U.S. patent application number 10/463569 was filed with the patent office on 2003-11-06 for preparation of acariogenic sugar substitutes.
This patent application is currently assigned to Sudzucker Aktiengesellschaft. Invention is credited to Klein, Kathrin, Kunz, Markwart, Mattes, Ralf, Munir, Mohammed, Schiweck, Hurbert.
Application Number | 20030207437 10/463569 |
Document ID | / |
Family ID | 27511724 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030207437 |
Kind Code |
A1 |
Mattes, Ralf ; et
al. |
November 6, 2003 |
Preparation of acariogenic sugar substitutes
Abstract
The invention relates to sucrose isomerases, to DNA sequences
that code for sucrose isomerases, and to novel processes for the
production of non-cariogenic sugars.
Inventors: |
Mattes, Ralf; (Stuttgart,
DE) ; Klein, Kathrin; (Stuttgart, DE) ;
Schiweck, Hurbert; (Worms, DE) ; Kunz, Markwart;
(Worms, DE) ; Munir, Mohammed; (Kindenheim,
DE) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Assignee: |
Sudzucker
Aktiengesellschaft
Mannheim/Oschsenfurt
DE
|
Family ID: |
27511724 |
Appl. No.: |
10/463569 |
Filed: |
June 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10463569 |
Jun 18, 2003 |
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10061269 |
Feb 4, 2002 |
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10061269 |
Feb 4, 2002 |
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09168720 |
Oct 9, 1998 |
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09168720 |
Oct 9, 1998 |
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08785396 |
Jan 21, 1997 |
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5985622 |
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08785396 |
Jan 21, 1997 |
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08374155 |
Jan 18, 1995 |
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5786140 |
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Current U.S.
Class: |
435/233 ;
435/252.33; 435/320.1; 435/6.16; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/90 20130101; C12N
9/2451 20130101; C12P 19/24 20130101; C12P 19/16 20130101 |
Class at
Publication: |
435/233 ; 435/6;
435/69.1; 435/320.1; 435/252.33; 536/23.2 |
International
Class: |
C12N 009/90; C12Q
001/68; C07H 021/04; C12P 021/02; C12N 001/21; C12N 015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 19, 1994 |
DE |
P 44 01 451.1 |
Apr 22, 1994 |
DE |
44 14 185.8 |
Claims
Applicants claim:
1. An isolated or purified protein with sucrose isomerase activity,
wherein the protein is recombinant and is encoded by a DNA sequence
comprising (a) A nucleotide sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
9, SEQ ID NO: 11, SEQ ID NO: 13, and any of these sequences without
the signal peptide-coding region; (b) a nucleotide sequence
corresponding to the sequences from (a) within the scope of the
degeneracy of the genetic code, or (c) a nucleotide sequence that
hybridizes with a sequence from (a), (b), or both (a) and (b),
wherein a positive hybridization signal is still observed after
washing with 1.times.SSC and 0.1% SDS at 55.degree. C. for one
hour.
2. An isolated or purified protein as claimed in claim 1, wherein
the protein is recombinant and comprises (a) an amino acid sequence
selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5,
SEQ ID NO; 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and any
of these sequences without the signal peptide region; or (b) an
amino acid sequence that it is at least 80% homologous with the
sequences from (a).
3. An isolated or purified protein as claimed in claim 1, wherein
the protein is recombinant and has an amino acid sequence that is
at least 90% homologous to the amino acid sequences from (a) amino
acid 51-149, (b) amino acid 168-181, (c) amino acid 199-250, (d)
amino acid 351-387, or (e) amino acid 390-420 of the amino acid
sequence shown in SEQ ID NO: 4.
4. A method for isolating nucleic acids that code for a protein
with a sucrose isomerase activity comprising (a) preparing a gene
bank from a donor organism that contains a DNA sequence coding for
a protein with a sucrose isomerase activity in a suitable host
organism, (b) screening the clones of the gene bank, and (c)
isolating the clones which contain a nucleic acid coding for a
protein with sucrose isomerase activity.
5. A method as claimed in claim 4, wherein E. coli is used as host
organism.
6. A method as claimed in claim 4, wherein the steps of preparing a
gene bank, screening the clones, and isolating the clones are
performed in an E. coli strain that does not utilize galactose.
7. A method as claimed in claim 4, wherein the clones in the gene
bank are screened using nucleic acid probes that are derived from
the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
8. A method as claimed in claim 7, wherein a DNA fragment which has
been obtained by PCR amplification of the DNA from the donor
organism using the oligonucleotide mixtures 5'-TGGTGGM (A,G) GA
(A,G) GCTGT-3' (SEQ ID NO: 17) and 5'-TCCCAGTTCAG (A,G) TCCGGCTG-3'
(SEQ ID NO: 18) as primers is used as nucleic acid probe.
9. Protein with palatinase activity, trehalulase activity, or both,
that is encoded by a DNA sequence comprising (a) one of the
nucleotide sequences shown in SEQ ID NO: 7 or SEQ ID NO: 15, (b) a
nucleotide sequence characterized in that it corresponds to the
sequence from (a) within the scope of the degeneracy of the genetic
code, or (c) a nucleotide sequence characterized in that it
hybridizes with the sequences from (a), (b), or both (a) and
(b).
10. The protein as claimed in claim 9, comprising the amino acid
sequence shown in SEQ ID NO: 8 or SEQ ID NO: 16.
Description
[0001] This is a Continuation in Part of U.S. application Ser. No.
09/168,720, filed Oct. 9, 1998, which is a Divisional Application
of U.S. application Ser. No. 08/785,396, now U.S. Pat. No.
5,985,622, filed Jan. 21, 1997, which is a Divisional Application
of U.S. application Ser. No. 08/374,155, now U.S. Pat. No.
5,786,140, filed Jan. 18, 1995, which claims priority of DE P 44 01
451.1, filed Jan. 19, 1994 and DE 44 14 185.8, filed Apr. 22, 1994.
All of these documents are expressly incorporated by reference. All
patents and other publications listed herein are expressly
incorporated by reference.
[0002] The present invention relates to an improved process for the
preparation of non-cariogenic sugars, in particular trehalulose
and/or palatinose, using recombinant DNA technology.
[0003] The acariogenic sugar substitutes palatinose (isomaltulose)
and trehalulose are produced on a large scale from sucrose by an
enzymatic rearrangement using immobilized bacterial cells (for
example of the species Protaminobacter rubrum, Erwinia rhapontici,
Serratia plymuthica). This entails the .alpha.1-.beta.2 glycosidic
linkage existing between the two monosaccharide units of the
disaccharide sucrose being isomerized to an .alpha.1-6 linkage in
palatinose and to an .alpha.1-.alpha.1 linkage in trehalulose. This
rearrangement of sucrose to give the two acariogenic disaccharides
takes place with catalysis by the bacterial enzyme sucrose
isomerase, also called sucrose mutase. Depending on the organism
used, this reaction results in a product mixture which, besides the
desired acariogenic disaccharides palatinose and trehalulose, also
contains certain proportions of unwanted monosaccharides (glucose
and/or fructose). These monosaccharide contents are a considerable
industrial problem because elaborate purification procedures
(usually fractional crystallizations) are necessary to remove
them.
[0004] For example EP-0 028 900 describes a method for producing
palantinose in a bioreactor by using immobilized sucrose isomerase,
which was purified and immobilized from a raw extract by
selectively binding to an anionic carrier matrix. The product
composition obtained by this method contains, apart from the
desired acariogenic disaccharides palantinose and trehalulose,
2.1-2.5% of the unwanted monosaccharide fructose and 0.6-1.0% of
the unwanted monosaccharide glucose.
[0005] Further, EP-0 483 755 describes a method for producing
trehalulose and palatinose, wherein a sucrose solution is contacted
with at least one trehalulose-forming enzyme system of a
trehalulose-forming microorganism at a temperature of 10-35.degree.
C., wherein a mostly tetrahalulose-containing product mixture is
obtained, which, however, contains low amounts of the unwanted
monosaccharides fructose and glucose. It could further be shown
that the amount of unwanted monosaccharides drastically increases
by using higher incubation temperatures preferred in large-scale
technical methods and, in addition, a rapid thermal inactivation of
the enzyme preparations occurred.
[0006] One object on which the present invention is based was thus
to suppress as far as possible the formation of monosaccharides in
the isomerization of sucrose to trehalulose and/or palatinose.
Another object on which the present invention is based was to
provide organisms which produce palatinose and/or trehalulose in a
higher yield than do known organisms.
[0007] To achieve these objects, recombinant DNA molecules,
organisms transformed with recombinant DNA molecules, recombinant
proteins and an improved process for the preparation of
non-cariogenic sugars, in particular of palatinose and/or
trehalulose, are provided.
[0008] The invention relates to a DNA sequence which codes for a
protein with a sucrose isomerase activity and comprises
[0009] (a) one of the nucleotide sequences shown in SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID
NO: 13, where appropriate without the signal peptide-coding
region,
[0010] (b) a nucleotide sequence corresponding to the sequences
from (a) within the scope of the degeneracy of the genetic code,
or
[0011] (c) a nucleotide sequence which hybridizes with the
sequences from (a) and/or (b).
[0012] In the context of the present invention, the term "protein
with a sucrose isomerase activity" is intended to embrace those
proteins which are able to isomerize sucrose to other disaccharides
with conversion of the .alpha.1-.beta.2 glycosidic linkage between
glucose and fructose in sucrose into another glycosidic linkage
between two monosaccharide units, in particular into an .alpha.1-6
linkage and/or an .alpha.1-.alpha.1 linkage. The term "protein with
a sucrose isomerase activity" therefore particularly preferably
relates to a protein which is able to isomerize sucrose to
palatinose and/or trehalulose. Moreover, the proportion of
palatinose and trehalulose in the total disaccharides formed by
isomerization of sucrose is preferably .gtoreq.2%, particularly
preferably .gtoreq.20% and most preferably .gtoreq.50%.
[0013] The nucleotide sequence shown in SEQ ID NO: 1 codes for the
complete sucrose isomerase from the microorganism Protaminobacter
rubrum (CBS 547.77) including the signal peptide region. The
nucleotide sequence shown in SEQ ID NO: 2 codes for the N-terminal
section of the sucrose isomerase from the microorganism Erwinia
rhapontici (NCPPB 1578) including the signal peptide region. The
nucleotide sequence shown in SEQ ID NO: 3 codes for a section of
the sucrose isomerase from the microorganism SZ 62 (Enterobacter
spec.).
[0014] The region which codes for the signal peptide in SEQ ID NO:
1 extends from nucleotide 1-99. The region coding for the signal
peptide in SEQ ID NO: 2 extends from nucleotide 1-108. The DNA
sequence according to the present invention also embraces the
nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 without
the region coding for the signal peptide because the signal peptide
is, as a rule, necessary only for correct localization of the
mature protein in a particular cell compartment (for example in the
periplasmic space between the outer and inner membrane, in the
outer membrane or in the inner membrane) or for extracellular
export, but not for the enzymatic activity as such. The present
invention thus furthermore embraces sequences which also code for
the mature protein (without signal peptide) and are operatively
linked to heterologous signal sequences, in particular to
prokaryotic signal sequences as described, for example, in E. L.
Winnacker, Gene und Klone, Eine Einfuhrung in die Gentechnologie,
VCH-Verlagsgesellschaft Weinheim, Germany (1985), p. 256.
[0015] Nucleotide sequence SEQ ID NO: 9 codes for a variant of the
isomerase from Protaminobacter rubrum. Nucleotide sequence SEQ ID
NO: 11 codes for the complete isomerase from the isolate SZ 62.
Nucleotide sequence SEQ ID NO: 13 codes for most of the isomerase
from the microorganism MX-45 (FERM 11808 or FERM BP 3619).
[0016] Besides the nucleotide sequences shown in SEQ ID NO: 1, SEQ
ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO:
13, and nucleotide sequences corresponding to one of these
sequences within the scope of the degeneracy of the genetic code,
the present invention also embraces a DNA sequence which hybridizes
with one of these sequences, provided that it codes for a protein
which is able to isomerize sucrose. The term "hybridization"
according to the present invention is used as in Sambrook et al.
(Molecular Cloning. A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1989), 1.101-1.104). According to the present
invention, hybridization is the word used when a positive
hybridization signal is still observed after washing for 1 hour
with 1.times.SSC and 0.1% SDS at 55.degree. C., preferably at
62.degree. C. and particularly preferably at 68.degree. C., in
particular for 1 hour in 0.2.times.SSC and 0.1% SDS at 55.degree.
C., preferably at 62.degree. C. and particularly preferably at
68.degree. C. A nucleotide sequence which hybridizes under such
washing conditions with one of the nucleotide sequences shown in
SEQ ID NO: 1 or SEQ ID NO: 2, or with a nucleotide sequence which
corresponds thereto within the scope of the degeneracy of the
genetic code, is a nucleotide sequence according to the
invention.
[0017] The DNA sequence according to the invention preferably
has
[0018] (a) one of the nucleotide sequences shown in SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID
NO: 13, where appropriate without the signal peptide-coding region,
or
[0019] (b) a nucleotide sequence which is at least 70% homologous
with the sequences from (a).
[0020] The DNA sequence according to the invention preferably also
has an at least 80% homologous nucleotide sequence to the conserved
part-regions of the nucleotide sequences shown in SEQ ID NO: 1, SEQ
ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO:
13. These conserved part-regions are, in particular, from
nucleotide 139-186, nucleotide 256-312, nucleotide 328-360,
nucleotide 379-420 and/or nucleotide 424-444 in the nucleotide
sequence shown in SEQ ID NO: 1.
[0021] In a particularly preferred embodiment, the DNA sequence
according to the invention has an at least 80% homologous, in
particular an at least 90% homologous, nucleotide sequence to the
part-regions
[0022] (a) nucleotide 139-155 and/or
[0023] (b) nucleotide 625-644
[0024] of the nucleotide sequence shown in SEQ ID NO: 1.
[0025] Oligonucleotides derived from the above sequence regions
have proved suitable as primers for PCR amplification of isomerase
fragments from the genomic DNA of a large number of tested
microorganisms, for example Protaminobacter rubrum (CBS 547.77),
Erwinia rhapontici (NCPPB 1578), isolate SZ 62 and Pseudomonas
mesoacidophila MX-45 (FERM 11808).
[0026] Particularly preferably used for this purpose are the
following oligonucleotides, where appropriate in the form of
mixtures, where the bases in parentheses can be present as
alternatives:
[0027] Oligonucleotide I (17 nt):
1 5-TGGTGGAA(A,G)GA(G,A)GCTGT-3' (SEQ ID NO:17)
[0028] Oligonucleotide II (20 nt):
2 5'-TCCCAGTTCAG(G,A)TCCGGCTG-3' (SEQ ID NO:18)
[0029] Oligonucleotide I is derived from nucleotides 139-155 of SEQ
ID NO: 1, and oligonucleotide II is derived from the sequence,
complementary to nucleotides 625-644, of SEQ ID NO: 1. The
differences between the homologous part-regions of the DNA
sequences according to the invention and the sequences called
oligonucleotide I and oligonucleotide II are preferably in each
case not more than 2 nucleotides and particularly preferably in
each case not more than 1 nucleotide.
[0030] In another particularly preferred embodiment of the present
invention, the DNA sequence has an at least 80% homologous, in
particular an at least 90% homologous, nucleotide sequence to the
part-regions of
[0031] (c) nucleotide 995-1013 and/or
[0032] (d) nucleotide 1078-1094
[0033] of the nucleotide sequence shown in SEQ ID NO: 1.
[0034] Oligonucleotides derived from the above sequence regions
hybridize with sucrose isomerase genes from the organisms
Protaminobacter rubrum and Erwinia rhapontici. The following
oligonucleotides, where appropriate in the form of mixtures, are
particularly preferably used, where the bases indicated in
parentheses may be present as alternatives:
[0035] Oligonucleotide III (19 nt):
3 AAAGATGGCG(G,T)CGAAAAGA (SEQ ID NO:19)
[0036] Oligonucleotide IV (17 nt):
4 5'-TGGAATGCCTT(T,C)TTCTT-3' (SEQ ID NO:20)
[0037] Oligonucleotide III is derived from nucleotides 995-1013 of
SEQ ID NO: 1, and oligonucleotide IV is derived from nucleotides
1078-1094 of SEQ ID NO: 1. The differences between the homologous
part-regions of the DNA sequences according to the invention and
the sequences called oligonucleotide III and IV are preferably in
each case not more than 2 nucleotides and particularly preferably
in each case not more than 1 nucleotide.
[0038] Nucleotide sequences according to the invention can be
obtained in particular from microorganisms of the genera
Protaminobacter, Erwinia, Serratia, Leuconostoc, Pseudomonas,
Agrobacterium and Klebsiella. Specific examples of such
microorganisms are Protoaminobacter rubrum (CBS 547.77), Erwinia
rhapontici (NCPPB 1578), Serratia plymuthica (ATCC 15928), Serratia
marcescens (NCIB 8285), Leuconostoc mesenteroides NRRL B-521f (ATCC
10830a), Pseudomonas mesoacidophila MX-45 (FERM 11808 or FERM BP
3619), Agrobacterium radiobacter MX-232 (FERM 12397 or FERM BP
3620), Klebsiella subspecies and Enterobacter species. The
nucleotide sequences according to the invention can be isolated in
a simple manner from the genome of the relevant microorganisms, for
example using oligonucleotides from one or more of the conserved
regions of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 9,
SEQ ID NO. 11 and SEQ ID NO. 13, by standard techniques of
amplification and/or hybridization, and be characterized. The
nucleotide sequences according to the invention are preferably
obtained by PCR amplification of the genomic DNA of the relevant
organism using oligonucleotides I and II. A part-fragment of the
relevant sucrose isomerase gene is obtained in this way and can
subsequently be used as hybridization probe for isolating the
complete gene from a gene bank of the relevant microorganism.
Alternatively, the nucleotide sequences can be obtained by
producing a gene bank from the particular organism and direct
screening of this gene bank with oligonucleotides I, II, III and/or
IV.
[0039] The present invention further relates to a vector which
contains at least one copy of a DNA sequence according to the
invention. This vector can be any prokaryotic or eukaryotic vector
on which the DNA sequence according to the invention is preferably
under the control of an expression signal (promoter, operator,
enhancer, etc.). Examples of prokaryotic vectors are chromosomal
vectors such as, for example, bacteriophages (for example
bacteriophage .lambda.) and extrachromosomal vectors such as, for
example, plasmids, with circular plasmid vectors being particularly
preferred. Suitable prokaryotic vectors are described, for example,
in Sambrook et al., supra, Chapters 1-4.
[0040] A particularly preferred example of a vector according to
the invention is the plasmid pHWS 88 which harbors a sucrose
isomerase gene from Protaminobacter rubrum (with the sequence shown
in SEQ ID NO: 1 ) under the control of the regulatable tac
promoter. The plasmid pHWS 88 was deposited on Dec. 16, 1993, at
the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM),
Mascheroder Weg 1b, 38124 Braunschweig, Germany, under the deposit
number DSM 8824 in accordance with the provisions of the Budapest
Treaty.
[0041] Two further preferred examples of a vector according to the
invention are the plasmids pHWG314 and pHWG315, which harbor a
sucrose isomerase gene from Protaminobacter rubrum and Pseudomonas
mesoacidophila MX-45, respectively, under the control of a
regulatable rhamnose promoter. This promoter is described in Wiese
et al. (Arch. Microbiol. 176 (2001):187-196) and Wiese et al.
(Appl. Microbiol. 55 (2001), 750-757).
[0042] In another preferred embodiment of the present invention,
the vector according to the invention is a plasmid which is present
in the host cell with a copy number of less than 10, particularly
preferably with a copy number of 1 to 2 copies per host cell.
Examples of vectors of this type are, on the one hand, chromosomal
vectors such as, for example, bacteriophage .lambda. or F plasmids.
F plasmids which contain the sucrose isomerase gene can be
prepared, for example, by transformation of an E. coli strain which
contains an F plasmid with a transposon containing the sucrose
isomerase gene, and subsequent selection for recombinant cells in
which the transposon has integrated into the F plasmid. One example
of a recombinant transposon of this type is the plasmid pHWS 118
which contains the transposon Tn 1721 Tet and was prepared by
cloning a DNA fragment containing the sucrose isomerase gene from
the above-described plasmid PHWS 88 into the transposon pJOE 105
(DSM 8825).
[0043] On the other hand, the vector according to the invention can
also be a eukaryotic vector, for example a yeast vector (for
example YIp, YEp, etc.) or a vector suitable for higher cells (for
example a plasmid vector, viral vector, plant vector). Vectors of
these types are familiar to the person skilled in the area of
molecular biology so that details thereof need not be given here.
Reference is made in this connection in particular to Sambrook et
al., supra, Chapter 16.
[0044] The present invention further relates to a cell which is
transformed with a DNA sequence according to the invention or a
vector according to the invention. In one embodiment, this cell is
a prokaryotic cell, preferably a Gram-negative prokaryotic cell,
particularly preferably an enterobacterial cell. It is moreover
possible on the one hand to use a cell which contains no sucrose
isomerase gene of its own, such as, for example, E. coli, but it is
also possible, on the other hand, to use cells which already
contain such a gene on their chromosome, for example the
microorganisms mentioned above as source of sucrose isomerase
genes. Preferred examples of suitable prokaryotic cells are E.
coli, Protaminobacter rubrum or Erwinia rhapontici cells. The
transformation of prokaryotic cells with exogenous nucleic acid
sequences is familiar to a person skilled in the area of molecular
biology (see, for example, Sambrook et al., supra, Chapter
1-4).
[0045] In another embodiment of the present invention, the cell
according to the invention may, however, also be a eukaryotic cell
such as, for example, a fungal cell (for example yeast), an animal
or a plant cell. Methods for the transformation or transfection of
eukaryotic cells with exogenous nucleic acid sequences are likewise
familiar to the person skilled in the area of molecular biology and
need not be explained here in detail (see, for example, Sambrook et
al., Chapter 16).
[0046] The invention also relates to a protein with a sucrose
isomerase activity as defined above, which is encoded by a DNA
sequence according to the invention. This protein preferably
comprises
[0047] (a) one of the amino-acid sequences shown in SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID
NO: 14, where appropriate without the signal peptide region or
[0048] (b) an amino-acid sequence which is at least 80% homologous
with the sequences from (a).
[0049] The amino-acid sequence shown in SEQ ID NO: 4 comprises the
complete sucrose isomerase from Protaminobacter rubrum. The signal
peptide extends from amino acid 1-33. The mature protein starts at
amino acid 34. The amino-acid sequence shown in SEQ ID NO: 5
comprises the N-terminal section of the sucrose isomerase from
Erwinia rhapontici. The signal peptide extends from amino acid
1-36. The mature protein starts at amino acid 37. The amino-acid *
sequence shown in SEQ ID NO: 6 comprises a section of the sucrose
isomerase from the microorganism SZ 62. FIG. 1 compares the
amino-acid sequences of the isomerases from P. rubrum, E.
rhapontici and SZ 62.
[0050] Amino-acid sequence SEQ ID NO: 10 comprises a variant of the
isomerase from P. rubrum. Amino-acid sequence SEQ ID NO: 12
comprises the complete isomerase from SZ 62. This enzyme has a high
activity at 37.degree. C. and produces only a very small proportion
of monosaccharides. Amino-acid sequence SEQ ID NO: 14 comprises a
large part of the isomerase from MX-45. This enzyme produces about
85% trehalulose and 13% palatinose.
[0051] The protein according to the invention particularly
preferably has an at least 90% homologous amino-acid sequence to
conserved part-regions from the amino-acid sequences shown in SEQ
ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12
or SEQ ID NO: 14, especially in part-regions from
[0052] (a) amino acid 51-149,
[0053] (b) amino acid 168-181,
[0054] (c) amino acid 199-250,
[0055] (d) amino acid 351-387 and/or
[0056] (e) amino acid 390-420
[0057] of the amino-acid sequence shown in SEQ ID NO: 4.
[0058] It is possible by means of the above mentioned DNA
sequences, vectors, transformed cells and proteins to provide a
sucrose isomerase activity in a simple manner without interfering
additional enzymatic activities. Preferably, the sucrose isomerase
activity is >30 units/mg and more preferably >45 units/mg.
Most preferably the sucrose isomerase activity lies in the range of
from 45 units/mg to 150 units/mg.
[0059] It is possible for this purpose on the one hand to obtain
the sucrose isomerase by recombinant DNA technology as constituent
of an extract from the host organism or in isolated and purified
form (for example by expression in E. coli). This preferably
purified and isolated sucrose isomerase enzyme can be used, for
example, in immobilized form, for the industrial production of
acariogenic sugars such as, for example, trehalulose and/or
palatinose by reaction of sucrose in an enzyme reactor. The
immobilization of enzymes is familiar to a skilled person and need
not be described in detail here.
[0060] On the other hand, the production of acariogenic sugars from
sucrose can also take place in a complete microorganism, preferably
in immobilized form. Cloning of the abovementioned sucrose
isomerase gene into an organism without or with reduced palatinose
and/or trehalulose metabolism (that is to say in an organism which
is unable significantly to degrade the abovementioned sugars)
allows generation of a novel organism which, owing to the
introduction of exogenous DNA, is able to produce acariogenic
disaccharides with negligible formation of monosaccharides. Thus,
suitable for introducing the sucrose isomerase gene is, on the one
hand, an organism which is unable to utilize palatinose and/or
trehalulose (for example E. coli, bacillus, yeast) and, on the
other hand, an organism which would in principle be able to utilize
palatinose and/or trehalulose but has reduced palatinose and/or
trehalulose metabolism owing to undirected or directed
mutation.
[0061] The term "reduced palatinose and/or trehalulose metabolism"
means for the purpose of the present invention that a whole cell of
the relevant organism produces, on utilization of sucrose as C
source, acariogenic disaccharides but is able to utilize the latter
to only a small extent in metabolism, for example by degrading them
to monosaccharides. The organism preferably produces less than
2.5%, particularly preferably less than 2%, most preferably less
than 1%, of glucose plus fructose based-on the total of acariogenic
disaccharides and monosaccharide degradation products at a
temperature of 15-65.degree. C., in particular of 25-55.degree.
C.
[0062] The present invention thus further relates to a cell which
contains at least one DNA sequence coding for a protein with a
sucrose isomerase activity, and has a reduced palatinose and/or
trehalulose metabolism as defined above. Preferably the cell
according to the invention exhibits such a sucrose isomerase
expression rate that the amount of sucrose isomerase expressed in
the cell is >10%, preferably >15% and particularly >25% of
the total amount of proteins of the cell. A cell of this type
produces larger proportions of the non-cariogenic disaccharides
trehalulose and/or palatinose and reduced amounts of the
interfering byproducts glucose and fructose.
[0063] It is possible in one embodiment of the present invention to
reduce the palatinose and/or trehalulose metabolism by partial or
complete inhibition of the expression of invertase and/or
palatinase genes which are responsible for the intracellular
degradation of palatinose and/or trehalulose. This inhibition of
gene expression can take place, for example, by site-directed
mutagenesis and/or deletion of the relevant genes. A site-directed
mutation of the palatinase gene shown in SEQ ID NO: 7 or of-the
palatinose hydrolase gene shown in SEQ ID NO: 15 can take place,
for example, by introduction of a vector which is suitable for
homologous chromosomal recombination and which harbors a mutated
palatinase gene, and selection for organisms in which such a
recombination has taken place. The principle of selection by
genetic recombination is explained in E. L. Winnacker, Gene und
Klone, Eine Einfuhrung in die Gentechnologie (1985),
VCH-Verlagsgesellschaft Weinheim, Germany, pp. 320 et seq.
[0064] It is furthermore possible to obtain organisms according to
the invention with reduced palatinose and/or trehalulose metabolism
by non-specific mutagenesis from suitable starting organisms and
selection for palatinase-deficient mutants. One example of a
palatinase-deficient mutant of this type is the Protaminobacter
rubrum strain SZZ 13 which was deposited on Mar. 29, 1994, at the
Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM),
Mascheroder Weg 1b, 38124 Braunschweig, Germany, under deposit
number DSM 9121 in accordance with the provisions of the Budapest
Treaty. This microorganism was prepared by non-specific mutagenesis
of P. rubrum wild-type cells with N-methyl-N'-nitro-N-nitroso-
guanidine and is distinguished in that it is no longer able to
cleave the non-cariogenic sugars trehalulose and palatinose to
glucose and fructose. Selection for such mutants can take place,
for example, by using MacConkey palatinose media or minimal salt
media with palatinose or glucose as sole C source. The mutants
which are white on MacConkey palatinose medium (MacConkey Agar Base
from Difco Laboratories, Detroit, Mich., USA (40 g/l) and 20 g/l
palatinose) or which grow on minimal salt media with glucose as
sole C source but not on corresponding media with palatinose as
sole C source are identified as palatinase-deficient mutants.
[0065] The present invention furthermore relates to a method for
isolating nucleic acid sequences which code for a protein with a
sucrose isomerase activity, wherein a gene bank from a donor
organism which contains a DNA sequence coding for a protein with a
sucrose isomerase activity is set up in a suitable host organism,
the clones of the gene bank are examined, and the clones which
contain a nucleic acid coding for a protein with sucrose isomerase
activity are isolated. The nucleic acids which are isolated in this
way and code for sucrose isomerase can in turn be used for
introduction into cells as described above in order to provide
novel producer organisms of acariogenic sugars.
[0066] In this method, the chosen host organism is preferably an
organism which has no functional genes of its own for palatinose
metabolism, in particular no functional palatinase and/or invertase
genes. A preferred host organism is E. coli. To facilitate
characterization of palatinose-producing clones it is possible on
examination of the clones in the gene bank for sucrose-cleaving
clones and the DNA sequences which are contained therein and
originate from the donor organism to be isolated and transformed in
an E. coli strain which does not utilize galactose and which is
used as screening strain for the clones in the gene bank.
[0067] On the other hand, the examination of the clones in the gene
bank for DNA sequences which code for a protein with a sucrose
isomerase activity can also take place using nucleic acid probes
derived from the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
3, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 which code for the
sucrose isomerase genes from Protaminobacter rubrum, Erwinia
rhapontici and the isolate SZ 62. A DNA fragment obtained by PCR
reaction with oligonucleotides I and II as primers, or the
oligonucleotides III and/or IV, are particularly preferably used as
probes.
[0068] The present invention further relates to a process for the
production of non-cariogenic sugars, in particular trehalulose
and/or palatinose, which comprises using for the production of the
sugars
[0069] (a) a protein with sucrose isomerase activity in isolated
form,
[0070] (b) an organism which is transformed with a DNA sequence
which codes for protein with sucrose isomerase activity, or with a
vector which contains at least one copy of this DNA sequence,
[0071] (c) an organism which contains at least one DNA sequence
coding for a protein with a sucrose isomerase activity, and has a
reduced palatinose and/or trehalulose metabolism, and/or
[0072] (d) an extract from such a cell or from such an
organism.
[0073] The process is generally carried out by contacting the
protein, the organism or the extract in a suitable medium with
sucrose under conditions such that the sucrose is at least partly
converted by the sucrose isomerase into acariogenic disaccharides.
Subsequently, the acariogenic disaccharides are obtained from the
medium or the organism and purified in a known manner.
[0074] In a preferred embodiment of this process, the organism, the
protein or the extract is used in immobilized form. Proteins (in
pure form or in extracts) are preferably immobilized by coupling of
reactive side groups (for example NH.sub.2 groups) to a suitable
carrier. Immobilization of cells takes place, for example, in a
sodium alginate/calcium chloride solution. A review of suitable
methods for immobilizing cells and proteins is given, for example,
in I. Chibata (Immobilized Enzymes, John Wiley and Sons, New York,
London, 1978).
[0075] It is possible on use of a cell transformed with the sucrose
isomerase gene to increase the rate of production of acariogenic
sugars by comparison with known organisms by increasing the number
of gene copies in the cell and/or by increasing the expression rate
in a combination with strong promoters. It is furthermore possible
by transformation of a cell which is unable or able to only a
limited extent to utilize acariogenic sugars with the sucrose
isomerase gene to produce a transformed cell with whose aid it is
possible to obtain acariogenic sugars, in particular palatinose
and/or trehalulose, without or with fewer byproducts.
[0076] On use of a microorganism with reduced palatinose and/or
trehalulose metabolism, which already contains a functional sucrose
isomerase gene, transformation with an exogenous sucrose isomerase
gene is not essential but may be carried out to improve the
yields.
[0077] Finally, the present invention also relates to a DNA
sequence which codes for a protein with palatinase or palatinose
hydrolase activity and comprises
[0078] (a) one of the nucleotide sequences shown in SEQ ID NO: 7 or
SEQ ID NO: 15,
[0079] (b) a nucleotide sequence which corresponds to the sequence
from (a) within the scope of the degeneracy of the genetic code
or
[0080] (c) a nucleotide sequence which hybridizes with the
sequences from (a) and/or (b).
[0081] The invention further relates to a vector which contains at
least one copy of the above mentioned DNA sequence and to a cell
which is transformed with a DNA sequence or a vector as mentioned
above. The invention likewise embraces a protein with palatinase
activity which is encoded by a DNA sequence as indicated above and
which preferably has one of the amino-acid sequences shown in SEQ
ID NO: 8 or SEQ ID NO: 16.
[0082] The palatinase from P. rubrum shown in SEQ ID NO: 8 differs
from known sucrose-cleaving enzymes in that it cleaves the sucrose
isomers which are not cleaved by known enzymes, in particular
palatinose.
[0083] The amino acid sequence shown in SEQ ID NO: 16 comprises a
palatinose hydrolase from MX-45, which cleaves palatinose to form
fructose and glucose. The gene-coding for this enzyme is shown in
SEQ ID NO: 15 and is located in the genome of MX-45 on the 5' side
of the isomerase gene shown in SEQ ID NO: 13.
[0084] The invention is further described by the following sequence
listings and figures:
[0085] SEQ ID NO: 1 shows the nucleotide sequence of the gene
coding for the sucrose isomerase from Protaminobacter rubrum. The
sequence coding for the signal peptide terminates at nucleotide No.
99.
[0086] SEQ ID NO: 2 shows the N-terminal section of the nucleotide
sequence of the gene coding for the sucrose isomerase of Erwinia
rhapontici. The sequence coding for the signal peptide terminates
at the nucleotide with No. 108.
[0087] SEQ ID NO: 3 shows a section of the nucleotide sequence of
the gene coding for the sucrose isomerase from the isolate SZ
62.
[0088] SEQ ID NO: 4 shows the amino-acid sequence of the sucrose
isomerase from Protaminobacter rubrum.
[0089] SEQ ID NO: 5 shows the N-terminal section of the amino-acid
sequence of the sucrose isomerase from Erwinia rhapontici.
[0090] SEQ ID NO: 6 shows a section of the amino-acid sequence of
the sucrose isomerase from the isolate SZ 62.
[0091] SEQ ID NO: 7 shows the nucleotide sequence for the
palatinase gene from Protaminobacter rubrum.
[0092] SEQ ID NO: 8 shows the amino-acid sequence of the palatinase
from Protaminobacter rubrum.
[0093] SEQ ID NO: 9 shows the nucleotide sequence of a variant of
the sucrose isomerase gene from P. rubrum.
[0094] SEQ ID NO: 10 shows the corresponding amino-acid
sequence.
[0095] SEQ ID NO: 11 shows the complete nucleotide sequence of the
sucrose isomerase gene from SZ 62.
[0096] SEQ ID NO: 12 shows the corresponding amino-acid
sequence.
[0097] SEQ ID NO: 13 shows most of the sucrose isomerase gene from
Pseudomonas mesoacidophila (MX-45).
[0098] SEQ ID NO: 14 shows the corresponding amino acid
sequence.
[0099] SEQ ID NO: 15 shows the palatinose hydrolase gene from
Pseudomonas mesoacidophila (MX-45).
[0100] SEQ ID NO: 16 shows the corresponding amino-acid
sequence.
[0101] FIG. 1 shows a comparison of the amino-acid sequences of the
sucrose isomerases from Protaminobacter rubrum, Erwinia rhapontici
and the isolate SZ 62,
[0102] FIG. 2 shows the cloning diagram for the preparation of the
recombinant plasmid pHWS 118 which contains the sucrose isomerase
gene on the transposon Tn 1721,
[0103] FIG. 3 shows the diagram for the preparation of E. coli
transconjugants which contain the sucrose isomerase gene of a F
plasmid and
[0104] FIG. 4 shows a comparison between the saccharides produced
by P. rubrum wild-type cells and cells of the P. rubrum mutant SZZ
13.
[0105] FIG. 5 shows plasmid pHWG314.
[0106] FIG. 6 shows plasmid pHWG315.
[0107] The following examples serve to illustrate the present
invention.
EXAMPLE 1
[0108] Isolation of the Sucrose Isomerase Gene from Protaminobacter
rubrum
[0109] Complete DNA from the organism Protaminobacter rubrum (CBS
574.77) was partially digested with Sau3A I. Collections of
fragments with a size of about 10 kBp were obtained from the
resulting fragment mixture by elution after fractionation by gel
electrophoresis and were ligated into a derivative, which had been
opened with BamHI, of the lambda EMBL4 vector derivative .lambda.
RESII (J. Altenbuchner, Gene 123 (1993), 63-68). A gene bank was
produced by transfection of E. coli and transformation of the
phages into plasmids according to the above reference. Screening of
the kanamycin-resistant colonies in this gene bank was carried out
with the radiolabeled oligonucleotide S214 which was derived from
the sequence of the N-terminus of the mature isomerase by
hybridization:
5 S214: 5'-ATCCCGAAGTGGTGGAAGGAGGC-3' (SEQ ID NO:21) T A A A A
[0110] Subsequently, the plasmid DNA was isolated from the colonies
with a positive reaction after appropriate cultivation. After a
restriction map had been drawn up, suitable subfragments were
sequenced from a plasmid pKAT 01 obtained in this way, and thus the
complete nucleotide sequence, which is shown in SEQ ID NO: 1, of
the DNA coding for isomerase was obtained. The amino-acid sequence
derived therefrom corresponds completely to the peptide sequence of
the mature isomerase obtained by sequencing (Edmann degradation). A
cleavage site for SacI is located in the non-coding 3' region of
this isomerase gene, and a cleavage site for HindIII is located in
the non-coding 5' region. This makes it possible to subclone the
intact isomerase gene into the vector pUCBM 21 (derivative of the
vector pUC 12, Boehringer Mannheim GmbH, Mannheim, Germany) which
had previously been cleaved with the said enzymes. The resulting
plasmid was called pHWS 34.2 and confers on the E. coli cells
harboring it the ability to synthesize sucrose isomerase.
[0111] A variant of the sucrose isomerase gene from P. rubrum has
the nucleotide sequence shown in SEQ ID NO: 9.
EXAMPLE 2
[0112] Cloning and Expression of the Sucrose Isomerase from P.
rubrum in E. coli
[0113] 1. Preparation of the Plasmid pHWS88
[0114] The non-coding 5' region of the sucrose isomerase gene was
deleted from the plasmid pHWS 34.2, using an oligonucleotide S434
with the sequence 5'-CGGMTTCTTATGCCCCGTCAAGGA-3' (SEQ ID NO: 22),
with simultaneous introduction of an EcoRI cleavage site (GAATTC).
The isomerase gene derivative obtained in this way was treated with
BstE II, the protruding BstE II end was digested off with S1
nuclease and subsequently digestion with EcoRI was carried out. The
isomerase gene treated in this way was cloned into the vector
pBTacI (Boehringer Mannheim GmbH, Mannheim, Germany) which had been
pretreated with EcoRI and SmaI. The resulting vector PHWS 88 (DSM
8824) contains the modified isomerase gene with a preceding EcoRI
restriction site in front of the ATG start codon, and the 3' region
of the isomerase gene up to the S1-truncated BstE II cleavage site.
On induction with IPTG, this vector confers on the cells harboring
this plasmid the ability to produce isomerase and resistance to
ampicillin (50 to 100 .mu.g/ml). Preferably used for producing
isomerase are E. coli host cells which overproduce the lac
repressor.
[0115] 2. Preparation of the Plasmid pHWS118::Tn1721Tet
[0116] The gene cassette for the sucrose mutase was incorporated
into a transposon.
[0117] This took place by cloning an SphI/HindIII DNA fragment from
the plasmid pHWS88, which harbors the sucrose mutase gene under the
control of the tac promoter, into the plasmid pJOE105 on which the
transposon Tn 1721 is located. The plasmid pJOE105 was deposited on
Dec. 16, 1993, at the DSM under the deposit number DSM 8825 in
accordance with the provisions of the Budapest Treaty. The
resulting plasmid pHWS118, on which the sucrose mutase gene is
under the control of the regulatable tac promoter, was used to
transform a E. coli strain containing an F' plasmid. FIG. 2 shows
the cloning diagram for the preparation of pHWS 118 from pHWS88 and
pJOE 105.
[0118] E. coli transconjugants containing the sucrose mutase gene
were prepared as described in the diagram in FIG. 3. For this
purpose, firstly the F'-harboring E. coli strain CSH36 (J. H.
Miller, Experiments in Molecular Genetics, Cold Spring Harbor
Laboratory (1972), p. 18), which carries the Lac+ phenotype
mediated by the F' plasmid, was crossed with the E. coli strain
JM108 which is resistant to nalidixic acid (Sambrook et al., supra,
p. A9-A13). Selection on minimal medium to which lactose, proline
and nalidixic acid were added resulted in an F'-Lac-harboring
transconjugant. This was additionally transformed with the Iq
plasmid FDX500 (Brinkmann et al., Gene 85 (1989), 109-114) in order
to permit control of the sucrose mutase gene by the tac
promoter.
[0119] The transconjugant prepared in this way was transformed with
the transposon plasmid pHWS118 harboring the sucrose mutase gene.
For selection of transconjugants, crossing into the
streptomycin-resistant E. coli strain HB101 (Boyer and
Roulland-Dussoix, J. Mol. Biol 41 (1969), 459472) was carried out.
Transfer of the tetracycline resistance mediated by the transposon
was possible only after transposition of the modified Tn1721Tet
from the plasmid pHWS118, which is not capable of conjugation or
mobilization, to the F' plasmid which is capable of conjugation.
Transmission of the F' plasmid with the modified transposon in
HB101 was selected on LB plates containing streptomycin and
tetracycline, and retested on ampicillin and nalidixic acid
plates.
[0120] 3. Expression of the Sucrose Isomerase in E. coli
[0121] Examination of the enzyme production by such F'
plasmid-harboring E. coli cells showed that it was possible to
produce sucrose mutase protein. F' plasmid-containing HB101 cells
which harbored no additional Lac repressor plasmid (for example
K1/1 or K1/10) produced sucrose mutase protein in identical amounts
with and without the inducer isopropyl .beta.-D-thiogalactoside
(IPTG). The productivities of three transconjugants K1/1, K1/10 and
K1/4 are shown in Table 1.
[0122] It was possible to observe normal growth of the E. coli
cells during production of sucrose mutase protein.
[0123] Introduction of the sucrose mutase gene into the F plasmid
in the presence of the repressor-encoded plasmid pFDX500 (see
transconjugants K1/4) made it possible to control enzyme production
with the inducer IPTG. Whereas no enzymatic activity was measured
without IPTG, production of about 1.6 U/mg sucrose mutase protein
was obtainable after induction for 4 hours.
[0124] No adverse effect on cell growth was observable. The
plasmid-harboring E. coli cells reached a density of about 3
OD.sub.600 after induction for 4 hours.
[0125] Up to 1.6 U/mg sucrose mutase activity were measured in
transformed E. coli. The synthetic performance is comparable to
that of P. rubrum. Analysis of the produced enzyme by SDS gel
electrophoresis provides no evidence of inactive protein
aggregates. The band of the sucrose mutase protein was only weakly
visible with Coomassie staining and was detectable clearly only in
a Western blot. It was possible to correlate the strength of the
protein band and the measured enzymatic activity in the production
of sucrose mutase in E. coli.
EXAMPLE 3
[0126] Isolation of the sucrose isomerase gene from Erwinia
rhapontici
[0127] A gene bank was produced by restriction cleavage of the
complete DNA from Erwinia rhapontici (NCPPB 1578) in the same way
as described in Example 1.
[0128] Using the primer mixtures
6 5'-TGGTGGAAAGAAGCTGT-3' (SEQ ID NO:23) G G
[0129] and
7 5'-TCCCAGTTCAGGTCCGGCTG-3' (SEQ ID NO:24), A
[0130] PCR amplification resulted in a DNA fragment with whose aid
it is possible to identify colonies containing the mutase gene by
hybridization.
[0131] In this way, a positive clone pSST2023 which contains a
fragment, 1305 nucleotides long, of the Erwinia isomerase gene was
found. The nucleotide sequence of this fragment is depicted in SEQ
ID NO: 2.
[0132] Sequence comparison with the Protaminobacter gene reveals an
identity of 77.7% and a similarity of 78% for the complete gene
section including the signal peptide region, and an identity of
83.4% and a similarity of 90.3% at the amino-acid level.
[0133] The sequence differences are mainly concentrated in the
signal peptide region. For this reason, only the enzyme-encoding
region responsible for the actual mutase activity, without the
signal peptide, should be considered for comparison. From these
viewpoints, the identity or similarity at the nucleotide level
emerges as 79%. Comparison of the amino-acid sequences (FIG. 1) in
this section shows 87.9% identical amino acids. Of 398 amino acids
(this corresponds to 71% of the complete enzyme) in the Erwinia
mutase, 349 are the same as in Protaminobacter. 25 of 48 exchanged
amino acids show strong similarity so that the overall similarity
at the AA level emerges as 94%. The M exchanges are mainly
concentrated in the region between amino acid 141 and 198. In front
of this region there is a sequence of 56 conserved amino acids.
Other sections also exhibit particularly high conservation (see
FIG. 1).
[0134] These data show that, for the section cloned and sequenced
to date, overall there is very extensive conservation of the two
mutases from Erwinia and Protaminobacter.
[0135] Identity of the Cloned Mutase Gene from Erwinia
[0136] The probe chosen for a rehybridization experiment with
genomic Erwinia DNA was the SspI/EcoRI fragment, which is about 500
bp in size, from pSST2023. This fragment was used, after
digoxigenin labeling, for hybridization with Erwinia DNA with high
stringency (68.degree. C.). Complete Erwinia DNA cut with
SspI/EcoRI showed a clear hybridization signal with the expected
size of about 500 bp. Erwinia DNA cut only with SspI showed a
hybridization signal of about 2 kb.
[0137] It was possible to verify by the successful rehybridization
of pSST2023 with genomic Erwinia DNA that the mutase region cloned
into pSST2023 originates from Erwinia rhapontici.
[0138] Cloning of the C-Terminal Part-Fragment of the Erwinia
Mutase
[0139] The N-terminal part-fragment of the Erwinia mutase gene
which has been cloned to date has a size of 1.3 kb and has the
nucleotide sequence shown in SEQ ID NO: 2. Since it can be assumed
that the complete Erwinia gene is virtually identical in size to
the known Protaminobacter gene (1.8 kb), a section of about 500 bp
is missing from the C-terminal region of the Erwinia gene.
[0140] The SspI fragment which is about 2 kb in size from the
complete Erwinia DNA was selected for cloning of the Erwinia
C-terminus. In a Southern blot, this fragment provides a clear
signal with a digoxigenin-labeled DNA probe from pSST2023. This 2
kb SspI fragment overlaps by about 500 bp at the 3' end with the
region already cloned in pSST2023. Its size ought to be sufficient
for complete cloning of the missing gene section of about 500 bp.
The digoxigenin-labeled fragment probe SspI/EcoRI from pSST2023 is
suitable for identifying clones which are sought.
EXAMPLE 4
[0141] Preparation of a Protaminobacter Palatinase-Deficient
Mutant
[0142] Cells of Protoaminobacter rubrum (CBS 547, 77) were
mutagenized with N-methyl-N'-nitro-N-nitroso-guanidine by the
method of Adelberg et al. (Biochem. Biophys. Research Commun. 18
(1965), 788) as modified by Miller, J., (Experiments in Molecular
Genetics, Cold Spring Harbor Laboratory, 125-179 (1972)).
Palatinase-deficient mutants were selected using MacConkey
palatinose medium (MacConkey Agar Base (Difco Laboratories,
Detroit, Mich., USA), 40 g/l with the addition of 20 g/l
palatinose, sterilized by filtration, 25 mg/l kanamycin) and
minimal salt media (10.5 g of K.sub.2HPO.sub.4, 4.5 g of
KH.sub.2PO.sub.4, 1 g of (NH.sub.4).sub.2SO.sub.4, 0.5 g of sodium
citrate 2 H.sub.2O, 0.1 g of MgSO.sub.4.7H.sub.2O), 1 mg of
thiamine, 2 g of palatinose or glucose, 25 mg of kanamycin and 15 g
of agar per liter, pH 7.2). Mutants of P. rubrum which are white on
MacConkey palatinose medium or grow on minimal salt medium with
glucose in contrast to the same medium with palatinose are
identified as palatinase-deficient mutants. The enzyme activity of
cleaving palatinose to glucose and fructose (palatinase activity)
cannot, in contrast to the wild-type, be detected in cell extracts
from these mutants. On cultivation of these cells in minimal salt
medium with 0.2% sucrose as sole C source there is, in contrast to
the wild-type cells in which palatinose can be detected only
transiently in the time from 4 to 11 hours after starting the
culture, a detectable continuous accumulation of palatinose
(isomaltulose). Overnight cultures in the same medium contain no
palatinose in the case of the wild-type cells but contain >0.08%
palatinose in the case of the mutant SZZ 13 (DSM 9121) prepared in
this way (see FIG. 4).
EXAMPLE 5
[0143] Immobilization of Microorganism Cells
[0144] Cells are rinsed off a subculture of the appropriate strain
using 10 ml of a sterile nutrient substrate composed of 8 kg of
concentrated juice from a sugar factory (dry matter content=65%), 2
kg of corn steep liquor, 0.1 kg of (NH.sub.4).sub.2HPO.sub.4 and
89.9 kg of distilled water, pH 7.2. This suspension is used as
inoculum for preculture in 1 l flasks containing 200 ml of nutrient
solution of the above composition in shaking machines. After an
incubation time of 30 hours at 29.degree. C., 10 flasks (total
contents 2 l) are used to inoculate 18 l of nutrient solution~of
the above composition in a 30 l small fermenter, and fermentation
is carried out at 29.degree. C. and a stirring speed of 350 rpm
introducing 20 l of air per minute.
[0145] After organism counts above 5.times.10.sup.9 organisms per
ml are reached, the fermentation is stopped and the cells are
harvested from the fermenter solution by centrifugation. The cells
are then suspended in a 2% strength sodium alginate, solution and
immobilized by dropwise addition of the suspension to a 2% strength
calcium chloride solution. The resulting immobilizate beads are
washed with water and can be stored at +4.degree. C. for several
weeks.
[0146] Cells of the palatinase-deficient mutant SZZ 13 (DSM 9121)
show better catalytic properties in respect of their product
composition than do comparable cells from the known microorganisms
Protaminobacter rubrum (CBS 547.77) and Erwinia rhapontici (NCPPB
1578).
[0147] Whole cells and crude extracts of SZZ 13, and an
immobilizate of SZZ 13 in calcium alginate prepared as above, were
evaluated in respect of product composition in an activity assay.
Before the actual activity assay, the immobilizate was swollen in
0.1 mol/l potassium phosphate buffer, pH 6.5.
[0148] The activity measurements at 25.degree. C. revealed that no
fructose and glucose were found with the mutant SZZ 13, while with
P. rubrum wild-type cells 2.6% fructose and glucose (based on the
total of mono- and disaccharides) were found in whole cells and
12.0% were found in the crude extract. In the case of E.
rhapontici, 4% glucose and fructose were found in whole cells, and
41 % in the crude extract.
EXAMPLE 6
[0149] Isolation of the Sucrose Isomerase Gene from Other
Microorganisms
[0150] Partial digestion of genomic DNA from the isolate SZ62
(Enterobacter spec.), the organism Pseudomonas mesoacidophila
(MX-45) or from another microorganism and insertion of the
resulting fragments into suitable E. coli vectors and
transformation result in a gene bank whose clones contain genomic
sections between 2 and 15 kb of the donor organism.
[0151] Those E. coli cells which harbor these plasmids and which
display a red coloration of the colony are selected by plating on
McConkey palatinose medium. The plasmid DNA contained in these
cells is transferred into an E. coli mutant which is unable to grow
on galactose as sole C source (for example ED 8654, Sambrook et
al., supra, pages A9-A13).
[0152] This transformed cell line is able to identify palatinose
producers in the gene bank which has been prepared as described
above from DNA of the donor organism.
[0153] To identify the palatinose-producing clones which are
sought, the cells of the gene bank are isolated and cultured on
minimal salt media containing galactose and sucrose. After replica
plating of the colonies on plates containing the same medium, the
cells are killed by exposure to toluene vapor. Subsequently, cells
of the screening strain are spread as lawn in minimal salt soft
agar without added C source over the colonies of the gene bank and
incubated. Significant growth of the cells of the screening strain
appears only at the location of cells in the gene bank which have
produced palatinose. The isomerase content emerges on testing the
cells of the replica control.
[0154] These E. coli clones identified in this way are unable to
grow on palatinose as sole C source in the medium, show no ability
to cleave sucrose in a test on whole cells or on cell extracts, but
on cultivation under these conditions and without addition of
sucrose to the medium produce palatinose.
[0155] Alternatively, isomerase clones can also be identified using
a PCR fragment prepared by the procedure of Example 3.
[0156] Use of plasmid DNA from the E. coli clones identified in
this way as probes for hybridization on filters with immobilized
DNA from the donor organism allows the gene regions which harbor
isomerase genes to be detected and specifically made available.
[0157] A clone which contains the nucleotide sequence shown in SEQ
ID NO: 3, with the amino-acid sequence which, is derived therefrom
and shown in SEQ ID NO: 6, was identified in this way. In the same
way an isomerase clone from DNA of the bacterial strain Pseudomonas
mesoacidophila MX-45 (FERM 11808) was found.
[0158] The complete nucleotide sequence and amino-acid sequence of
the sucrose isomerase from SZ 62 are depicted in SEQ ID NO: 11 and
12. A large part of the nucleotide sequence and amino-acid sequence
of the sucrose isomerase from MX-45 are depicted in SEQ ID NO: 13
and 14.
EXAMPLE 7
[0159] Cloning of a Palatinase Gene
[0160] The Protaminobacter rubrum gene bank prepared in Example 1
was screened with the radiolabeled oligonucleotide mixture S433
which was derived from the sequence of the N-terminus of the
isolated palatinase and had the sequence
8 CA(G,A)TT(C,T)GG(T,C)TA(C,T)GG-3'. (SEQ ID NO:25)
[0161] A positive clone was found, and a plasmid named pKAT 203 was
isolated therefrom.
[0162] E. coli cells which harbor the plasmid pKAT 203 are able to
metabolize palatinose. The cleavage of palatinose to glucose and
fructose which is detectable in the activity assay suggests that
there is a "palatinase".
[0163] It is possible by sequencing pKAT203 DNA with the
oligonucleotide S433 as primer to obtain a DNA sequence from which
it was possible to read off, after translation into amino-acid
sequence data, the N-terminal amino acids known to us. An open
reading frame was obtained by a subsequent sequencing step.
[0164] Determination of the Sequence of the "Palatinase" Gene
[0165] For further sequencing of the "palatinase" gene,
part-fragments from the plasmid pKAT 203 were selected on the basis
of the restriction map and subcloned in the M13 phage system, and a
sequencing of the single-stranded phage DNA was carried out with
the universal primer 5'-GTTTTCCCAGTCACGAC-3' (SEQ ID NO: 26).
[0166] Combination of the resulting DNA sequence data for the
individual fragments taking account of overlapping regions allows a
continuous reading frame of 1360 base pairs to be determined for
the "palatinase" (SEQ ID NO: 7).
[0167] Translation of this DNA sequence into amino-acid data
reveals a protein with 453 amino acids (SEQ ID NO: 8) and a
molecular weight, which can be deduced therefrom, of about 50,000
Da. This is consistent with the finding that a protein fraction
which had a band at about 48,000 Da in the SDS gel was obtainable
by concentration of the "palatinase" activity. In the native gel,
the palatinose-cleaving activity was attributable to a band with a
size of about 150,000 Da.
[0168] Comparisons of Homology with Other Known Proteins
[0169] Comparison of the amino-acid sequence derivable from the DNA
sequence with data stored in a gene bank (SwissProt) revealed a
homology with melibiase from E. coli (MeIA) (in two parts: identity
32%).
EXAMPLE 8
[0170] Cloning of a Palatinose Hydrolase Gene from P.
mesoacidophila MX-45
[0171] A gene with the nucleotide sequence shown in SEQ ID NO: 15
was isolated from the gene bank prepared from the microorganism P.
mesoacidophila MX-45 in Example 6. This gene codes for a protein
with the amino-acid sequence shown in SEQ ID NO: 16. The protein is
a palatinose hydrolase which catalyzes the cleavage of palatinose
to form fructose and glucose.
EXAMPLE 9
[0172] Cloning and Expression of the Sucrose Isomerase from
Protaminobacter rubrum and Pseudomonas mesoacidophila MX-45 in
E.coli
[0173] 1. Preparation of the Plasmids pHWG314 and pHWG315
[0174] The plasmids were prepared by inserting the gene modules
into the vector pJOE2702 (Wiese et al. (2001) supra) digested with
NdeI/HindIII (314) and NdeI/BamHI (315), respectively. The plasmids
carry the entire sequence coding for each mutase. FIGS. 5 and 6
show the restriction maps of both plasmids. E. coli JM109 was used
as host.
[0175] 2. Expression of the Sucrose Isomerase in E.coli and
Isolation
[0176] The production of the enzyme was induced by adding 0.2%
rhamnose to the medium. The cells were harvested via centrifugation
and sonification. Standard conditions were applied, as described in
the other examples. For the purification from E. coli a
chromatography of the raw extract was performed using a cation
exchange chromatography column, e.g. a MonoS column (Pharmacia).
The material was loaded on the column in the presence of 10 mM
Ca-acetate, pH 6.5. The elution was carried out with a NaCl
gradient of from 0-100 mM. Then the fractions were tested for their
protein content. All results are contained in Table 2.
[0177] 3. Isolation of the Sucrose Isomerase from Protaminobacter
rubrum and Pseudomonas mesoacidophila MX-45
[0178] The enzymes from the (wild-type) strains Protaminobacter
rubrum and Pseudomonas mesoacidophila were not purified.
Purification according to the above protocol was successful only
regarding the enzymes produced in E. coli.
[0179] After cultivation in a suitable complete medium containing
2% sucrose the cells of the wild-type strains were harvested at
30.degree. C. and decomposed. Then the enzymatic activity and the
protein content were determined.
[0180] Surprisingly, the recombinant sucrose isomerase can be
separated by a one-step procedure from homologous proteins in the
E. coli extract exhibiting high yield and purity. Apparently the
recombinant protein has a different charge composition compared to
the isomerase from native organisms.
[0181] A comparison of the results shown in Table 2 shows that with
the help of the recombinant E.coli strains significantly higher
sucrose isomerase yields can be obtained than with the wild-type
strains Pseudomonas mesoacidophila MX-45 and Protominobacter
rubrum. The amount of recombinant sucrose isomerase expressed in
the E.coli strains is about 15.6 and 28.9%, respectively, of the
total amount of proteins in the cell. Contrary thereto, the amount
of sucrose isomerase formed in the wild-type strains Pseudomonas
mesoacidophila MX-45 and Protominobacter rubrum was so small that
it could not be detected by using conventional detection methods.
Furthermore, the activity of the recombinant sucrose isomerase is
about 10-times higher than the activity of the sucrose isomerase
formed in the respective wild-type strains. Consequently, the
protein isolated from the wild-type strains must have been
inactivated to a great extent when isolated from the wild-type
strains.
9TABLE 1 Sucrose mutase activity in E. coli HB101 (F'::Tn1721
[Mutase]) U/mg mutase after 4 hours U/mg mutase after 4 hours
Strain without induction induction with 50 .mu.M IPTG K1/1 1.0 1.2
K1/10 0.9 1.1 K1/4 0 1.6
[0182]
10TABLE 2 The results obtained under items 3 and 4 of example 9 are
summarized in the following table: Expression in E. coli Culture mg
U in pure U in (plasmid) Wild-type strain Gene Vol. (ml) OD.sub.600
U ml.sup.-1 ml.sup.-1 mg ml.sup.-1 U (total) mg.sup.-1 % cell
protein P. mesoacido- mutB 400 4.0 4.3 0.48 9.0 1.720 n.m. u.m.
phila MX-45 pHWG315 mutB 400 3.8 45.2 0.46 98.6 18.080 340 28.9% P.
rubrum smuA 400 3.0 1.6 0.36 4.4 640 n.m. u.m. pHWG314 smuA 400 2.8
16.5 0.34 48.5 6.600 310 15.6% P. mesoacido- mutB 3000 27.2 28.4
3.26 8.7 85.200 n.m. u.m. phila MX-45 pHWG315 mutB 3000 26.6 308.5
3.19 96.7 925.500 n.m. n.m. P. rubrum smuA 3000 29.5 14.9 3.54 4.2
44.700 n.m. u.m. pHWG314 smuA 3000 26.1 149.6 3.13 47.8 448.800
n.m. n.m. n.m. = not measured u.m. = unmeasurable
[0183]
Sequence CWU 1
1
* * * * *