Carrot antifreeze polypeptides

Details Carrot antifreeze polypeptides Carrot antifreeze polypeptides

1999-12-30

2004-09-28

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S012200, C530S350000, C530S326000, C530S325000, C530S300000, C426S100000, C426S049000, C426S565000, C426S660000

Reexamination Certificate

active

06797690

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The invention relates to anti-freeze polypeptides (AFPs) and food product containing AFPs.
BACKGROUND TO THE INVENTION
Anti-freeze polypeptides (AFPs) have been suggested for improving the freezing tolerance of foodstuffs.
For the purpose of the invention, the term AFP has the meaning as well-known in the art, namely those proteins which inhibit the growth of ice-crystals. See for example U.S. Pat. No. 5,111,792.
WO 90/13571 discloses, antifreeze peptides produced chemically or by recombinant DNA techniques from slants. The AFPs can suitably be used in food-products such as ice-cream. Example 3B shows modified ice-crystal shapes if a water-ice mixture is frozen into a film in combination with 0.01 wt % of AFP.
WO 92/22581 discloses AFPs from plants which can be used for controlling ice crystal shape in ice-cream. This document also describes a process for extracting a polypeptide composition from intercellular spaces of plants by infiltrating leaves with an extraction medium without rupturing the plant cells.
WO 94/03617 discloses the production of AFPs from yeast and their possible use in ice-cream. WO 96/11586 describes fish AFPs produced by microbes.
Up till now, however the use of AFPs has not been applied to commercially available consumer products. One reason for this are the high costs and complicated process for obtaining AFPs. Another problem is that sources of the AFPs are either difficult to obtain in sufficient quantities (e.g. fish containing AFPs) or are not directly suitable for use in food products.
The present invention aims to provide novel antifreeze polypeptides which have the advantage that they can easily be obtained from an abundant natural source and which provide good properties to products in which they are used.
It has been found that antifreeze polypeptides which possess good recrystallisation inhibition properties can be obtained from carrots. In particular it has been found that antifreeze polypeptides obtained from carrots show markedly better properties as compared to polypeptides isolated from other root vegetables. In particular the antifreeze polypeptides of the invention are capable of providing good recrystallisation inhibition properties without significantly changing the crystal shape of the ice-crystals, therewith possible leading to more favourable properties e.g. soft ice-cream.
Applicants have found that the effective antifreeze polypeptides from carrots are generally characterised by an apparent Molecular Weight on SDS-PAGE of 36 kDa. Accordingly in a first aspect the invention relates to antifreeze polypeptides which can be obtained from carrots and which have an apparent molecular weight on SDS-PAGE of 36 kDa.
In this context it will be clear to the skilled person that due to variation e.g. in SDS PAGE, the apparent molecular weight can only be determined with some variation in the results. For the purpose of the invention these variations e.g. from 30 to 40 kDa or from 34 to 39 kDa are also embraced within the scope of the term “apparent Molecular Weight of 36 kDa”.
Applicants also have found that the effective anti-freeze polypeptides according to the invention comprise fragments having an amino acid sequence as represented in the examples.
Accordingly in a second aspect the invention relates to polypeptides comprising one or more fragments (A-E) having an amino add sequence as follows:
SEQ ID NOS. 1-5, respectively, in order of appearance.
(A) LEU-PRC-ASN-LEU-PHE-GLY-LYS
(B) ILE-PRO-GLU-GLU-ILE-SER-ALA-LEU-LYS
(C) LEU-THR-X-LEU-ASP-LEU-SER-PHE-ASN-LYS
(D) SER-LEU-ARG-LEU-SER-SER-THR-SER-LEU-SER-GLY-PRO-VAL-PRO-LEU-PHE-PHE-PRO-GLN-LEU-X-LYS
(E) X-X-GLU-VAL-ILE-PRO-X-GLN-LEU-SER-THR-LEU-PRO-ASN-LEU-LYS
Preferably the AFPs of the invention comprise all of the partial sequences (A-E).
The complete amino acid sequence of the preferred AFP of the invention is represented below. Accordingly, in a third aspect the invention relates to an anti-freeze protein having an amino acid sequence as shown in Listing 1:
ATGAATATTGAATCATCTTTCTGCCCTATTTTGTGCATATGCATGATTTTCCTCTGCCTT
13
-------+---------+---------+---------+---------+---------+--
  72
a
M  N  I  E  S  S  F  C  P  I  L  C  I  C  M  I  F  L  C  L
-

CCAAACCTCTCTGCATCACAAAGATGCAACAACAACGACAAGCAAGCTTTACTCCAAATC
73
-------+---------+-------------------+---------+---------+--
 132
a
P  N  L  S  A  S  Q  R  C  N  N  N  D  K  Q  A  L  L  Q  I
-

AAAACAGCCTTGAAAAACCCCACCATTACAGACTCATGGGTGTCAGACGACGATTGTTGT
133
-------+---------+---------+--------+----------+---------+--
 192
a
K  T  A  L  K  N  P  T  I  T  D  S  N  V  S  D  D  D  C  C
-

GGTTGGGACCTAGTCGAATGTGACGAAACCAGCAACCGCATAATTTCCCTCATAATTCAA
193
-------+---------+-------------------+---------+---------+--
 252
a
G  W  D  L  V  E  C  D  E  T  S  N  R  I  I  S  L  I  I  Q
-

GACGACGAAGCTCTCACCGGCCAAATCCCACCTCAGGTGGGAGACCTACCATACCTCCAA
253
-------+---------+---------+---------+--------+----------+--
 312
a
D  D  E  A  L  T  G  Q  I  P  P  Q  V  G  D  L  P  Y  L  Q
-

GCCTTATGGTTCCGTAAACTCCCCAATCTTTTCGGAAAAATCCCAGAAGAAATTTCTGCA
313
-------+---------+---------+---------+---------+---------+--
 372
a
A  L  W  F  R  K  L  P  N  L  F  G  K  I  P  E  E  I  S  A
-

CTCAAAGACCTAAAATCCCTCAGACTCAGCTCGACCAGTCTCAGTGGCCCTGTCCCTTTA
373
-------+---------+---------+---------+---------+---------+--
 432
a
L  K  D  L  K  S  L  R  L  S  S  T  S  L  S  G  P  V  P  L
-

TTCTTCCCTCAGCTTACGAAACTAACTTGTTTAGACTTATCGTTTAACAAACTTTTGGGT
433
-------+---------+-------------------+---------+---------+--
 492
a
F  F  P  Q  L  T  K  L  T  C  L  D  L  S  F  N  K  L  L  G
-

GTAATCCCTCCTCAGCTTTCCACTCTTCCGAACCTTAAAGCCCTGCACTTAGAACGTAAC
493
-------+---------+---------+---------+---------+---------+--
 552
a
V  I  P  P  Q  L  S  T  L  P  N  L  K  A  L  H  L  E  R  N
-

GAACTCACCGGTGAAATCCCCGATATCTTTGGGAATTTTGCTGGATCCCCGGACATATAT
553
-------+---------+---------+---------+---------+---------+--
 612
a
E  L  T  G  E  I  P  D  I  F &ems

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