U.S. patent application number 16/771752 was filed with the patent office on 2021-03-11 for peptidase and its uses.
The applicant listed for this patent is Centre national de la recherche scientifique, Commissariata I'energie atomique et aux energies alternatives, UNIVERSITE GRENOBLE ALPES. Invention is credited to Alexandre APPOLAIRE, Hind BASBOUS, Bruno FRANZETTI, Eric GIRARD.
Application Number | 20210071162 16/771752 |
Document ID | / |
Family ID | 1000005272960 |
Filed Date | 2021-03-11 |
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United States Patent
Application |
20210071162 |
Kind Code |
A1 |
FRANZETTI; Bruno ; et
al. |
March 11, 2021 |
PEPTIDASE AND ITS USES
Abstract
The invention relates to the uses of a new characterized TET
protein showed restricted to N-terminus glycine residues
exopeptidase. The invention also relates to a method comprising
said use of said new characterized TET protein as a N-terminus
glycine residues specific exopeptidase. The invention further
relates to a support wherein it is immobilized on said new
characterized TET protein as a N-terminus glycine residues specific
exopeptidase.
Inventors: |
FRANZETTI; Bruno;
(SASSENAGE, FR) ; GIRARD; Eric; (ROMANS-SUR-ISERE,
FR) ; APPOLAIRE; Alexandre; (GRENOBLE, FR) ;
BASBOUS; Hind; (MEYLAN, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Centre national de la recherche scientifique
Commissariata I'energie atomique et aux energies alternatives
UNIVERSITE GRENOBLE ALPES |
PARIS
PARIS
SAINT MARTIN D'HERES |
|
FR
FR
FR |
|
|
Family ID: |
1000005272960 |
Appl. No.: |
16/771752 |
Filed: |
December 12, 2018 |
PCT Filed: |
December 12, 2018 |
PCT NO: |
PCT/EP2018/084632 |
371 Date: |
June 11, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23J 3/341 20130101;
A23J 3/347 20130101; C12P 21/06 20130101; A23J 3/346 20130101; A23J
3/348 20130101; C12N 9/485 20130101 |
International
Class: |
C12N 9/48 20060101
C12N009/48; C12P 21/06 20060101 C12P021/06; A23J 3/34 20060101
A23J003/34 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 12, 2017 |
EP |
17306758.8 |
Claims
1. A method for providing a N-terminus glycine residues specific
exopeptidase, wherein said N-terminus glycine residues specific
exopeptidase is provided by a TET protein comprising the amino acid
sequence as set forth in SEQ ID NO: 1, or any homologous protein
derived from said TET protein as set forth in SEQ ID NO: 1 by
substitution, addition or deletion of at least one amino acid,
provided that the derived protein retains at least 70%, preferably
at least 79% of identity with the amino acid sequence as set forth
in SEQ ID NO: 1, and said derived protein retaining a N-terminus
glycine residues specific exopeptidase activity.
2. A method for the modification of all or part of the polypeptide
content of a substrate comprising peptides, polypeptides and/or
proteins harbouring a N-terminus glycine residue, wherein said
modification is performed by at least a TET protein harbouring at
least a N-terminus glycine residues specific exopeptidase activity,
said at least TET protein comprising the amino acid sequence as set
forth in SEQ ID NO: 1, or any homologous protein derived from said
at least TET protein as set forth in SEQ ID NO: 1 by substitution,
addition or deletion of at least one amino acid, provided that the
derived protein retains at least 70%, preferably at least 79% of
identity with the amino acid sequence as set forth in SEQ ID NO: 1,
and said derived protein retaining a N-terminus glycine residues
specific exopeptidase activity.
3. The method according to claim 1, wherein said TET protein or
said derived protein originates from an extremophilic microorganism
belonging to the Thermococcales order.
4. The method according to claim 3, wherein said extremophilic
microorganism is Pyrococcus horikoshii.
5. The method according to claim 2, wherein peptides, polypeptides
and/or proteins of said substrate are obtained from food industry
or health industry or chemical industry, preferably proteins from
fermented products or soya products or sea food products or cheese
products.
6. Method for degrading, from the N-terminus part, a polypeptide
harbouring a glycine residue at its N-terminal part, said method
comprising a step of contacting said polypeptide harbouring a
glycine residue at its N-terminal part with at least a TET protein
harbouring at least a N-terminus glycine residues specific
exopeptidase activity, said TET protein comprising the amino acid
sequence as set forth in SEQ ID NO: 1, or at least one homologous
protein derived from said TET protein as set forth in SEQ ID NO: 1
by substitution, addition or deletion of at least one amino acid,
provided that the derived protein retains at least 70%, preferably
at least 79% of identity with the amino acid sequence as set forth
in SEQ ID NO: 1, and said derived protein retaining a N-terminus
glycine residues specific exopeptidase activity, and possibly a
step of recovering the resulting N-terminal glycine free
peptides.
7. Method for modifying all or part of the polypeptide content of a
substrate comprising peptides, polypeptides and/or proteins,
wherein at least one of the peptides, polypeptides and/or proteins
of said substrate harbours a N-terminus glycine residue, said
method comprising a step of contacting said substrate with at least
a TET protein harbouring at least a N-terminus glycine residues
specific exopeptidase activity, said TET protein comprising the
amino acid sequence as set forth in SEQ ID NO: 1, or at least one
homologous protein derived from said TET protein as set forth in
SEQ ID NO: 1 by substitution, addition or deletion of at least one
amino acid, provided that the derived protein retains at least 70%,
preferably at least 79% of identity with the amino acid sequence as
set forth in SEQ ID NO: 1, and said derived protein retaining a
N-terminus glycine residues specific exopeptidase activity, and
possibly a step of recovering the modified polypeptide content of
said substrate.
8. The method according to claim 6, wherein said step of contacting
comprises the activation of the said TET protein or derived protein
using as enzyme cofactor at least one of the metal ions of the
group comprising: Ni.sup.2+, Co.sup.2+ and Mn.sup.2+, preferably
using Ni.sup.2+ as enzyme cofactor.
9. The method according to claim 6, wherein said step of contacting
is carried out from pH 9 to pH 10, preferably at pH 9.5.
10. The method according to claim 6, wherein said step of
contacting is carried out from 80.degree. C. to 100.degree. C.,
preferably at 85.degree. C.
11. The method according to claim 6, wherein said at least TET
protein or said derived protein is immobilized on a solid support,
preferably on a filter cartridge or on silica beads or on magnetic
beads or on organic polymeric materials, or on inorganic polymeric
materials or on membrane devices or in microcapsules.
12. A method for the modification of all or part of the polypeptide
content of a substrate comprising peptides, polypeptides and/or
proteins, wherein at least one of the peptides, polypeptides and/or
proteins of said substrate harbours a N-terminus glycine residue,
said method comprises the contact of said polypeptide content with
a solid support, and wherein is immobilized on said solid support
at least a TET protein harbouring at least a N-terminus glycine
residues specific exopeptidase activity, said TET protein
comprising the amino acid sequence as set forth in SEQ ID NO: 1, or
at least one homologous protein derived from said TET protein as
set forth in SEQ ID NO: 1 by substitution, addition or deletion of
at least one amino acid, provided that the derived protein retains
at least 70%, preferably at least 79% of identity with the amino
acid sequence as set forth in SEQ ID NO: 1, and said derived
protein retaining a N-terminus glycine residues specific
exopeptidase activity.
13. The method according to claim 12, wherein said solid support is
a filter cartridge or silica beads or magnetic beads or on organic
polymeric materials, or on inorganic polymeric materials or on
membrane devices or in microcapsules.
14. The method according to claim 12, wherein peptides,
polypeptides and/or proteins of said substrate are obtained from
food, chemical and health industries, or from biomass.
Description
[0001] The invention is related to peptidases and their use.
[0002] Peptidases are involved in digesting polypeptide chain of
peptides and proteins into shorter fragments by splitting the
peptide bonds that link amino acid residues. Some detach the
terminal amino acids from the protein chain and are called
exopeptidases, such as aminopeptidases, carboxypeptidase A; others
attack internal peptide bonds of a protein and are called
endopeptidases, such as trypsin, chymotrypsin, pepsin, papain,
elastase.
[0003] Aminopeptidases are enzymes that catalyse the cleavage of
amino acids from the amino terminus (N-terminus) of proteins or
peptides. They are widely distributed throughout the three-domain
system, i.e. archaea, bacteria, and eukaryote domains, and are
found in many subcellular organelles, in cytosol, and as membrane
components.
[0004] Aminopeptidases which are directed to glycine residues,
called glycine aminopeptidases (GAPs), are of great interest for
the industrial food. Indeed, GAPs are known to better degrade
peptides enriched in glycine, which can modify the taste of food
preparations from fermentation, like cheeses, the tofu or the sufu.
In particular, the release of glycine from the polypeptide chains
is of great interest for the Japanese industry because glycine is
known to be an enhancer of sweet tastes which are specific to the
Japanese gastronomy. The release of the glycine is also important
for the flavour of several dry cheeses (feta, parmesan, etc. . . .
).
[0005] However, glycine residues are hard to release for
aminopeptidases. Nowadays, only three aminopeptidases were found to
exhibit clear preference for glycine residues. One of these GAPs is
a Zn-dependent metallopeptidase from M61 family secreted by the
gram-negative bacteria Sphingomonas capsulata (Jamdar, S. N.
(2009)). Another one is a eukaryotic S12 family serine peptidase
found in the cytosol of Actinomucor oryzae (Marui, J., et al.
(2012)). The last one is the cytosolic glycyl aminopeptidase of
Actinomucor elegans, for which the residues implicated in enzymatic
mechanism are still ambiguous (Ito K et al. (2003)).
[0006] However, these GAPs are not restricted to the glycine
residues and show a significant amidolytic activity on other amino
acids. Further, these GAPs shows poor yield of production, and come
from mesophilic organisms which limits their scope of
application.
[0007] Therefore, there is a need to provide a new aminopeptidase
specific for N-terminus glycine residues, able to operate in
industrial conditions.
[0008] The aim of the invention is to obviate these drawbacks.
[0009] Thus, the invention relates to the use of a TET protein as a
N-terminus glycine residues specific exopeptidase, said TET protein
comprising the amino acid sequence as set forth in SEQ ID NO:
1,
[0010] or any homologous protein derived from said TET protein as
set forth in SEQ ID NO: 1 by substitution, addition or deletion of
at least one amino acid, provided that the derived protein retains
at least 70%, preferably at least 79% of identity with the amino
acid sequence as set forth in SEQ ID NO: 1, and said derived
protein retaining a N-terminus glycine residues specific
exopeptidase activity.
[0011] The invention also relates to the use of at least a TET
protein harbouring at least a N-terminus glycine residues specific
exopeptidase activity, for the modification of all or part of the
polypeptide content of a substrate comprising peptides,
polypeptides and/or proteins harbouring a N-terminus glycine
residue, said at least TET protein comprising the amino acid
sequence as set forth in SEQ ID NO: 1,
[0012] or any homologous protein derived from said at least TET
protein as set forth in SEQ ID NO: 1 by substitution, addition or
deletion of at least one amino acid, provided that the derived
protein retains at least 70%, preferably at least 79% of identity
with the amino acid sequence as set forth in SEQ ID NO: 1, and said
derived protein retaining a N-terminus glycine residues specific
exopeptidase activity.
[0013] The invention is based on the unexpected characterization by
the inventors of a new TET protein as an exopeptidase unambiguously
restricted to the cleavage of glycine residues. The inventors
surprisingly found that this peptidase is devoid of amidolytic
activity on all other amino acid residues except glycine residues.
Advantageously, the inventors discovered that this TET protein is a
thermophilic protein and can be activated by heat. This is of
interest in fermentation industry for example, where the processes
are carried out at high temperatures. As a TET protein, this
exopeptidase harbours biophysical properties favourable to its
immobilization on a support. Consequently, there is advantageously
no need for recovering said TET protein in the final preparation,
which allows reducing the cost of production of peptides devoid of
their glycine N-terminus residues.
[0014] Hereafter, the TET protein as set forth in SEQ ID NO: 1
corresponds to the protein PhTET4.
[0015] By "exopeptidase", it is meant in the invention a peptidase
that catalyses the cleavage of the terminal peptide bond of an
amino acid chain, starting either from the amino or carboxyl
terminal of the said amino acid chain.
[0016] By "peptide", it is meant in the invention an amino acid
chain comprising at least 2 amino acids. Peptides can be obtained
either from protein degradation or from chemical synthesis. By
"polypeptide", it is meant in the invention an amino acid chain
larger than a peptide and obtained from degradation of proteins and
not from chemical synthesis.
[0017] Peptides and polypeptides may harbour biological functions
within the context of a protein (signal peptide, death domain, bHLH
domain . . . ). By "protein", it is meant in the invention an amino
acid chain containing molecule harbouring biological function and
which is found naturally in an organism, said biological function
being part of a natural process of the cell.
[0018] By "at least 70% of identity with the sequence as set forth
in SEQ ID NO: 1", it is meant in the invention 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and
100% of identity with the sequence as set forth in SEQ ID NO: 1.
Regarding the percentage of identity, it is defined by the
percentage of amino acid residues of SEQ ID NO: 1 which align with
the same amino acid in the sequence of the homologous protein. The
sequence alignment is carried out using dedicated algorithms and
programs (such as ClustalW, for instance).
[0019] In the invention, the term "comprising" is meant to include
the terms "consisting essentially of" and "consisting of".
[0020] By "modification of all or part of the polypeptide", it is
meant in the invention that the modification of a peptide can
result in the removal of one or more amino acid from the peptide.
If the peptide contains only aromatic amino acids, the peptide can
be completely degraded, i.e. can be converted into the free amino
acids that constitute the peptide.
[0021] Moreover, the "modification of all or part of the
polypeptide", means also in the invention that, if a composition
contains two or more peptides, at least one peptide will be
degraded by contacted the TET protein according to the invention.
If only some peptides are degraded, the composition of peptide will
be considered to be partially modified. If all the peptides are
subjected to a degradation, the composition of peptide will be
considered to be totally modified.
[0022] In the invention, regarding a peptide, a polypeptide, a
protein or a polypeptide content, the terms "modification" and
"degradation" can be used uniformly.
[0023] Advantageously, said TET protein or said derived protein
originates from an extremophilic microorganism belonging to the
Thermococcales order and is isolated from this extremophilic
microorganism. By "extremophilic", it is meant in the invention an
organism that thrives in physically or geochemically extreme
conditions that are detrimental to most life on Earth. In contrast,
organisms that live in more moderate environments may be termed
mesophiles or neutrophiles. Among the hyperthermophilic archaea,
representatives of order Thermococcales form the most numerous
group to date. Members of this group are the most frequently
isolated hyperthermophilic archaea. They are heterotrophic and as
such regarded as the major constituents of organic matter within
marine hot water ecosystems. They belong to the branch of
Euryarchaeota that contains the methanogens, the genus
Thermoplasma, and the extremely halophilic archaea. The
Thermococcales order is actually represented by three genera:
Pyrococcus, Thermococcus and the newly described Paleococcus.
[0024] Advantageously, said extremophilic microorganism is
Pyrococcus horikoshii.
[0025] Advantageously, peptides, polypeptides and/or proteins of
said substrate are obtained from food industry or health industry
or chemical industry. Advantageously, proteins from fermented
products or soya products or sea food products or cheese
products.
[0026] The generation of protein-rich industrial wastes is very
high (only from sunflower, about one million tons in Spain). These
wastes are not used at all, or are underused in the form of low
added-value products. This type of by-products constitutes a
reservoir of proteins with a great economic potential.
[0027] The TET protein according to the invention, in view of its
activity, can be used in various domains for instance, but without
limitation: [0028] For the valorization of agriculture wastes:
proteins and peptides originating from agriculture can be recycled
for animal feed, or for producing feed additives. In order to be
used, the vegetal proteins have to be degraded to avoid any
antinutritional side effects. [0029] For the valorization of
chemical wastes [0030] For the valorization of food industry waste:
suitable proteins to be treated with the TET aminopeptidase
according to the invention may be for instance proteins obtained
from dairy products, fruit juices, beers, flours or cured products.
Moreover, the products from wine industry, from cheese industry,
and see food industry are particularly advantageous and can be
valorized by using the TET protein according to the invention.
[0031] Products from biomass: some alternative to common protein
sources are now emerging in view of the need to provide more and
more feed for the Earth population. For instance, algae and
microorganisms, along with insects, are very rich in protein that
can be used for providing new sources of amino acids or proteins
that can be eaten by animal and humans. Therefore, proteins or
peptides from algae or microorganisms can be relevant sources for
treatment by using the TET protein according to the invention.
[0032] Another kind of wastes are the wastes produced from health
industry. For instance, solid, regulated medical waste can includes
materials generated in the diagnosis, treatment, research, or
immunization of human beings or animals. Examples of regulated
medical waste includes: cultures and stocks, pathological wastes,
human blood and blood products, sharps, certain animal waste and
isolation wastes. The TET protein according to the invention may
help to valorize such kinds of wastes.
[0033] The invention also relates to a method for degrading, from
the N-terminus part, a polypeptide harbouring a glycine residue at
its N-terminal part, said method comprising a step of contacting
said polypeptide harbouring a glycine residue at its N-terminal
part with
[0034] at least a TET protein harbouring at least a N-terminus
glycine residues specific exopeptidase activity, said TET protein
comprising the amino acid sequence as set forth in SEQ ID NO: 1,
or
[0035] at least one homologous protein derived from said TET
protein as set forth in SEQ ID NO: 1 by substitution, addition or
deletion of at least one amino acid, provided that the derived
protein retains at least 70%, preferably at least 79% of identity
with the amino acid sequence as set forth in SEQ ID NO: 1, and said
derived protein retaining a N-terminus glycine residues specific
exopeptidase activity,
[0036] and possibly a step of recovering the resulting N-terminal
glycine free peptides.
[0037] The invention also relates to a method for modifying all or
part of the polypeptide content of a substrate comprising peptides,
polypeptides and/or proteins, wherein at least one of the peptides,
polypeptides and/or proteins of said substrate harbours a
N-terminus glycine residue, said method comprising a step of
contacting said substrate with
[0038] at least a TET protein harbouring at least a N-terminus
glycine residues specific exopeptidase activity, said TET protein
comprising the amino acid sequence as set forth in SEQ ID NO: 1,
or
[0039] at least one homologous protein derived from said TET
protein as set forth in SEQ ID NO: 1 by substitution, addition or
deletion of at least one amino acid, provided that the derived
protein retains at least 70%, preferably at least 79% of identity
with the amino acid sequence as set forth in SEQ ID NO: 1, and said
derived protein retaining a N-terminus glycine residues specific
exopeptidase activity,
[0040] and possibly a step of recovering the modified polypeptide
content of said substrate.
[0041] Advantageously, in the method for modifying all or part of
the polypeptide content of a substrate comprising peptides,
polypeptides and/or proteins harbouring a N-terminus glycine
residue, said step of contacting comprises the activation of the
said TET protein or derived protein using as enzyme cofactor at
least one of the metal ions of the group consisting of Ni.sup.2+,
Co.sup.2+ and Mn.sup.2+, preferably using Ni.sup.2+ as enzyme
cofactor. This aspect of the invention is interesting because
Ni.sup.2+ is a rare cofactor of aminopeptidase. Indeed, most of the
aminopeptidases are activated by Zn.sup.2+ cofactor. Consequently,
PhTET4 can be selectively activated in a pool of
aminopeptidases.
[0042] In particular, said step of contacting is carried out from
pH 9 to pH 10, provided that the aminopeptidase activity of said at
least TET protein or said derived protein being maintained to a
aminopeptidase activity of at least 80% of their maximum activity.
Advantageously, said step of contacting is carried out at least at
pH 9. By "at least at pH 9", it is meant in the invention pH 9, pH
9.1, pH 9.2, pH 9.3, pH 9.4, pH 9.5. Advantageously, said step of
contacting is carried out at most at pH 10. By "at most at pH 10",
it is meant in the invention pH 10, pH 9.9, pH 9.8, pH 9.7, pH 9.6.
Advantageously said step of contacting is carried out at pH
9.5.
[0043] Advantageously, said step of contacting is carried out from
80.degree. C. to 100.degree. C., provided that the aminopeptidase
activity of said at least TET protein or said derived protein being
maintained to an aminopeptidase activity of at least 80% of their
maximum activity. Advantageously, said step of contacting is
carried out at least at 80.degree. C. By "at least at 80.degree.
C.", it is meant in the invention, 80.degree. C., 80.1.degree. C.,
80.2.degree. C., 80.3.degree. C., 80.4.degree. C., 80.5.degree. C.,
80.6.degree. C., 80.7.degree. C., 80.8.degree. C., 80.9.degree. C.,
81.degree. C., 81.1.degree. C., 81.2.degree. C., 81.3.degree. C.,
81.4.degree. C., 81.5.degree. C., 81.6.degree. C., 81.7.degree. C.,
81.8.degree. C., 81.9.degree. C., 82.degree. C., 82.1.degree. C.,
82.2.degree. C., 82.3.degree. C., 82.4.degree. C., 82.5.degree. C.,
82.6.degree. C., 82.7.degree. C., 82.8.degree. C., 82.9.degree. C.,
83.degree. C., 83.1.degree. C., 83.2.degree. C., 83.3.degree. C.,
83.4.degree. C., 83.5.degree. C., 83.6.degree. C., 83.7.degree. C.,
83.8.degree. C., 83.9.degree. C., 84.degree. C., 84.1.degree. C.,
84.2.degree. C., 84.3.degree. C., 84.4.degree. C., 84.5.degree. C.,
84.6.degree. C., 84.7.degree. C., 84.8.degree. C., 84.9.degree. C.,
85.degree. C. Advantageously, said step of contacting is carried
out at most at 100.degree. C. By "at most at 100.degree. C.", it is
meant in the invention 100.degree. C., 99.9.degree. C.,
99.8.degree. C., 99.7.degree. C., 99.6.degree. C., 99.5.degree. C.,
99.4.degree. C., 99.3.degree. C., 99.2.degree. C., 99.1.degree. C.,
99.degree. C., 98.9.degree. C., 98.8.degree. C., 98.7.degree. C.,
98.6.degree. C., 98.5.degree. C., 98.4.degree. C., 98.3.degree. C.,
98.2.degree. C., 98.1.degree. C., 98.degree. C., 97.9.degree. C.,
97.8.degree. C., 97.7.degree. C., 97.6.degree. C., 97.5.degree. C.,
97.4.degree. C., 97.3.degree. C., 97.2.degree. C., 97.1.degree. C.,
97.degree. C., 96.9.degree. C., 96.8.degree. C., 96.7.degree. C.,
96.6.degree. C., 96.5.degree. C., 96.4.degree. C., 96.3.degree. C.,
96.2.degree. C., 96.1.degree. C., 96.degree. C., 95.9.degree. C.,
95.8.degree. C., 95.7.degree. C., 95.6.degree. C., 95.5.degree. C.,
95.4.degree. C., 95.3.degree. C., 95.2.degree. C., 95.1.degree. C.,
95.degree. C., 94.9.degree. C., 94.8.degree. C., 94.7.degree. C.,
94.6.degree. C., 94.5.degree. C., 94.4.degree. C., 94.3.degree. C.,
94.2.degree. C., 94.1.degree. C., 94.degree. C., 93.9.degree. C.,
93.8.degree. C., 93.7.degree. C., 93.6.degree. C., 93.5.degree. C.,
93.4.degree. C., 93.3.degree. C., 93.2.degree. C., 93.1.degree. C.,
93.degree. C., 92.9.degree. C., 92.8.degree. C., 92.7.degree. C.,
92.6.degree. C., 92.5.degree. C., 92.4.degree. C., 92.3.degree. C.,
92.2.degree. C., 92.1.degree. C., 92.degree. C., 91.9.degree. C.,
91.8.degree. C., 91.7.degree. C., 91.6.degree. C., 91.5.degree. C.,
91.4.degree. C., 91.3.degree. C., 91.2.degree. C., 91.1.degree. C.,
91.degree. C., 90.9.degree. C., 90.8.degree. C., 90.7.degree. C.,
90.6.degree. C., 90.5.degree. C., 90.4.degree. C., 90.3.degree. C.,
90.2.degree. C., 90.1.degree. C., 90.degree. C., 89.9.degree. C.,
89.8.degree. C., 89.7.degree. C., 89.6.degree. C., 89.5.degree. C.,
89.4.degree. C., 89.3.degree. C., 89.2.degree. C., 89.1.degree. C.,
89.degree. C., 88.9.degree. C., 88.8.degree. C., 88.7.degree. C.,
88.6.degree. C., 88.5.degree. C., 88.4.degree. C., 88.3.degree. C.,
88.2.degree. C., 88.1.degree. C., 88.degree. C., 87.9.degree. C.,
87.8.degree. C., 87.7.degree. C., 87.6.degree. C., 87.5.degree. C.,
87.4.degree. C., 87.3.degree. C., 87.2.degree. C., 87.1.degree. C.,
87.degree. C., 86.9.degree. C., 86.8.degree. C., 86.7.degree. C.,
86.6.degree. C., 86.5.degree. C., 86.4.degree. C., 86.3.degree. C.,
86.2.degree. C., 86.1.degree. C., 86.degree. C., 85.9.degree. C.,
85.8.degree. C., 85.7.degree. C., 85.6.degree. C., 85.5.degree. C.,
85.4.degree. C., 85.3.degree. C., 85.2.degree. C., 85.1.degree. C.
Advantageously said step of contacting is carried out at 85.degree.
C. Carried out amidolytic activity at high temperature is
interesting in industry, because one use of the TET protein is
about fermentation which is also carried out at high temperature.
Moreover, the TET protein is heat activatable and can be therefore
specifically activated during the fermentation step of an
industrial process.
[0044] Compared to other TET family member peptidases, the TET
protein according to the invention is active at a pressure varying
from 0.1 MPa to 350 MPa.
[0045] Advantageously, said at least TET protein or said derived
protein is immobilized on a solid support. As abovementioned, the
biophysical properties of a TET protein allow its use on a support.
Consequently, in an industrial process for a final preparation, the
TET protein can be put into contact with the substrate without
mixing them together so that there is no need to recover said TET
protein in the final preparation. Advantageously, said at least TET
protein or said derived protein is immobilized on a filter
cartridge or on silica beads or on magnetic beads or on organic
polymeric materials, or on inorganic polymeric materials or on
membrane devices or in microcapsules. Membrane devices include
hollow fibers.
[0046] In view of the robustness of the TET protein, and the
stability of the structure, it is possible to immobilized the TET
protein on a support. Such a support is advantageous and allows to
carry out a peptide, polypeptide or peptide degradation and recover
the resulting degraded peptide, polypeptides and protein, easily
without additional step of separation of the enzyme and the
resulting product. Current enzyme immobilization methods are
described in the review of Bilal et al. (Bilal M, Iqbal H M, Guo S,
Hu H, Wang W, Zhang X (2017) State-of-the-art protein engineering
approaches using biological macromolecules: A review from
immobilization to implementation view point. Int J Biol Macromol.
November 2.).
[0047] The invention is further related to the use of a solid
support for the modification of all or part of the polypeptide
content of a substrate comprising peptides, polypeptides and/or
proteins, wherein at least one of the peptides, polypeptides and/or
proteins of said substrate harbours a N-terminus glycine residue,
and wherein is immobilized on said solid support
[0048] at least a TET protein harbouring at least a N-terminus
glycine residues specific exopeptidase activity, said TET protein
comprising the amino acid sequence as set forth in SEQ ID NO: 1,
or
[0049] at least one homologous protein derived from said TET
protein as set forth in SEQ ID NO: 1 by substitution, addition or
deletion of at least one amino acid, provided that the derived
protein retains at least 70%, preferably at least 79% of identity
with the amino acid sequence as set forth in SEQ ID NO: 1, and said
derived protein retaining a N-terminus glycine residues specific
exopeptidase activity.
[0050] Advantageously, said solid support is a filter cartridge or
silica beads or magnetic beads or on organic polymeric materials,
or on inorganic polymeric materials or on membrane devices or in
microcapsules. Membrane devices include hollow fibers.
[0051] Advantageously, peptides, polypeptides and/or proteins of
said substrate are obtained from food, chemical and health
industries, or from biomass, as mentioned above. Advantageously,
proteins from fermented products or soya products or sea food
products or cheese products.
[0052] Alternatively, said at least TET protein or said derived
protein is immobilized as cross-linked enzyme aggregates
(CLEAs).
[0053] CLEAs are developed by precipitation of the enzyme from a
solution by adding salt, such as ammonium sulphate, or
water-miscible organic solvent, followed by cross-linking with a
bifunctional reagent (Bilal M, Iqbal H M, Guo S, Hu H, Wang W,
Zhang X (2017) State-of-the-art protein engineering approaches
using biological macromolecules: A review from immobilization to
implementation view point. Int J Biol Macromol. November 2.).
[0054] The invention will be better understood from the following
example and the 8 following figures.
LEGEND TO THE FIGURES
[0055] FIG. 1 is a sequence alignment of PhTET1, 2, 3 and 4.
Conserved residues are in a grey box and similar residues are
boxed. The numbering and the secondary structure elements are those
of PhTET1 (PDB ID: 2WYR). Light grey stars and heavy grey starts
highlight the metal-binding and active residues, respectively.
[0056] FIGS. 2A and 2B are respectively an elution profile of
PhTET4 on size-exclusion chromatography column (superdex200 10/300
GL), and a micrograph of the said eluted PhTET4 observed by
negative strain electron microscopy. In FIG. 2A, X-axis represents
the volume (ml) of exclusion and Y-axis represents the absorbance
(mAU) at 280 nm.
[0057] FIG. 3 is a graph representing the variation of PhTET4
specific activity in the absence (WM) of metal, or in the presence
of ions Calcium (Ca), Cobalt (Co), Fer (Fell), Magnesium (Mg),
Nickel (Ni), and Zinc (Zn). Y-axis represent the specific activity
of PhTET4 (.mu.mol (pNA) mg-1 (PhTET4) min-1). pNA stands for
p-Nitroaniline.
[0058] FIG. 4 is a graph representing the evolution of PhTET4
specific activity from pH 6 to pH 11. Y-axis represent the specific
activity of PhTET4 (.mu.mol (pNA) mg-1 (PhTET4) min-1). pNA stands
for p-Nitroaniline.
[0059] FIG. 5 is a graph representing the evolution of PhTET4
specific activity from 20.degree. C. to 95.degree. C. Y-axis
represent the specific activity of PhTET4 (.mu.mol (pNA) mg-1
(PhTET4) min-1). pNA stands for p-Nitroaniline.
[0060] FIG. 6 represents the size exclusion chromatography of
PhTET4 in absence or presence of EDTA. The continuous line
represents the size exclusion chromatography of PhTET4 in absence
of EDTA. The discontinuous line represents the size exclusion
chromatography of PhTET4 in presence of EDTA. X-axis represent the
volume (ml) of exclusion. Y-axis represents the absorbance (mAU) at
280 nm.
[0061] FIG. 7 is a graph representing the relative amidolytic
activity of PhTET4 on L-Alanine (A), D-Alanine (D-A), L-Aspartate
(D), L-Glutamate (E), L-Phenylalanine (F), L-Glycine (G),
L-Histidine (H), L-Isoleucine (I), L-Lysine (K), L-Leucine (L),
L-Methionine (M), L-Asparagine (N), L-Proline (P), L-Glutamine (Q),
L-Arginine (R), L-Serine (S), L-Threonine (T), L-Valine (V),
L-Tryptophan (W), and L-Tyrosine (Y). Y-axis represents the
relative activity (%) of PhTET4.
[0062] FIG. 8A represents the size exclusion chromatography of GLM.
FIG. 8B represents the size exclusion chromatography of GMDSLAFSGGL
(SEQ ID NO: 6) peptides, respectively, after incubation with
PhTET4. In both figures, the continuous line represents the size
exclusion chromatography of the intact peptide, and the
discontinuous line represents size exclusion chromatography of the
said peptide cleaved by PhTET4. X-axis represents the elution time
(sec). Y-axis represents the Absorbance (mAU) at 214 nm.
EXAMPLE
[0063] Methods
[0064] PhTET4 Expression and Purification
[0065] The gene encoding for PH0737 (SEQ ID NO: 5) was cloned in
pET-41c vector GeneCust Europe, Luxembourg. Recombinant protein was
overexpressed in Escherichia coli BL21 (DE3)-RIL strain during 4
hours at 37.degree. C. by induction with 0.1 mM IPTG in 1 L of
lysogeny broth medium. The cell pellet was conserved at -80.degree.
C. until using. The cells were re-suspended in 25 mL Tris-HCl 50 mM
pH 8, NaCl 90 mM and Triton X-100 0.1%, supplemented with 6.25 mg
lysozyme (Euromedex), 1.25 mg DNase I grade II (Roche), 5 mg RNase
(Roche), 25 mg Pefabloc SC (Roche) and 0.25 mL MgSO4 at 2 M. Cells
were disrupted by sonication at 30 watts with 5 cycles on/off of 30
sec each at 4.degree. C., and then heated for 20 min at 75.degree.
C. to eliminate most mesophilic proteins of host strain. The lysate
was clarified by centrifugation at 12,000 rpm, 4.degree. C. for 1
hour with JA20 rotor (Beckman), and the supernatant was loaded on
ResourceQ column (GE healthcare) equilibrated with 20 mM Tris-HCl
pH 7.5, 100 mM NaCl. The 30 mL of the flow-through were retained
and diluted with 20 mL Tris-HCl pH 7.5 to a final concentration of
105 mM NaCl. The new supernatant volume was loaded a second time on
ResourceQ column. After washing the column with 20 mM Tris-HCl pH
7.5, 100 mM NaCl, bound proteins were eluted with linear salt
gradient (154-290 mM NaCl). The protein-contained fractions were
pooled together and loaded on Superdex200 10/300 GL size exclusion
column (GE healthcare) equilibrated with 20 mM Tris-HCl pH 7.5, 150
mM NaCl. PhTET4 protein was eluted at 11 mL of column exclusion
volume. The peak fractions were combined and concentrated with
Amicon Ultra 30 kDa cut-off and stored at 4.degree. C. The purity
of prepared protein was checked by SDS-PAGE. 3 mg of PhTET4 were
produced from 1 L of culture.
[0066] PhTET4 Negative Stain Electron Microscopy
[0067] After the size exclusion chromatography step, 4 .mu.l of
PhTET4 at 0.05 mg/ml were deposited onto carbon-coated 400-mesh
copper grids. The samples were stained using uranyl acetate 2% and
air-dried. Images were taken under low-dose conditions in a T12FEI
electron microscope working at 120 kV and with a nominal
magnification of 40,000 using an Orius SC1000 CCD camera.
[0068] PhTET4 Substrate Specificity
[0069] The hydrolytic activity of PhTET4 was determined by using
chromogenic and fluorogenic compounds: aminoacyl-pNAs
(p-Nitroaniline) and aminoacyl-AMCs (7-Amino-4-methylcoumarin),
respectively. Reactions were initiated by addition of 4 .mu.g/ml
(final concentration) of PhTET4 to 400 .mu.l of pre-warmed mixture
containing 5 mM chromogenic or fluorogenic substrate in 50 mM
PIPES, 150 mM KCl, pH 7.5 at 80.degree. C. Since all tetrahedral
aminopeptidases of P. horikoshii are activated by cobalt, the
inventors started by using 0.1 mM CoCl2 as metal activator for
PhTET4 enzymatic activity. Then, the experimentation was later
repeated using the determined optimal conditions (0.1 mM NiCl2, pH
9.5 and at 85.degree. C.). In order to avoid water evaporation, the
total volume was covered by a layer of mineral oil. Catalytic
activity was followed during 10 min by measuring the absorbance of
released pNA at 405 nm or the AMC fluorescence using excitation and
emission wavelengths of 355 and 460 nm, respectively.
[0070] PhTET4 Metallic Cofactor
[0071] In presence of cobalt low activity was identified against
Gly-pNA. So, in order to enhance the enzymatic activity of PhTET4,
several divalent metals were tested at 0.1 mM final concentration
in a reaction volume containing 4 .mu.g/ml PhTET4, 50 mM CHES, 150
mM KCl and 5 mM Gly-pNA, pH 9.5. The reaction was followed at
80.degree. C. by measuring the absorbance of released pNA at 405 nm
during 10 min.
[0072] PhTET4 Optimal pH
[0073] The effect of pH on PhTET4 enzymatic activity was studied by
using different buffers: PIPES, pH 6-7.5; CHES, pH 8.2-10 and CAPS,
pH 10.5-11. All buffers were used at 50 mM final concentration in
presence of 4 .mu.g/ml PhTET4, 150 mM KCl, 0.1 mM NiCl2 and 5 mM
Gly-pNA at 80.degree. C. The incubation was done for 10 min by
measuring the absorbance of released pNA at 405 nm.
[0074] PhTET4 Optimal Temperature
[0075] The temperature impact on the enzymatic activity of PhTET4
was measured in a range from 20 to 95.degree. C. In all cases, 4
.mu.g/ml of PhTET4 were incubated with 50 mM CHES, 150 mM KCl, 0.1
mM NiCl.sub.2 and 5 mM Gly-pNA, pH 9.5 during 10 min. The reaction
was assessed as described previously.
[0076] Peptide Substrates
[0077] More enzymatic studies were performed to decipher PhTET4
cleavage activity by using peptide substrates (GI; GL; GLM;
GMDSLAFSGGL (SEQ ID NO: 6); LGG). 6 .mu.g/ml (final concentration)
of PhTET4 were added to a pre-warmed mixture of 3 mM peptide, 50 mM
CHES, 150 mM KCl, 0.1 mM NiCl.sub.2, pH 9.5. To avoid water
evaporation, 20 .mu.l of mineral oil were added on the top of the
total volume. The reaction incubation was done at 85.degree. C.
during 6 min. Aliquot of 80 .mu.l was then removed and added to 220
.mu.l of 2% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA).
Proteins were removed by centrifugation at 13,000 rpm during 15
min. 100 .mu.l of the supernatant were retrieved and injected on
Nova-Pak C18 column, (4 .mu.m, 3.9.times.300 mm, (Waters)) in a
HPLC purifier system (PerkinElmer), equilibrated with 2% ACN, 0.1%
TFA. The elution of peptide products was achieved with a linear ACN
gradient (2-33.2%) and followed by measuring the absorbance at 214
nm. Chromatographic runs were carried out at room temperature. The
separated fragments were collected and submitted to N-terminal
sequence analysis.
[0078] PhTET4 Inhibition
[0079] PhTET4 was incubated in presence of 5 mM EDTA, 50 mM CHES,
150 mM KCl, pH 9.5 at 85.degree. C. during 15 min. Then, the
oligomeric state of inhibited protein was evaluated by using
exclusion chromatography column (Superdex200) equilibrated with the
same incubation buffer: 50 mM CHES, 150 mM KCl, pH 9.5.
[0080] Results
[0081] In all cell types, metallo-aminopeptidases play crucial
roles in energy metabolism, protein maturation and degradation and
in the regulation of biologically active peptides by removing the
N-terminal amino acid from proteins and oligopeptides. Overall,
metalloenzymes all have one feature in common, namely that the
metal ion is bound to the protein with one labile coordination
site. As with all enzymes, the shape of the active site is crucial.
The metal ion is usually located in this active site or in the
catalytic pocket. In most of case, metallo-aminopeptidases operate
beyond the action of an endoprotease and their activity is limited
to small peptides.
[0082] Several aminopeptidases assemble as large dodecameric
particles, first discovered in archaea and named TET due to their
peculiar tetrahedral shapes. The 13 nm hollow dodecahedrons enclose
12 active sites distributed in 4 funnel-shaped chambers located in
the apices of the particles and four large access holes, formed by
the junction of six subunits, situated in the facets. This
organization strongly distinguishes TET from the other cytosolic
compartmentalized peptidases that mostly adopt a barrel-shaped
architecture.
[0083] Biochemical and structural studies of TET peptidases have
accumulated over the past 10 years. They revealed that TET
dodecamers represent a common scaffold for an efficient polypeptide
capture and processing system. In all TET, the aminopeptidase
activity is based on co-catalytic dinuclear metal active site
belonging to M18 or M42 peptidase families according to MEROPS
peptidase database. Bounding peptides are cleaved following common
mechanism involving water molecule and glutamate residue. The
nature of the metal occupying the bimetallic active site has been
shown to modulate TET enzymatic activity and Co.sup.2+ ions appear
to be the best activators for almost all archaeal and bacterial M42
TET.
[0084] TET machines are widespread and were found in the three life
domains. Interestingly, in prokaryotes, 1 to 4 different types of
TET complexes can co-exist in the cytosol depending upon the cell
type. These enzymes can be categorized according to their
preference for the chemical structure of the N-terminal amino acid
residues present in the polypeptide chains. Three main categories
have been identified so far: glutamyl/aspartyl aminopeptidases,
lysine aminopeptidases and leucine aminopeptidases, these later
exhibiting the broader specificities. In eukarya, M18 TET complexes
displayed aspartyl aminopeptidase activity. In bacteria, M42 TET
peptidases, from Clostridium thermocellum and Thermotoga maritima,
were assigned as leucyl aminopeptidases. Two M42 enzymes from the
pathogens Streptococcus pneumoniae and Mycoplasma hyopneumoniae
displayed glutamyl-aminopeptidase activities. In archaea, the
common TET structural scaffold can harbour disparate functions. So
far, the unique TET complex described in the halophilic archaeon
Haloarcula marismortui displayed the broader specificities with a
preference for neutral and basic residues.
[0085] 1. Characterisation of a New TET Protein, PhTET4
[0086] Pyrococcus horikoshii is a deep-sea hyperthermophilic
archaeon belonging to the Thermococcales order that was isolated at
1395 m depth from a hydrothermal vent. P. horikoshii, and other
related hyperthermophilic Thermococcales are distinguished by the
fact that they possess three different TET complexes: PhTET1 (SEQ
ID NO: 2), a glutamyl/aspartyl aminopeptidase, PhTET2 (SEQ ID NO:
3), a leucyl aminopeptidase with a broad activity against neutral
amino-acids and PhTET3 (SEQ ID NO: 4), a lysyl aminopeptidase with
a clear preference for positively charged residues. The analysis of
their activities on synthetic peptides of different sizes and
compositions using reverse phase HPLC indicated that the TET
peptidases degrade oligopeptides in a sequential manner and
displayed strict aminopeptidase behaviour.
[0087] An analysis of the genomes of P. horikoshii revealed the
existence of a conserved gene PH0737 encoding for an unassigned
peptidase of MH clan in MEROPS peptidase database. The protein
encoded by PH0737 gene shares 20.6%, 22.5% and 22% of sequence
identity with the three characterized aminopeptidases PhTET1,
PhTET2 and PhTET3 respectively (FIG. 1). The residues involved in
the coordination of metal ions in M42 peptidase family are well
conserved between PhTET1, 2, 3 and the said unassigned peptidase.
Moreover, the unassigned peptidase comprises two regions (the
catalytic domain and the little .beta. sheet domain localized on
the top of the former) that conferring the ability of M42
peptidases to form large multimers. Most likely, a shift was
occurred for the two latest putative ligands of the unassigned
peptidase, Asp231 and His311, which are highlighted by light grey
triangle in comparison with the conserved position of PhTET1, 2 and
3 ligands. Hence, said unassigned peptidase was named PhTET4.
PhTET4 refers to SEQ ID NO: 1.
[0088] 2. Determination of the Three-Dimensional Structure of
PhTET4
[0089] The three-dimensional structure of PhTET4 was assessed by
producing the recombinant PhTET4 in E. coll. The cellular extract
was clarified by heat shock precipitation and the recombinant
protein was purified by ion-exchange and size-exclusion
chromatographies. At the final step, PhTET4 was eluted at the same
exclusion volume (11 mL) than PhTET1 to 3, suggesting that it forms
large molecular weight assembly similar to the .about.500 kDa TET
dodecameric complexes (FIG. 2A). Negative stain electron microscopy
micrographs realized on PhTET4 peak fractions, showed that the
protein self-assembled in a hollow tetrahedral-shaped complex of
homogenous size (FIG. 2B). The dimensions and the shape of PhTET4
are analogous to the three P. horikoshii tetrahedral TET edifices:
PhTET1, PhTET2 and PhTET3.
[0090] 3. Characterisation of PhTET4 Amidolytic Activity
[0091] To determine PhTET4 functional identity the inventors tested
its amidolytic activity toward the 20 amino acids by using a broad
array of chromogenic p-nitroaniline (pNA) or fluorogenic
7-amino-4-methylcoumarin (AMC) conjugated aminoacyl compounds.
Because of the homologies between phTET4 with PhTET1, 2 and 3, the
inventors first test the amidolytic activity of PhTET4 using the
known operative conditions of PhTET1, 2 and 3. Co.sup.2+ being the
main activating metal of the 3 TET enzymes from P. horikoshii, the
inventors first assayed PhTET4 activity in the presence of 0.1 mM
CoCl.sub.2 as enzyme cofactor, at 80.degree. C. and in identical
buffer conditions (5 mM substrate, 50 mM PIPES, 150 mM KCl, pH
7.5). Surprisingly, the results showed that no hydrolysis was
observed against all tested substrates with the notable exception
of Gly-pNA toward which PhTET4 exhibited a weak catalytic
activity.
[0092] 4. Characterisation of PhTET4 Operatives Conditions
[0093] a) Metal Cofactors
[0094] Since metal cofactors have been shown to be essential to
control the activity and the oligomeric state of the various TET
edifices characterized so far, the inventors tested the influence
of various metal ions on PhTET4 glycyl aminopeptidase activity. The
results are shown on FIG. 3. Surprisingly, the inventors revealed
that Ni.sup.2+ shows the most important stimulating effect on
PhTET4 cleavage activity, with 12 times greater activity compared
with control assay where no metallic ion (WM) was added to the
reaction volume. Co.sup.2+ and Mn.sup.2+ also stimulated PhTET4
activity but less efficiently than Ni.sup.2+ (3 and 1.4 fold
activation, respectively). Interestingly, Zn.sup.2+, Ca.sup.2+,
Fe.sup.2+ and Mg.sup.2+ were found to inhibit PhTET4 hydrolytic
activity, and total inhibition was observed in presence of
Ca.sup.2+ ions. This is the first time that Ni.sup.2+ ions have
been described as an essential activating cofactor of an
aminopeptidase from the M42 family. This is an advantage and allow
user to selectively activate PhTET4 in a pull of peptidases.
[0095] b) pH Conditions
[0096] In order to determine the influence of pH on PhTET4
enzymatic behaviour, the amidolytic activity was measured between
pH 6 and pH 11, against Gly-pNA at 80.degree. C. The results are
shown on FIG. 4. The optimal activity was found at pH 9.5 and a
significant percentage of activity (beyond 80% of the maximal
activity) was observed from pH 9 to pH 10. Consequently, a
significant percentage of activity is maintained at elevated pH.
These experiments revealed that, compared to the other 3 PhTETs
enzymes, PhTET4 can be defined as an alkaline peptidase.
[0097] c) Temperature Conditions
[0098] To assess the hyperthermophilic properties of the enzyme,
the temperature dependence of PhTET4 activity was studied at
different temperatures varying from 20 to 95.degree. C. The results
are shown on FIG. 5. PhTET4 enzymatic activity increases in
parallel with the augmentation of heating temperature, with a
maximal activity observed at 95.degree. C. in the measurable
temperature range. A significant percentage of activity (beyond 80%
of the maximal activity) was observed from 80.degree. C. to
95.degree. C. Thus PhTET4 displays a high hyperthermophilic
behaviour comparable to the ones reported for the 3 other PhTETs
aminopeptidases present in Pyrococcus horikoshii cells. Therefore,
PhTET4 can advantageously work at high temperature required by
industrial fermentation process.
[0099] d) Inhibition of PhTET4 Amidolytic Activity Using EDTA
[0100] In case of PhTET1, 2 and 3 aminopeptidases, it has been
shown that the two metal ions present in the catalytic site are
essential both for catalysis and for assembling the dodecameric
edifice. For this reason, treatment with the divalent ions
chelating agent EDTA leads to the dissociation of the TET
quaternary structures and to enzymes inactivation. Indeed, in the
optimal conditions for PhTET4 activity, the addition of 5 mM EDTA
completely inhibited the glycine aminopeptidase activity. However,
analysis of the oligomeric state of EDTA-treated PhTET4 samples by
size-exclusion chromatography revealed that the PhTET4 dodecameric
edifice remained unaffected by the EDTA treatment unlike what was
reported for all the other TET enzymes (FIG. 6). This suggests that
the contribution of the metals ions situated in position M1 and/or
M2 for PhTET4 oligomerization is not as important as for the other
TET peptidases.
[0101] e) Amidolytic Activity of PhTET4 Using Optimal Operation
Conditions
[0102] The initial characterization of PhTET4 aminopeptidase
activity indicated that the enzyme displayed narrow substrate
specificity with a strong preference for glycine residues. In order
to consolidate this finding, the experiments were repeated in the
presence of nickel and in the optimal temperature and pH conditions
defined above (0.1 mM NiCl.sub.2, pH 9.5 and at 85.degree. C.), and
results are represented in FIG. 7. These results show unambiguously
that the enzyme acted only on Gly-pNA. No hydrolytic activity could
be detected toward all other amino acids, even with long incubation
times. This confirms the previous results.
[0103] The inventors also tried to investigate if PhTET4 exhibits
high D-stereospecificity against D-Alanine as shown for Aspergillus
oryzae glycine aminopeptidase (Marui, J., et al. (2012)). For this,
D-Ala-pNA was used as chromogenic substrate in optimal activity
conditions (0.1 mM NiCl.sub.2, pH 9.5 and 85.degree. C.) and the
results are reported in FIG. 7. The experiment showed that PhTET4
is unable to cleave residue alanine in D-conformation, thus
demonstrating that PhTET4 is devoid from D-stereospecificity.
[0104] 5. Endopeptidase and Exopeptidase Activities of PhTET4
[0105] In aminopeptidases, it is known that the catalytic
activities and specificities can be affected by the length and
N-terminal amino acid composition of the peptide substrate.
Consequently, the inventors tested if PhTET4 maintains its narrow
specificity toward glycine residues in a peptide context. For this,
the inventors measured PhTET4 capacity to cleave N-terminal residue
of the following peptides: GI; GL; GLM; GMDSLAFSGGL and LGG. After
incubation of PhTET4 with the peptide substrates in optimal
activity conditions (0.1 mM NiCl.sub.2, pH 9.5 and 85.degree. C.),
the reaction products were separated by reverse phase HPLC and
identified by N-terminal sequencing.
[0106] The HPLC chromatographic profiles of the degradation of GLM
and GMDSLAFSGGL peptides are shown on FIGS. 8A and 8B,
respectively. The sequences of the detected accumulating peptides
were determined and the results clearly demonstrated that PhTET4
does not exert amidolytic activity beyond the N-ter glycine in a
peptide context. Therefore, PhTET4 is exclusively an exopeptidase.
Overall, the results of these experiments showed that no enzymatic
activity was detected against peptide that do not start by glycine
residue even if a glycine residue is present at P1' position as
demonstrated with the LGG tripeptide.
[0107] To assess if PhTET4 can process consequently several glycine
residues in a peptide sequence, the inventors tested the enzymatic
activity against the chromogenic peptide Gly-Gly-pNA in the same
conditions as described previously. In the results, PhTET4
displayed significant activity against this substrate,
corresponding to 10% of the total activity exhibited in presence of
monoacyl compound Gly-pNA. Taken together, these experiments
clearly mark PhTET4 as an aminopeptidase strictly specialized in
the hydrolysis of N-terminal glycine residues.
BIBLIOGRAPHY
[0108] Jamdar, S. N. (2009) A novel aminopeptidase from
Burkholderia cepacia specific for acidic amino acids. FEMS
Microbiol Lett 295, 230-237 [0109] Marui, J., Matsushita-Morita,
M., Tada, S., Hattori, R., Suzuki, S., Amano, H., Ishida, H.,
Yamagata, Y., Takeuchi, M., and Kusumoto, K. (2012) Enzymatic
properties of the glycine D-alanine [corrected] aminopeptidase of
Aspergillus oryzae and its activity profiles in liquid-cultured
mycelia and solid-state rice culture (rice koji). Applied
microbiology and biotechnology 93, 655-669 [0110] Ito K, Ma X, Azmi
N, Huang H S, Fujii M, Yoshimoto T. (2003) Novel aminopeptidase
specific for glycine from Actinomucor elegans. Biosci Biotechnol
Biochem. January; 67(1):83-8
Sequence CWU 1
1
61336PRTPYROCOCCUS HORIKOSHII 1Met Glu Arg Ile Val Lys Ile Leu Arg
Glu Ile Leu Glu Ile Pro Ser1 5 10 15Pro Thr Gly Tyr Thr Lys Glu Val
Met Ser Tyr Leu Glu Lys Phe Leu 20 25 30Lys Glu Asn Glu Val Asn Phe
Tyr Tyr Thr Asn Lys Gly Ala Leu Ile 35 40 45Ala Gly Asn His Pro Lys
Pro Glu Leu Val Val Ile Ala His Val Asp 50 55 60Thr Leu Gly Ala Met
Val Lys Glu Ile Leu Pro Asp Gly His Leu Ala65 70 75 80Phe Ser Arg
Ile Gly Gly Leu Val Leu Pro Thr Phe Glu Gly Glu Tyr 85 90 95Cys Thr
Ile Ile Thr Arg Lys Gly Lys Lys Phe Arg Gly Thr Leu Leu 100 105
110Leu Arg Asn Pro Ser Ala His Val Asn Arg Glu Val Gly Lys Lys Glu
115 120 125Arg Lys Glu Glu Asn Met Tyr Ile Arg Leu Asp Glu Leu Val
Glu Lys 130 135 140Arg Glu Asp Thr Glu Lys Leu Gly Ile Arg Pro Gly
Asp Phe Ile Ala145 150 155 160Phe Asp Pro Lys Phe Glu Tyr Val Asn
Gly Phe Val Lys Ser His Phe 165 170 175Leu Asp Asp Lys Ala Ser Val
Ala Ala Ile Leu Asp Leu Ile Ile Asp 180 185 190Met Lys Asp Glu Leu
Glu Lys Tyr Pro Val Ala Phe Phe Phe Ser Pro 195 200 205Tyr Glu Glu
Val Gly His Gly Gly Ser Ala Gly Tyr Pro Pro Thr Thr 210 215 220Lys
Glu Leu Leu Val Val Asp Met Gly Val Val Gly Glu Gly Val Ser225 230
235 240Gly Lys Glu Thr Ala Val Ser Ile Ala Ala Lys Asp Thr Thr Gly
Pro 245 250 255Tyr Asp Tyr Asp Met Thr Asn Arg Leu Ile Glu Leu Ala
Glu Glu Asn 260 265 270Asn Ile Pro Tyr Val Val Asp Val Phe Pro Tyr
Tyr Gly Ser Asp Gly 275 280 285Ser Ala Ala Leu Arg Ala Gly Trp Asp
Phe Arg Val Ala Leu Ile Gly 290 295 300Pro Gly Val His Ala Ser His
Gly Met Glu Arg Thr His Val Lys Gly305 310 315 320Leu Leu Ala Thr
Lys Glu Leu Ile Arg Ala Tyr Ile Lys Trp Lys Gly 325 330
3352332PRTPYROCOCCUS HORIKOSHII 2Met Met Ser Met Ile Glu Lys Leu
Lys Lys Phe Thr Gln Ile Pro Gly1 5 10 15Ile Ser Gly Tyr Glu Glu Arg
Ile Arg Glu Glu Ile Ile Arg Glu Ile 20 25 30Lys Asp Phe Ala Asp Tyr
Lys Val Asp Ala Ile Gly Asn Leu Ile Val 35 40 45Glu Leu Gly Glu Gly
Glu Glu Arg Ile Leu Phe Met Ala His Met Asp 50 55 60Glu Ile Gly Leu
Leu Ile Thr Gly Ile Thr Asp Glu Gly Lys Leu Arg65 70 75 80Phe Arg
Lys Val Gly Gly Ile Asp Asp Arg Leu Leu Tyr Gly Arg His 85 90 95Val
Asn Val Val Thr Glu Lys Gly Ile Leu Asp Gly Val Ile Gly Ala 100 105
110Thr Pro Pro His Leu Ser Leu Glu Arg Asp Lys Ser Val Ile Pro Trp
115 120 125Tyr Asp Leu Val Ile Asp Ile Gly Ala Glu Ser Lys Glu Glu
Ala Leu 130 135 140Glu Leu Val Lys Pro Leu Asp Phe Ala Val Phe Lys
Lys His Phe Ser145 150 155 160Val Leu Asn Gly Lys Tyr Val Ser Thr
Arg Gly Leu Asp Asp Arg Phe 165 170 175Gly Val Val Ala Leu Ile Glu
Ala Ile Lys Asp Leu Val Asp His Glu 180 185 190Leu Glu Gly Lys Val
Ile Phe Ala Phe Thr Val Gln Glu Glu Val Gly 195 200 205Leu Lys Gly
Ala Lys Phe Leu Ala Asn His Tyr Tyr Pro Gln Tyr Ala 210 215 220Phe
Ala Ile Asp Ser Phe Ala Cys Cys Ser Pro Leu Thr Gly Asp Val225 230
235 240Lys Leu Gly Lys Gly Pro Val Ile Arg Ala Val Asp Asn Ser Ala
Ile 245 250 255Tyr Ser Arg Asp Leu Ala Arg Lys Val Trp Ser Ile Ala
Glu Lys Asn 260 265 270Gly Ile Glu Ile Gln Ile Gly Val Thr Gly Gly
Gly Thr Asp Ala Ser 275 280 285Ala Phe Gln Asp Arg Ser Lys Thr Leu
Ala Leu Ser Val Pro Ile Lys 290 295 300Tyr Leu His Ser Glu Val Glu
Thr Leu His Leu Asn Asp Leu Glu Lys305 310 315 320Leu Val Lys Leu
Ile Glu Ala Leu Ala Phe Glu Leu 325 3303353PRTPYROCOCCUS HORIKOSHII
3Met Glu Val Arg Asn Met Val Asp Tyr Glu Leu Leu Lys Lys Val Val1 5
10 15Glu Ala Pro Gly Val Ser Gly Tyr Glu Phe Leu Gly Ile Arg Asp
Val 20 25 30Val Ile Glu Glu Ile Lys Asp Tyr Val Asp Glu Val Lys Val
Asp Lys 35 40 45Leu Gly Asn Val Ile Ala His Lys Lys Gly Glu Gly Pro
Lys Val Met 50 55 60Ile Ala Ala His Met Asp Gln Ile Gly Leu Met Val
Thr His Ile Glu65 70 75 80Lys Asn Gly Phe Leu Arg Val Ala Pro Ile
Gly Gly Val Asp Pro Lys 85 90 95Thr Leu Ile Ala Gln Arg Phe Lys Val
Trp Ile Asp Lys Gly Lys Phe 100 105 110Ile Tyr Gly Val Gly Ala Ser
Val Pro Pro His Ile Gln Lys Pro Glu 115 120 125Asp Arg Lys Lys Ala
Pro Asp Trp Asp Gln Ile Phe Ile Asp Ile Gly 130 135 140Ala Glu Ser
Lys Glu Glu Ala Glu Asp Met Gly Val Lys Ile Gly Thr145 150 155
160Val Ile Thr Trp Asp Gly Arg Leu Glu Arg Leu Gly Lys His Arg Phe
165 170 175Val Ser Ile Ala Phe Asp Asp Arg Ile Ala Val Tyr Thr Ile
Leu Glu 180 185 190Val Ala Lys Gln Leu Lys Asp Ala Lys Ala Asp Val
Tyr Phe Val Ala 195 200 205Thr Val Gln Glu Glu Val Gly Leu Arg Gly
Ala Arg Thr Ser Ala Phe 210 215 220Gly Ile Glu Pro Asp Tyr Gly Phe
Ala Ile Asp Val Thr Ile Ala Ala225 230 235 240Asp Ile Pro Gly Thr
Pro Glu His Lys Gln Val Thr His Leu Gly Lys 245 250 255Gly Thr Ala
Ile Lys Ile Met Asp Arg Ser Val Ile Cys His Pro Thr 260 265 270Ile
Val Arg Trp Leu Glu Glu Leu Ala Lys Lys His Glu Ile Pro Tyr 275 280
285Gln Leu Glu Ile Leu Leu Gly Gly Gly Thr Asp Ala Gly Ala Ile His
290 295 300Leu Thr Lys Ala Gly Val Pro Thr Gly Ala Leu Ser Val Pro
Ala Arg305 310 315 320Tyr Ile His Ser Asn Thr Glu Val Val Asp Glu
Arg Asp Val Asp Ala 325 330 335Thr Val Glu Leu Met Thr Lys Ala Leu
Glu Asn Ile His Glu Leu Lys 340 345 350Ile4354PRTPYROCOCCUS
HORIKOSHII 4Met Asp Leu Lys Gly Gly Glu Ser Met Val Asp Trp Lys Leu
Met Gln1 5 10 15Glu Ile Ile Glu Ala Pro Gly Val Ser Gly Tyr Glu His
Leu Gly Ile 20 25 30Arg Asp Ile Val Val Asp Val Leu Lys Glu Val Ala
Asp Glu Val Lys 35 40 45Val Asp Lys Leu Gly Asn Val Ile Ala His Phe
Lys Gly Ser Ser Pro 50 55 60Arg Ile Met Val Ala Ala His Met Asp Lys
Ile Gly Val Met Val Asn65 70 75 80His Ile Asp Lys Asp Gly Tyr Leu
His Ile Val Pro Ile Gly Gly Val 85 90 95Leu Pro Glu Thr Leu Val Ala
Gln Arg Ile Arg Phe Phe Thr Glu Lys 100 105 110Gly Glu Arg Tyr Gly
Val Val Gly Val Leu Pro Pro His Leu Arg Arg 115 120 125Gly Gln Glu
Asp Lys Gly Ser Lys Ile Asp Trp Asp Gln Ile Val Val 130 135 140Asp
Val Gly Ala Ser Ser Lys Glu Glu Ala Glu Glu Met Gly Phe Arg145 150
155 160Val Gly Thr Val Gly Glu Phe Ala Pro Asn Phe Thr Arg Leu Asn
Glu 165 170 175His Arg Phe Ala Thr Pro Tyr Leu Asp Asp Arg Ile Cys
Leu Tyr Ala 180 185 190Met Ile Glu Ala Ala Arg Gln Leu Gly Asp His
Glu Ala Asp Ile Tyr 195 200 205Ile Val Gly Ser Val Gln Glu Glu Val
Gly Leu Arg Gly Ala Arg Val 210 215 220Ala Ser Tyr Ala Ile Asn Pro
Glu Val Gly Ile Ala Met Asp Val Thr225 230 235 240Phe Ala Lys Gln
Pro His Asp Lys Gly Lys Ile Val Pro Glu Leu Gly 245 250 255Lys Gly
Pro Val Met Asp Val Gly Pro Asn Ile Asn Pro Lys Leu Arg 260 265
270Ala Phe Ala Asp Glu Val Ala Lys Lys Tyr Glu Ile Pro Leu Gln Val
275 280 285Glu Pro Ser Pro Arg Pro Thr Gly Thr Asp Ala Asn Val Met
Gln Ile 290 295 300Asn Lys Glu Gly Val Ala Thr Ala Val Leu Ser Ile
Pro Ile Arg Tyr305 310 315 320Met His Ser Gln Val Glu Leu Ala Asp
Ala Arg Asp Val Asp Asn Thr 325 330 335Ile Lys Leu Ala Lys Ala Leu
Leu Glu Glu Leu Lys Pro Met Asp Phe 340 345 350Thr
Pro51011DNAPYROCOCCUS HORIKOSHII 5atggaaagga tagtcaagat cttaagggaa
atcttagaga taccttctcc aacgggctac 60acgaaggagg taatgagtta cctagaaaaa
tttctaaagg aaaatgaagt aaacttttac 120tatacgaaca agggggccct
aatagccggt aatcatccaa agcctgagct cgttgttata 180gcccacgtag
acacgcttgg ggcaatggtt aaggagatac taccagacgg acacttagca
240ttttcaagga taggagggct cgttctacct acgtttgaag gcgaatactg
tactataata 300acgagaaaag gaaagaagtt tagaggaacg ctcctcctta
gaaatccgag cgctcatgta 360aatagggaag taggtaaaaa ggagagaaaa
gaggagaata tgtatataag attggacgag 420ctcgtggaga agagagagga
tacagaaaag ctggggataa ggccaggaga cttcatagct 480tttgatccca
aatttgaata cgtaaacggc tttgttaaat cacacttcct agatgacaag
540gctagcgtag ctgcaatact cgatctaata atagatatga aggatgaact
cgagaagtat 600ccagttgcat tcttcttctc accgtatgag gaagttggcc
acggaggctc agctggctac 660ccaccaacga ctaaggaact gctcgtggtt
gatatgggag tagtgggtga aggtgtttca 720ggaaaagaaa ccgccgtatc
tatagcggcc aaggatacaa ctgggcctta tgactatgac 780atgacgaaca
ggttaataga gcttgctgaa gagaacaata tcccatatgt agttgacgtg
840ttcccctact atggttccga tggttcagct gcactaagag ctggatggga
tttcagggtt 900gccctaattg ggccaggtgt gcacgcaagc cacggaatgg
agagaaccca cgttaaggga 960ttgttggcaa ctaaagagct tataagggct
tacataaaat ggaaggggta a 1011611PRTArtificial SequenceGenerated
peptide for amidolytic activity assay 6Gly Met Asp Ser Leu Ala Phe
Ser Gly Gly Leu1 5 10
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