Title: Mutant Lactobacillus bulgaricus strains free from .beta.-galactoside activity
Abstract: The invention concerns mutant L. bulgaricus strains bearing a nonsense mutation, in at least one of the sequences coding for the lactose operon, and free from .beta.-galactosidase activity, and lactic starters comprising said strains. Said strains and starters can be used to obtain fermented milk products from glucose-added milk.
Patent Number: 6,875,601 Issued on 04/05/2005 to Benbadis, et al.
| Inventors:
|
Benbadis; Laurent (Anthony, FR);
Brignon; Pierre (Strasbourg, FR);
Gendre; Francois (Strasbourg, FR)
|
| Assignee:
|
Compagnie Gervais Danone (Levallois-Perret, FR)
|
| Appl. No.:
|
700687 |
| Filed:
|
February 14, 2001 |
| PCT Filed:
|
May 17, 1999
|
| PCT NO:
|
PCT/FR99/01165
|
| 371 Date:
|
February 14, 2001
|
| 102(e) Date:
|
February 14, 2001
|
| PCT PUB.NO.:
|
WO99/61627 |
| PCT PUB. Date:
|
December 2, 1999 |
Foreign Application Priority Data
| Current U.S. Class: |
435/252.9; 435/253.4; 424/439; 424/780; 426/42; 426/43 |
| Intern'l Class: |
C12N 001//20; A61K 035//74; A61K 047//00; A23C 009//12 |
| Field of Search: |
435/252.9,253.4
424/439,780
426/42,43
|
References Cited [Referenced By]
U.S. Patent Documents
| 5382438 | Jan., 1995 | Hottinger et al.
| |
| 5639648 | Jun., 1997 | Mainzer et al.
| |
| 5691185 | Nov., 1997 | Dickely et al.
| |
| Foreign Patent Documents |
| WO 90 05459 | May., 1990 | WO.
| |
Other References
Yoast et al., "Generation and characterization of environmentally sensitive
variants of the beta-galactosidase from L. bulgaricus", Applied and
Environmental Microbiology, 1994, vol. 60, No. 4, p. 1221-1226--abstract.*
Mollet et al., "A beta-galactosidase deletion mutant of L. bulgaricus
reverts to generate an active enzyme by internal DNA seq duplication", Mol
Gen Genet, 1991, 227(1), 17-21--abstract.
|
Primary Examiner: Lankford, Jr.; Leon B.
Assistant Examiner: Davis; Ruth A.
Attorney, Agent or Firm: Alston & Bird LLP
Claims
What is claimed is:
1. A mutant strain of L. bulgaricus which was deposited on Jan. 14, 1998
with the CNCM under the number I-1968, said strain lacking
.beta.-galactosidase activity and carrying a non-sense mutation in at
least one of the coding sequences of the lactose operon.
2. A mutant strain of L. bulgaricus deposited on Jan. 14, 1998 with the
CNCM under the number I-1968.
3. A lactic ferment which comprises at least one strain of L. bulgaricus as
claimed in claim 2.
4. The lactic ferment as claimed in claim 3, wherein said strain of L.
bulgaricus is combined with at least one strain of S. thermophilus.
5. A method for preparing a fermented dairy product, which comprises a step
wherein milk is fermented using a lactic ferment comprising at least one
strain of L. bulgaricus as claimed in claim 2, in the presence of at least
one sugar which can be assimilated by said strain.
6. The method as claimed in claim 5, wherein said sugar which can be
assimilated is glucose.
7. The method as claimed in claim 5, wherein the arrest of fermentation is
carried out without cooling of said dairy product.
8. A fermented dairy product obtained by the method as claimed in claim 5.
9. The fermented dairy product as claimed in claim 8 wherein said product
is a yogurt.
Description
These strains and ferments can be used for obtaining fermented dairy
products from milk supplemented with glucose.
The present invention relates to novel variants of bulgaricus and to their
use for preparing fermented dairy products.
Yogurts are conventionally obtained by fermentation of milk with a
combination of Streptococcus thermophilus and Lactobacillus bulgaricus.
During the fermentation, which is carried out at a temperature of
approximately 40 to 45.degree. C., these bacteria use mainly lactose as an
energetic substrate, and produce lactic acid which causes the milk to
coagulate; when the pH reaches a value of approximately 4.8 to 4.5, this
fermentation step (also named "acidification") is terminated by cooling
the product. This product is then kept in the cold during the remainder of
the manufacturing and packaging process, and until its consumption.
However, the cooling does not completely stop the lactic acid fermentation;
even when the product is kept at 4.degree. C., a gradual increase in its
acidity is observed over time.
This phenomenon, known as postacidification, is responsible for degradation
of the organoleptic qualities of the product during its conservation.
The postacidification results essentially from the use by the bacteria, and
mainly by L. bulgaricus, of the lactose remaining in the product at the
end of the controlled acidification step. In order to avoid it, it has
been proposed to use strains of L. bulgaricus which ferment lactose hardly
or not at all.
One of the enzymes which are essential for the fermentation of lactose is
.beta.-galactosidase, which hydrolyzes lactose into glucose and galactose.
It has therefore been proposed, in order to obtain non-postacidifying
strains of L. bulgaricus, to produce artificial mutants, or to select
natural mutants, in which the activity of this enzyme is affected.
For example, patent EP 402 450 in the name of GENENCOR describes the
production, by localized mutagenesis of the .beta.-galactosidase gene, of
conditional mutants of L. bulgaricus, in which the .beta.-galactosidase,
which is active during the fermentation at 40.degree. C., loses its
activity at the temperature or at the pH corresponding to the conditions
of conservation of fermented dairy products.
Application JP 90053437 describes the production of an artificial mutant of
L. bulgaricus which has completely lost the capacity to ferment lactose,
and the selection of a natural mutant with decreased lactose fermentation
capacity; these mutants are however both capable of developing and
acidifying normally in the presence of S. thermophilus, on condition that
the medium is supplemented with glucose. The subcultures of these mutants
conserve their acidification characteristics, in milk lacking glucose,
after 10 subculturings.
Patent EP 0518 096, in the name of the SOCIETE DES PRODUITS NESTLE,
proposes to use, for manufacturing yogurt, poorly postacidifying mutants
of Lactobacillus bulgaricus which have been preselected on the criterion
of the deletion of a fragment of the .beta.-galactosidase gene. The
screening and characterization of these mutants are facilitated due to the
fact that the presence of this deletion can be easily verified on
restriction profiles. In addition, the deletions are known to be
irreversible mutations, which makes it possible to easily obtain stable
mutant strains from the parent strain. Patent EP 0518 096 describes two
types of weakly postacidifying mutants selected in this way. The first
have a deletion which affects only the .beta.-galactosidase gene; when
they are combined with S. thermophilus and cultured on milk, they exhibit,
even without the addition of glucose, growth and acidification properties
which are comparable to those of the wild-type strain from which they are
derived. The second have a larger deletion, stretching over at least 1 kb
downstream of the .beta.-galactosidase gene; when they are combined with
S. thermophilus, they grow more slowly and acidify much less than the
wild-type strain from which they are derived; the addition of glucose to
the culture medium has only a slight influence on their acidification and
postacidification properties.
Natural mutants in which the .beta.-galactosidase is inactive are much more
difficult to select and to maintain as pure cultures in the case of point
mutations than in the case of deletion mutants; this is explained by the
lower probability of a point mutation producing an inactive protein, by
the greater difficulty in localizing and characterizing the point
mutations using restriction profiles, and by the very high reversion rate.
The applicant has now found other natural mutants of L. bulgaricus, which
do not carry a deletion in the gene encoding .beta.-galactosidase, and
which have advantageous technological characteristics. In the context of
the present invention, a non-sense mutant, which is incapable of
assimilating lactose, has been isolated from a culture of a wild-type L.
bulgaricus. When combined with S. thermophilus, in culture on milk, it
grows and acidifies much more slowly than the wild-type strain from which
it is derived. Conversely, its growth and its acidification are virtually
normal when the milk is supplemented with glucose.
A subject of the present invention is a mutant strain of L. bulgaricus
lacking .beta.-galactosidase activity, characterized in that it carries a
mutation which introduces a non-sense codon into one of the coding
sequences of the lactose operon, and in particular the sequence encoding
.beta.-galactosidase.
A strain of L. bulgaricus in accordance with the invention was deposited
according to the Treaty of Budapest, on Jan. 14, 1998, with the CNCM
(Collection Nationale de Cultures de Microorganisms [National Collection
of Microorganism Cultures]) held by the Pasteur Institute, 25 rue du
Docteur Roux, in Paris, under the number I-1968.
This strain has the following morphological and biochemical
characteristics:
Morphology: Gram-positive microorganism, immobile, isolated or short-chain,
asporogenic, pleomorphic, thin bacilli.
Metabolism: homofermentative, catalase (-).
Fermentation of sugars: D-glucose (+), D-fructose (+), D-mannose (+),
esculine (+).
The inventors have sequenced the lactose operon in the I-1968 mutant. The
corresponding sequence is represented in the appended sequence listing
under the number SEQ ID No: 1. The sequences of the translation products
(permease and .beta.-galactosidase) are represented under the numbers SEQ
ID No: 2 and SEQ ID No: 3, respectively.
The analysis of this sequence reveals two point mutations: one, in the
permease gene (position 122 of the sequence SEQ ID No: 1), induces an
amino acid change (Lys.fwdarw.Asn); the other, in the .beta.-galactosidase
gene (position 4519 of the sequence SEQ ID No: 1), introduces a stop
codon. Although conserving its active sites (positions 464 and 531), the
.beta.-galactosidase produced by this mutant is inactive. The inventors
have also noted that this mutation remains stable after several series of
subculturing, on a culture medium containing glucose. On the other hand,
on a culture medium without glucose, this non-sense mutation reverts very
rapidly at a rate of approximately 10.sup.-6.
The present invention also encompasses mutant strains which are incapable
of assimilating lactose and which are derived from the I-1968 strain. Such
strains can, for example, be obtained by inducing other mutations in the
lactose operon of the I-1968 strain, by site-directed mutagenesis.
A subject of the present invention is also a lactic ferment, in particular
a yogurt ferment, characterized in that it comprises at least one strain
of L. bulgaricus in accordance with the invention as defined above,
preferably combined with at least one strain of S. thermophilus.
For the production of a ferment in accordance with the invention, any
strain of S. thermophilus which is suitable for manufacturing yogurt can
be used; the choice of one or more strains of S. thermophilus can be made
as a function of the additional characteristics that it is desired
optionally to confer on the finished product.
By way of example of strains of S. thermophilus which can be used in
combination with a strain of L. bulgaricus in accordance with the
invention, mention may be made of the following strains, deposited with
the CNCM (Collection Nationale de Cultures de Microorganismes [National
Collection of Microorganism Cultures]) held by the Pasteur Institute, 25
rue du Docteur Roux, in Paris:
the strain deposited on Aug. 25, 1994, under the number I-1470, and the
strain deposited on Aug. 23, 1995, under the number I-1620; these two
strains are described in the European Application published under the
number 96/06924;
the strains deposited on Dec. 30, 1994, under the numbers I-1520 and
I-1521; these 2 strains are described in PCT international application WO
96/20607;
the strain deposited on Oct. 24, 1995 under the number I-1630; the
characteristics of this strain are described in PCT international
application WO 96/01701.
These strains can be combined mutually or with one or more other industrial
strains of S. thermophilus.
The strain(s) of S. thermophilus is (are) combined with the strain(s) of L.
bulgaricus in accordance with the invention, in the same way and in the
same proportions as in conventional yogurt ferments; the population of L.
bulgaricus bacteria in accordance with the invention may, for example,
represent between 10 and 90%, preferably between 20 and 50%, of the total
bacterial population.
A subject of the present invention is also a method for preparing a
fermented dairy product, characterized in that it comprises a step during
which milk is fermented using a ferment comprising at least one strain of
L. bulgaricus in accordance with the invention, in the presence of at
least one sugar which can be assimilated by said strain; it can be in
particular fructose, mannose and, preferably, glucose. Advantageously,
said fermented dairy product is a yogurt.
The method in accordance with the invention is similar to conventional
methods for preparing yogurt with regard to the main methods of
implementation of the controlled acidification step; in particular, this
acidification is carried out at a temperature of between 20 and 45.degree.
C., and preferably between 30 and 45.degree. C., and "batchwise", i.e. in
a single step and using a single fermentation tank.
The duration of this controlled acidification step is generally about 6 to
24 hours, and preferably about 6 to 16 hours; it is therefore longer than
in the case of conventional methods for preparing yogurt (in which it is 3
to 5 hours at 44.degree. C.). Specifically, the strains of L. bulgaricus
in accordance with the invention, even combined with S. thermophilus, grow
and acidify much more slowly than the wild-type strains.
In addition, the rate of growth and acidification of the strains of L.
bulgaricus in accordance with the invention varies very significantly
depending on the amount of glucose added to the milk. This property makes
it possible to control their growth and their acidification, by simply
adding the desired amount of glucose at the start of fermentation.
The inventors have also observed that, when strains of L. bulgaricus or
ferments in accordance with the invention are used, the acidification
slows down considerably when the pH reaches the range of 4.8 to 4.5 (which
corresponds to the pH range at which acidification is stopped in the case
of a conventional method), and stabilizes, even if the milk is maintained
at fermentation temperature, at a minimum pH. The value of this minimum pH
depends essentially on the amount of glucose added.
This property makes it possible to reduce, or even to eliminate, the
cooling phase used in conventional methods for manufacturing yogurt to
stop the fermentation. It also eliminates the necessity of measuring the
pH to determine the optimum moment for stopping the fermentation; for a
given ferment and amount of added glucose, it is possible, without risk of
overacidification, to stop the fermentation at the end of a given period,
calculated as a function of the time required to reach the minimum pH.
This makes it possible to have better control of the regularity of the
final pH and of the texture for the product at the end of fermentation.
Advantageously, for the implementation of the method in accordance with the
invention, and depending on the degree of acidification that it is desired
to reach, the amount of glucose added to the milk prior to the
fermentation is between 0.5 and 10 g/l, preferably between 0.5 and 5 g/l.
The fermented product obtained in this way can be conserved for several
hours at a temperature close to the fermentation temperature, without a
drop in pH, thereby making it possible to eliminate the installations for
intermediate cold storage, and to increase the capacity of the
fermentation tanks.
The implementation of the method in accordance with the invention makes it
possible to reduce the postacidification in the fermented products during
their longer term conservation. The degree of postacidification can vary
depending on the composition of the ferment and the amount of glucose
used. However, the postacidification is always clearly lower than that
observed in the case of yogurts obtained with conventional ferments and
methods.
For example, experiments carried out by the inventors have shown that,
under the same conservation conditions (28 days of conservation at
10.degree. C.), the .DELTA.pH (difference between the pH at D0 and the pH
at D28) is between 0.05 and 0.4 in the case of the products obtained using
a ferment in accordance with the invention, whereas it is always greater
than 0.7 in the case of control ferments in which the strain of L.
bulgaricus in accordance with the invention is replaced with a wild-type
strain.
This weak postacidification is accompanied by good survival of the strains
of the ferment; the population of L. bulgaricus, at the end of
conservation, in the fermented product obtained in accordance with the
invention is only slightly smaller than that of the control product.
A subject of the present invention is also the fermented dairy products
which can be obtained by implementing a method in accordance with the
invention.
These products can be conserved for a longer time and at higher
temperatures than the products obtained using conventional methods, and
have organoleptic properties which remain stable during conservation.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a graph showing the galactosidase activities of the LbS parent
strain and of the I-1968 mutant in accordance with the invention, measured
as a function of time.
EXAMPLE 1
Biochemical Assaying of the .beta.-Galactosidase Activity of a Mutant in
Accordance with the Invention
The .beta.-galactosidase activity of the I-1968 strain was compared with
that of the wild-type strain of L. bulgaricus (hereafter termed LbS) from
which it is derived.
The bacteria are cultured overnight on MRS agar medium (MERCK) at
37.degree. C., in an anaerobiosis jar (MERCK) in the presence of an oxygen
fixer (AnaerocultA, MERCK).
A 10-microliter loop (NUNC) of bacteria is resuspended in 1 milliliter of
sterile water. The bacteria are lyzed with 2 cycles of vigorous shaking,
20 seconds at 5000 rotations per minute in the presence of glass
microbeads (0.5 mm in diameter, BIOSPEC PRODUCTS), and then addition of
0.15 ml of chloroform. The mixture is shaken for 30 minutes at 37.degree.
C., and the volume is made to 2 ml with sterile water at 4.degree. C. The
beta-galactosidase activity is then measured: starting with 0.2 ml of the
cell suspension, 1.2 ml of 0.067M NaH.sub.2 PO.sub.4 buffer, pH 6.8; 0.05
ml of L-cysteine (SIGMA) at t0 0.05 ml of
O-nitrophenyl-beta-D-galactopyranoside (SIGMA) are added. The enzymatic
reaction is stopped after 0, 2, 5 or 10 min, with 1 ml of 10% Na.sub.2
CO.sub.3 buffer, and, after centrifugation of the reaction medium, a
measurement of the OD at 400 nanometers is performed on the supernatant.
The galactosidase activities of the LbS parent strain and of the I-1968
mutant in accordance with the invention, measured as a function of time,
are given in FIG. 1.
These results show that the .beta.-galactosidase is totally inactive in the
mutant in accordance with the invention.
EXAMPLE 2
Stability of the I-1968 Mutant of L. Bulgaricus
The stability of the I-1968 mutant was tested in media containing, as
carbon sources, either a mixture of glucose and of lactose, or lactose
only.
An I-1968 culture obtained on MRS medium containing glucose is subcultured
on sterilized milk which is supplemented with yeast autolyzate (2 g/l) and
which may or may not be supplemented with glucose (20 g/l). When a pH of
5.2 (coagulation of the milk) is reached, samples of each subculturing are
taken, on which the capacity of the bacteria to ferment sugars, as well as
the presence of .beta.-galactosidase activity (X-gal plate assay: white
colonies=.beta.-galactosidase minus; blue colonies=.beta.-galactosidase
plus), and analyzed.
The results are given in Table 1 below.
TABLE I
Milk + glucose
Medium (20 g/l) Milk
Time to reach 6 h 00 20 h 00
pH 5.2
Fermentation of glucose, fructose, lactose, glucose,
sugars mannose fructose, mannose
X-gal plate 100% white 20% white colonies
assay colonies 80% blue colonies
These results show that, in the presence of glucose, the I-1968 strain does
not revert toward a strain capable of using lactose. Conversely, in a
medium containing lactose as the only carbon source, rapid reversion of
the I-1968 strain toward the original state is observed.
EXAMPLE 3
Acidification, Postacidification and Survival Properties of the I-1968
Variant of L. Bulgaricus in Symbiosis with S. Thermophilus: the Case of a
Method for Manufacturing a Set Yogurt (Fermentation in a Ventilated Oven)
Yogurt ferments are prepared combining the I-1968 strain in accordance with
the invention with various industrial strains of S. thermophilus (the
strains of S. thermophilus used are hereafter termed ST1, ST2 and ST3).
By way of comparison, the ferments are prepared combining the LbS parent
strain and the same strains of S. thermophilus.
For preparing the ferments, the strains are seeded separately and at 1% on
the following composition:
Composition for 1 Liter:
135 g of skimmed milk powder
2 g of yeast autolyzate
920 ml of distilled water
20 g of glucose (for the I-1968 strain only)
Hydration: 10 min
Pasteurization: 30 min at 95.degree. C.
The milk is then cooled to 44.degree. C. and inoculated, and then incubated
at 44.degree. C. until an acidity of 85.degree. D (degrees Dornic) for the
streptococci and of 80.degree. D for the lactobacilli is obtained.
The cultures are then cooled so as to obtain a ferment consisting of 80%
Streptococcus thermophilus and of 20% Lactobacillus bulgaricus.
The ferments thus obtained are used to inoculate the following preparation:
Composition for 1 Liter:
99% of milk
0, 1, or 2 g/l of glucose
Hydration: 10 min
Pasteurization: 10 min at 95.degree. C.
The milk is then cooled to 44.degree. C. and inoculated at 1%.
For each experiment, the composition of the ferment and the amount of
glucose added are given in Table II below:
TABLE II
Experiment Glucose g/l Strains Percentage
1 0 ST 3 64%
ST 2 16%
LbS 20%
2 0 ST 3 64%
ST 2 16%
I-1968 20%
3 1 ST 3 64%
ST 2 16%
I-1968 20%
4 0 ST 1 80%
LbS 20%
5 0 ST 1 80%
I-1968 20%
6 2 ST 1 80%
I-1968 20%
After inoculation, the milk is distributed into round-bottomed flasks and
incubated at a temperature of 44.degree. C. The acidification profile is
monitored during the incubation. The products are uncurdled at pH 4.6 by
cooling in a cold unit (16 hours at 4.degree. C.).
The products are then subjected to a conservation test at 10.degree. C. In
this test, the pH and Dornic acidity are measured after 1, 14, 21 and 28
days of conservation.
The acidification results (time to reach a pH of 4.6 and pH value at 24 h)
are given in Table III below:
TABLE III
Time to Time to
reach pH 4.6 reach pH 4.5
Experiment (min) (min) pH at 24 h
1 215 236 3.67
2 550 778 4.33
3 416 507 4.26
4 225 241 3.67
5 660 >1500 4.54
6 390 465 4.35
The results of the conservation test at 10.degree. C. (monitoring of the pH
and of the Dornic acidity) and the survival test (S. thermophilus and L.
bulgaricus populations) at 28 days are given in Table IV below:
TABLE IV
Storage Streptococcus Lactobacillus
time Dornic thermophilus bulgaricus
Experiment (days) pH acidity cells/ml cells/ml
1 1 4.41 101 7.25E + 08 3.35E + 08
1 14 3.98 140 ND ND
1 21 3.95 145 ND ND
1 28 3.9 148 7.35E + 08 3.30E + 08
2 1 4.5 93 5.60E + 08 2.90E + 07
2 14 4.23 110 Nd ND
2 21 4.18 112 ND ND
2 28 4.19 114 5.6SE + 08 1.87E + 07
3 1 4.49 96 6.90E + 08 7.45E + 07
3 14 4.14 115 ND ND
3 21 4.15 117 ND ND
3 28 4.15 120 5.65E + 08 6.30E + 07
4 1 4.39 105 6.30E + 07 4.40E + 08
4 14 3.91 145 &D ND
4 21 3.9 151 ND ND
4 28 3.85 157 4.70E + 08 6.30E + 08
5 1 4.6 85 9.05E + 08 6.70E + 07
5 14 4.58 80 ND ND
5 21 4.53 80 ND ND
5 28 4.61 79 9.40E + 08 7.00E + 07
6 1 4.51 89 1.05E + 09 1.96E + 08
6 14 4.38 90 ND ND
6 21 4.39 96 ND ND
6 28 4.42 90 1.62E + 09 1.91E + 08
ND = Not Determined
These results show that the yogurts produced using the symbioses combining
the I-1968 strain with one or two strains of S. thermophilus show
extremely reduced postacidification with respect to the same symbioses
with the LbS parent strain, while at the same time conserving an abundant
population at the end of fermentation and good survival for 28 days at
10.degree. C.
Stopping the acidification and maintaining the pH at around 4.6 to 4.5 for
at least 24 hours at 44.degree. C. makes it possible, in the context of
manufacturing stirred yogurt, to reduce or even eliminate the phase of
cooling in a tank, which is conventionally used.
SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 3
<210> SEQ ID NO 1
<211> LENGTH: 5059
<212> TYPE: DNA
<213> ORGANISM: Lactobacillus bulgaricus
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (122)..(1873)
<221> NAME/KEY: CDS
<222> LOCATION: (1877)..(4519)
<400> SEQUENCE: 1
gcttgtctca cgcttgtcgt acgcggccgg tgcctttggc aacgacgtct tctacgcgac 60
tctgtcaacc tactttatcg tcttcgtcac cacccacctc tttaatgccg gtgaccacaa 120
g atg atc ttt atc atc acc aac ttg atc acc gcc atc cgg atc ggg gaa 169
Met Ile Phe Ile Ile Thr Asn Leu Ile Thr Ala Ile Arg Ile Gly Glu
1 5 10 15
gtc ctg ctc gac ccc ttg atc ggt aac gcc atc gac cgg acc gaa agc 217
Val Leu Leu Asp Pro Leu Ile Gly Asn Ala Ile Asp Arg Thr Glu Ser
20 25 30
cgg tgg ggg aag ttc aag ccc tgg gtt gtg ggc ggg ggg atc atc agc 265
Arg Trp Gly Lys Phe Lys Pro Trp Val Val Gly Gly Gly Ile Ile Ser
35 40 45
tca tta gcc ctc tta gcc ctc ttt acc gac ttt ggc ggc att aac caa 313
Ser Leu Ala Leu Leu Ala Leu Phe Thr Asp Phe Gly Gly Ile Asn Gln
50 55 60
agc aac ccc gtt gtt tac tta gta atc ttc ggt att gtt tac ttg att 361
Ser Asn Pro Val Val Tyr Leu Val Ile Phe Gly Ile Val Tyr Leu Ile
65 70 75 80
atg gat atc ttc tac tca ttt aaa gac act ggc ttc tgg gcc atg atc 409
Met Asp Ile Phe Tyr Ser Phe Lys Asp Thr Gly Phe Trp Ala Met Ile
85 90 95
ccg gcc ttg tcc ctg gat tcc cgg gaa aga gag aag acc tcc acc ttc 457
Pro Ala Leu Ser Leu Asp Ser Arg Glu Arg Glu Lys Thr Ser Thr Phe
100 105 110
gcc aga gtc ggc tcc acc atc ggg gcc aac ctg gtc ggg gta gtc atc 505
Ala Arg Val Gly Ser Thr Ile Gly Ala Asn Leu Val Gly Val Val Ile
115 120 125
acc cca atc atc ctc ttc ttc tcg gcc agc aag gcc aac ccc aac ggg 553
Thr Pro Ile Ile Leu Phe Phe Ser Ala Ser Lys Ala Asn Pro Asn Gly
130 135 140
gat aag cag ggc tgg ttc ttc ttt gcc ttg atc gtg gcc att gtc ggc 601
Asp Lys Gln Gly Trp Phe Phe Phe Ala Leu Ile Val Ala Ile Val Gly
145 150 155 160
atc ttg acc tca att acc gtt ggt ctt ggt act cac gaa gta aaa tcc 649
Ile Leu Thr Ser Ile Thr Val Gly Leu Gly Thr His Glu Val Lys Ser
165 170 175
gcc ctg cgg gaa agc aat gaa aag acc act ttg aag cag gtc ttt aag 697
Ala Leu Arg Glu Ser Asn Glu Lys Thr Thr Leu Lys Gln Val Phe Lys
180 185 190
gtc ctg ggg caa aac gac cag ctc ctc tgg ctg gcc ttt gcc tac tgg 745
Val Leu Gly Gln Asn Asp Gln Leu Leu Trp Leu Ala Phe Ala Tyr Trp
195 200 205
ttt tac ggc ctg ggt atc aac acc ctg aac gct ctg caa ctt tac tac 793
Phe Tyr Gly Leu Gly Ile Asn Thr Leu Asn Ala Leu Gln Leu Tyr Tyr
210 215 220
ttc tca tac atc tta ggc gat gcc cgc ggc tac agc ctg ctt tac acc 841
Phe Ser Tyr Ile Leu Gly Asp Ala Arg Gly Tyr Ser Leu Leu Tyr Thr
225 230 235 240
atc aac acc ttt gtc ggt tta atc tct gca tcc ttc ttc cca tca ctg 889
Ile Asn Thr Phe Val Gly Leu Ile Ser Ala Ser Phe Phe Pro Ser Leu
245 250 255
gcc aag aag ttc aac aga aat cgc ctc ttc tac gcc tgc atc gcg gtg 937
Ala Lys Lys Phe Asn Arg Asn Arg Leu Phe Tyr Ala Cys Ile Ala Val
260 265 270
atg ctg tta ggg atc ggg gtc ttc tcc gtg gcc agc ggt tct ctg gcc 985
Met Leu Leu Gly Ile Gly Val Phe Ser Val Ala Ser Gly Ser Leu Ala
275 280 285
ctg tcc ctt gtt ggg gca gaa ttc ttc ttt att ccg cag cct ctg gcc 1033
Leu Ser Leu Val Gly Ala Glu Phe Phe Phe Ile Pro Gln Pro Leu Ala
290 295 300
ttc ctg gtc gtt ttg atg atc atc tct gac gct gtt gaa tac ggc cag 1081
Phe Leu Val Val Leu Met Ile Ile Ser Asp Ala Val Glu Tyr Gly Gln
305 310 315 320
ctg aaa act ggc cac aga gac gaa gct ttg acc ctg tct gtc cgg cca 1129
Leu Lys Thr Gly His Arg Asp Glu Ala Leu Thr Leu Ser Val Arg Pro
325 330 335
ttg gtc gat aag ctg ggc ggg gcc ttg tcc aac tgg ttt gtt tcc ttg 1177
Leu Val Asp Lys Leu Gly Gly Ala Leu Ser Asn Trp Phe Val Ser Leu
340 345 350
att gcc tta act gcc ggc atg acc act ggg gcg act gcc tca aca att 1225
Ile Ala Leu Thr Ala Gly Met Thr Thr Gly Ala Thr Ala Ser Thr Ile
355 360 365
aca gct cat ggc cag atg gtc ttc aag tta gct atg ttt gcc tta ccg 1273
Thr Ala His Gly Gln Met Val Phe Lys Leu Ala Met Phe Ala Leu Pro
370 375 380
gca gtc atg ctc ttg atc gct gtt tct att ttc gcc aaa aag gtc ttc 1321
Ala Val Met Leu Leu Ile Ala Val Ser Ile Phe Ala Lys Lys Val Phe
385 390 395 400
ttg act gaa gaa aag cac gcg gaa atc gtc gac cag ctg gaa act caa 1369
Leu Thr Glu Glu Lys His Ala Glu Ile Val Asp Gln Leu Glu Thr Gln
405 410 415
ttc agc caa agc cat gcc caa aag ccg gcg caa gct gaa agc ttc act 1417
Phe Ser Gln Ser His Ala Gln Lys Pro Ala Gln Ala Glu Ser Phe Thr
420 425 430
ttg gcc agc cca gtc tcc gga caa tta atg aac ctg gac atg gtt gac 1465
Leu Ala Ser Pro Val Ser Gly Gln Leu Met Asn Leu Asp Met Val Asp
435 440 445
gac ccg gtc ttt gcc gac aaa aag tta ggc gac ggc ttt gcc ctg gtg 1513
Asp Pro Val Phe Ala Asp Lys Lys Leu Gly Asp Gly Phe Ala Leu Val
450 455 460
cca gca gac ggt aag gtc tac gcg cca ttt gcc ggt act gtc cgc cag 1561
Pro Ala Asp Gly Lys Val Tyr Ala Pro Phe Ala Gly Thr Val Arg Gln
465 470 475 480
ctg gcc aag acc cgg cac tcg atc gtc ctg gaa aat gaa cat ggg gtc 1609
Leu Ala Lys Thr Arg His Ser Ile Val Leu Glu Asn Glu His Gly Val
485 490 495
ttg gtc ttg att cac ctt ggc ctg ggc acg gtc aaa tta aac ggg act 1657
Leu Val Leu Ile His Leu Gly Leu Gly Thr Val Lys Leu Asn Gly Thr
500 505 510
ggc ttt gtc agc tat gtt gaa gag ggc agc cag gta gaa gcc ggc cag 1705
Gly Phe Val Ser Tyr Val Glu Glu Gly Ser Gln Val Glu Ala Gly Gln
515 520 525
cag atc ctg gaa ttc tgg gac ccg gcg atc aag cag gcc aag ctg gac 1753
Gln Ile Leu Glu Phe Trp Asp Pro Ala Ile Lys Gln Ala Lys Leu Asp
530 535 540
gac acg gta atc gtg acc gtc atc aac agc gaa act ttc gca aat agc 1801
Asp Thr Val Ile Val Thr Val Ile Asn Ser Glu Thr Phe Ala Asn Ser
545 550 555 560
cag atg ctc ttg ccg atc ggc cac agc gtc caa gcc ctg gat gat gta 1849
Gln Met Leu Leu Pro Ile Gly His Ser Val Gln Ala Leu Asp Asp Val
565 570 575
ttc aag tta gaa ggg aag aat tag aaa atg agc aat aag tta gta aaa 1897
Phe Lys Leu Glu Gly Lys Asn Met Ser Asn Lys Leu Val Lys
580 585 590
gaa aaa aga gtt gac cag gca gac ttg gcc tgg ctg act gac ccg gaa 1945
Glu Lys Arg Val Asp Gln Ala Asp Leu Ala Trp Leu Thr Asp Pro Glu
595 600 605
gtt tac gaa gtc aat aca att ccc ccg cac tcc gac cat gag tcc ttc 1993
Val Tyr Glu Val Asn Thr Ile Pro Pro His Ser Asp His Glu Ser Phe
610 615 620
caa agc cag gaa gaa ctg gag gag ggc aag tcc agt tta gtg cag tcc 2041
Gln Ser Gln Glu Glu Leu Glu Glu Gly Lys Ser Ser Leu Val Gln Ser
625 630 635
ctg gac ggg gac tgg ctg att gac tac gct gaa aac ggc cag gga cca 2089
Leu Asp Gly Asp Trp Leu Ile Asp Tyr Ala Glu Asn Gly Gln Gly Pro
640 645 650 655
gtc aac ttc tat gca gaa gac ttt gac gat agc aat ttt aag tca gtc 2137
Val Asn Phe Tyr Ala Glu Asp Phe Asp Asp Ser Asn Phe Lys Ser Val
660 665 670
aaa gta ccc ggc aac ctg gaa ctg caa ggc ttt ggc cag ccc cag tat 2185
Lys Val Pro Gly Asn Leu Glu Leu Gln Gly Phe Gly Gln Pro Gln Tyr
675 680 685
gtc aac gtc caa tat cca tgg gac ggc agt gag gag att ttc ccg ccc 2233
Val Asn Val Gln Tyr Pro Trp Asp Gly Ser Glu Glu Ile Phe Pro Pro
690 695 700
caa att cca agc aaa aat ccg ctc gct tct tat gtc aga tac ttt gac 2281
Gln Ile Pro Ser Lys Asn Pro Leu Ala Ser Tyr Val Arg Tyr Phe Asp
705 710 715
ctg gat gaa gct ttc tgg gac aag gaa gtc agc ttg aag ttt gac ggg 2329
Leu Asp Glu Ala Phe Trp Asp Lys Glu Val Ser Leu Lys Phe Asp Gly
720 725 730 735
gcg gca aca gcc atc tat gtc tgg ctg aac ggc cac ttc gtc ggc tac 2377
Ala Ala Thr Ala Ile Tyr Val Trp Leu Asn Gly His Phe Val Gly Tyr
740 745 750
ggg gaa gac tcc ttt acc cca agc gag ttt atg gtt acc aag ttc ctc 2425
Gly Glu Asp Ser Phe Thr Pro Ser Glu Phe Met Val Thr Lys Phe Leu
755 760 765
aag aaa gaa aat aac cgc ctg gca gtg gct ctc tac aag tat tct tcc 2473
Lys Lys Glu Asn Asn Arg Leu Ala Val Ala Leu Tyr Lys Tyr Ser Ser
770 775 780
gcc tcc tgg ctg gaa gac cag gac ttc tgg cgc atg tct ggt ttg ttc 2521
Ala Ser Trp Leu Glu Asp Gln Asp Phe Trp Arg Met Ser Gly Leu Phe
785 790 795
aga tca gtg act ctt cag gcc aag ccg cgt ctg cac ttg gag gac ctt 2569
Arg Ser Val Thr Leu Gln Ala Lys Pro Arg Leu His Leu Glu Asp Leu
800 805 810 815
aag ctt acg gcc agc ttg acc gat aac tac caa aaa gga aag ctg gaa 2617
Lys Leu Thr Ala Ser Leu Thr Asp Asn Tyr Gln Lys Gly Lys Leu Glu
820 825 830
gtc gaa gcc aat att gcc tac cgc ttg cca aat gcc agc ttt aag ctg 2665
Val Glu Ala Asn Ile Ala Tyr Arg Leu Pro Asn Ala Ser Phe Lys Leu
835 840 845
gaa gtg cgg gat agt gaa ggt gac ttg gtt gct gaa aag ctg ggc cca 2713
Glu Val Arg Asp Ser Glu Gly Asp Leu Val Ala Glu Lys Leu Gly Pro
850 855 860
atc aga agc gag cag ctg gaa ttc act ctg gct gat ttg cca gta gct 2761
Ile Arg Ser Glu Gln Leu Glu Phe Thr Leu Ala Asp Leu Pro Val Ala
865 870 875
gcc tgg agc gcg gaa aag cct aac ctt tac cag gtc cgc ctg tat tta 2809
Ala Trp Ser Ala Glu Lys Pro Asn Leu Tyr Gln Val Arg Leu Tyr Leu
880 885 890 895
tac cag gca ggc agc ctc tta gag gtt agc cgg cag gaa gtg ggt ttc 2857
Tyr Gln Ala Gly Ser Leu Leu Glu Val Ser Arg Gln Glu Val Gly Phe
900 905 910
cgc aac ttt gaa cta aaa gac ggg att atg tac ctt aac ggc cag cgg 2905
Arg Asn Phe Glu Leu Lys Asp Gly Ile Met Tyr Leu Asn Gly Gln Arg
915 920 925
atc gtc ttc aag ggg gcc aac cgg cac gaa ttt gac agt aag ttg ggc 2953
Ile Val Phe Lys Gly Ala Asn Arg His Glu Phe Asp Ser Lys Leu Gly
930 935 940
cgg gct atc aca gaa gag gat atg atc tgg gat atc aag acc atg aag 3001
Arg Ala Ile Thr Glu Glu Asp Met Ile Trp Asp Ile Lys Thr Met Lys
945 950 955
cga agc aac atc aat gct gtc cgc tgc tct cac tac ccg aac cag tcc 3049
Arg Ser Asn Ile Asn Ala Val Arg Cys Ser His Tyr Pro Asn Gln Ser
960 965 970 975
ctc ttt tac cgg ctc tgt gac aag tac ggc ctt tac gtc att gat gaa 3097
Leu Phe Tyr Arg Leu Cys Asp Lys Tyr Gly Leu Tyr Val Ile Asp Glu
980 985 990
gct aac ctg gaa agc cac ggc acc tgg gaa aaa gtg ggg ggg cac gaa 3145
Ala Asn Leu Glu Ser His Gly Thr Trp Glu Lys Val Gly Gly His Glu
995 1000 1005
gat cct agc ttc aat gtt cca ggc gat gac cag cat tgg ctg gga gcc 3193
Asp Pro Ser Phe Asn Val Pro Gly Asp Asp Gln His Trp Leu Gly Ala
1010 1015 1020
agc tta tcc cgg gtg aag aac atg atg gct cgg gac aag aac cat gct 3241
Ser Leu Ser Arg Val Lys Asn Met Met Ala Arg Asp Lys Asn His Ala
1025 1030 1035
tca atc ctg atc tgg tct tta ggc aat gag tct tac gcc ggc act gtc 3289
Ser Ile Leu Ile Trp Ser Leu Gly Asn Glu Ser Tyr Ala Gly Thr Val
1040 1045 1050 1055
ttt gcc caa atg gct gat tac gtc cgg aag gct gat ccg acc cgg gtt 3337
Phe Ala Gln Met Ala Asp Tyr Val Arg Lys Ala Asp Pro Thr Arg Val
1060 1065 1070
cag cac tat gaa ggg gtg acc cac aac cgg aag ttt gac gac gcc acc 3385
Gln His Tyr Glu Gly Val Thr His Asn Arg Lys Phe Asp Asp Ala Thr
1075 1080 1085
cag att gaa agc cgg atg tat gct ccg gcc aag gta att gaa gaa tac 3433
Gln Ile Glu Ser Arg Met Tyr Ala Pro Ala Lys Val Ile Glu Glu Tyr
1090 1095 1100
ttg acc aat aaa cca gcc aag cca ttt atc tca gtt gaa tac gct cac 3481
Leu Thr Asn Lys Pro Ala Lys Pro Phe Ile Ser Val Glu Tyr Ala His
1105 1110 1115
gcc atg ggc aac tcc gtc ggt gac ctg gcc gcc tac acg gcc ctg gaa 3529
Ala Met Gly Asn Ser Val Gly Asp Leu Ala Ala Tyr Thr Ala Leu Glu
1120 1125 1130 1135
aaa tac ccc cac tac cag ggc ggc ttc atc tgg gac tgg att gac caa 3577
Lys Tyr Pro His Tyr Gln Gly Gly Phe Ile Trp Asp Trp Ile Asp Gln
1140 1145 1150
gga ctg gaa aaa gac ggg cac ctg ctt tat ggg ggc gac ttc gat gac 3625
Gly Leu Glu Lys Asp Gly His Leu Leu Tyr Gly Gly Asp Phe Asp Asp
1155 1160 1165
cgg cca acc gac tat gaa ttc tgc ggg aac ggc ctg gtc ttt gct gac 3673
Arg Pro Thr Asp Tyr Glu Phe Cys Gly Asn Gly Leu Val Phe Ala Asp
1170 1175 1180
cgg act gaa tcg ccg aaa ctg gct aat gtc aag gcc ctt tac gcc aac 3721
Arg Thr Glu Ser Pro Lys Leu Ala Asn Val Lys Ala Leu Tyr Ala Asn
1185 1190 1195
ctt aag tta gaa gta aaa gat ggg cag ctc ttc ctc aaa aac gac aat 3769
Leu Lys Leu Glu Val Lys Asp Gly Gln Leu Phe Leu Lys Asn Asp Asn
1200 1205 1210 1215
tta ttt acc aac agc tca tct tac tac ttc ttg act agt ctt ttg gtc 3817
Leu Phe Thr Asn Ser Ser Ser Tyr Tyr Phe Leu Thr Ser Leu Leu Val
1220 1225 1230
gat ggc aag ttg acc tac cag agc cgg cct ctg acc ttt ggc ctg gag 3865
Asp Gly Lys Leu Thr Tyr Gln Ser Arg Pro Leu Thr Phe Gly Leu Glu
1235 1240 1245
cct ggc gaa tcc ggg acc ttt gcc ctg cct tgg ccg gaa gtc gct gat 3913
Pro Gly Glu Ser Gly Thr Phe Ala Leu Pro Trp Pro Glu Val Ala Asp
1250 1255 1260
gaa aaa gga gag gtc gtc tac cgg gta acg gcc cac tta aaa gaa gac 3961
Glu Lys Gly Glu Val Val Tyr Arg Val Thr Ala His Leu Lys Glu Asp
1265 1270 1275
ttg cct tgg gcg gat gag ggc ttc act gtg gct gaa gca gaa gaa gta 4009
Leu Pro Trp Ala Asp Glu Gly Phe Thr Val Ala Glu Ala Glu Glu Val
1280 1285 1290 1295
gct caa aag ctg ccg gaa ttt aag ccg gaa ggg cgg cca gat tta gtt 4057
Ala Gln Lys Leu Pro Glu Phe Lys Pro Glu Gly Arg Pro Asp Leu Val
1300 1305 1310
gat tcc gac tac aac cta ggc ctg aaa gga aat aac ttc caa att ctc 4105
Asp Ser Asp Tyr Asn Leu Gly Leu Lys Gly Asn Asn Phe Gln Ile Leu
1315 1320 1325
ttc tcc aag gtc aag ggc tgg ccg gtt tcc ctc aag tat gcc ggt agg 4153
Phe Ser Lys Val Lys Gly Trp Pro Val Ser Leu Lys Tyr Ala Gly Arg
1330 1335 1340
gaa tac ttg aag cgg ctg ccg gaa ttt acc ttc tgg cgg gcc ctg acg 4201
Glu Tyr Leu Lys Arg Leu Pro Glu Phe Thr Phe Trp Arg Ala Leu Thr
1345 1350 1355
gac aac gac cgg gga gct ggt tac ggc tat gat ctg gcc cgg tgg gaa 4249
Asp Asn Asp Arg Gly Ala Gly Tyr Gly Tyr Asp Leu Ala Arg Trp Glu
1360 1365 1370 1375
aat gcc ggc aag tat gcc cgc ttg aaa gac atc agc tgc gag gtc aag 4297
Asn Ala Gly Lys Tyr Ala Arg Leu Lys Asp Ile Ser Cys Glu Val Lys
1380 1385 1390
gaa gac tcc gtt ttg gtc aag act gcc ttt acg ttg cct gtc gcc tta 4345
Glu Asp Ser Val Leu Val Lys Thr Ala Phe Thr Leu Pro Val Ala Leu
1395 1400 1405
aag ggt gat tta act gtg acc tat gaa gtc gat gga cgg ggc aag att 4393
Lys Gly Asp Leu Thr Val Thr Tyr Glu Val Asp Gly Arg Gly Lys Ile
1410 1415 1420
gct gta aca gct gac ttc cca ggc gcg gaa gaa gcc ggt ctc ttg cca 4441
Ala Val Thr Ala Asp Phe Pro Gly Ala Glu Glu Ala Gly Leu Leu Pro
1425 1430 1435
gcc ttt ggc ttg aac ctg gcc ctg cca aaa gaa ctg acc gat tac cgc 4489
Ala Phe Gly Leu Asn Leu Ala Leu Pro Lys Glu Leu Thr Asp Tyr Arg
1440 1445 1450 1455
tac tat ggt ctg gga cct aat gag agc taa ccagaccgct tggaaggtaa 4539
Tyr Tyr Gly Leu Gly Pro Asn Glu Ser
1460 1465
ttacctgggc atctaccagg gagcggtaaa aaagaacttt agcccatacc tgcgtccgca 4599
ggaaacgggc aaccggagca aggttcgctg gtaccagctc tttgatgaaa agggcggctt 4659
ggaatttacg gccaatgggg cagacttgaa cttgtctgct ttgccatatt ctgccgccca 4719
aattgaagca gcggaccacg cttttgaact gactaacaat tacacttggg ttagagcctt 4779
aagcgcccag atgggggtcg gcggggatga ctcctggggg cagaaggtcc acccggaatt 4839
ctgcctggat gctcaaaaag cccgccagct ccgcctggtg attcagcccc ttttactaaa 4899
ataaatgcta caattgactt aacaggatga aattttagta aaagcaaagc gagtgaggaa 4959
gatggcaacg atcagagaag tgccaaggca gccggcgtgt cgctagcgac ggtttcccgc 5019
gtcttgaact atgaccagac cctgtcagtc aatgaggcaa 5059
<210> SEQ ID NO 2
<211> LENGTH: 583
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus bulgaricus
<400> SEQUENCE: 2
Met Ile Phe Ile Ile Thr Asn Leu Ile Thr Ala Ile Arg Ile Gly Glu
1 5 10 15
Val Leu Leu Asp Pro Leu Ile Gly Asn Ala Ile Asp Arg Thr Glu Ser
20 25 30
Arg Trp Gly Lys Phe Lys Pro Trp Val Val Gly Gly Gly Ile Ile Ser
35 40 45
Ser Leu Ala Leu Leu Ala Leu Phe Thr Asp Phe Gly Gly Ile Asn Gln
50 55 60
Ser Asn Pro Val Val Tyr Leu Val Ile Phe Gly Ile Val Tyr Leu Ile
65 70 75 80
Met Asp Ile Phe Tyr Ser Phe Lys Asp Thr Gly Phe Trp Ala Met Ile
85 90 95
Pro Ala Leu Ser Leu Asp Ser Arg Glu Arg Glu Lys Thr Ser Thr Phe
100 105 110
Ala Arg Val Gly Ser Thr Ile Gly Ala Asn Leu Val Gly Val Val Ile
115 120 125
Thr Pro Ile Ile Leu Phe Phe Ser Ala Ser Lys Ala Asn Pro Asn Gly
130 135 140
Asp Lys Gln Gly Trp Phe Phe Phe Ala Leu Ile Val Ala Ile Val Gly
145 150 155 160
Ile Leu Thr Ser Ile Thr Val Gly Leu Gly Thr His Glu Val Lys Ser
165 170 175
Ala Leu Arg Glu Ser Asn Glu Lys Thr Thr Leu Lys Gln Val Phe Lys
180 185 190
Val Leu Gly Gln Asn Asp Gln Leu Leu Trp Leu Ala Phe Ala Tyr Trp
195 200 205
Phe Tyr Gly Leu Gly Ile Asn Thr Leu Asn Ala Leu Gln Leu Tyr Tyr
210 215 220
Phe Ser Tyr Ile Leu Gly Asp Ala Arg Gly Tyr Ser Leu Leu Tyr Thr
225 230 235 240
Ile Asn Thr Phe Val Gly Leu Ile Ser Ala Ser Phe Phe Pro Ser Leu
245 250 255
Ala Lys Lys Phe Asn Arg Asn Arg Leu Phe Tyr Ala Cys Ile Ala Val
260 265 270
Met Leu Leu Gly Ile Gly Val Phe Ser Val Ala Ser Gly Ser Leu Ala
275 280 285
Leu Ser Leu Val Gly Ala Glu Phe Phe Phe Ile Pro Gln Pro Leu Ala
290 295 300
Phe Leu Val Val Leu Met Ile Ile Ser Asp Ala Val Glu Tyr Gly Gln
305 310 315 320
Leu Lys Thr Gly His Arg Asp Glu Ala Leu Thr Leu Ser Val Arg Pro
325 330 335
Leu Val Asp Lys Leu Gly Gly Ala Leu Ser Asn Trp Phe Val Ser Leu
340 345 350
Ile Ala Leu Thr Ala Gly Met Thr Thr Gly Ala Thr Ala Ser Thr Ile
355 360 365
Thr Ala His Gly Gln Met Val Phe Lys Leu Ala Met Phe Ala Leu Pro
370 375 380
Ala Val Met Leu Leu Ile Ala Val Ser Ile Phe Ala Lys Lys Val Phe
385 390 395 400
Leu Thr Glu Glu Lys His Ala Glu Ile Val Asp Gln Leu Glu Thr Gln
405 410 415
Phe Ser Gln Ser His Ala Gln Lys Pro Ala Gln Ala Glu Ser Phe Thr
420 425 430
Leu Ala Ser Pro Val Ser Gly Gln Leu Met Asn Leu Asp Met Val Asp
435 440 445
Asp Pro Val Phe Ala Asp Lys Lys Leu Gly Asp Gly Phe Ala Leu Val
450 455 460
Pro Ala Asp Gly Lys Val Tyr Ala Pro Phe Ala Gly Thr Val Arg Gln
465 470 475 480
Leu Ala Lys Thr Arg His Ser Ile Val Leu Glu Asn Glu His Gly Val
485 490 495
Leu Val Leu Ile His Leu Gly Leu Gly Thr Val Lys Leu Asn Gly Thr
500 505 510
Gly Phe Val Ser Tyr Val Glu Glu Gly Ser Gln Val Glu Ala Gly Gln
515 520 525
Gln Ile Leu Glu Phe Trp Asp Pro Ala Ile Lys Gln Ala Lys Leu Asp
530 535 540
Asp Thr Val Ile Val Thr Val Ile Asn Ser Glu Thr Phe Ala Asn Ser
545 550 555 560
Gln Met Leu Leu Pro Ile Gly His Ser Val Gln Ala Leu Asp Asp Val
565 570 575
Phe Lys Leu Glu Gly Lys Asn
580
<210> SEQ ID NO 3
<211> LENGTH: 880
<212> TYPE: PRT
<213> ORGANISM: Lactobacillus bulgaricus
<400> SEQUENCE: 3
Met Ser Asn Lys Leu Val Lys Glu Lys Arg Val Asp Gln Ala Asp Leu
1 5 10 15
Ala Trp Leu Thr Asp Pro Glu Val Tyr Glu Val Asn Thr Ile Pro Pro
20 25 30
His Ser Asp His Glu Ser Phe Gln Ser Gln Glu Glu Leu Glu Glu Gly
35 40 45
Lys Ser Ser Leu Val Gln Ser Leu Asp Gly Asp Trp Leu Ile Asp Tyr
50 55 60
Ala Glu Asn Gly Gln Gly Pro Val Asn Phe Tyr Ala Glu Asp Phe Asp
65 70 75 80
Asp Ser Asn Phe Lys Ser Val Lys Val Pro Gly Asn Leu Glu Leu Gln
85 90 95
Gly Phe Gly Gln Pro Gln Tyr Val Asn Val Gln Tyr Pro Trp Asp Gly
100 105 110
Ser Glu Glu Ile Phe Pro Pro Gln Ile Pro Ser Lys Asn Pro Leu Ala
115 120 125
Ser Tyr Val Arg Tyr Phe Asp Leu Asp Glu Ala Phe Trp Asp Lys Glu
130 135 140
Val Ser Leu Lys Phe Asp Gly Ala Ala Thr Ala Ile Tyr Val Trp Leu
145 150 155 160
Asn Gly His Phe Val Gly Tyr Gly Glu Asp Ser Phe Thr Pro Ser Glu
165 170 175
Phe Met Val Thr Lys Phe Leu Lys Lys Glu Asn Asn Arg Leu Ala Val
180 185 190
Ala Leu Tyr Lys Tyr Ser Ser Ala Ser Trp Leu Glu Asp Gln Asp Phe
195 200 205
Trp Arg Met Ser Gly Leu Phe Arg Ser Val Thr Leu Gln Ala Lys Pro
210 215 220
Arg Leu His Leu Glu Asp Leu Lys Leu Thr Ala Ser Leu Thr Asp Asn
225 230 235 240
Tyr Gln Lys Gly Lys Leu Glu Val Glu Ala Asn Ile Ala Tyr Arg Leu
245 250 255
Pro Asn Ala Ser Phe Lys Leu Glu Val Arg Asp Ser Glu Gly Asp Leu
260 265 270
Val Ala Glu Lys Leu Gly Pro Ile Arg Ser Glu Gln Leu Glu Phe Thr
275 280 285
Leu Ala Asp Leu Pro Val Ala Ala Trp Ser Ala Glu Lys Pro Asn Leu
290 295 300
Tyr Gln Val Arg Leu Tyr Leu Tyr Gln Ala Gly Ser Leu Leu Glu Val
305 310 315 320
Ser Arg Gln Glu Val Gly Phe Arg Asn Phe Glu Leu Lys Asp Gly Ile
325 330 335
Met Tyr Leu Asn Gly Gln Arg Ile Val Phe Lys Gly Ala Asn Arg His
340 345 350
Glu Phe Asp Ser Lys Leu Gly Arg Ala Ile Thr Glu Glu Asp Met Ile
355 360 365
Trp Asp Ile Lys Thr Met Lys Arg Ser Asn Ile Asn Ala Val Arg Cys
370 375 380
Ser His Tyr Pro Asn Gln Ser Leu Phe Tyr Arg Leu Cys Asp Lys Tyr
385 390 395 400
Gly Leu Tyr Val Ile Asp Glu Ala Asn Leu Glu Ser His Gly Thr Trp
405 410 415
Glu Lys Val Gly Gly His Glu Asp Pro Ser Phe Asn Val Pro Gly Asp
420 425 430
Asp Gln His Trp Leu Gly Ala Ser Leu Ser Arg Val Lys Asn Met Met
435 440 445
Ala Arg Asp Lys Asn His Ala Ser Ile Leu Ile Trp Ser Leu Gly Asn
450 455 460
Glu Ser Tyr Ala Gly Thr Val Phe Ala Gln Met Ala Asp Tyr Val Arg
465 470 475 480
Lys Ala Asp Pro Thr Arg Val Gln His Tyr Glu Gly Val Thr His Asn
485 490 495
Arg Lys Phe Asp Asp Ala Thr Gln Ile Glu Ser Arg Met Tyr Ala Pro
500 505 510
Ala Lys Val Ile Glu Glu Tyr Leu Thr Asn Lys Pro Ala Lys Pro Phe
515 520 525
Ile Ser Val Glu Tyr Ala His Ala Met Gly Asn Ser Val Gly Asp Leu
530 535 540
Ala Ala Tyr Thr Ala Leu Glu Lys Tyr Pro His Tyr Gln Gly Gly Phe
545 550 555 560
Ile Trp Asp Trp Ile Asp Gln Gly Leu Glu Lys Asp Gly His Leu Leu
565 570 575
Tyr Gly Gly Asp Phe Asp Asp Arg Pro Thr Asp Tyr Glu Phe Cys Gly
580 585 590
Asn Gly Leu Val Phe Ala Asp Arg Thr Glu Ser Pro Lys Leu Ala Asn
595 600 605
Val Lys Ala Leu Tyr Ala Asn Leu Lys Leu Glu Val Lys Asp Gly Gln
610 615 620
Leu Phe Leu Lys Asn Asp Asn Leu Phe Thr Asn Ser Ser Ser Tyr Tyr
625 630 635 640
Phe Leu Thr Ser Leu Leu Val Asp Gly Lys Leu Thr Tyr Gln Ser Arg
645 650 655
Pro Leu Thr Phe Gly Leu Glu Pro Gly Glu Ser Gly Thr Phe Ala Leu
660 665 670
Pro Trp Pro Glu Val Ala Asp Glu Lys Gly Glu Val Val Tyr Arg Val
675 680 685
Thr Ala His Leu Lys Glu Asp Leu Pro Trp Ala Asp Glu Gly Phe Thr
690 695 700
Val Ala Glu Ala Glu Glu Val Ala Gln Lys Leu Pro Glu Phe Lys Pro
705 710 715 720
Glu Gly Arg Pro Asp Leu Val Asp Ser Asp Tyr Asn Leu Gly Leu Lys
725 730 735
Gly Asn Asn Phe Gln Ile Leu Phe Ser Lys Val Lys Gly Trp Pro Val
740 745 750
Ser Leu Lys Tyr Ala Gly Arg Glu Tyr Leu Lys Arg Leu Pro Glu Phe
755 760 765
Thr Phe Trp Arg Ala Leu Thr Asp Asn Asp Arg Gly Ala Gly Tyr Gly
770 775 780
Tyr Asp Leu Ala Arg Trp Glu Asn Ala Gly Lys Tyr Ala Arg Leu Lys
785 790 795 800
Asp Ile Ser Cys Glu Val Lys Glu Asp Ser Val Leu Val Lys Thr Ala
805 810 815
Phe Thr Leu Pro Val Ala Leu Lys Gly Asp Leu Thr Val Thr Tyr Glu
820 825 830
Val Asp Gly Arg Gly Lys Ile Ala Val Thr Ala Asp Phe Pro Gly Ala
835
