useful in controlling microbial contamination as well as retrogradation of starchy foods because they have low water activity and high moisture-retaining capacity (Kweon et al., 1994; Komoto et al., 1993). Branched oligosaccharides are of benefit in in preventing dental caries (Glor et al., 1988) and effective for the growth of Bifidobacteria (Park et al., 1992) in human intestinal tract. They are hardly digested by human body. Therefore, the low calorie content of branched oligosaccharides has a great appeal to low calorie dieters. Due to these useful properties, the production of BOS mixtures has been increased by 50-100% per year during last few years. Generally, branched oligosaccharides are manufactured by a 2-step procedure that uses a-amylase, [~-amylase, and transglycosidase on starch solution (Takaku, 1988). The starch slurry is liquefied to a degree of
44
0
0
0
t~ 0 t~
c~
~r~
0 0
0 0 t~
0
0
~ ~o.~
~
,
~
~
0
0
'
o~
~
o b~
o
-
o.o
0~
o
~0
0 0 0
'
0 0 0
,
0
~
+
o ~
r~D
0 0 0
+
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~D
"~
o
t~O
0 0 0
c~ o
O o O
0 0 0
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o
~
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t~O
o 00
0 0 0
o
o
~0
o.
0 0 0
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"--'
0 0 0
i
~
~
r~O
E-,
45 hydrolysis (DE) 6-10 by the action of a thermostable c~-amylase, then saccharified by t r e a t m e n t with soybean ~-amylase and transglycosidase of AspergiUus niger at 60~ for 72 hours. It is a time-consuming procedure. A series of amylolytic enzymes including a thermostable a-amylase (BLTA), a maltogenic amylase (BLMA), a CDase, and a CGTase were isolated from two Bacillus species,B, licheniformis and alkalophilic Bacills I-5 strain, in our laboratory. The genes responsible for them were cloned in E. coli and the physicochemical properties and the action patterns of these amylases on various carbohydrates were characterized. Recently, we developed a one-step procedure which simplified and shortened the reaction for the production of branched oligosaccharides. It applies BLTA and BLMA to starch solution simultaneously (Kim et al., 1994). The two amylases were prepared from an E. coli transformant harboring a recombinant plasmid which carried both of genes for the enzymes. The reaction was carried out for 24 hours and 45% of the product was branched oligosaccharides of various lengths. The amount of branched oligosaccharides in the mixture was comparable to that of the Alo (anomalously linked oligosaccharide) mixture in market (Takaku, 1988). However, it contained much less glucose than the Alo mixture (27% vs. 40%). Development of a continuous process of branched oligosaccharides production using immobilized BLMA and yeast cells would also improve the efficiency of the whole process and the quality of the product. Modification of the enzymes by recombinant DNA techniques would make application of the amylases to food industry more diverse and effective. Improving thermostability and inducing secretion of BLMA would be of significant benefit in applying the enzyme to the production of branched oligosaccharides. The predicted amino acid sequences indicated that they share homology with various amylolytic enzymes at four conserved active domains (Ihara et al., 1985; Ryoichi et al., 1986). Based on amino acid sequence comparison to that of Taka amylase, whose tertiary structure is known (Matsuura et al., 1984), BLTA and BLMA are likely to have the structure of 8 (~/a) barrels with intervening loops (MacGreger, 1993; Jespersen et al., 1993). Mutagenesis of the genes responsible for the enzymes is under investigation as an effort to improve the process for the production of oligosaccharides. Results and Discussion
Catalytic properties of starch degrading enzymes Physicochemical properties of the starch hydrolyzing enzymes isolated in the laboratory and those reported by others are summarized in Table 1. Substrate specificity of BLMA was compared with that of BLTA. Soluble starch, pullulan, and cyclodextrin were hydrolyzed by BLMA, while BLTA hydrolyzed soluble starch only. Optimum temperature of BLTA was 70~ and it was increased to 90~ in the presence of 5raM Ca +* (Kim, 1991). Optimum temperature of BLMA was 50~ (Kim et al., 1992). The activity of BLMA was inhibited by most of divalent cations such as Ca ++, Zn §247and Mn §247 CGTase produced a-, B-, ~-cyclodextrins at the ratio of 0.7 : 3.3 : 1.0
46 from starch in the mother strain. The ratio among various CD products was changed when the enzyme prepared from recombinant DNA in E. coli (see below) was used. CDase hydrolyzed cyclodextrin very rapidly into maltose. BLMA and CDase exhibited transferring activity in addition to hydrolyzing activity in the presence of excess glucose. A BOS mixture is prepared from liquefied starch using the hydrolyzing and the transferring activities of BLMA (Kim et al., 1994). The BOS mixture was analyzed by various methods including high performance liquid chromatography (HPLC) and found to contain various small branched sugars such as isomaltose, panose, and isopanose etc. Based on the results, a model of BLMA action mode was proposed as shown in Fig. 1. BLMA is likely to synthesize branched oligosaccharides in a complicated m a n n e r : it hydrolyzes liquefied starch further to glucose and maltose, and at the same time transfers the resulting molecules onto cleavage sites of other sugar moieties by forming c~-l,6-1inkages. The whole reaction is completed by repeating the coupled hydrolysis and transfer reactions, thereby creating a new population of branched oligosaccharide molecules.
Pullulan
4, Starch
s ~
/ t ~' "" " ~ l
%
i
Figure 1. A proposed model of BLMA action mode involved in the production of branched oligosaccharides from pulllulan or maltooligosaccharide. BLMA hydrolyze pullulan to panose and the resulting molecule is transferred to acceptor (in this case glucose) by forming a-l,6-glycosidic linkage. Maltooligosaccharide is hydrolyzed mainly to maltose, but also to glucose or maltotriose etc. They are then transferred to acceptor molecules such as glucose, maltose, or maltotriose. Resulting branched molecules are likely to be hydrolyzed further by BLMA.
47
Continuous production of the BOS mixture The procedure for continuous production of the BOS mixture and a highly concentrated BOS (high BOS) mixture is illustrated in Fig. 2. BLMA has an optimal temperature of 50~ and its thermostability could be enhanced by immobilization on CPC Silica. Decimal reduction time of immobilized BLMA at 55~ was 96.8 min, while that of free BLMA at the same temperature was only 20.5 min. Thirty percents (w/v) corn starch suspension in 50mM maleate-NaOH buffer (pH 6.8) was liquefied by thermostable c~-amylase (Termamyl ; Novo Nordisk, Denmark). The sample was heated in a boiling water bath for 10 min and the reaction was stopped by autoclaving the hydrolysate for 10 min when the DE value was about 20-25. Five millimoles of EDTA was added to the liquefied solution and its final pH was adjusted to 6.8 using 1N NaOH. For the synthesis of the BOS mixture, the liquefied starch was run through a column of immobilized BLMA at 45-50~ Residence time of the
Starch slurry
l
30% Soluble starch
Liquefaction
~-
alpha-Amylase 100~ 10min
Saccharification
--
45~
}_
~-
BLMA
15hrs
Filtration
BOS
i
Yeast fermentation ~-
I_.
Immobilized Yeast
27~ 2days Freeze drying
High B O S
Figure 2. mixtures.
Procedure
for
the
production
of the
BOS
and
high
BOS
48 liquefied starch solution in the column was 2.5 hours. It took 24 hours at 40~ to prepare a BOS mixture with similar composition when free BLMA was used. The reaction was stopped by boiling, filtered through a W h a t m a n paper No. 5, and then dried using a freeze-drier. The BOS mixture was analyzed by HPLC (Fig. 3) and its composition is shown in Table 2. It contained BOS over 60% including panose, branched DP4, and branched DP5 etc. The shoulder of the maltose peak indicated that isomaltose might be present in the mixture. Further analysis of the mixture using ion-chromatography showed that isomaltose and isopanose were also present in the mixture (data will be discussed elsewhere). The BOS mixture contained about 30% glucose and maltose. In order to get rid of sweet taste of the mixture, it was fermented by yeast. As the result of glucose and maltose fermentation by yeast, BOS would be concentrated to make a high BOS mixture. For the preparation of a high BOS mixture, the BOS mixture (30%; w/v) prepared as described above was fermented by Saccharomyces cerevisiae var. ellipsoideus that was immobilized on sodium-alginate matrix. Fermentation was carried out at 27-28~ for 2 days to remove glucose and maltose contained in the BOS mixture. Upon the completion of fermentation, the mixture was filtered and dried as described above. As the result of fermentation, almost all of glucose and maltose was removed and the BOS content increased to over 90% (Table 2).
o-o +
0
aD --"
N
Retention Time (min)
A
Retention Time (min)
B
Figure 3. High performance liquid chromatography analysis of the BOS (A) and high BOS (B) mixtures. Saccharide which is likely to constitute each peak is shown above the peaks. HPLC analysis was carried out as described previously (Kim et al., 1992).
49 Table 2 The compositions of the BOS and the high BOS mixtures Saccharide
BOS mixture
High BOS mixture
Glucose
10.2 %
1.3 %
Maltose
18.9 %a
_
Isomaltose
-
2.7 %
Maltotriose
6.8 %
7.2 %
Panose b
15.4 %
20.4 %
Branched tetraose
30.4 %
43.8 %
Branched pentaose
18.3 %
24.6 %
> Branched pentaose
trace
trace
Total a m o u n t of BOS
64 %
91.5 %
a : m a y contain isomaltose b : m a y contain isopanose
Application of the BOS mixture to bread as h u m e c t a n t for starchy foods was a t t e m p t e d . It retarded starch retrogradation and lowered w a t e r activity (manuscript submitted). The high BOS mixture would be of great benefit to apply it as h u m e c t a n t to food.
Comparison of amino acid sequences of various amylases The
genes
responsible
for
BLTA
or
BLMA
were
isolated
from
B.
licheniformis by shotgun cloning EcoRI or BamHI/EcoRI genomic DNA digests into an E. coli vector, pBR322. A CDase gene was isolated from an alkalophilic Bacillus strain I-5 using the same method. The positive clones were screened for their starch hydrolyzing phenotype in E. coli HB101 using iodine test. The starch hydrolyzing activity of BLMA or CDase was observed only when cell m e m b r a n e of the t r a n s f o r m a n t carrying one of the genes was disrupted by D-cycloserine. This indicated the two enzymes were cytoplasmic proteins. In order to isolate a CGTase gene from Bacillus I-5 strain, two oligonucleotides were synthesized based on homology among CGTase genes. An 1.1kb DNA fragment was amplified from I-5 genomic DNA and used as a probe to screen a genomic DNA library constructed using EMBL3 k phage DNA (Amersham, U.S.A.). The insert of a plaque with positive signal was subcloned into pUC18. The CGTase positive phenotype of the clone was confirmed by a phenolphthalein tested (Park et al., 1989) and HPLC analysis of the product produced by the enzyme p r e p a r e d from the clone. The restriction m a p s of pIJ322, pTA322, pCGTJ322, and pTJ3, which
50 contain the BLMA, BLTA, CGTase, and CDase gene, respectively, are shown in Fig. 4. Each gene was expressed from its own promoter and stably m a i n t a i n e d in E. coli. The gene products were characterized in detail and three of them, BLMA, BLTA, and CGTase, had properties t h a t were in good correlation with those found in the mother strains. The activity of CDase has not been detected in the mother strain, alkalophilic Bacillus I-5. To analyze the structure of the BLTA and BLMA genes, nucleotide sequencing was carried out. Nucleotide sequences of the two B. licheniformis amylases were determined by sequencing the plasmids
EcoRI
EcoRI
Pvull
Hindlll SlII
/ X
c.,
cloning
m"'
Pstl
Pvull
~
J J
Hlndlll Pvull
EcoRI
EcoRI
hmHI
BIImHI
EcoRI
Sail
oa,
BamHI
pBR322
BImHI
Smal
Hindlll
EcoRI
Figure 4. Restriction maps of pTA322, pIJ322, pTJ3, and pCGTJ322. pTA322 carries the BLTA gene on a 3.1kb insert; pIJ322, the BLMA gene on a 3.5kb insert; pTJ322, a CDase gene on a 3.2kb insert; pCGTJ322, a CGTase gene on a 4.8 kb insert. All of them are cloned on an E. coli vector, pBR322, and selected by resistance to ampicillin in E. coli.
51 carrying the genes according to Sanger's chain t e r m i n a t i o n method (Sanger et al., 1977) and using Sequenase kit purchased from U.S.B. Corp. The DNA f r a g m e n t s were sequenced in both strands. The BLTA gene coded for a 55 kD protein of 483 amino acids. The m a t u r e BLTA protein was proceeded by 29 amino acids which are likely to function as a signal sequence for secretion (Kim, 1991; Yuuki et al., 1986). The protein was localized in both periplasmic and cytoplasmic spaces. The BLMA gene was capable of encoding 584 amino acids from a promoter located 5' flanking region and the molecular weight of the gene product was predicted to be 66.5 kD. There was no signal sequence like sequence found at the amino t e r m i n a l of the gene. The protein was found only in cytoplasm of E. coli and the mother strain. The deduced amino acid sequences were aligned with those of various a m y l a s e s reported previously (Jespersen et al., 1993) and four highly conserved regions were found in Table 3. Conserved regions III and IV are considered to include active sites of amylases. The specificity of the anti-BLTA antibody was tested using Ouchterlony double immunodiffusion analysis (Jang et al., 1994). The antibody formed a precipitate with Taka-amylase, which is a thermostable amylase. However, neither pullulanase nor glucoamylase cross-reacted with the antibody. Termamyl (Novo Inc., Denmark) formed a precipitate with the antibody (data not shown). In a recent study, the antibody raised in the laboratory against another amylase of B. licheniformis, BLMA, did not cross-reacted with BLTA (Shim, 1994). Pullulanase, Termamyl, and glucoamylase are also Table 3 Comparison of amino acid sequences in the conserved domains Domains
I
II
III
IV
Reference
Enzymes
DAVINH GFRLDAAKH
EVIH
FVDNHD
BLTA
DVVINH GFRLDAVKH
EYWQ
FVDNHD Kim, 1991
BLMA
DAVFNH GWRLDVANE
EIWH
LLDSHD Kim et al., 1991
Neopullulanase
DAVFNH GWRLDVANE
EIWH
LLGSHD Kuriki & Imanaka, 1989
Pullulanase
DVVYNH GFRFDLMGI
EGWD
YVESHD Nakajima et al., 1985
CGTase
DFADNH GIRVDAVKH
EYHQ
FIDNHE Kaneko et al., 1988
ct-amylase a
DAVFNH GWRLDVANE
EIWH
LLDSHD Tonozuka et al., 1993
a: neopullulanse-type a-amyalse of T. vulgaris
52 known to have four active domains conserved among various amylases (Kim et al., 1992). The results obtained in this study suggest that the antigenic epitope determinant is located on a different portion of the protein than the conserved domains. Also, it is likely that the two amylases (BLTA and BLMA) of B. licheniformis should be easily separated from each other by immunoaffinity chromatography due to the specificity of the antibodies. AP BLTA BP DG GD IA KP BSMA BLMA CD NP OG PP TA
62 10 35 17 28 21 28 31 36 36 36 17 5 28
27 37 41 27 37 46 103 18 27 27 27 26 34 34
15 8
I105
1~7
9
:3
12
21 ' lO 53 I 27 101 151 151 15 , 19 ' 25
5
o
8
~4
20 5 3 6 12 9 9
' 72 1 261
156 48 50 60 61 60 "/6 75 6,3
g
20
t
[
i.4
~
27 2O
271
9
34 37 45
27 27 14 53 27
~'JP~ R ~
Sttarcls
9 i
BLTA BP DG GD IA KP BLMA BSMA CD NP OG PP TA
5 160 g 74 177 423 130 134 134 134 8 9 9
4
4
6
4
4
3 2 1
6 34 6
7
5 2 4
4 4 4
7 4
4
7 2
6
4
4
5
4
1 1 1 1 1 2 1
5 6 6 6 6 3 6
3 3 3 3 6 15 3
5 5
4 4
1
~0
4
9 g
1 4 4
9
4
5
5
6
4
8
2
To
G-t~minai domtms
Figure 5. Comparison of numbers of amino acid residues in loops between the 8 (~/c0 barrels in BLTA, BLMA, and other amylolytic enzymes. The presence of 8 (B/a) barrels in BLTA and BLMA was predicted by aligning the amino acid sequences to that of Taka-amylase. Numbers in the boxes represent the number of amino acid residues in loops between B-strand (El-E8) and c~-helix (HI-H8 and H) or vice versa. Abbreviations for the enzymes are : AP, c~-amylase-pullulanase from C. thermohydrosulfuricum; BA, BLTA ; BP, pullulanase from B. stearothermophilus; DG, dextran glucosidase; GD, gycogen debranching enzyme; IA, isoamylase; KP, pullulanase from K. pseudomonas; MA, BLMA; MB, maize branching enzyme; NP, neopullulanase; OG, oligo-1,6-glucosidase from B. cereus; PP, porcine pancreatic mamylase; TA, Taka amyalse A. The format of the figure was cited from Jespersen et al., 1993.
53
Mutagenesis of the BLTA and BLMA genes The c~-amyalse family are known to have catalytic domain consisted of a barrel of eight parallel ~-strands surrounded by eight helices (MacGreger, 1993; Jespersen et. al., 1993). Differences in specificities of starch metabolizing enzymes are in the numbers of subsites at active and catalytic sites. Structure of such (~/c0s-barrel was likely to be present in BLTA and BLMA based on circular dichroism a n a l y s i s , sequence comparison analysis, and prediction according to Chou and Fasman's method (Fig. 5). Enzymes specific for forming a-l,6-1inkage might resemble a-amylases at subsites of active site but differ from them in loops 4 and 5. The lysine-histidine residues of loop 4 (in conserved domain II) should be important for specificity of amylases, since they are absent from enzymes that hydrolyze or synthesize a-l,6-glucosidic bonds. Therefore, mutation in one or more residues might alter the ratio of a-l,4-bond hydrolysis to a-l,6-bond hydrolysis. Among the amylolytic enzymes, only a-amylase from Aspergillus oryzae (Taka-amylase A ; Matsuura et al., 1984) and from porcine pancreas (Buisson et al., 1987) have been investigated by X-ray crystallographic analysis. Three-dimensional structures of enzymes acting on a-l,6-glucosidic linkages are yet to be elucidated. Deletion mutation of the BLTA gene Spacing among the four conserved regions of various amylolytic enzymes is compared in Table 4. A large variation among the enzymes in the spacing between conserved regions I and II was found. Spacing between the conserved region I and II of BLTA was changed using Bal 31 exonuclease after linearizing the plasmid carrying the BLTA gene at the unique KpnI site occurring between the first and the second conserved domains (Fig. 6). The DNA fragments with various lengths of deletion Table 4 Comparison of the spacing between the conserved domains Domain N-terminal 1st-2nd-3rd-- 4th/C-terminal Enzyme'~'-----_~ BLTA 101 127 33 63 159 BSA a
101
129
34
62
189
BLMA
244
78
33
62
168
Neopullulanase
242
82
33
62
169
Pullulanase GG producing amylase G4 producing amyalse
281
67
33
83
194
107
133
92
112
80
95
a: c~-amylase of Bacillus stearothermophilus (Ihara et al., 1985)
54 were ligated and transformed into E. coli. Mutants were tested for their starch hydrolyzing activity on a starch agar plate by staining with iodine solution. Four of them showed less activity than wild type BLTA. These m u t a n t s were less stable at 75~ and had narrower pH range for stability than wild type BLTA. Also, the sizes of starch hydrolysis products were changed. The mutants produced glucose and maltose from starch, while wild type BLTA produced oligosaccharides of G l-G6. This draws a hypothesis : the size of the reaction product might be closely related to the spacing between the first and second conserved regions. The spacings between the conserved domains could reflect the chemical nature of the reaction and product specificity of an enzyme.
pUCNF18 RI
Kpnl
r--''
Hd RIm i Hd
i
l
"
Hindlll deletion
pUCNF18 A Hd RI
Kpnl
Hd
Ba131 deletion Hd
! pUCNF~I
n
i ~ pUCNF~.2 "
pUCNF~3
Figure 6. Construction of BLTA deletion mutants using Bal31 exonuclease. The 3.1kb EcoRi fragment of pTA322 harboring the BLTA gene was subcloned into pUC18 (pUNCF18). The KpnI site on the polylinker was removed by simply digesting pUCNF18 with HindIII and religating it. The resulting plasmid, pUCNF18AHd, was linearized at the unique KpnI site and treated with Ba131 to create deletion between the 1st and 2nd conserved domains, the open box represents the BLTA coding region; the line, the vector; the dark boxes, four conserved domains. The restriction sites shown are : RI, EcoRi; Hd, HindIII.
55
Figure. 7. TLC analysis of starch hydrolyzed by wild type BLTA or by the m u t a n t s (A1-A4).
Site directed mutagenesis of the BLMA gene The BLMA gene was mutated by synthetic oligonucleotide mediated site directed mutagenesis (Kunkel, 1985; Vieira and Messing, 1987). The resulting mutations were confirmed by DNA sequence analysis and activities of the mutants were tested by DNS method (Kim et. al., 1992). Three of the mutants are listed in Table 5. Glu-356 in the conserved domain III of BLMA was assumed to be an active site and was replaced with Asp by site directed mutagenesis. The m u t a n t did not exhibit the activity of wild type BLMA at all. The result indicated t h a t Glu-356 may constitute the active center of the Table 5 BLMA m u t a n t s created by site-directed mutagenesis Conserved Domain I II BLMA Wild type
DAVFNH
H250Q
.....
GWRLDVANE
III
EIWH
IX"
LLDSHD
Q
E356D D420G
- -G-
- -
55 enzyme as known for other enzymes (Kuriki et al., 1991). The mutant, H250Q, in which the histidine residue at the position 250 was substituted with glutamine retained only 10% cyclodextrin hydrolyzing activity of the wild type BLMA. The mutant, D420G, in which the aspartic acid residue at the position 420 was substituted with glycine showed only 5% cyclodextrin hydrolyzing activity of the wild type BLMA. The conserved histidine residue at 250 is known to be one of the substrate binding sites. Substitution of the residue with another basic amino acid caused significant loss of enzyme activity. Neopullulanase of B. stearothermophilus (Imanaka and Kuriki, 1989) carries out c~-l,6-glucosidic linkage hydrolysis and c~-l,4-transglycosylation reactions as well as the a-l,4-glucosidic linkage hydrolysis and a-l,6-transglycosylation reactions. BLMA shares significant homology (over 80%) with neoplullanase at the amino acid sequence level, especially around the four conserved regions. All conserved residues except one amino acid, aspartic acid at 420 (glycine in neoplullanase), are identical in these enzymes (Kuriki and Imanaka, 1989). The aspartic acid was substituted with glycine to test how the BLMA activity would be changed. In addition to decrease in activity, change of substrate specificity was observed in these mutants. The wild type BLMA hydrolyzed cyclodextrin best, then pullulan, and then soluble starch. The relative activity of the wild type BLMA on these substrate was 8 : 1 : 0.4. The ratio was changed to 10 : 1 : 1.4 and 27 : 1 : 1.1 by H250Q and D420G, respectively. The two m u t a n t s did not lose the transglycosylation activity of BLMA. More BOS was produced by the mutants (Table 6) when equal amount of pullulan hydrolyzing activity of the wild type BLMA or the m u t a n t s was added to 15% (w/v) liquefied starch solution. Especially, increase of branched DP4 molecules in the product was significant. This could be due to less efficient hydrolysis of oligosaccharides by the m u t a n t BLMA enzymes. More mutants have been created and they are under investigation in the laboratory.
Table 6 Production of BOS by the BLMA mutants Reaction 12 hours a time Enzyme BOS (%)
BDP4 (%)
36 hours a BOS (%)
BDP4 (%)
Wild Type
55.6
19.2
40.4
8.7
H250Q
54.1
19.2
56.2
17.3
D420G
56.9
25.3
58.8
25.9
a : the reaction was carried out using 15% (w/v) liquefied starch solution
57
Conclusions Four genes encoding various amylolytic enzymes were cloned from two strains. Two of them, the BLTA and BLMA genes were characterized in detail and their gene products were applied to the production of BOS. Immobilization of BLMA enabled continuous production of BOS at higher temperature than the process using free BLMA. Fermentation of the BOS mixture using immobilized yeast was useful to remove glucose and maltose, and the resulting high BOS mixture contained 91.5% BOS. DNA sequencing analysis of the BLTA and the BLMA genes suggested that the enzymes are likely to have the (~/~)s structure common to many amylolytic enzymes. Deletion mutation of the BLTA and BLMA enzymes indicated that the spacing between conserved domain I and II is important in determining the enzyme activity and the reaction product. Histidine at 250 and aspartic acid at 420 of BLMA were important for the hydrolysis activity of the enzyme. However, the transferase activity of the enzyme was not changed by the mutation introduced to the residues. The m u t a t e d enzymes showed preference to starch over pullulan as substrate. This would be of benefit for controlling the production of oligosaccharides.
Bacillus
References Bender, H., and K. Wallenfels. 1961. Untersuchungen an pullulan. II. Spezifisher abbau durch ein basterielles enzyme. Biochem. Z. 334 : 1913-1920 Buisson, G., E. Du~e, R. Haser, and F. Payan. 1987. Three dimensional structure of porcine pancreatic a-amylase at 2.9/k resolution, role of calcium in structure and activity. EMBO J. 6:3909-3916. David, M. H., H. Gunter, and H. Roper. 1987. 39:436-440
Catalytic properties of
Bacillus megaterium amylase. Starch
Glor, E. B., C. H. Miller, and D. F. Spandan. 1988. Degradation of starch and hydrolytic products by oral bacteria. J. Dent. Res. 67 : 75-81. Ihara, H., T. Sasaki, A. Tsuboi, H. Yamakata, N. Tsukagoshi, and S. Udaka. 1985. Complete nucleotide sequence of a thermophilic a-amyalse gene : homology between procaryotic and eukaryotic a-amyalses at the active site. J. Biochem. 9 8 : 9 5 - 1 0 3 . Imanaka,
T.
and
T.
Kuriki.
1989.
Pattern of action of Bacillus J. Bacteriol. 171 : 369-374.
stearothermophilus neopullulanase on pullulan.
Jang, S. Y., T. K. Cheong, W. Shim. J. W. Kim, and K. H. Park. 1994. Purification of Bacillus licheniformis thermostable c~-amylase by immunoaffinity chromatography. Korean Biochem. J. 27 : 38-41.
58 Jespersen, H. M., E. A. MacGregor, B. Henrissat, M. R. Sierks, and B. Svensson. 1993. Starch- and glycogen-debranching and branching enzymes : prediction of structural features of the catalytic (B/c0s barrel domain and evolutionary relationship to other amylolytic enzymes. J. Prot. Chem. 12 : 791-805 Kainuma, K. 1988. a-Amylases which produce specific oligosaccharides. In "Handbook of Amylases and Related Enzymes: Their Sources, Isolation Methods, Properties and Applications," The Amylase Research Society of Japan, Osaka, Japan (Ed.). pp 50-62, Pergamon Press, New York Kaneko, T., T. Hamamoto, and K. Horikoshi. 1988. Molecular cloning and nucleotide sequence of the cyclomaltodextrin glucanotransferase gene from the alkalophilic Bacillus sp. strain No. 38-2. J. Gen. Micro. 134 : 97-103. Kaneko, T., T. Kato, N. Nakamura, and K. Horikoshi. 1987. Spectrophotometric determination of cyclization activity of beta-cyclodextrin-forming cyclomaltodextrin glucanotransferase. J. Japan. Soci. Starch. Sci. 3 4 : 45-48. Kim, I. C. 1991. Molecular cloning of thermostable a-amylase and maltogenic amylase from Bacillus licheniformis and characterization of their enzymatic properties. PhD thesis, Seoul National University Kim, I. C., J. H. Cha, J. R. Kim, S. Y. Jang, B. C. Seo, T. K. Cheong, D. S. Lee, Y. D. Choi, and K. H. Park. 1992. Catalytic properties of the cloned amylase from Bacillus licheniformis. J. Biol. Chem. 267: 22108-22114. Kim, I. C., S. H. Yoo, S. J. Lee, B. H. Oh, J. W. Kim, and K. W. Park. 1994. Synthesis of branched oligosaccharides from starch by two amylases cloned from Bacillus licheniformis. Biosci. Biotech. Biochem. 58 : 416-418 Kunkel, T. A. 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection Proc. Nat. Acad. Sci. 82:488-492 Kuriki, T., H. Takata, S. Okata, and T. Imanaka. 1991. Analysis of the active center of Bacillus stearothermophilus neopullulanase. J. Bacteriol. 173:6147-6152. Kuriki, T., T. Imanaka. 1989. Nucleotide sequence of the neopullulanase gene from Bacillus stearothermophilus. J. Gen. Microbiol. 135 : 1521-1528. Kweon, M. R., C. S. Park, J. H. Auh, B. M. Cho, N. S. Yang, and K. H. Park. 1994. Phospholipid hydrolysate and antistaling amylase effects on retrogradation of starch in bread. J. Food. Sci. 59 : 3953-3958 MacGreger, E. A. 1993. Relationships between structure and activity in the a-amylase family of starch-metabolizing enzymes, Starch 45: 232-237.
59 Matssura, Y., M. Kusunoki, W. Harada, and M. Kakudo. 1984. Structure and possible catalytic residues of Taka-amylase. J. Biochem., 95 : 697-702. Matsumoto, A., and K. Matsuda. 1983. Role of branching enzyme in glycogen biosynthesis in Neurospora crassa. J. J a p a n Soc. Starch Sci., 30 : 212-222. Nakajima, R., T. Imanaka, and S. Aiba. 1985. Nucleotide sequence of the Bacillus stearothermophilus ~-amylase gene. J. Bacteriol., 163 : 401-406. Nakamura, N. and K. Horikoshi. 1976. Purification and properties of neutral cyclodextrin glucanotransferase of an alkalophilic Bacillus sp. Agri. Biol. Chem. 40 : 1785-1790. Osaki, S., Z. Yoshino, Y. Tsujisaka, H. Takaku. 1988. Manufacture of Oligosaccharides. In "Handbook of Amylases and Related Enzymes: Their Sources, Isolation Methods, Properties and Applications," The Amylase Research Society of Japan, Osaka, Japan (Ed.). pp 210-217, Pergamon Press, New York Park, C. S., K. H. Park and S. H. Kim. 1989. A rapid screening method for alkalophilic ~-cyclodextrin glucanotransferase using phenolphthalein methylorange containing solid medium. Agri. Biol. Chem. 53: 1167-1169. Park, J. H., J. Y. Yoo, O. H. Shin, H. K. Shin, S. J. Lee, and K. W. Park. 1992. Growth effect of branched oligosaccharides on principal intestinal bacteria. Kor. J. Appl. Microbiol. Biotechnol. 20 : 237-242. Pazur, J. H., and T. Ando. 1960. The hydrolysis oligosaccharide with a-D-(1,4) and a-D-(1,6) bonds amyloglucosidase J. Biol. Chem. 235 : 297-302.
of by
glycosyl fungal
Robyt, J. R. and R. J. Ackerman. 1971. Isolation, purification, and characterization of a maltotetraose-producing amylase from Pseudomonas stutzeri. Arch. Biochem. Biophys. Acta. 145 : 105-114. Ryoichi, N., T. Imanaka, and S. Aiba. 1986. Comparison of amino acid sequence of eleven different a-amylase. Appl. Microbiol. Biotechnol. 23 : 355-360 Sakano, Y., N. Masuda, and T. Kobayashi. 1971. Hydrolysis of pullulan by a novel enzyme from Aspergillus niger. Agric. Biol. Chem. 35:971-973 Sanger, F., S. Nicklen and A. R. Coulson. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74:5463-5467 Shim, W. 1994. Purification of Bacillus licheniformis maltogenic amylase by Immunoaffinity chromatography, MS thesis, Seoul National University.
60 Takaku, H. 1988. in "Handbook of Amylases and Related Enzymes: Their Sources, Isolation Methods, Properties and Applications," The Amylase Research Society of Japan, Osaka, Japan (Ed.), Pergamon Press, New York, 1988, pp.215-217. Takasaki, Y. 1985. An amylase producing maltotetraose and maltopentaose from Bacillus circulans. Agric. Biol. Chem. 47 : 2193-2199. Tonozuka, T., M Ohtsuka, S, Mogi, H, Sakai, T. Ohta, and Y. Sakano. 1993. A neopullulanase-type ~-amylase gene from Thermoactinomyces vulgaris R-47. Biosci. Biotech. Biochem. 57 : 395-401 Vieira, J. and J. Messing. 1987. Production of single-stranded plasmid DNA. Methods in Enzymology. 153 : 3-11. Yuki, T., Nomura, T., Tezuka, H., Tsuboi, A., Yamagata, H., Tsukagoshi, N., and Udaka, S. 1985. Complete nucleotide sequence of a gene coding for heat- and pH-stable a-amylase of Bacillus licheniformis : comparison of the amino acid sequences of three bacterial liquefying a-amylase deduced from the DNA sequences. J. Biochem. 98:1147-1156.