Note: Descriptions are shown in the official language in which they were submitted.
CA 02199904 1997-03-18
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to cyclodextrin
glycosyl transferases (CGTases) EC 2.4.1.19 for producing
~-~yclodextrin, to processes for preparing ~-cyclodextrin
gl~cosyl transferases, and to their use.
2. The Prior Art
As a rule, cyclodextrins are prepared from starch or
starch-like substrates. In these preparations, CGTases are used
to convert starch enzymically into cyclodextrin (CD). For
thermodynamic reasons, the starch is mainly converted into ~-CD,
independently of the CGTase used for the reaction, if the
reaction is carried out until the thermodynamic equilibrium is
reached (maximum CD yield). However, in the initial phase, at
the beginning of the starch conversion reaction, the enzymes
which are used for the conversion differ in the composition of
the primary product mixture. ~ or ~-CGTases are
di~ferentiated depending upon the product, ~ -, or ~-CD, which
is chiefly formed by the enzyme in this initial phase.
CA 02199904 1997-03-18
These enzymes, which are suitable, and have also
already been used, for the industrial production of CD, have
hi-therto only been detected in bacteria. ~-CGTases have hitherto
only been identified in Bacillus macerans, Bacillus
stearothermophilus and ~lebsiella oxytoca. ~-CGTases have been
detected, for example, in ~acillus circulans, Bacillus
me~aterium, Bacillus ohbensis, Micrococcus sp. and alkalophilic
Bacillae which have not been precisely classified taxonomically,
such as Bacillus sp. 38-2, 17-1, 1011 or 1-1. Naturally
occurring enzymes having an initially high
~-CD-forming activity have been reported in Bacillus subtilis
313, Bacillus sp. A1-6 and Bacillus sp. 290-3.
Since the CGTases which are used in the industrial
preparation of cyclodextrins always yield mixtures of several
cyclodextrins when converting starch into cyclodextrins, various
processes have been developed for isolating pure cyclodextrins
(~, ~ or ~). These are described below:
Defined CDs can be separated out chromatographically
from the product mixtures, e.g. on the basis of differences in
their molecular weights (described, for example, in US Patent No.
4,~08,232).
-- 3
CA 02199904 1997-03-18
As a rule, when starch is converted enzymically into
cyclodextrins, complexing agents are added which only react with
one defined CD and with this CD form an insoluble complex, for
example, which can then be separated out from the reaction
mixture by physical means. Subsequently, the complex is resolved
and the homogeneous CD is isolated (described, for example, in EP
0291067).
When a ~-CGTase is used, the product composition can
be displaced in the ~-CD direction by adding an organic solvent,
such as ethanol, to the reaction mixture fJ. ~erm. Bioeng. (1990)
70 (3), pp. 150-154J.
In each of the processes, those CGTases are
optimally used which possess an initial product formation
preference which is as high as possible for the CD which is to be
prepared in pure form.
The specificity of the previously known ~- and
~-CGTases is adequate for industrial production of the
corresponding cyclodextrins. By contrast, none of the known,
na~urally occurring ~-CGTases possesses a product specificity
which permits a comparable industrial production of ~-CD.
CA 02199904 1997-03-18
In order to prepare ~-CD, therefore, it was
proposed, in CA 115:157165, that ~- and/or ~-cyclodextrins be
converted enzymically into ~-CD by adding the ~-CD-specific
complexing agent glycosyl glycyrrhizin, maltose and a CGTase.
Another option for preparing ~-CD consists in
increasing the ~-CD specificity of ~-CGTase, by means of
exchanging defined amino acid residues, to such a degree that the
mutagenized enzyme produces ~-CD to an increased extent and can
consequently be used for preparing ~-CD on an industrial scale.
Appropriate mutations are known and described, for example, in DE
43 24 650 A1 (corresponds to US Patent No. 5,474,917),
Biochemistry (1994) 33 (33J, pp. 9929-9936, Biochemistry (1995J
34 (lOJ, pp. 3368-3376 and J. Biotech. (1994J 3Z, pp. 283-288.
Such CGTase derivatives, which have been produced by
mutagenizing ~-CGTases, possess an increased specificity for ~-CD
and are consequently, on the basis of their product spectra,
suited, in principle, for the industrial preparation of ~-CD.
However, a disadvantage is that the specific actïvities of the
starting enzymes which are used for the mutagenesis are reduced
by introducing the relevant mutations. In dependence on the amino
acid residues which are introduced, mutated enzymes having an
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CA 02199904 1997-03-18
increased specificity for ~-CD only possess between 25% and 50%
of the CD-forming activity of the starting enzyme (Biochemistry
(1994) 33 (33J, pp. 9929-9936, Biochemistry (1995) 34 flO), pp.
33 68-33 76) .
SU~nMARY OF THE INVENTION
It is an object of the present invention to provide
cyclodextrin glycosyl transferases (CGTases) which, when
converting starch or starch-like substrates into CD, produce ~-CD
to an increased extent and which still exhibit at least 60% of
the specific total CGTase activity of the starting CGTase which
was used for preparing the enzyme concerned.
An additional object of the present invention is to
provide processes for preparing the said CGTases.
A further object of the present invention is to
provide a process for producing ~-CD.
The first-mentioned object is achieved by CGTases
whose amino acid sequence differs from the amino acid sequence of
wi:Ld-type CGTases by the deletion of at least one amino acid in
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CA 02199904 1997-03-18
the region from amino acid position 155 up to and including amino
acid position 195, where position 1 of the protein sequence is
the beginning of the signal peptide of the CGTase and the
deletion increases the ~-CGTase activity of the protein.
Within the meaning of the invention, increases in
the ~-CGTase activity is understood to mean that the quotient
quantity of ~-CD formed
(quantity of ~-CD formed ~ quantity of ~-CD formed)
becomes greater in the product mixture which arises when starch
or starch-like substrates are reacted with CGTases.
Preferably, the amino acid sequences of CGTases
according to the invention differ from the amino acid sequences
of known CGTases by between four and eight amino acid residues
being deleted in the region between amino acid position 155 and
amino acid position 195 of their protein sequence, where position
1 of the protein sequence is the beginning of the signal peptide
of the CGTase and the deletion increases the ~-CGTase activity of
the protein.
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Particularly preferably, the amino acid sequences of
CGTases according to the invention differ from the amino acid
sequences of known CGTases by six amino acid residues being
deleted in the region between amino acid position 155 and amino
acid position 195 of their protein sequence, where position 1 of
the protein sequence is the beginning of the signal peptide of
the CGTase and the deletion increases the
~-CGTase activity of the protein.
It also applies for each of the other amino acid
positions mentioned in the application that position 1 of the
protein sequence is the beginning of the signal peptide of the
CG~ase.
In addition, CGTases are in particular preferred
whose amino acid sequences differ from the amino acid sequences
of the CGTases specified in Table 1 and FIG. 1 at least by the
deletion of the amino acid residues which are in each case
printed in bold, with the remaining amino acid sequence of the
respective CGTase according to the invention being homologous to
the amino acid sequence of the CGTase specified in Table 1 and
FI~. 1 to the extent that the sequence exhibits CGTase activity
without the deletion according to the invention.
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Examples of CGTases according to the invention are
CGTases which are obtained from the CGTases listed in Table 1 and
FIG. 1, or from other CGTases, by deleting individual amino acid
residues in the region between the amino acid residues 155 and
195. CGTases are preferred in which from four to eight residues
have been deleted from the said region. CGTases are particularly
preferred in which the six amino acids marked by bold type in
Ta~le 1 and FIG. 1 have been deleted from the said region.
Further examples of CGTases according to the
in~ention are enzymes from which the amino acids which are
homologous to the amino acids specified in Table 1 and FIG. l
have been deleted, with these enzymes exhibiting CGTase activity
wi~hout the deletion according to the invention.
Further, examples of CGTases according to the
invention are enzymes in which the amino acid residues which are
in each case printed in bold in FIG. 1 have been deleted from the
re~ion between amino acid position 155 and amino acid position
195, with the remaining amino acid sequence of the CGTases
according to the invention being homologous to the amino acid
sequence of the CGTase from the microorganism which is in each
case specified in FIG. 1 and Table 1 to the extent that the
CA 02199904 1997-03-18
enzyme whose sequence does not contain the deletion according to
the invention exhibits CGTase activity.
The six amino acids residue of (SEQ ID NO:5) is that
d~leted portion found in (SEQ ID NO:4). The six amino acids
residue of (SEQ ID NO:7) is that deleted portion found in (SEQ ID
NO:3). The six amino acids residue of (SEQ ID NO:9) is that
deleted portion found in (SEQ IS NO:2). The six amino acids
residue of (SEQ ID NO:10) is that delete dportion found in (SEQ
ID NO:1).
The CGTase protein of the invention has an amino
acid sequence which differs from the amino acid sequences of the
CGTases specified in FIG. 1 and Table 1 at least by the deletion
of the amino acid residues which are in each case printed in bold
in FIG. 1, and Table 1 and is selected from the group consisting
of:
(SEQ ID NO:5) deleted from (SEQ ID NO:4);
(SEQ ID NO:7) deleted from (SEQ ID NO:3);
(SEQ ID NO:9) deleted from (SEQ ID NO:2); and
(SEQ ID NO:10) deleted from (SEQ ID NO:1).
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The remaining amino acid sequence of the CGTase
according to the invention is homologous to the amino acid
sequence of the CGTase from the microorganism which is in each
case specified in FIG. 1 to the extent that the sequence exhibits
CGTase activity without the deletion according to the invention.
TABLE 1:
Six Deleted o
~-CGTase from Position Amino acid sequence Amino Acid Residue ~O
Bacillus ohbensis 160 (SEQ ID NO:l) (SEQ ID NO:10) r
Bacillus macerans 165 (SEQ ID NO:2) (SEQ ID NO:9) ~
Bacillus 5p. 1-1 160 (SEQ ID NO:3) (SEQ ID NO:7) 00
Bacillus circulans #8 172 (SEQ ID NO:4) (SEQ ID NO:5)
-- 12 -- _ .
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Unexpectedly, the CGTases according to the invention
possess a higher ~-CD specificity than that of the starting
CGTases which were used for their preparation while, at the same
time, the mutated enzyme only exhibits an insignificant reduction
in specific total CGTase activity as compared with that of the
starting CGTase.
When converting starch or starch-like substrates,
the CGTases according to the invention consequently produce CDs
in a product distribution in which the quotient of ~-CD and the
sum of ~-CD and ~-CD is greater than the quotlent of these
products which is obtained when starch is converted using the
respective unaltered starting CGTase.
The list shown in Table 1 and in FIG. 1 shows, using
a few CGTases by way of example, the homologous amino acid
se~uence region which is generally present in CGTases and the six
amino acid residues within this sequence region which are in each
case relevant for modifying the product specificity.
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CA 02199904 1997-03-18
The four amino acid sequences shown in FIG. 1 are
the same respectively as the four amino acid sequences shown in
Table 1.
The number of the first amino acid of each of the
amino acid sequences depicted in Table 1 and in FIG. 1 is
designated as the position, with the first amino acid of the
signal peptide of the particular CGTase sequence having been
counted as position 1. The corresponding sequence region can be
found in all CGTases using well known standard methods. This can
be done, for example, using known algorithms which calculate
multiple sequence alignments. An example of a suitable computer
al~orithm is the "pileup" program from the commercially available
Wisconsin Sequence Analysis package (Genetic Computer Group,
Madison, Wisconsin) sequence analysis program.
By means of mutagenizing the depicted region in
CGTases, enzymes according to the invention can be prepared from
any CGTases using known standard methods, as explained, by way of
CA 02199904 1997-03-18
,
example, in the present application. For this purposet a gene
encoding a CGTase is as a rule mutated in such a way that it then
encodes a CGTase according to the invention.
The invention consequently also relates to
pFocesses for preparing mutated CGTase genes which encode CGTases
according to the invention, wherein the DNA sequence of a gene
encoding a starting CGTase is mutated, by means of mutagenesis
methods which are known per se, such that the amino acid sequence
in the region between amino acid positions 155 and 195, which is
encoded by the DNA sequence of the mutated gene, differs from the
amino acid sequence which is encoded by the DNA of the unmutated
gene by the deletion of at least one amino acid residue.
- Preferably, in the process according to the
in~ention, the DNA sequence of a gene encoding a starting CGTase
is mutated, by means of mutagenesis methods which are known per
se, such that the amino acid sequence encoded by the DNA sequence
of the mutated gene differs from the amino acid sequence encoded
,
CA 02199904 1997-03-18
by the DNA of the unmutated gene by the deletion of from four to
eight amino acid residues from the region between amino acid
positions 155 and 195.
Particularly preferably, in the process according to
the invention, the DNA sequence of a gene encoding a starting
CGTase is mutated, by means of mutagenesis methods which are
known per se, such that the amino acid sequence encoded by the
DN~ sequence of the mutated gene differs from the amino acid
se~uence encoded by the DNA of the unmutated gene by the deletion
of six amino acid residues from the region between amino acid
positions 155 and 195.
The invention furthermore relates to processes for
preparing ~-CGTases, wherein at least one of the described DNA
sequences is expressed in a microorganism.
The genes of all CGTases (starting CGTases) are
suitable for preparing the CGTases according to the invention.
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While starting CGTases can be all naturally occurring CGTases,
they can also be CGTases which are obtained by mutagenesis, for
example those CGTases in which the product formation ratio has
already been altered by another mutation which is not in
accordance with the invention (e.g.: as in DE 43 24 650 Al, which
corresponds to US Patent No. 5,474,917) . Starting CGTases are
preferably those CGTases in which the product formation ratio has
already been altered by another mutation which is not in
accordance with the invention (e.g.: as described in D~ 43 24 650
AlJ.
The gene encoding a starting CGTase is isolated
usîng known methods and the mutation according to the invention
is introduced into the gene of the CGTase by "in-vivo" or "in-
vitro" mutagenesis methods. These methods are likewise well known
in the state of the art.
"In-vivo" mutagenesis methods are to be understood
as being, in particular, those methods in which microorganisms
CA 02199904 1997-03-18
which chromosomally and/or episomally contain a gene encoding a
CGTase are mutagenized in a non-specific manner with a mutagen
such as W light, nitrosoguanidine or ethyl methyl sulfonate.
Such a method is described, for example, by Miller J. H. in
(1972) Experiments in Molecular Genetics; Cold Spring Harbor
Laboratory; Cold Spring Harbor, N.Y..
Subsequently, known methods, such as sequence
analysis in accordance with the chain termination method
described by Sanger et al. in PNAS 74 (1977) 5463-5467 are used
to identify mutants in which at least one codon of the CGTase
gene, encoding an amino acid residue, has been deleted from the
region between amino acid residues 155 and 195 of the
corresponding CGTase.
Those mutants are preferably selected in accordance
with the invention in which from four to eight codons have been
deleted from the said region.
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Mutants are particularly preferably selected in
which six codons have been deleted, with those mutants once again
preferably being selected in which the six codons have been
deleted which encode the amino acid residues which are printed in
bold in FIG. 1 or encode the amino acid residues which are
homologous to these residues in other CGTases.
Within the meaning of the invention, "in-vitro"
mutagenesis methods are to be understood as being those methods
in which an isolated CGTase gene, or a fragment of a CGTase gene,
is modified, in a manner known per se, such that a gene is
produced which encodes a CGTase enzyme in which at least one
codon of the CGTase gene, encoding one of the amino acid residues
in the region between amino acid residues 155 and 195, has been
deleted.
Mutants are preferred which have been modified such
that from four to eight codons have been deleted in the said
re~ion. Mutants are particularly preferred which have been
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CA 02199904 1997-03-18
modified such that six codons have been deleted in the said
region. In particular, mutants are particularly preferred which
have been modified such that, in the said region, the six codons
have been deleted which encode the amino acid residues which are
printed in bold in FIG. 1 or encode the amino acid residues which
are homologous to these residues in other CGTases.
The invention consequently also relates to DNA
sequences which encode ~-CGTases according to the invention.
Examples of methods for "in-vitro" mutagenesis which
are known from the state of the art are specific (BioTechniques
(1992) 13 (3), pp. 342-346J or non-specific f~echnique (1989J 1
fl), pp. 11-15) mutagenesis methods which use the "PCR"
te~hnique. Methods are also known in which the mutation is
introduced into the target gene in a directed manner using a
synthetic oligonucleotide. This can take place either using so-
called "single-strand methods~ (Ausubel F.M. et al. (1987)
Cu~rent Protocols in Molecular Biology, Green Publishing
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CA 02199904 1997-03-18
Associates) or using "double-strand methods" (Promega 1992-1993
Catalogue, 150) or using other methods as described, for example,
in Ann. Rev. Genet. (1985) l9, pp. 423-462.
The main area of application for the CGTase
according to the invention is its use for isolating ~-CD from
starch. The CGTases according to the invention can be employed
for this purpose using current preparation methods.
The invention consequently also relates to processes
for preparing ~-CD by converting starch using a CGTase, wherein
at least one CGTase according to the invention is employed as the
CGTase.
Current preparation methods for producing ~-CD, in
which the CGTases according to the invention can be employed in
place of the CGTases which are specified in these methods, are
described, for example, in:
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- Journal of Fermentation and Bioengineering
(199OJ 70 (3), pp. 190-192: The preparation of ~-CD using the ~-
and ~-CD-forming CGTase from Baclllus sp. AL-6 in the presence of
ethanol, which results in an increased production of ~-CD.
- CA 107:57466 describes the preparation of ~-CD
using the ~-CGTase from Bacillus sp. 313.
- EP Z91,067: Preparation of ~-CD using the
CGTase from Bacillus macerans. Product specificity for ~-CD is
achieved by adding a complexing agent, for example cyclohexadec-
8-en-1-one.
- DE 40 09 822 (corresponds to US Patent No.
5,409,824): Production of ~-CD using the ~-CGTase from Bacillus
sp. 290-3.
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Both in comparison to ~-CD and in comparison to
~-CD, ~-CD possesses specific advantages which identify it as the
only possible CD, or the most suitable CD, for a series of
applications.
In comparison to ~-CD, which is made of six glucose
units, ~-CD, which consists of eight glucose units, possesses a
larger hydrophobic cavity which also makes it possible to complex
guest molecules which, for steric reasons, cannot be complexed by
~-~D.
In comparison to ~-CD (solubility in water at room
temperature: approx. 18.5 g/l), ~-CD possesses a substantially
higher solubility in water (at room temperature: approx.
232.0 g/l) and is consequently more suited than ~-CD for
complexing reactions in aqueous solutions. A further advantage of
~-CD, when compared with ~-CD and modified ~-CD derivatives, is
its low toxicity. In an animal model, ~-CD derivatives and ~-CD
- 23 -
CA 02199904 1997-03-18
derivatives are more toxic than ~-CD when administered either
orally or intravenously.
Other objects and features of the present invention
will become apparent from the drawing and from the following
Examples, which disclose the embodiments of the present
invention. It should be understood, however, that the drawing
and the Examples are designed for the purpose of illustration
only and not as a definition of the limits of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawing, FIG. 1 shows the homologous amino
acid sequence region which is generally present in CGTases and
the six amino acid residues in bold print within this sequence
region which are in each case relevant for modifying the product
specificity.
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DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
Mutaqenesis of the Bacillus circulans #8 (DSM 10559~ CGTase
Deletion of any amino acid residues in the region,
according to the invention, of amino acid residues 155-195, in
particular deletion of the six amino acid residues
tSEQ ID NO:5) at positions 179-184 in the ~-CGTase from Bacillus
circulans #8 (see Table l; deposited March 2, 1996, in the DSMZ
Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH
(German Collection of Microorganisms and Cell Cultures) in
Mascheroder Weg lb, D-38124 Braunschweig, Germany, under number
DS~ 10559 according to the Budapest Treaty), is achieved by
deleting, in a manner which is known per se to a person skilled
in the art, the base triplets of the CGTase structural gene which
encode the corresponding amino acid residues.
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For the mutagenesis, the ~-CGTase gene from Bacillus
circulans ~8 was first of all cloned into the commercially
available E. coli vector pUC19 (Boehringer, Mannheim). For this
purpose, chromosomal DNA was isolated from Bacillus circulans #8
(Appl. Microbiol. Biotechnol. (1990) 33: pp. 542-546) as
described in Ausubel F.M., Current Protocols In Molecular
Biology, Vol. 1; Greene Publishing Associates & Wiley -
Interscience, N.Y. and cleaved with the restriction endonucleases
HindIII and XbaI (Boehringer, Mannheim). Fragments in a size
range of between two and five kb were isolated and incubated, at
16~C for 12 hours, together with pUC19 DNA, which had been
cleaved with the restriction endonucleases HindIII and XbaI
(Boehringer, Mannheim), and with T4 DNA ligase. The ligation
mixture was used to transform E. coli K 12 cells which had been
rendered competent for DNA uptake by means of known methods
(Maniatis, Molecular Cloning, A Laboratory Manual; Cold Spring
Harbor Laboratory (1982), N.Y.). The recombinant plasmid, which
carries the gene for the Bacillus circulans #8 ~-CGTase, was
isolated from those E. coli cells which, following the
CA 02199904 1997-03-18
transformation, formed starch degradation haloes on starch-
containing indicator plates (Maniatis, Molecular Cloning, A
Laboratory Manual; Cold Spring Harbor Laboratory (1982), N.Y.,
pp. 86-92).
This gene was mutagenized using the oligonucleotide-
directed in vitro mutagenesis system, version 2.1, which is
marketed by Amersham (Braunschweig) and based on a method
developed by Eckstein (NUcl. Acids. Res. (1986) 14, pp. 9679-9698
and Nucl. Acids. Res. (1988) 16, pp. 791-802). The mutagenesis
was carried out exactly in accordance with the protocol which is
enclosed with this Amersham mutagenesis system. The method is
summarized below. Details can be obtained from the protocol of
this mutagenesis system.
That part of the pUC19-cloned gene for the Bacillus
circulans #8 ~-CGTase which encoded the region, according to the
in~ention, from amino acid residue 155 to amino acid residue 195
of the CGTase was cloned into the commercially available vector
CA 02199904 1997-03-18
M13 (New England Biolabs) using commercially available enzymes
such as restriction endonucleases and T4 DNA ligase (Boehringer,
Mannheim). A 1.6 kb AccI fragment is an example of such a
fragment. This fragment was cloned into the AccI-cleaved M13
vector.
Single-stranded, recombinan~ M13 DNA (initial DNA)
was isolated, in accordance with the experimental protocol
supplied by Amersham together with the above mentioned
mutagenesis system, from those E. coli host cells which had taken
up the recombinant M13 vector.
For the actual mutagenesis, chemically de~ined
mut:agenesis oligonucleotides were synthesized which in each case
possessed the desired sequence. Such oligonucleotides can, for
example, be obtained commercially from MWG (Ebersberg). The
se~uence of the mutagenesis oligonucleotide was chosen such that
the order of the bases in the mutagenesis oligonucleotide was
inversely complementary to that part of the nucleotide sequence
- 28 -
CA 02i99904 1997-03-18
o~ the initial DNA which flanked, for 15 bases upstream and 15
bases downstream, the base triplets of the Bacillus circulans #8
~-CGTase which were contained in the initial DNA and which were
to be deleted.
The sequence of the mutagenesis oligonucleotide
which was used is shown in Table 2.
TABLE Z
5'-(SEO ID NO:6)-3'
Use of the mutagenesis oligonucleotide shown in
Table 2 resulted in a ~-CGTase gene fraction which encodes an
amino acid fragment from which the six amino acid residues (SEQ
ID NO:5) have been deleted.
The mutagenesis oligonucleotide was phosphorylated
at its 5' end using T4 polynucleotide kinase and ATP (Amersham).
The phosphorylated mutagenesis oligonucleotide was bound to the
CA 02199904 1997-03-18
homologous regions of the initial DNA. For this, 5 ~g of single-
stranded initial DNA were incubated, at 70~C for three minutes
a~d then at 37~C for 30 minutes, together with approximately
4 pmol of the phosphorylated mutagenesis oligonucleotide. A DNA
strand which was complementary to the initial DNA, with the -
exception of the nucleotides to be deleted, was then synthesized,
with the mutagenesis oligonucleotide which was bound to the
initial DNA serving as the starting point for the synthesis and
the initial DNA serving as the template for the new synthesis of
the mutated DNA strand. The synthesis itself was carried out,
after adding the Klenow fragment of DNA polymerase (Amersham), a
T4 DNA ligase and a nucleotide mix which contains the nucleotides
dATP, dGTP and dTTP and, in place of dCTP, the thionucleotide
dCTPS (Amersham), at 16~C for 15 hours.
Remaining molecules of single-stranded initial DNA
were removed from this synthesis mixture. For this, the mixture
wa~ treated with NaCl and filtered through a nitrocellulose
filter (Amersham) which specifically binds single-stranded DNA.
- 30 -
CA 02199904 1997-03-18
The double-stranded hybrid DNA, which remained in the flow-
through, was concentrated and desalted by precipitation with
EtOH. The hybrid DNA was then incubated with NciI (Amersham), a
restriction endonuclease which recognizes the nucleotide sequence
CC(G/C)GG but only cleaves native DNA strands and not those which
contain the nucleotide analog dCTPS, at 37~C for 90 minutes in a
suitable incubation buffer (Amersham). This treatment only
introduced breaks into the non-mutagenized strand.(initial DNA).
The initial DNA was then removed during a 30-minute
treatment, at 37~C, with exonuclease III (Amersham), which is an
enzyme which degrades DNA strands starting at free ends. After
the exonuclease III had been inactivated thermally (70~C for 15
minutes), the remaining, single-stranded and mutagenized DNA
strand was incubated with DNA polymerase I (Amersham), T4 DNA
li~ase and the nucleotides dATP, dTTP, dCTP and dGTP at 16~C for
3 hours. This made the mutagenized single-stranded DNA into
double-stranded DNA. Following a further EtOH precipitation for
- 31 -
CA 02199904 1997-03-18
purification purposes, the mutagenized DNA was transformed into
competent E. coli ~12 cells.
The success of the mutagenesis procedure was checked
by sequence analysis of the relevant region of the recombinant
DNA from five clones which were obtained in the transformation.
The DNA fragment which was originally cloned into M13 for the
mutagenesis was excised from a vector, which was confirmed to
possess a mutation, using the restriction enzymes XhoI and NdeI.
Subsequently, the corresponding, but
non-mutagenized, XhoI/NdeI fragment was excised from the
pUC19-based plasmid for expressing Bacillus circulans #8
~-CGTase and replaced with the mutagenized fragment using T4 DNA
ligase.
CA 02199904 1997-03-lX
EXAMPLE 2
Mutaqenesis of the Bacillus sp. 1-1 ~-CGTase
The six codons of the Bacillus sp. 1~ CGTase gene
encoding the amino acid residues (SEQ ID N0:7) at position 167-
172 of the corresponding CGTase (see Table 1) were deleted, in
analogy with the method described in Example 1, using the
oligonucleotide shown in Table 3 ( A) in Ex. 8, Comparison 2).
The same deletion was also introduced into a derivative of this
CGTase which was described in
DE 43 24 650 A1 and whose ~-CD specificity had already been
increased, as compared with that of the wild-type enzyme, by an
amino acid exchange (Tyr => Trp) ( B) in Ex. 8,
Comparison 2).
TABLE 3
5'-(SEQ ID N0:8)-3'
- 33 -
CA 02199904 1997-03-18
~ EXAMPLE 3
Production of Bacillus circulans #8 B-CG~ase
and its derivatives accordinq to the invention, in E. coli
The pUC19-based expression plasmids which were
described in Example 1 were transformed into a secretory E. coli
strain for the purpose of producing the Bacillus circulans #8 ~-
CGTase and its derivative which was prepared as described in Ex.
1. E. coli WCM105 was used as the secretory E. coli strain. This
strain was prepared from E. coli DS 410 as described in
EP 338,410.
For the purpose of producing the Bacillus circulans
#8 ~-CGTase, or its derivative, therefore, ~. coli WC~105 cells
harboring suitable CGTase expression plasmids were incubated in
LB medium (Maniatis, Molecular Cloning, A Laboratory ~anual; Cold
Spring Harbor Laboratory (1982), N.Y.), containing 10 g/l lactose
and 0.1 g/l ampicillin at 30~C for 72 hours in a shaking water
bath (revolution rate: 250 rpm). The cells were then separated
- 34 -
CA 02199904 1997-03-lX
off by centrifuging at 5000 x g. The cell-free culture
supernatant contains the ~-CGTase or its derivatives.
EXAMPLE 4
Production of the Bacil l us sp . 1-1 ~-CGTase,
and its derivatives accordinq to the invention, in E. col i
Production was effected utilizing a procedure
analogous to that described in Example 3 using the expression
plasmids described in Example 2.
EXAMPLE 5
Purification of CGTases by means of
adsorPtion to carrier-bound ~-cyclodextrin
CGTases are purified in a specific and mild manner
by means of affinity purification using SEPHAROSE~-coupled ~-CD
molecules.
CA 02199904 1997-03-18
1 g of epoxy-activated SEPHAROSE~ 6B (Sigma) is
washed with 3 x 10 ml of H,O and then with 1 x 5 ml of 0.1 N
NaOH. The SEPHAROSE~ 6B is subsequently suspended in 2 ml of a
2.8~ (w/v) solution of ~-CD in 0.1 N NaOH, and this suspension is
incubated at 450C for 20 h while being shaken gently. The
ccupling product, consisting of ~-CD and SEPHADEX~ 6B, is then
washed with 2 x 5 ml of H20. After having suspended the washed
material in a 2.5~ (w/v) solution of glucose in H70, the
suspension is incubated at RT for 1 h in order to saturate the
free coupling sites which remain. The coupling product is then
washed successively with 2 x 5 ml of H,O, 2 x 5 ml of 0.1 M
bo:rate buffer, pH 8.0, 2 x 5 ml of 0.1 M acetate buffer, pH 4.0,
and 2 x 2 ml of 20 mM triethanolamine/Cl, pH 7.2. The coupling
product is treated with 0.2 ml of 20 mM triethanolamine/Cl,
pH 7.2 (final volume, approximately 2-2.5 ml) and stored until
used at 4~C.
In order to specifically bind CGTases to the
SEPHAROSE~ 6B-coupled ~-CD (CD-SEPHAROSE~), the cell-free,
- 36 -
CA 02199904 1997-03-18
CGTase-containing culture supernatants which were obtained in
accordance with Example 3 or 4 are treated with 0.Z ml of the CD-
Sepharose and incubated at 4OC for 1.5 h while being shaken
gently. During this period, the enzyme couples to the
CD-SEPHAROSE~. The enzyme/CD-SEPHAROSE~ complex is isolated by
centrifugation (5 min at 4000 x g) and washed with 2 x 10 ml of
20 mM triethanolamine/cl~ pH 7.2. The CGTase enzyme is
subsequently eluted by incubating the complex, at 4~C for 1.5 h,
with 2 ml o~ a 1% solution of ~-CD in 20 mM triethanolamine/Cl,
pH 7.2. After a final centrifugation (5 min at 4000 x g), the
supernatant containing the purified CGTase is removed.
Before characterizing the CGTases which have been
purified in this way, the ~-CD which is contained in the
solution, and which was used for the elution, still has to be
removed. To do this, a commercially obtainable PD-10 column
(SEPHADEX~ G-25 M; Pharmacia) is equilibrated with 35 ml of 20 mM
Tris/HCl, pH 7.2 and 5 mM CaCl2 (TC buffer). The
- 37 -
CA 02199904 1997-03-18
,B~CD-containing solution is made up to a volume of 2.5 ml with TC
buffer and loaded onto the column. The column is subsequently
eluted with 3.5 ml of TC buf~er. The eluate which is obtained in
this way contains the purified, ~-CD-free CGTase.
EXAMPLE 6
Conversion of starch into cyclodextrins
The CGTase activities were determined using the
method described in Eur. J. Biochem. (1990) 191, pp. 17~-185.
Different quantities of a CGTase solution to be
tested were incubated at 45~C, for a defined time, with a 5%
solution of a soluble starch (Merck, Darmstadt) in a buffer
consisting of 20 mM Tris/HCl, pH 7.2, and S mM CaCl,. After the
defined time, the reaction was terminated by adding 1.5 parts by
volume of methanol. Unreacted residual starch was precipitated by
incubating at 4~C for one hour and separated off by
centrifugation (10 min at 12000 x g). The resulting products were
- 38 -
,
CA 02199904 1997-03-18 ~..
determined by HPLC on a NUCLEOSIL~ 10-NH, column (Macherey &
Nagel, Duren), with defined cyclodextrins (Sigma, Munich) serving
as standards. One unit (1 U) is defined as the quantity of enzyme
which forms 1 ~M of cyclodextrins per minute from starch under
the conditions described.
EXAMPLE 7
Determination of the s~ecific total
CGTase activities of Purified CGTases
The specific total CGTase activity of purified
CGTases is defined as the volume activity per quantity of protein
(U/mg)
The CGTase volume activity (U/ml) of an enzyme
sample is determined as described in Example 6.
The protein content (mg/ml) of a solution of the
purified CGTase is determined using the method described by M.
CA 02199904 1997-03-18
Bradford (Anal. Biochem. (1976) 72, pp. 248 ff). The chemicals
which are required for this purpose are obtained from Bio-Rad.
The following specific total CGTase activities
(spec. activity) were determined for the Bacillus Circulans #8
and Bac1llus sp. 1~ CGTases and the deletion mutants which
were produced (as described in Example 1 and Example 2) from
them:
Enzyme Rel. activity (~) SPec. activity (U/mq)
Wild-type CGTase from
Bacillus circulans #8 100 106
Deletion mutant derived
from it 77 82
Wild-type CGTase from
Bacillus sp. 1-1 100 120
Deletion mutant derived
from it 65 78
Mutated derivative
tTyrTrp) of Bacillus sp.
1-1 CGTase lO0 23
Deletion mutant derived
from it 73 17
- 40 -
CA 02199904 1997-03-18
EXAMPLE 8
Conversion of starch usinq the non-mutaqenized
~-CGTases from Bacillus circulans #8 and Bacillus sp. 1-1 and
the derivatives Prepared as described in Exs. 3 and 4.
In order to compare the product spectra of the
non-muta~enized CGTases with those of the derivatives which were
in each case obtained from them by deletion mutagenesis, as
described in Examples 1 and 2, identical enzyme activities were
employed for converting the starch (Example 6). At defined times,
the product composition was investigated as described. The
following results were obtained:
- 41 -
COMPARISON 1: BACILLUS CIRCULANS ~8
Nild-Type CGTase Deletion mut~nt of this g
Time ~-CD ~-CD ~y-CD ~ +~ CD ,B-CD ~-CD ~+~3)
(min) (%) (%) (%) (%) (%) (%)
S 13 70 17 0 . 2 9 58 . 0 33 0 . 49 ~
13.5 70.3 16.2 0.19 8.2 60.5 31.3 0.46 r~
11.7 66.3 22.0 0.28 9.4 62.0 28.6 0.40
-- 42 --
¦ COMPARISON 2: BACILLVS SP. 1-1
A) J
Wild-Type CGTase Deletion mutant of this
Time ~-CD ~-CD ~-CD ~ +~) ~-CD ~-CD ~-CD ~ +~)
(min) (%) (%) (%) (%) (%) (%)
0 100 0 0 68 32 33 0.47
~ 10 0 88 12 0.14 0 6~ 32 0.47 O
1 15 0 88 12 0.14 0 67.7 32.3 0.48
B)
¦ Wild-Type CGTase Deletion mutant of th: s ~
Time ~-CD ~-CD ~-CD ~ t~ CD ~-CD ~-CD ~ t~) X
(min) (%) (%) (%) (%) (%) (%)
0 27 73 2.7 0 0 100
0 3l 69 2.23 0 22 78 3.54
0 32.S 67.5 2.05 0 16.6 8~.4 S.02
CA 02199904 1997-03-18
While several embodiments of the present invention
have been shown and described, it is to be understood that many
changes and modifications may be made thereunto without departing
from the spirit and scope of the invention as defined in the
appended claims.
CA 02199904 1997-03-18
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICAN~:
Consortium fur elektrochemische Industrie
GmbH whose full post office address is
Zielstattstrasse 20, 81379 Munchen, Germany
(ii) TITLE OF I~V~;N'1'1ON:
Cyclodextrin Glycosyl Transferases
for Producing y-Cyclodextrin
(iii) NUMBER OF SEQUENCES: 10
(iv) CORRESPONDENCE ADDRESS:
225 Metcalfe Street
Suite 606
Ottawa, Ontario
KlP lP9
Canada
(v) COM~Ul~ RE~n~RT~ FORM:
a) COM~Ul~: IBM PC compatible
b) OPERATING SYSTEM: PC-DOS/MS-DOS
c) SOFTWARE: WordPerfect Version 5.1 for DOS
(vi) CURRENT APPLICATION DATA:
a) APPLICATION NUMBER: Not Yet Known
b) FILING DATE: March 13, 1997
c) CLASSIFICATION. Not Yet Known
(vii) PRIOR APPLICATION DATA:
a) APPLICATION NUMBER: DE 19615336
b) FILING DATE: 18-APR-1996
c) CLASSIFICATION: Unknown
(viii) ATTORNEY/AGENT INFORMATION:
a) NAME: McFadden, Fincham
b) REFERENCE NUMBER: 1546-281
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
a) LENGTH: 24 Amino acids
b) TYPE: Amino acid
c) STRANDEDNESS:
d) TOPOLOGY: Linear
(ii) MOLECULE TYPE: Peptide
(iii) HYPOTHETICAL: No
(v) FRAGMENT TYPE: Internal fragment
- 45 -
CA 02199904 1997-03-18
~Vi) ORIGINAL SOURCE:
a) ORGANISM: Bacillus ohbensis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Pro Asn His Ser Ser Pro Ala Leu Glu Thr Asp Pro Ser Tyr Ala Glu
1 5 10 15
Asn Gly Ala Val Tyr Asn Asp Gly
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
a) LENGTH: 24 Amino acids
b) TYPE: Amino acid
c) STRANDEDNESS:
d) TOPOLOGY: Linear
(ii) MOLECULE TYPE: Peptide
(iii) HYPOTHETICAL: No
(v) FRAGMENT TYPE: Internal fragment
(vi) ORIGINAL SOURCE:
a) ORGANISM: Bacillus macerans
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Pro Asn His Thr Ser Pro Ala Asp Arg Asp Asn Pro Gly Phe Ala Glu
1 5 10 15
Asn Gly Gly Met Tyr Asp Asn Gly
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
a) LENGTH: 24 Amino acids
b) TYPE: Amino acid
c) STRANDEDNESS:
d) TOPOLOGY: Linear
(ii) MOLECULE TYPE: Peptide
(iii) HYPOTHETICAL: No
(v) FRAGMENT TYPE: Internal fragment
(vi) ORIGINAL SOURCE:
a) ORGANISM: Bacillus sp. 1-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Pro Asn His Ser Ser Pro Ala Leu Glu Thr Asn Pro Asn Tyr Val Glu
1 5 10 15
Asn Gly Ala Ile Tyr Asp Asn Gly
- 46 -
CA 02199904 1997-03-18
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
a LENGTH: 24 Amino acids
b TYPE: Amino acid
c STRANDEDNESS:
d TOPOLOGY: Linear
(ii) MOLECULE TYPE; Peptide
(iii) HYPOTHETICAL: No
(v) F~ NT TYPE: Internal fragment
(vi) ORIGINAL SOURCE:
a) ORGANISM: Bacillus circulans #8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Pro Asn His Thr Ser Pro Ala Met Glu Thr Asp Thr Ser Phe Ala Glu
1 5 10 . 15
Asn Gly Arg Leu Tyr Asp Asn Gly
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
a LENGTH: 6 Amino acids
b TYP~: Amino acid
c STRANDEDNESS: Single
d, TOPOLOGY: Linear
(ii) MOLECULE TYPE: Peptide
(iii) HYPOTHETICAL: No
(iv) ANTISENSE: No
(v) F~A~.M~T TYPE: Internal fragment
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met Glu Thr Asp Thr Ser
1 5
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
a) LENGTH: 30 Base pairs
b) Type: Nucleotide
c) STRANDEDNESS: Single
d) TOPOLOGY: Linear
(ii) MOLECULE TYPE: Other nucleic acid
a) DESCRIPTION: /desc = "synthetic"
CA 02199904 1997-03-18
~iii) HYPOTHETICAL: No
(iv) ANTISENSE: No
(viii) POSITION IN THE GENOME:
c) UNITS: bp
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CACACCTCTC CAGCGTTTGC C~.AAAATGGC 30
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
a) LENGTH: 6 Amino acids
b) TYPE: Amino acid
c) STRANDEDNESS: Single
d) TOPOLOGY: Linear
(ii) MOLECULE TYPE: Peptide
(iii) HYPOTHETICAL: No
(iv) ANTISENSE: No
(v) FRAGMENT TYPE: Internal fragment
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Leu Glu Thr Asn Pro Asn
1 5
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
a) LENGTH: 30 base pairs
b) TYPE: Nucleotide
c) STRANDEDNESS: Single
d) TOPOLOGY: Linear
(ii) MOLECULE TYPE: Other nucleic acid
a) DESCRIPTION: /desc = ~synthetic~
(iii) HYPOTHETICAL: No
(iv) ANTISENSE: No
', (viii) POSITION IN THE GENOME:
c) UNITS: bp
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
, CATTCATCAC CGGCATATGT T~-AAAA~GGG 30
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
a) LENGTH: 6 Amino acids
- 48 -
CA 02199904 1997-03-18
b) TYPE: Amino acid
c) STRANDEDNESS: Single
d) TOPOLOGY: Linear
(ii) MOLECULE TYPE: Peptide
(iii) HY~O~ CAL: No
(iv) ANTISENSE: No ..
(v) FRAGMENT TYPE: Internal fragment
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Asp Arg Asp Asn Pro Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
a) LENGTH: 6 Amino acids
b) TYPE: Amino acid
c) STRANDEDNESS: Single
d) TOPOLOGY: Linear
(ii) MOLECULE TYPE: Peptide
(iii) HYPOTHETICAL: No
(iv) ANTISENSE: No
(v) FRAGMENT TYPE: Internal fragment
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Leu Glu Thr Asp Pro Ser
1 5
- 49 -
