U.S. patent application number 16/812405 was filed with the patent office on 2021-08-26 for novel esterases and uses thereof.
The applicant listed for this patent is CARBIOS. Invention is credited to ISABELLE ANDRE, SOPHIE BARBE, MARIE-LAURE DESROUSSEAUX, SOPHIE DUQUESNE, ALAIN MARTY, HEL NE TEXIER, CHRISTOPHER TOPHAM, VINCENT TOURNIER.
Application Number | 20210261931 16/812405 |
Document ID | / |
Family ID | 1000005764841 |
Filed Date | 2021-08-26 |
United States Patent
Application |
20210261931 |
Kind Code |
A9 |
TOPHAM; CHRISTOPHER ; et
al. |
August 26, 2021 |
NOVEL ESTERASES AND USES THEREOF
Abstract
The present invention relates to novel esterase, more
particularly to esterase variants having improved thermostability
compared to the esterase of SEQ ID No 1 and the uses thereof for
degrading polyester containing material, such as plastic products.
The esterases of the invention are particularly suited to degrade
polyethylene terephthalate, and material containing polyethylene
terephthalate.
Inventors: |
TOPHAM; CHRISTOPHER;
(LAVAUR, FR) ; TEXIER; HEL NE; (EAUNES, FR)
; TOURNIER; VINCENT; (TOULOUSE, FR) ;
DESROUSSEAUX; MARIE-LAURE; (LOMPRET, FR) ; DUQUESNE;
SOPHIE; (TOULOUSE, FR) ; ANDRE; ISABELLE;
(TOULOUSE, FR) ; BARBE; SOPHIE; (GOYRANS, FR)
; MARTY; ALAIN; (TOULOUSE, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CARBIOS |
SAINT-BEAUZIRE |
|
FR |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20200270591 A1 |
August 27, 2020 |
|
|
Family ID: |
1000005764841 |
Appl. No.: |
16/812405 |
Filed: |
March 9, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16317160 |
Jan 11, 2019 |
10584320 |
|
|
PCT/EP2017/067582 |
Jul 12, 2017 |
|
|
|
16812405 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/18 20130101; C08J
11/105 20130101; C12Y 301/01 20130101; C12Y 301/01074 20130101 |
International
Class: |
C12N 9/18 20060101
C12N009/18; C08J 11/10 20060101 C08J011/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 12, 2016 |
EP |
16305898.5 |
Claims
1. An esterase variant which (i) has at least 75% identity to the
full length amino acid sequence set forth in SEQ ID NO: 1, and (ii)
has one or more amino acid modifications as compared to SEQ ID NO:
1 at position(s) selected from D203+S248, E173, L202, N204,
A172+A209, V28, S29, R30, L31, S32, V33, S34, G35, F36, G37, G38,
G39, A103, L82, G53, L104, L107, L119, A121, L124, I54, M56, L70,
L74, A127, V150, L152, L168, V170, P196, V198, V200, V219, Y220,
T221, S223, W224, M225, L239, T252, N253, H256, S1, Y4, Q5, R6, N9,
S13, T16, S22, T25, Y26, S34, Y43, S48, T50, R72, S98, N105, R108,
S113, N122, S145, K147, T160, N162, S181, Q189, N190, S193, T194,
S212, N213, N231, T233, R236, Q237, N241, N243, N254, R255 and Q258
or at least one amino acid substitution selected from V177I,
Y92G/P, F208I/W, T61M, wherein the positions are numbered by
reference to the amino acid sequence set forth in SEQ ID NO: 1.
2. The esterase variant of claim 1, which has one or more amino
acid modifications, as compared to SEQ ID NO: 1, at position(s)
selected from D203+S248, E173, L202, N204, V170, V219, S212, N213,
N241 and N243 or at least one amino acid substitution selected from
V177I, Y92G/P, F208VW, T61M.
3. The esterase variant of claim 1, comprising at least the amino
acid substitutions D203C+S248C.
4. The esterase variant of claim 1, comprising at least the
replacement of amino acids V28 to G39 of SEQ ID NO: 1 with an amino
acid sequence consisting of E-G-P-S-C or A-G-P-S-C, and the
substitution L82A and/or A103C.
5. The esterase variant of claim 3, further comprising at least one
substitution at a position selected from E173, L202, N204 and F208,
wherein the positions are numbered by reference to the amino acid
sequence set forth in SEQ ID NO: 1.
6. The esterase variant of claim 5, wherein the at least one
substitution is selected from E173A/R, L202R, N204D and
F208W/I.
7. The esterase variant of claim 1, comprising the combination of
substitutions at positions selected from D203+S248+E173,
D203+S248+F208, and D203+S248+E173+N204+L202.
8. The esterase variant of claim 1, which comprises at least one
substitution or combination of substitutions selected from the
group consisting of V177I, Y92G, Y92P, F208W, Y92P+F208W, T61M,
V170I+F208W, D203C+S248C, D203C+S248C+E173R, D203C+S248C+E173A,
D203C+S248C+F208W, D203C+S248C+F208I, F208W+D203C+S248C+E173A,
F208I+D203C+S248C+E173A and D203C+S248C+E173R+N204D+L202R wherein
the positions are numbered by reference to the amino acid sequence
set forth in SEQ ID NO: 1.
9. The esterase variant of claim 1, which is further glycosylated,
at a position selected from N9, N143, N162, N204, N231, or
combinations thereof.
10. A nucleic acid encoding an esterase as defined in claim 1.
11. An expression cassette or vector comprising the nucleic acid of
claim 10.
12. A host cell comprising the nucleic acid of claim 10.
13. A method of producing an esterase comprising: (a) culturing the
host cell according to claim 12 under conditions suitable to
express the nucleic acid encoding the esterase; and (b) recovering
said esterase from the cell culture.
14. A composition comprising an esterase according to claim 1 and
one or several excipients or additives.
15. A method of degrading a plastic product containing at least one
polyester comprising (a) contacting the plastic product with an
esterase according to claim 1, thereby degrading the plastic
product.
16. The method of claim 15, further comprising (b) recovering
monomers and/or oligomers resulting from the degradation of the at
least one polyester.
17. The method of claim 15, wherein the plastic product comprises
at least one polyester selected from polyethylene terephthalate
(PET), polytrimethylene terephthalate (PTT), polybutylen
terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT),
polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene
succinate (PBS), polybutylene succinate adipate (PBSA),
polybutylene adipate terephthalate (PBAT), polyethylene furanoate
(PEF), Polycaprolactone (PCL), poly(ethylene adipate) (PEA),
polyethylene naphthalate (PEN) and blends/mixtures of these
materials.
18. The esterase variant of claim 1 which exhibits a polyester
degrading activity
19. A polyester containing material comprising an esterase variant
according to claim 1 and/or a host cell expressing said esterase
variant.
20. A plastic compound comprising at least one polyester and an
esterase variant according to claim 1 and/or a host cell expressing
said esterase variant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/317,160, filed Jan. 11, 2019, now U.S. Pat. No. 10,584,320,
which is the U.S. national stage application of International
Patent Application No. PCT/EP2017/067582, filed Jul. 12, 2017.
[0002] The Sequence Listing for this application is labeled
"Seq-List.txt" which was created on Jan. 3, 2019 and is 3 KB. The
entire content of the sequence listing is incorporated herein by
reference in its entirety.
[0003] The present invention relates to novel esterases, more
particularly to esterases having improved thermostability compared
to a parent esterase and the uses thereof for degrading polyester
containing material, such as plastic products. The esterases of the
invention are particularly suited to degrade polyethylene
terephthalate, and material containing polyethylene
terephthalate.
BACKGROUND
[0004] Esterases are able to catalyze the hydrolysis of a variety
of polymers, including polyesters. In this context, esterases have
shown promising effects in a number of industrial applications,
including as detergents for dishwashing and laundry applications,
as degrading enzymes for processing biomass and food, as
biocatalysts in detoxification of environmental pollutants or for
the treatment of polyester fabrics in the textile industry. In the
same way, the use of esterases as degrading enzymes for hydrolyzing
polyethylene terephthalate (PET) is of particular interest. Indeed,
PET is used in a large number of technical fields, such as in the
manufacture of clothes, carpets, or in the form of a thermoset
resin for the manufacture of packaging or automobile plastics or
other parts, and PET accumulation in landfills becomes an
increasing ecological problem.
[0005] Among esterases, cutinases, also known as cutin hydrolases
(EC 3.1.1.74), are of particular interest. Cutinases have been
identified from various fungi (P. E. Kolattukudy in "Lipases", Ed.
B. Borg-strom and H. L. Brockman, Elsevier 1984, 471-504), bacteria
and plant pollen. Recently, metagenomics approaches have led to
identification of additional esterases.
[0006] The enzymatic degradation is considered as an interesting
solution to decrease such plastic waste accumulation. Indeed,
enzymes may accelerate hydrolysis of polyester containing material,
and more particularly of plastic products, even up to the monomer
level. Furthermore, the hydrolysate (i.e., monomers and oligomers)
can be recycled as material for synthesizing new polymers.
[0007] In this context, several esterases have been identified as
candidate degrading enzymes. For instance, several variants of the
esterase (cutinase) of Fusarium solani pisi have been published
(Appl. Environm. Microbiol. 64, 2794-2799, 1998; Proteins:
Structure, Function and Genetics 26,442-458,1996).
[0008] However, most of these esterases are not efficient at an
industrial level, because of their poor resistance to high
temperatures. Accordingly, there is still a need for esterases with
improved thermostability that may be used for degrading polyester
at an industrial level and with high yield.
SUMMARY OF THE INVENTION
[0009] The present invention provides new variants of esterase
exhibiting increased thermostability compared to a parent, or
wild-type esterase. These esterases are particularly useful in
processes for degrading plastic material and product, such as
plastic material and product containing PET. More particularly, the
present invention provides variants of an esterase having the amino
acid sequence as set forth in SEQ ID No 1, that corresponds to the
amino acids 36 to 293 of the amino acid sequence of the
metagenome-derived cutinase described in Sulaiman et al., Appl
Environ Microbiol. 2012 March, or to the amino acids 36 to 293 of
the amino acid sequence referenced G9BY57 in SwissProt.
[0010] In this regard, it is an object of the invention to provide
an esterase which (i) has at least 75%, 80%, 85%, 90%, 95% or 99%
identity to the full length amino acid sequence set forth in SEQ ID
No 1, (ii) contains at least one amino acid modification as
compared to SEQ ID No 1, (iii) has a polyester degrading activity
and (iv) exhibits increased thermostability as compared to the
esterase of SEQ ID No 1.
[0011] More particularly, the esterase of the invention comprises
one or more amino acid modification(s) as compared to SEQ ID No 1
at position(s) selected from D203+5248, E173, L202, N204, F208,
A172+A209, G39, A103, L82, G53, L104, L107, L119, A121, L124, 154,
M56, L70, L74, A127, V150, L152, L168, V170, P196, V198, V200,
V219, Y220, T221, S223, W224, M225, L239, T252, N253, H256, S1, Y4,
Q5, R6, N9, S13, T16, S22, T25, Y26, S34, Y43, S48, T50, R72, S98,
N105, R108, S113, N122, S145, K147, T160, N162, S181, Q189, N190,
S193, T194, N204, S212, N213, N231, T233, R236, Q237, N241, N243,
N254, R255 and Q258 wherein the positions are numbered by reference
to the amino acid sequence set forth in SEQ ID No 1.
[0012] In a particular embodiment, the variant esterase of the
invention comprises one or more amino acid substitution(s) as
compared to SEQ ID No 1 at position(s) selected from D203+5248,
E173, N204, L202, F208, and V170. Preferably, the variant esterase
of the invention comprises at least amino acid substitution(s) as
compared to SEQ ID No 1 at position(s) selected from D203+5248 and
F208.
[0013] In another particular embodiment, the variant esterase of
the invention comprises one or more amino acid substitution(s) as
compared to SEQ ID No 1 at a position selected from T61, Y92 and
V177, wherein the positions are numbered by reference to the amino
acid sequence set forth in SEQ ID No 1, and wherein the
substitutions are different from T61A/G, Y92A and V177A.
Preferably, the variant esterase of the invention comprises one or
more amino acid substitution(s) as compared to SEQ ID No 1,
selected from V177I, Y92G, Y92P, Y92P+F208W and T61M.
[0014] In a particular embodiment, the variant esterases of the
invention may comprise, as compared to the esterase of SEQ ID No 1:
[0015] at least one additional disulphide bridge; and/or [0016] at
least one additional salt bridge; and/or [0017] at least one
mutation of an amino acid residue located in a void
solvent-excluded cavity of the esterase; and/or [0018] a
suppression of at least one N- and/or C-terminal amino acid
residue.
[0019] It is another object of the invention to provide a nucleic
acid encoding an esterase of the invention. The present invention
also relates to an expression cassette or an expression vector
comprising said nucleic acid, and to a host cell comprising said
nucleic acid, expression cassette or vector.
[0020] It is a further object of the invention is to provide a
method of producing an esterase comprising:
[0021] (a) culturing the host cell according to the invention under
suitable conditions to express the nucleic acid encoding the
esterase; and optionally
[0022] (b) recovering said esterase from the cell culture.
[0023] The present invention also relates to a method of degrading
a plastic product containing at least one polyester comprising
[0024] (a) contacting the plastic product with an esterase or the
host cell according to the invention, thereby degrading the plastic
product; and optionally
[0025] (b) recovering monomers and/or oligomers.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0026] The present disclosure will be best understood by reference
to the following definitions.
[0027] Herein, the terms "peptide", "polypeptide", "protein",
"enzyme", refer to a chain of amino acids linked by peptide bonds,
regardless of the number of amino acids forming said chain. The
amino acids are herein represented by their one-letter or
three-letters code according to the following nomenclature: A:
alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E:
glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H:
histidine (His); I: isoleucine (Ile); K: lysine (Lys); L: leucine
(Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro);
Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T:
threonine (Thr); V: valine (Val); W: tryptophan (Trp) and Y:
tyrosine (Tyr).
[0028] The term "esterase" refers to an enzyme which belongs to a
class of hydrolases classified as EC 3.1.1 according to Enzyme
Nomenclature that catalyzes the hydrolysis of esters into an acid
and an alcohol. The term "cutinase" or "cutin hydrolase" refers to
an esterase classified as EC 3.1.1.74 according to Enzyme
Nomenclature that is able to catalyse the chemical reaction of
production of cutin monomers from cutin and water.
[0029] The terms "wild-type protein" or "parent protein" are used
interchangeably and refer to the non-mutated version of a
polypeptide as it appears naturally. In the present case, the
parent esterase refers to the esterase having the amino acid
sequence as set forth in SEQ ID No 1.
[0030] Accordingly, the terms "mutant" and "variant" may be used
interchangeably to refer to polypeptides derived from SEQ ID No 1
and comprising a modification or an alteration, i.e., a
substitution, insertion, and/or deletion, at one or more (e.g.,
several) positions and having a polyester degrading activity. The
variants may be obtained by various techniques well known in the
art. In particular, examples of techniques for altering the DNA
sequence encoding the wild-type protein, include, but are not
limited to, site-directed mutagenesis, random mutagenesis and
synthetic oligonucleotide construction.
[0031] The term "modification" or "alteration" as used herein in
relation to a position or amino acid means that the amino acid in
the particular position has been modified compared to the amino
acid of the wild-type protein.
[0032] A "substitution" means that an amino acid residue is
replaced by another amino acid residue. Preferably, the term
"substitution" refers to the replacement of an amino acid residue
by another selected from the naturally-occurring standard 20 amino
acid residues, rare naturally occurring amino acid residues (e.g.
hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylysine,
N-ethylglycine, N-methylglycine, N-ethylasparagine,
allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine,
aminobutyric acid, ornithine, norleucine, norvaline), and
non-naturally occurring amino acid residue, often made
synthetically, (e.g. cyclohexyl-alanine). Preferably, the term
"substitution" refers to the replacement of an amino acid residue
by another selected from the naturally-occurring standard 20 amino
acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E,
D, S and T). The sign "+" indicates a combination of substitutions.
In the present document, the following terminology is used to
designate a substitution: L82A denotes that amino acid residue
(Leucine, L) at position 82 of the parent sequence is changed to an
Alanine (A). A121V/I/M denotes that amino acid residue (Alanine, A)
at position 121 of the parent sequence is substituted by one of the
following amino acids: Valine (V), Isoleucine (I), or Methionine
(M). The substitution can be a conservative or non-conservative
substitution. Examples of conservative substitutions are within the
groups of basic amino acids (arginine, lysine and histidine),
acidic amino acids (glutamic acid and aspartic acid), polar amino
acids (glutamine, asparagine and threonine), hydrophobic amino
acids (methionine, leucine, isoleucine, cysteine and valine),
aromatic amino acids (phenylalanine, tryptophan and tyrosine), and
small amino acids (glycine, alanine and serine).
[0033] The term "deletion", used in relation to an amino acid,
means that the amino acid has been removed or is absent.
[0034] The term "insertion" means that one or more amino acids have
been added.
[0035] Unless otherwise specified, the positions disclosed in the
present application are numbered by reference to the amino acid
sequence set forth in SEQ ID No 1.
[0036] As used herein, the term "sequence identity" or "identity"
refers to the number (or fraction expressed as a percentage %) of
matches (identical amino acid residues) between two polypeptide
sequences. The sequence identity is determined by comparing the
sequences when aligned so as to maximize overlap and identity while
minimizing sequence gaps. In particular, sequence identity may be
determined using any of a number of mathematical global or local
alignment algorithms, depending on the length of the two sequences.
Sequences of similar lengths are preferably aligned using a global
alignment algorithms (e.g. Needleman and Wunsch algorithm;
Needleman and Wunsch, 1970) which aligns the sequences optimally
over the entire length, while sequences of substantially different
lengths are preferably aligned using a local alignment algorithm
(e.g. Smith and Waterman algorithm (Smith and Waterman, 1981) or
Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)).
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software available on internet web sites such as
http://blast.ncbi.nlm.nih.gov/ or
http://www.ebi.ac.uk/Tools/emboss/). Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the full
length of the sequences being compared. For purposes herein, %
amino acid sequence identity values refers to values generated
using the pair wise sequence alignment program EMBOSS Needle that
creates an optimal global alignment of two sequences using the
Needleman-Wunsch algorithm, wherein all search parameters are set
to default values, i.e. Scoring matrix=BLOSUM62, Gap open=10, Gap
extend=0.5, End gap penalty=false, End gap open=10 and End gap
extend=0.5.
[0037] The terms "disulphide bridge", "disulphide bond" and "S-S
bond" are used interchangeably and refer to a covalent bond between
sulphur atoms of two cysteines.
[0038] The term "salt bridge", or "ion-pair", refers to a
noncovalent electrostatic interaction between two residues with
opposite charges in a protein. A salt bridge most often is formed
between the anionic carboxylate (RCOO.sup.-) of either aspartic
acid or glutamic acid and the cationic ammonium (RNH.sub.3.sup.+)
of lysine or the guanidinium (RNHC(NH.sub.2).sub.2.sup.+) moiety of
arginine. Other amino acid residues with ionizable side chains,
such as histidine, tyrosine, serine, threonine and cysteine can
also be part of a salt bridge.
[0039] The term "glycosylated", in relation to a polypeptide, means
that one or several glycans are attached to at least one amino acid
residue of the polypeptide. In the context of the invention,
glycosylation encompasses N-linked glycans, attached to the amide
nitrogen of asparagine residue, O-linked glycans attached to the
hydroxyl oxygen of serine or tyrosine residues, C-linked glycans
attached to a carbon of a tryptophan residue.
[0040] The "protein conformation" or "crystal structure" refers to
the three dimensional structure of the protein.
[0041] The term "recombinant" refers to a nucleic acid construct, a
vector, a polypeptide or a cell produced by genetic
engineering.
[0042] The term "expression", as used herein, refers to any step
involved in the production of a polypeptide including, but being
not limited to, transcription, post-transcriptional modification,
translation, post-translational modification, and secretion.
[0043] The term "expression cassette" denotes a nucleic acid
construct comprising a coding region, i.e. a nucleic acid of the
invention, and a regulatory region, i.e. comprising one or more
control sequences, operably linked.
[0044] As used herein, the term "expression vector" means a DNA or
RNA molecule that comprises an expression cassette of the
invention. Preferably, the expression vector is a linear or
circular double stranded DNA molecule.
[0045] A "polymer" refers to a chemical compound or mixture of
compounds whose structure is constituted of multiple monomers
(repeat units) linked by covalent chemical bonds. Within the
context of the invention, the term polymer includes natural or
synthetic polymers, constituted of a single type of repeat unit
(i.e., homopolymers) or of a mixture of different repeat units
(i.e., copolymers or heteropolymers). According to the invention,
"oligomers" refer to molecules containing from 2 to about 20
monomers.
[0046] In the context of the invention, a "polyester containing
material" or "polyester containing product" refers to a product,
such as plastic product, comprising at least one polyester in
crystalline, semi-crystalline or totally amorphous forms. In a
particular embodiment, the polyester containing material refers to
any item made from at least one plastic material, such as plastic
sheet, tube, rod, profile, shape, film, massive block etc., which
contains at least one polyester, and possibly other substances or
additives, such as plasticizers, mineral or organic fillers. In
another particular embodiment, the polyester containing material
refers to a plastic compound, or plastic formulation, in a molten
or solid state, suitable for making a plastic product.
[0047] In the present description, "polyesters" encompass but is
not limited to polyethylene terephthalate (PET), polytrimethylene
terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene
isosorbide terephthalate (PEIT), polylactic acid (PLA),
polyhydroxyalkanoate (PHA), polybutylene succinate (PBS),
polybutylene succinate adipate (PBSA), polybutylene adipate
terephthalate (PBAT), polyethylene furanoate (PEF),
polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene
naphthalate (PEN) and blends/mixtures of these polymers.
Novel Esterases with Improved Thermostability
[0048] The present invention provides novel esterases with improved
thermostability. More particularly, the inventors have developed
different ways to improve the stability of esterases at high
temperatures, and advantageously at temperature above 50.degree.
C., which allow design of novel enzymes having superior properties
for use in industrial processes.
[0049] With the aim to improve the stability and/or activity of
esterases in conditions where industrial degradation of plastic
products can be performed, the inventors have developed novel
esterases derived from the esterase of SEQ ID No 1 that show high
resistance to temperature. The esterases of the invention are
particularly suited to degrade plastic product containing PET.
[0050] The invention shows that, by creating new disulphide
bridge(s) and/or salt bridge(s) in the crystal structure of the
protein; by reducing protein mobility and/or solvent-excluded
volume of intern cavities; and/or by reducing the N-terminal or
C-terminal extremity, novel proteins are obtained which exhibit
polyester degrading activity with improved thermostability.
[0051] It is thus an object of the present invention to provide an
esterase which (i) has at least 75%, 80%, 85%, 90%, 95% or 99%
identity to the full length amino acid sequence set forth in SEQ ID
No 1, (ii) contains at least one amino acid modification as
compared to SEQ ID No 1, (iii) has a polyester degrading activity
and (iv) exhibits increased thermostability as compared to the
esterase of SEQ ID No 1.
[0052] Within the context of the invention, the term "increased
thermostability" indicates an increased ability of the enzyme to
resist to changes in its chemical and/or physical structure at high
temperatures, and more particularly at temperature between
50.degree. C. and 90.degree. C., as compared to the esterase of SEQ
ID No 1. Such an increase is typically of about 1-fold, 2-fold,
3-fold, 4-fold, 5-fold, or more. Particularly, the esterases of the
present invention may exhibit an increased melting temperature (Tm)
as compared to the esterase of SEQ ID No 1. In the context of the
present invention, the melting temperature refers to the
temperature at which half of the protein/enzyme population
considered is unfolded or misfolded. Typically, the esterase of the
invention shows an increased Tm of about 1.degree. C., 2.degree.
C., 3.degree. C., 4.degree. C., 5.degree. C., 10.degree. C. or
more, as compared to the Tm of the esterase of SEQ ID No 1.
[0053] In particular, the esterases of the present invention can
have an increased half-life at a temperature between 50.degree. C.
and 90.degree. C., as compared to the esterase of SEQ ID No 1.
Furthermore, at such temperature, the esterases of the invention
may exhibit greater degrading activity as compared to the esterase
of SEQ ID No 1.
[0054] The thermostability of a protein may be evaluated by the one
skilled in the art, according to methods known per se in the art.
For instance, thermostability can be assessed by analysis of the
protein folding using circular dichroism. Alternatively or in
addition, thermostability can be assessed by measuring the residual
esterase activity and/or the residual polyester depolymerization
activity of the enzyme after incubation at different temperatures.
The ability to perform multiple rounds of polyester's
depolymerization assays at different temperatures can also be
evaluated. A rapid and valuable test may consist on the evaluation,
by halo diameter measurement, of the enzyme ability to degrade a
solid polyester compound dispersed in an agar plate after
incubation at different temperatures. Preferably, a Differential
Scanning Fluorimetry (DSF) is performed to assess the
thermostability of a protein/enzyme. More particularly, the DSF may
be used to quantify the change in thermal denaturation temperature
of a protein and thereby to determine its melting temperature (Tm).
In the context of the invention, and unless specific indications,
the Tm is measured using DSF as exposed in the experimental part.
In the context of the invention, comparisons of Tm are performed
with Tm that are measured under same conditions (e.g. pH, nature
and amount of polyesters, etc.).
[0055] In a particular embodiment, the variants of the invention
have both an improved thermostability and an increased polyester
degrading activity as compared to the esterase of SEQ ID No 1.
[0056] Within the context of the invention, the term "increased
activity" or "increased degrading activity" indicates an increased
ability of the enzyme to degrade a plastic product or material, and
more particularly a polyester containing plastic product or
material, as compared to the esterase of SEQ ID No 1. Such an
increase is typically of about 1-fold, 2-fold, 3-fold, 4-fold,
5-fold, or more. Particularly, the esterase variant has a polyester
degrading activity at least 10% greater than the polyester
degrading activity of the esterase of SEQ ID No 1, preferably at
least 20%, 50%, 100%, 200%, 300%, or greater.
[0057] The activity of a protein may be evaluated by the one
skilled in the art, according to methods known per se in the art.
For instance, the activity can be assessed by the measurement of
the specific esterase activity rate, the measurement of the
specific polyester's depolymerization activity rate, the
measurement of the rate to degrade a solid polyester compound
dispersed in an agar plate, or the measurement of the specific
polyester's depolymerization activity rate in reactor.
[0058] Within the context of the invention, the terms "specific
activity" or "specific degrading activity" designate the initial
rate of oligomers and/or monomers released under suitable
conditions of temperature, pH and buffer, when contacting the
polyester containing plastic product with a degrading enzyme, such
as an esterase according to the invention. As an example, the
specific activity of PET hydrolysis corresponds to umol of PET
hydrolysed/min or mg of equivalent TA produced/hour and per mg of
enzyme as determined in the linear part of the hydrolysis
curve.
[0059] The ability of a protein to adsorb on a substrate may be
evaluated by the one skilled in the art, according to methods known
per se in the art. For instance, the proteic content or the
residual esterase activity, residual polyester's depolymerization
activity, residual degradation of a solid polyester compound
dispersed in an agar plate, or residual polyester's
depolymerization activity in reactor can be measured from a
solution containing the esterase of the invention and wherein the
esterase has been previously incubated with a substrate under
suitable conditions where no enzymatic reaction can occur.
[0060] The esterases of the invention may comprise one or several
modifications as disclosed below.
[0061] In one embodiment, the esterase of the invention has at
least 75%, 80%, 85%, 90%, 95% or 99% identity to the full length
amino acid sequence set forth in SEQ ID No 1 and comprises at least
one additional disulphide bridge as compared to the esterase of SEQ
ID No 1.
[0062] In a particular embodiment, the esterase variant comprises
substitutions at positions A172+A209, wherein the positions are
numbered by reference to the amino acid sequence set forth in SEQ
ID No 1.
[0063] In another particular embodiment, the esterase variant
comprises at least one mutation at a position selected from V28 to
G39, L82 and A103, wherein the positions are numbered by reference
to the amino acid sequence set forth in SEQ ID No 1.
[0064] Particularly, the esterase variant exhibits a deletion of
the amino acid residues V28 to S34 of SEQ ID No 1, and
substitutions as compared to SEQ ID No 1, at a position selected
from G35, F36, G37, G38, G39, L82 and/or A103, and more
particularly substitutions consisting of
G35E/A+F36G+G37P+G38S+G39C, L82A and/or A103C.
[0065] Alternatively, the esterase variant exhibits a deletion of
the amino acids V33 to G39 of SEQ ID No 1, and substitutions as
compared to SEQ ID No 1 consisting of V28E+S29G+R30P+L31S+S32C or
V28A+S29G+R30P+L31S+S32C and substitution L82A and/or A103C.
[0066] Alternatively, the esterase variant comprises the
replacement of the amino acids V28 to G39 of SEQ ID No 1 with the
amino acid sequence consisting of E-G-P-S-C or A-G-P-S-C, and
eventually substitution L82A and/or A103C.
[0067] Alternatively, the esterase variant exhibits a deletion of
the amino acids V33 to G39 of SEQ ID No 1, and substitutions
consisting of V28E+S29G+R30P+L31S+S32C or V28A+S29G+R30P+L31S+S32C
and substitutions L82A, A103C, A172C and A209C.
[0068] In another particular embodiment, the esterase variant
comprises substitutions at positions D203+S248, wherein the
positions are numbered by reference to the amino acid sequence set
forth in SEQ ID No 1. Preferably, the substitutions consist of
D203C+S248C. In a particular embodiment, such esterase variant with
substitutions at positions D203+S248, further comprises at least
one substitution at position selected from E173, L202, N204 and
F208. Preferably, the additional substitutions are selected from
E173R, E173A, F208W or F208I. More particularly, the esterase
variant comprises the substitutions selected from
D203C+S248C+E173R, D203C+S248C+E173A, D203C+S248C+F208W and
D203C+S248C+F208I. In a particular embodiment, esterase variant
with substitutions at positions D203+S248 further comprises at
least two substitutions at positions selected from E173, L202, N204
and F208. For instance, the variant comprises at least the
substitutions D203C+S248C+E173R+N204D+L202R,
F208W+D203C+S248C+E173A and F208I+D203C+S248C+E173A.
[0069] It is an object of the invention to provide an esterase
having a polyester degrading activity which has at least 75%, 80%,
85%, 90%, 95% or 99% identity to the full length amino acid
sequence set forth in SEQ ID No 1 and comprises, as compared to SEQ
ID No 1, at least one mutation of an amino acid residue located in
a void solvent-excluded cavity of the protein. Particularly, such
mutation enables to reduce the solvent-excluded volume of the
cavity.
[0070] In a particular embodiment, the esterase variant comprises
at least one substitution at a position selected from G53, L104,
L107, L119, A121, L124, 154, M56, L70, L74, A127, V150, L152, L168,
V170, P196, V198, V200, V219, Y220, T221, S223, W224, M225, L239,
T252, N253, H256, by reference to SEQ ID No 1. Advantageously, the
esterase variant comprises at least two, three, four, five or more
amino acid substitutions among said positions.
[0071] In an embodiment, at least one of said amino acids is
replaced with a bulkier (i.e., more voluminous) amino acid.
[0072] Advantageously, the esterase variant comprises at least one
substitution selected from G53A/I, I54L, M56I, L70M/I, L74M, L104M,
L107M, L119M/A, A121V/I/M/Y, L124I/R/Q, A127V/I, V1501, L1521,
L1681, V170I, V1981, V2191, Y220F/P/M, T221A/V/L/I/M, S223A,
W2241/M, and T252S/D.
[0073] In a particular embodiment, the esterase variant comprises
substitutions at positions V170+F208. Advantageously, the esterase
variant comprises substitutions V170I+F208W.
[0074] In a particular embodiment, the esterase variant comprises
amino acid substitutions at positions belonging to at least two of
the groups (i)-(v) below. Advantageously, the esterase variant
comprises at least substitution at a position in each group
(i)-(v).
(i) G53, L104, L107, L119, A121, L124;
(ii) 154, M56, L70, L74, A127, V150, L152, T221, M225;
[0075] (iii) V150, L152, L168, V170, V200, T221;
(iv) P196, V198, W224, T252, N253, H256;
(v) V219, Y220, S223, L239.
[0076] It is an object of the invention to provide an esterase
having a polyester degrading activity which has at least 75%, 80%,
85%, 90%, 95% or 99% identity to the full length amino acid
sequence set forth in SEQ ID No 1, and comprises, as compared to
SEQ ID No 1, at least one additional salt bridge. Preferably, the
esterase comprises at least one additional surface salt bridge,
located on the extern surface of the protein structure.
[0077] To this aim, the esterase variant advantageously comprises
at least one amino acid substitution at a position selected from
51, Y4, Q5, R6, N9, S13, T16, S22, T25, Y26, S34, Y43, S48, T50,
R72, S98, N105, R108, S113, N122, S145, K147, T160, N162, E173,
S181, Q189, N190, S193, T194, D203, N204, S212, N213, N231, T233,
R236, Q237, N241, N243, N254, R255, Q258.
[0078] Advantageously, the esterase variant has at least one salt
bridge between two amino acids of said positions.
[0079] Most often, salt bridges are formed by interaction between
an anionic charge of either aspartic acid (D) or glutamic acid (E),
and a cationic charge of either lysine (K) or arginine (R).
Accordingly, the salt bridge in the esterase of the invention is
advantageously obtained by substituting at least one amino acid of
at least one pair of target amino acids as listed in Table 1, with
D or E and/or K or R, depending on the nature of the pair of target
amino acid considered.
TABLE-US-00001 TABLE 1 Combination of pairs of amino acid positions
(1.sup.st and 2scd) to target to form salt bridge 1.sup.st amino
acid position 2scd amino acid position S1 Q5 or N9 or S48 or N231
or T233 or R236 or Q237 Y4 R6 or S48 or T50 or N122 or N231 Q5 N9
or N231 or T233 or Q237 R6 S48 or T50 N9 N231 or T233 or Q237 T16
N213 or S13 S22 Y43 or R72 T25 R72 Y26 Y43 or S113 S34 S98 or N105
Y43 S48 or S113 T50 S48 R108 N105 or S145 or N122 S113 S48 N122 R6
or T50 or S145 K147 Y4 or N122 or S145 or N231 T160 N190 N162 N190
or S193 or T194 E173 S181 or D203 or N204 Q189 S181 or S193 S193
T160 or N190 or N254 or R255 T194 R255 S212 R72 N231 N122 R236 T233
or N254 or Q258 Q237 N241 or N243 N243 R236 or N254 or Q258 Q258
N241 or N254 or R255
[0080] Advantageously, when an amino acid residue of the targeted
pair of amino acids is R or K, solely the second amino acid of the
targeted pair is substituted with D or E. As an example, the
esterase variant of the invention may comprise the amino acid
substitution Y4D and thereby may exhibit a salt bridge between said
mutated amino acid residue and R6. In the same way, when an amino
acid residue of the targeted pair of amino acids is D or E, solely
the second amino acid of the targeted pair is substituted with R or
K. As an example, the esterase variant of the invention may
comprise the amino acid substitution N204R and thereby may exhibit
a salt bridge between said mutated amino acid residue and E173.
[0081] It is also an object of the invention to provide a esterase
having a polyester degrading activity which has at least 75%, 80%,
85%, 90%, 95% or 99% identity to the full length amino acid
sequence set forth in SEQ ID No 1, which comprises a suppression of
at least one N- and/or C-terminal amino acid residue as compared to
SEQ ID No 1, and preferably, the suppression of at least one
N-terminal amino acid residue.
[0082] In a particular embodiment, the variant esterase of the
invention comprises one or more amino acid substitution(s) as
compared to SEQ ID No 1 at a position selected from T61, Y92 and
V177, wherein the positions are numbered by reference to the amino
acid sequence set forth in SEQ ID No 1, and wherein the
substitutions are different from T61A/G, Y92A and V177A.
Preferably, the variant esterase of the invention comprises one or
more amino acid substitution(s) as compared to SEQ ID No 1,
selected from V177I, Y92G/P, and T61M.
[0083] In another particular embodiment, the variant esterase of
the invention comprises at least one substitution as compared to
SEQ ID No 1 at the position F208. According to the invention F208
may be substituted by any one of the 19 other amino acids.
Preferably, the substitution is F208W.
[0084] In another particular embodiment, the esterase variant
comprises at least two substitutions at positions selected from
T61, Y92, V177 and F208 as compared to SEQ ID No 1, preferably at
least two substitutions at positions F208 and Y92.
[0085] In a particular embodiment, the variant comprises at least
the substitutions Y92P+F208W.
[0086] In another particular embodiment, the variant comprises at
least the substitutions V170I+F208W.
[0087] According to the invention, the esterase variants may be
further glycosylated to further increase the thermostability of the
enzyme as compared to the enzyme of SEQ ID No 1.
[0088] In a particular embodiment, the esterase variant comprises a
glycosylated moiety on at least one asparagine residue of the
enzyme, said asparagine residue being preferably at a position
selected from N9, N143, N162, N204, N231, by reference to SEQ ID No
1, more preferably selected from N9, N162 and N231. In an
embodiment, esterase variant comprises a glycosylated moiety on N9,
N162 and N231.
[0089] For instance, a N-linked glycan moiety is attached to a
nitrogen of at least one of said asparagine residues.
[0090] Alternatively or in addition, the esterase variant of the
invention may further comprise one or more insertions of proline
residue and/or one or more deletions of glycine.
[0091] In a particular embodiment, the esterase variant of the
invention comprises one or several modifications and/or mutations
as listed above.
Novel Esterases with Both Improved Thermostability and Activity
[0092] It is a further object of the invention to provide novel
esterases that exhibit both increased thermostability and increased
polyester degrading activity as compared to the esterase of SEQ ID
No 1.
[0093] It is thus another object of the invention to provide an
esterase which (i) has at least 75%, 80%, 85%, 90%, 95% or 99%
identity to the full length amino acid sequence set forth in SEQ ID
No 1, (ii) contains at least one amino acid modification as
compared to SEQ ID No 1, and (iii) exhibits both an increased
thermostability and an increased activity as compared to the
esterase of SEQ ID No 1.
[0094] In a particular embodiment, the esterase variant comprises
at least one mutation as disclosed above and at least one
additional substitution selected from F208I or F208W by reference
to SEQ ID No 1.
[0095] In another particular embodiment, the variant comprises at
least one substitution selected from T61M, Y92G/P, F208W,
Y92P+F208W, and F208W+V170I and exhibits both an increased
thermostability and an increased activity as compared to the
esterase of SEQ ID No 1.
[0096] In a particular embodiment, the variant comprises at least
the substitution(s) selected from F208W+D203C+S248C and
F208I+D203C+S248C and exhibits both an increased thermostability
and an increased activity as compared to the esterase of SEQ ID No
1.
Polyester Degrading Activity
[0097] It is an object of the invention to provide new enzymes
having an esterase activity. In a particular embodiment, the enzyme
of the invention further exhibits a cutinase activity.
[0098] In a particular embodiment, the esterase of the invention
has a polyester degrading activity, preferably a polyethylene
terephthalate (PET) degrading activity.
[0099] In another particular embodiment, the esterase of the
invention also has a PBAT degrading activity.
[0100] Advantageously, the esterase variant of the invention
exhibits a polyester degrading activity at least in a range of
temperatures from 20.degree. C. to 90.degree. C., preferably from
40.degree. C. to 80.degree. C., more preferably from 50.degree. C.
to 70.degree. C., even more preferably from 60.degree. C. to
70.degree. C., even more preferably at 65.degree. C. In a
particular embodiment, the esterase variant of the invention
exhibits a polyester degrading activity at 70.degree. C. In another
particular embodiment, the polyester degrading activity is still
measurable at a temperature between 60.degree. C. and 90.degree.
C.
[0101] In a particular embodiment, the esterase variant of the
invention has an increased half-life at a given temperature,
compared to the esterase of SEQ ID No 1, and more particularly at a
temperature between 40.degree. C. and 80.degree. C., more
preferably between 50.degree. C. and 70.degree. C., even more
preferably between 60.degree. C. and 70.degree. C., even more
preferably at 65.degree. C. In a particular embodiment, the
esterase variant has a half-life at 65.degree. C. at least 5%
greater than the half-life of the esterase of SEQ ID No 1,
preferably at least 10%, 20%, 50%, 100%, 200%, 300%, or
greater.
[0102] In another particular embodiment, the esterase variant of
the invention has an increased melting temperature (Tm), as
compared to the esterase of SEQ ID No 1. Advantageously, the
esterase variant of the invention has an melting temperature (Tm)
increased of about 1.degree. C., 2.degree. C., 3.degree. C.,
4.degree. C., 5.degree. C., 10.degree. C. or more as compared to
the esterase of SEQ ID No 1.
[0103] In a particular embodiment, the esterase variant of the
invention exhibits a measurable esterase activity at least in a
range of pH from 5 to 11, preferably in a range of pH from 6 to 9,
more preferably in a range of pH from 6.5 to 9, even more
preferably in a range of pH from 6.5 to 8.
Nucleic Acids, Expression Cassette, Vector
[0104] It is a further object of the invention to provide a nucleic
acid encoding an esterase as defined above.
[0105] As used herein, the term "nucleic acid", "nucleic sequence,"
"polynucleotide", "oligonucleotide" and "nucleotide sequence" are
used interchangeably and refer to a sequence of
deoxyribonucleotides and/or ribonucleotides. The nucleic acids can
be DNA (cDNA or gDNA), RNA, or a mixture of the two. It can be in
single stranded form or in duplex form or a mixture of the two. It
can be of recombinant, artificial and/or synthetic origin and it
can comprise modified nucleotides, comprising for example a
modified bond, a modified purine or pyrimidine base, or a modified
sugar. The nucleic acids of the invention can be in isolated or
purified form, and made, isolated and/or manipulated by techniques
known per se in the art, e.g., cloning and expression of cDNA
libraries, amplification, enzymatic synthesis or recombinant
technology. The nucleic acids can also be synthesized in vitro by
well-known chemical synthesis techniques, as described in, e.g.,
Belousov (1997) Nucleic Acids Res. 25:3440-3444.
[0106] The invention also encompasses nucleic acids which
hybridize, under stringent conditions, to a nucleic acid encoding
an esterase as defined above. Preferably, such stringent conditions
include incubations of hybridization filters at about 42.degree. C.
for about 2.5 hours in 2.times.SSC/0.1% SDS, followed by washing of
the filters four times of 15 minutes in 1.times.SSC/0.1% SDS at
65.degree. C. Protocols used are described in such reference as
Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold
Spring Harbor Press, Cold Spring Harbor N.Y. (1988)) and Ausubel
(Current Protocols in Molecular Biology (1989)).
[0107] The invention also encompasses nucleic acids encoding an
esterase of the invention, wherein the sequence of said nucleic
acids, or a portion of said sequence at least, has been engineered
using optimized codon usage.
[0108] Alternatively, the nucleic acids according to the invention
may be deduced from the sequence of the esterase according to the
invention and codon usage may be adapted according to the host cell
in which the nucleic acids shall be transcribed. These steps may be
carried out according to methods well known to one skilled in the
art and some of which are described in the reference manual
Sambrook et al. (Sambrook et al., 2001).
[0109] Nucleic acids of the invention may further comprise
additional nucleotide sequences, such as regulatory regions, i.e.,
promoters, enhancers, silencers, terminators, signal peptides and
the like that can be used to cause or regulate expression of the
polypeptide in a selected host cell or system.
[0110] The present invention further relates to an expression
cassette comprising a nucleic acid according to the invention
operably linked to one or more control sequences that direct the
expression of said nucleic acid in a suitable host cell. Typically,
the expression cassette comprises, or consists of, a nucleic acid
according to the invention operably linked to a control sequence
such as transcriptional promoter and/or transcription terminator.
The control sequence may include a promoter that is recognized by a
host cell or an in vitro expression system for expression of a
nucleic acid encoding an esterase of the present invention. The
promoter contains transcriptional control sequences that mediate
the expression of the enzyme. The promoter may be any
polynucleotide that shows transcriptional activity in the host cell
including mutant, truncated, and hybrid promoters, and may be
obtained from genes encoding extracellular or intracellular
polypeptides either homologous or heterologous to the host cell.
The control sequence may also be a transcription terminator, which
is recognized by a host cell to terminate transcription. The
terminator is operably linked to the 3'-terminus of the nucleic
acid encoding the esterase. Any terminator that is functional in
the host cell may be used in the present invention. Typically, the
expression cassette comprises, or consists of, a nucleic acid
according to the invention operably linked to a transcriptional
promoter and a transcription terminator.
[0111] The invention also relates to a vector comprising a nucleic
acid or an expression cassette as defined above.
[0112] The term "vector" refers to DNA molecule used as a vehicle
to transfer recombinant genetic material into a host cell. The
major types of vectors are plasmids, bacteriophages, viruses,
cosmids, and artificial chromosomes. The vector itself is generally
a DNA sequence that consists of an insert (a heterologous nucleic
acid sequence, transgene) and a larger sequence that serves as the
"backbone" of the vector. The purpose of a vector which transfers
genetic information to the host is typically to isolate, multiply,
or express the insert in the target cell. Vectors called expression
vectors (expression constructs) are specifically adapted for the
expression of the heterologous sequences in the target cell, and
generally have a promoter sequence that drives expression of the
heterologous sequences encoding a polypeptide. Generally, the
regulatory elements that are present in an expression vector
include a transcriptional promoter, a ribosome binding site, a
terminator, and optionally present operator. Preferably, an
expression vector also contains an origin of replication for
autonomous replication in a host cell, a selectable marker, a
limited number of useful restriction enzyme sites, and a potential
for high copy number. Examples of expression vectors are cloning
vectors, modified cloning vectors, specifically designed plasmids
and viruses. Expression vectors providing suitable levels of
polypeptide expression in different hosts are well known in the
art. The choice of the vector will typically depend on the
compatibility of the vector with the host cell into which the
vector is to be introduced.
[0113] It is another object of the invention to provide a host cell
comprising a nucleic acid, an expression cassette or a vector as
described above. The present invention thus relates to the use of a
nucleic acid, expression cassette or vector according to the
invention to transform, transfect or transduce a host cell. The
choice of the vector will typically depend on the compatibility of
the vector with the host cell into which it must be introduced.
[0114] According to the invention, the host cell may be
transformed, transfected or transduced in a transient or stable
manner. The expression cassette or vector of the invention is
introduced into a host cell so that the cassette or vector is
maintained as a chromosomal integrant or as a self-replicating
extra-chromosomal vector. The term "host cell" also encompasses any
progeny of a parent host cell that is not identical to the parent
host cell due to mutations that occur during replication. The host
cell may be any cell useful in the production of a variant of the
present invention, e.g., a prokaryote or a eukaryote. The
prokaryotic host cell may be any Gram-positive or Gram-negative
bacterium. The host cell may also be an eukaryotic cell, such as a
yeast, fungal, mammalian, insect or plant cell. In a particular
embodiment, the host cell is selected from the group of Escherichia
coli, Bacillus, Streptomyces, Trichoderma, Aspergillus,
Saccharomyces, Pichia or Yarrowia.
[0115] The nucleic acid, expression cassette or expression vector
according to the invention may be introduced into the host cell by
any method known by the skilled person, such as electroporation,
conjugation, transduction, competent cell transformation,
protoplast transformation, protoplast fusion, biolistic "gene gun"
transformation, PEG-mediated transformation, lipid-assisted
transformation or transfection, chemically mediated transfection,
lithium acetate-mediated transformation, liposome-mediated
transformation,
[0116] Optionally, more than one copy of a nucleic acid, cassette
or vector of the present invention may be inserted into a host cell
to increase production of the variant.
[0117] In a particular embodiment, the host cell is a recombinant
microorganism. The invention indeed allows the engineering of
microorganisms with improved capacity to degrade polyester
containing material. For instance, the sequence of the invention
may be used to complement a wild type strain of a fungus or
bacterium already known as able to degrade polyester, in order to
improve and/or increase the strain capacity.
Production of Esterase Variant
[0118] It is another object of the invention to provide a method of
producing the esterase variant of the invention, comprising
expressing a nucleic acid encoding the esterase and optionally
recovering the esterase.
[0119] In particular, the present invention relates to in vitro
methods of producing an esterase of the present invention
comprising (a) contacting a nucleic acid, cassette or vector of the
invention with an in vitro expression system; and (b) recovering
the esterase produced. In vitro expression systems are well-known
by the person skilled in the art and are commercially
available.
[0120] Preferably, the method of production comprises
(a) culturing a host cell that comprises a nucleic acid encoding an
esterase of the invention under conditions suitable to express the
nucleic acid; and optionally (b) recovering said esterase from the
cell culture.
[0121] Advantageously, the host cell is a recombinant Bacillus,
recombinant E. coli, recombinant Aspergillus, recombinant
Trichoderma, recombinant Streptomyces, recombinant Saccharomyces,
recombinant Pichia or recombinant Yarrowia lipolytica.
[0122] The host cells are cultivated in a nutrient medium suitable
for production of polypeptides, using methods known in the art. For
example, the cell may be cultivated by shake flask cultivation, or
small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or
industrial fermentors performed in a suitable medium and under
conditions allowing the enzyme to be expressed and/or isolated. The
cultivation takes place in a suitable nutrient medium, from
commercial suppliers or prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection).
[0123] If the esterase is excreted into the nutrient medium, the
esterase can be recovered directly from the culture supernatant.
Conversely, the esterase can be recovered from cell lysates or
after permeabilisation. The esterase may be recovered using any
method known in the art. For example, the esterase may be recovered
from the nutrient medium by conventional procedures including, but
not limited to, collection, centrifugation, filtration, extraction,
spray-drying, evaporation, or precipitation. Optionally, the
esterase may be partially or totally purified by a variety of
procedures known in the art including, but not limited to,
chromatography (e.g., ion exchange, affinity, hydrophobic,
chromatofocusing, and size exclusion), electrophoretic procedures
(e.g., preparative isoelectric focusing), differential solubility
(e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to
obtain substantially pure polypeptides.
[0124] The esterase may be used as such, in purified form, either
alone or in combinations with additional enzymes, to catalyze
enzymatic reactions involved in the degradation and/or recycling of
a polyester containing material, such as plastic products
containing polyester. The esterase may be in soluble form, or on
solid phase. In particular, it may be bound to cell membranes or
lipid vesicles, or to synthetic supports such as glass, plastic,
polymers, filter, membranes, e.g., in the form of beads, columns,
plates and the like.
Composition
[0125] It is a further object of the invention to provide a
composition comprising an esterase or a host cell of the invention.
In the context of the invention, the term "composition" encompasses
any kind of compositions comprising an esterase of the invention.
In a particular embodiment, the esterase is in isolated or at least
partially purified form.
[0126] The composition may be liquid or dry, for instance in the
form of a powder. In some embodiments, the composition is a
lyophilisate. For instance, the composition may comprise the
esterase and/or recombinant cells encoding the esterase of the
invention or extract thereof, and optionally excipients and/or
reagents etc. Appropriate excipients encompass buffers commonly
used in biochemistry, agents for adjusting pH, preservatives such
as sodium benzoate, sodium sorbate or sodium ascorbate,
conservatives, protective or stabilizing agents such as starch,
dextrin, arabic gum, salts, sugars e.g. sorbitol, trehalose or
lactose, glycerol, polyethyleneglycol, polyethene glycol,
polypropylene glycol, propylene glycol, sequestering agent such as
EDTA, reducing agents, amino acids, a carrier such as a solvent or
an aqueous solution, and the like. The composition of the invention
may be obtained by mixing the esterase with one or several
excipients.
[0127] The composition of the invention may comprise from 0.1% to
99.9%, preferably from 0.1% to 50%, more preferably from 0.1% to
30%, even more preferably from 0.1% to 5% by weight of the esterase
of the invention and from 0.1% to 99.9%, preferably from 50% to
99.9%, more preferably from 70% to 99.9%, even more preferably from
95% to 99.9% by weight of excipient(s). A preferred composition
comprises between 0.1 and 5% by weight of the esterase of the
invention.
[0128] In a particular embodiment, the composition may further
comprise additional polypeptide(s) exhibiting an enzymatic
activity. The amounts of esterase of the invention will be easily
adapted by those skilled in the art depending e.g., on the nature
of the polyester containing material to degrade and/or the
additional enzymes/polypeptides contained in the composition.
[0129] In a particular embodiment, the esterase of the invention is
solubilized in an aqueous medium together with one or several
excipients, especially excipients which are able to stabilize or
protect the polypeptide from degradation. For instance, the
esterase of the invention may be solubilized in water, eventually
with additional components, such as glycerol, sorbitol, dextrin,
starch, glycol such as propanediol, salt, etc. The resulting
mixture may then be dried so as to obtain a powder. Methods for
drying such mixture are well known to the one skilled in the art
and include, without limitation, lyophilisation, freeze-drying,
spray-drying, supercritical drying, down-draught evaporation,
thin-layer evaporation, centrifugal evaporation, conveyer drying,
fluidized bed drying, drum drying or any combination thereof.
[0130] In a further particular embodiment, the composition of the
invention comprises at least one recombinant cell expressing an
esterase of the invention, or an extract thereof. An "extract of a
cell" designates any fraction obtained from a cell, such as cell
supernatant, cell debris, cell walls, DNA extract, enzymes or
enzyme preparation or any preparation derived from cells by
chemical, physical and/or enzymatic treatment, which is essentially
free of living cells. Preferred extracts are enzymatically-active
extracts. The composition of the invention may comprise one or
several recombinant cells of the invention or extract thereof, and
optionally one or several additional cells.
[0131] In a particular embodiment, the composition consists or
comprises a lyophilized culture medium of a recombinant
microorganism expressing and excreting an esterase of the
invention. In a particular embodiment, the powder comprises the
esterase of the invention and a stabilizing/solubilizing amount of
glycerol, sorbitol or dextrin, such as maltodextrine and/or
cyclodextrine, starch, glycol such as propanediol, and/or salt.
Use of the Esterase of the Invention
[0132] It is a further object of the invention to provide methods
using an esterase of the invention for degrading in aerobic or
anaerobic conditions and/or recycling polyester containing
material, as plastic products made of or containing polyesters. The
variant esterases of the invention are particularly useful for
degrading a plastic product comprising PET.
[0133] It is therefore an object of the invention to use an
esterase of the invention, or corresponding recombinant cell or
extract thereof, or composition for the enzymatic degradation of a
polyester containing material, such as a PET containing
material.
[0134] It is another object of the invention to provide a method
for degrading a plastic product containing at least one polyester,
wherein the plastic product is contacted with an esterase or host
cell or composition of the invention, thereby degrading the plastic
product. Advantageously, polyester(s) of the polyester containing
material is (are) depolymerized up to monomers and/or
oligomers.
[0135] In an embodiment of the method of degradation, at least one
polyester is degraded to yield repolymerizable monomers and/or
oligomers, which are advantageously retrieved in order to be
reused.
[0136] In an embodiment, polyester(s) of the polyester containing
material is (are) fully degraded.
[0137] In a particular embodiment, the plastic product comprises at
least one polyester selected from polyethylene terephthalate (PET),
polytrimethylene terephthalate (PTT), polybutylen terephthalate
(PBT), polyethylene isosorbide terephthalate (PEIT), polylactic
acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate
(PBS), polybutylene succinate adipate (PBSA), polybutylene adipate
terephthalate (PBAT), polyethylene furanoate (PEF),
polycaprolactone (PCL), poly(ethylene adipate) (PEA), polyethylene
naphthalate (PEN) and blends/mixtures of these materials,
preferably polyethylene terephthalate. In a preferred embodiment,
the polyester containing material comprises PET, and at least
monomers such as monoethylene glycol or terephthalic acid, and/or
oligomers such as methyl-2-hydroxyethyl terephthalate (MHET),
bis(2-hydroxyethyl) terephthalate (BHET), 2-hydroxyethyl benzoate
(HEB) and dimethyl terephthalate (DMT) are recovered for recycling
or methanisation for instance.
[0138] The invention also relates to a method of producing monomers
and/or oligomers from a polyester containing material, comprising
exposing a polyester containing material to an esterase of the
invention, or corresponding recombinant cell or extract thereof, or
composition, and optionally recovering monomers and/or oligomers.
The method of the invention is particularly useful for producing
monomers selected from monoethylene glycol and terephthalic acid,
and/or oligomers selected from methyl-2-hydroxyethyl terephthalate
(MHET), bis(2-hydroxyethyl) terephthalate (BHET), 2-hydroxyethyl
benzoate (HEB) and dimethyl terephthalate (DMT).
[0139] The time required for degrading a polyester containing
material may vary depending on the polyester containing material
itself (i.e., nature and origin of the plastic product, its
composition, shape etc.), the type and amount of esterase used, as
well as various process parameters (i.e., temperature, pH,
additional agents, etc.). One skilled in the art may easily adapt
the process parameters to the polyester containing material.
[0140] Advantageously, the degrading process is implemented at a
temperature comprised between 20.degree. C. and 90.degree. C.,
preferably between 40.degree. C. and 80.degree. C., more preferably
between 50.degree. C. and 70.degree. C., more preferably between
60.degree. C. and 70.degree. C., even more preferably at 65.degree.
C. In another particular embodiment, the degrading process is
implemented at 70.degree. C. More generally, the temperature is
maintained below an inactivating temperature, which corresponds to
the temperature at which the esterase is inactivated and/or the
recombinant microorganism does no more synthesize the esterase.
Particularly, the temperature is maintained below the glass
transition temperature (Tg) of the polyester in the polyester
containing material. More particularly, the process is implemented
in a continuous way, at a temperature at which the esterase can be
used several times and/or recycled.
[0141] Advantageously, the degrading process is implemented at a pH
comprised between 5 and 11, preferably at a pH between 6 and 9,
more preferably at a pH between 6.5 and 9, even more preferably at
a pH between 6.5 to 8.
[0142] In a particular embodiment, the polyester containing
material may be pretreated prior to be contacted with the esterase,
in order to physically change its structure, so as to increase the
surface of contact between the polyester and the variant of the
invention.
[0143] Optionally, monomers and/or oligomers resulting from the
depolymerization may be recovered, sequentially or continuously. A
single type of monomers and/or oligomers or several different types
of monomers and/or oligomers may be recovered, depending on the
starting polyester containing material.
[0144] The recovered monomers and/or oligomers may be further
purified, using all suitable purifying methods and conditioned in a
re-polymerizable form. Examples of purifying methods include
stripping process, separation by aqueous solution, steam selective
condensation, filtration and concentration of the medium after the
bioprocess, separation, distillation, vacuum evaporation,
extraction, electrodialysis, adsorption, ion exchange,
precipitation, crystallization, concentration and acid addition
dehydration and precipitation, nanofiltration, acid catalyst
treatment, semi continuous mode distillation or continuous mode
distillation, solvent extraction, evaporative concentration,
evaporative crystallization, liquid/liquid extraction,
hydrogenation, azeotropic distillation process, adsorption, column
chromatography, simple vacuum distillation and microfiltration,
combined or not.
[0145] The repolymerizable monomers and/or oligomers may then be
reused for instance to synthesize polyesters. Advantageously,
polyesters of same nature are repolymerized. However, it is
possible to mix the recovered monomers and/or oligomers with other
monomers and/or oligomers, in order for instance to synthesize new
copolymers. Alternatively, the recovered monomers may be used as
chemical intermediates in order to produce new chemical compounds
of interest.
[0146] The invention also relates to a method of surface hydrolysis
or surface functionalization of a polyester containing material,
comprising exposing a polyester containing material to an esterase
of the invention, or corresponding recombinant cell or extract
thereof, or composition. The method of the invention is
particularly useful for increasing hydrophilicity, or water
absorbency, of a polyester material. Such increased hydrophilicity
may have particular interest in textiles production, electronics
and biomedical applications.
[0147] It is a further object of the invention to provide a
polyester containing material in which an esterase of the invention
and/or a recombinant microorganism expressing and excreting said
esterase is/are included. In a particular embodiment, such
polyester containing material may be a plastic compound. It is thus
an object of the invention to provide a plastic compound containing
an esterase of the invention and/or a recombinant cell and/or a
composition or extract thereof; and at least one polyester. In a
preferred embodiment, the polyester is PET.
EXAMPLES
Example 1--Construction, Expression and Purification of
Esterases
[0148] Construction
[0149] The esterase variants have been generated using the
plasmidic construction pET26b-LCC-His. This plasmid consists in
cloning a gene encoding the esterase of SEQ ID No 1, optimized for
Escherichia coli expression between NdeI and XhoI restriction
sites. Two site directed mutagenesis kits have been used according
to the recommendations of the supplier, in order to generate the
esterase variants: QuikChange II Site-Directed Mutagenesis kit and
QuikChange Lightning Multi Site-Directed from Agilent (Santa Clara,
Calif., USA).
[0150] Expression and Purification of the Esterases
[0151] The strains Stellar.TM. (Clontech, Calif., USA) and E. coli
One Shot.RTM. BL21 DE3 (Life technologies, Carlsbad, Calif., USA)
have been successively employed to perform the cloning and
recombinant expression in 50 mL LB-Miller medium or ZYM auto
inducible medium (Studier et al., 2005--Prot. Exp. Pur. 41,
207-234). The induction in LB-Miller medium has been performed at
16.degree. C., with 0.5 mM of isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG, Euromedex,
Souffelweyersheim, France). The cultures have been stopped by
centrifugation (8000 rpm, 20 minutes at 10.degree. C.) in an Avanti
J-26 XP centrifuge (Beckman Coulter, Brea, USA). The cells have
been suspended in 20 mL of Talon buffer (Tris-HCl 20 mM, NaCl 300
mM, pH 8). Cell suspension was then sonicated during 2 minutes with
30% of amplitude (2 sec ON and 1 sec OFF cycles) by FB 705
sonicator (Fisherbrand, Illkirch, France). Then, a step of
centrifugation has been realized: 30 minutes at 11000 rpm,
10.degree. C. in an Eppendorf centrifuge. The soluble fraction has
been collected and submitted to affinity chromatography. This
purification step has been completed with Talon.RTM. Metal Affinity
Resin (Clontech, Calif., USA). Protein elution has been carried out
with gradient of Talon buffer supplemented with imidazole. Purified
protein has been dialyzed against Talon buffer then quantified
using Bio-Rad protein assay according to manufacturer instructions
(Lifescience Bio-Rad, France) and stored at +4.degree. C.
Example 2--Evaluation of the Thermostability of the Esterases of
the Invention
[0152] The thermostability of the esterase variants has been
determined and compared to the thermostability of the esterase of
SEQ ID No 1.
[0153] Different methodologies have been used to estimate
thermostability:
(1) Circular dichroism of proteins in solution; (2) Residual
esterase activity after protein incubation in given conditions of
temperatures, times and buffers; (3) Residual polyester's
depolymerization activity after protein incubation in given
conditions of temperatures, times and buffers; (4) Ability to
degrade a solid polyester compound (such as PET or PBAT or
analogues) dispersed in an agar plate, after protein incubation in
given conditions of temperatures, times and buffers; (5) Ability to
perform multiple rounds of polyester's depolymerization assays in
given conditions of temperatures, buffers, protein concentrations
and polyester concentrations;
(6) Differential Scanning Fluorimetry (DSF).
[0154] Details on the protocol of such methods are given below.
[0155] 2.1 Circular Dichroism
[0156] Circular dichroism (CD) has been performed with a Jasco 815
device (Easton, USA) to compare the fusion temperature (T.sub.m) of
the esterase of SEQ ID No 1 and the esterase variants of the
invention. The T.sub.m corresponds to the temperature at which 50%
of the protein is denaturated.
[0157] Technically 4004, protein sample was prepared at 0.5 mg/mL
in Talon buffer and used for CD. A first scan from 280 to 190 nm
was realized to determine two maxima intensities of CD
corresponding to the correct folding of the protein. A second scan
was then performed from 25.degree. C. to 110.degree. C., at length
waves corresponding to such maximal intensities and providing
specific curves (sigmoid 3 parameters y=a/(1+e{circumflex over (
)}((x-x0)/b))) that were analyzed by Sigmaplot version 11.0
software, the Tm is determined when x=x0. The T.sub.m obtained
reflects the thermostability of the given protein. The higher the
T.sub.m is, the more stable the variant is at high temperature.
[0158] 2.2 Residual Esterase Activity
[0159] 1 mL of a solution of 40 mg/L (in Talon buffer) of the
esterase of SEQ ID No 1 or of an esterase variant was incubated at
different temperatures (65, 70, 75, 80 and 90.degree. C.) during 10
days. Regularly, a sample, was taken, diluted 1 to 500 times in a
0.1M potassium phosphate buffer pH 8.0 and para nitro
phenol-butyrate (pNP-B) assay was realized. 204, of sample are
mixed with 1754, of 0.1M potassium phosphate buffer pH 8.0 and 54,
of pNP-B solution in 2-methyl-2 butanol (40 mM). Enzymatic reaction
was performed at 30.degree. C. under agitation, during 15 minutes
and absorbance at 405 nm was acquired by microplate
spectrophotometer (Versamax, Molecular Devices, Sunnyvale, Calif.,
USA). Activity of pNP-B hydrolysis (initial velocity expressed in
.mu.mol of pNPB/min) was determined using a standard curve for the
liberated para nitro phenol in the linear part of the hydrolysis
curve. The half-life of the enzyme at a given temperature
corresponds to the time necessary to lose 50% of the initial
activity.
[0160] 2.3 Residual Polyester Depolymerizing Activity
[0161] Crushed Cristal preform were immersed in liquid nitrogen and
were micronized using an Ultra Centrifugal Mill ZM 200 system to a
fine powder <500 .mu.m size. Then, the obtained powder was
sieved. The fraction with a size between 250 .mu.m and 500 .mu.m
only has been used for the depolymerization test. The crystallinity
of this fraction was measured at 11.5% using a Mettler Toledo DSC 3
with heating rate of 10.degree. C./min.
[0162] 10 mL of a solution of 40 mg/L (in Talon buffer) of the
esterase of SEQ ID No 1 and of an esterase variant respectively
were incubated at different temperatures (65, 70, 75, 80 and
90.degree. C.) during 10 to 30 days. Regularly, a 1 mL sample was
taken, and transferred into a bottle containing 100 mg of amorphous
PET micronized at 250-500 .mu.m and 49 mL of 0.1M potassium
phosphate buffer pH 8.0 and incubated at 65.degree. C. 150 .mu.L of
buffer were sampled regularly. When required, samples were diluted
in 0.1 M potassium phosphate buffer pH 8. Then, 150 .mu.L of
methanol and 6.5 .mu.L of HCl 6 N were added to 150 .mu.L of sample
or dilution. After mixing and filtering on 0.45 .mu.m syringe
filter, samples were loaded on UHPLC to monitor the liberation of
terephthalic acid (TA), MHET and BHET. Chromatography system used
was an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Inc.
Waltham, Mass., USA) including a pump module, an autosampler, a
column oven thermostated at 25.degree. C., and an UV detector at
240 nm. The column used was a Discovery.RTM. HS C18 HPLC Column
(150.times.4.6 mm, 5 .mu.m, equipped with precolumn, Supelco,
Bellefonte, USA). TA, MHET and BHET were separated using a gradient
of MeOH (30% to 90%) in 1 mM of H.sub.2SO.sub.4 at 1 mL/min.
Injection was 20 .mu.L of sample. TA, MHET and BHET were measured
according to standard curves prepared from commercial TA and BHET
and in house synthesized MHET in the same conditions than samples.
Activity of PET hydrolysis (.mu.mol of PET hydrolysed/min or mg of
equivalent TA produced/hour) was determined in the linear part of
the hydrolysis curve. Equivalent TA corresponds to the sum of TA
measured and of TA contained in measured MHET and BHET. The
half-life of the enzyme at a given temperature corresponds to the
time required to lose 50% of the initial activity.
[0163] 2.4 Degradation of a Polyester Under Solid Form
[0164] 20 .mu.L of enzyme preparation was deposited in a well
created in an agar plate containing PET. Preparation of agar plates
was realized by solubilizing 500 mg of PET solubilized in HFIP, and
this medium is poured in a 250 mL aqueous solution. After HFIP
evaporation at 52.degree. C., the solution was mixed v/v with 0.2 M
potassium phosphate buffer pH 8 containing 3% agar. Around 30 mL of
the mixture is used to prepare each omnitray and stored at
4.degree. C.
[0165] The diameter of the halo formed due to the polyester
degradation was measured after 24 hours at 60 or 65.degree. C. The
half-life of the enzyme at a given temperature corresponds to the
time required to decrease by a 2-fold factor the diameter of the
halo.
[0166] 2.5 Multiple Rounds of Polyester's Depolymerization
[0167] The ability of the esterase to perform successive rounds of
polyester's depolymerization assays was evaluated in an enzymatic
reactor. A Minibio 500 bioreactor (Applikon Biotechnology B.V.,
Delft, The Netherlands) was started with 3 g of amorphous PET and
100 mL of 10 mM potassium phosphate buffer pH 8 containing 3 mg of
LC-esterase. Agitation was set at 250 rpm using a marine impeller.
Bioreactor was thermostated at 65.degree. C. by immersion in an
external water bath. pH was regulated at 8 by addition of KOH at 3
M. The different parameters (pH, temperature, agitation, addition
of base) were monitored thanks to BioXpert software V2.95. 1.8 g of
amorphous PET were added every 20 h. 500 .mu.L of reaction medium
was sampled regularly.
[0168] Amount of TA, MHET and BHET was determined by HPLC, as
described in example 2.3. Amount of EG was determined using an
Aminex HPX-87K column (Bio-Rad Laboratories, Inc, Hercules, Calif.,
United States) thermostated at 65.degree. C. Eluent was
K.sub.2HPO.sub.4 5 mM at 0.6 mLmin.sup.-1. Injection was 20 .mu.L.
Ethylene glycol was monitored using refractometer.
[0169] The percentages of hydrolysis were calculated based on the
ratio of molar concentration at a given time (TA+MHET+BHET) versus
the total amount of TA contained in the initial sample, or based on
the ratio of molar concentration at a given time
(EG+MHET+2.times.BHET) versus the total amount of EG contained in
the initial sample. Rate of degradation is calculated in mg of
total liberated TA per hour or in mg of total EG per hour.
[0170] Half-life of enzyme was evaluated as the incubation time
required to obtain a loss of 50% of the degradation rate.
[0171] 2.6 Differential Scanning Fluorimetry (DSF)
[0172] DSF was used to evaluate the thermostability of the
wild-type protein (SEQ ID No 1) and variants thereof by determining
their melting temperature (Tm), temperature at which half of the
protein population is unfolded. Protein samples were prepared at a
concentration of 14 .mu.M (0.4 mg/mL) and stored in buffer A
consisting of 20 mM Tris HCl pH 8.0, 300 mM NaCl. The SYPRO orange
dye 5000.times. stock solution in DMSO was first diluted to
250.times. in water. Protein samples were loaded onto a white clear
96-well PCR plate (Bio-Rad cat # HSP9601) with each well containing
a final volume of 25 .mu.l. The final concentration of protein and
SYPRO Orange dye in each well were 5 .mu.M (0.14 mg/ml) and
10.times. respectively. Loaded volumes per well were as follow: 15
.mu.L of buffer A, 9 .mu.L of the 0.4 mg/mL protein solution and 1
.mu.L of the 250.times. Sypro Orange diluted solution. The PCR
plates were then sealed with optical quality sealing tape and spun
at 2000 rpm for 1 min at room temperature. DSF experiments were
then carried out using a CFX96 real-time PCR system set to use the
450/490 excitation and 560/580 emission filters. The samples were
heated from 25 to 100.degree. C. at the rate of 1.1.degree. C./min.
A single fluorescence measurement was taken every 0.3.degree. C.
Melting temperatures were determined by performing a curve fit to
the Boltzmann equation.
[0173] Wild-type protein and variants were then compared based on
their Tm values. Due to high reproducibility between experiments on
the same protein from different productions, a .DELTA.Tm of
0.8.degree. C. was considered as significant to compare variants.
Tm values correspond to the average of at least 2 measurements.
[0174] Compared thermostabilities of esterase variants of the
invention are shown in Table 2 below, expressed in Tm values and
evaluated according to Example 2.6. The gain of Tm as compared to
the esterase of SEQ ID No 1 is indicated in brackets.
TABLE-US-00002 TABLE 2 Tm of the esterases of the invention Variant
of the invention Tm of variant of the invention D203C + S248C
94.20.degree. C. +/- 0.00.degree. C. (+9.50.degree. C.) D203C +
S248C + E173A 93.45.degree. C. +/- 0.21.degree. C. (+8.75.degree.
C.) D203C + S248C + E173R 92.25.degree. C. +/- 0.21.degree. C.
(+7.55.degree. C.) D203C + S248C + E173R + 92.80.degree. C. +/-
0.17.degree. C. (+8.10.degree. C.) N204D + L202R F208W
85.90.degree. C. +/- 0.17.degree. C. (+1.20.degree. C.) Y92P
85.80.degree. C. +/- 0.00.degree. C. (+1.10.degree. C.) V177I
86.10.degree. C. +/- 0.30.degree. C. (+1.40.degree. C.) T61M
87.40.degree. C. +/- 0.17.degree. C. (+2.70.degree. C.) Y92G
87.00.degree. C. +/- 0.00.degree. C. (+2.30.degree. C.) Y92P +
F208W 86.60.degree. C. +/- 0.17.degree. C. (+1.90.degree. C.) F208W
+ V170I 85.80.degree. C. +/- 0.00.degree. C. (+1.10.degree. C.)
F208W + D203C + S248C 94.80.degree. C. +/- 0.00.degree. C.
(+10.10.degree. C.) F208I + D203C + S248C 90.90.degree. C. +/-
0.00.degree. C. (+6.20.degree. C.) F208W + D203C + S248C +
94.20.degree. C. +/- 0.00.degree. C. (+9.50.degree. C.) E173A F208I
+ D203C + S248C + 89.40.degree. C. +/- 0.00.degree. C.
(+4.70.degree. C.) E173A
Example 3--Evaluation of the Thermostability and Activity of
Esterase Variants of the Invention
[0175] The specific degrading activity of esterase variants of the
invention has been evaluated on PET and compared with the specific
degrading activity of the esterase of SEQ ID No 1.
[0176] 100 mg of amorphous PET were weighted and introduced in a
100 mL glass bottle. 1 mL of esterase preparation (as reference
control) or of variant preparation, respectively, prepared at 0.02
or 0.03 mg/mL in Talon buffer (Tris-HCl 20 mM, NaCl 0.3M, pH 8) and
introduced in the glass bottle. Finally, 49 mL of 0.1 M potassium
phosphate buffer pH 8 was added.
[0177] The depolymerization started by incubating each glass bottle
at 65.degree. C. and 150 rpm in a Max Q 4450 incubator (Thermo
Fisher Scientific, Inc. Waltham, Mass., USA).
[0178] The initial rate of depolymerization reaction, in mg of
equivalent TA generated/hour, was determined by samplings performed
at different time during the first 24 hours and analyzed by Ultra
High Performance Liquid Chromatography (UHPLC). If necessary,
samples were diluted in 0.1 M potassium phosphate buffer pH 8.
Then, 150 .mu.L of methanol and 6.5 .mu.L of HCl 6 N were added to
150 .mu.L of sample or dilution. After mixing and filtering on 0.45
.mu.m syringe filter, samples were loaded on UHPLC to monitor the
liberation of terephthalic acid (TA), MHET and BHET. Chromatography
system used was an Ultimate 3000 UHPLC system (Thermo Fisher
Scientific, Inc. Waltham, Mass., USA) including a pump module, an
autosampler, a column oven thermostated at 25.degree. C., and an UV
detector at 240 nm. The column used was a Discovery.RTM. HS C18
HPLC Column (150.times.4.6 mm, 5 .mu.m, equipped with precolumn,
Supelco, Bellefonte, USA). TA, MHET and BHET were separated using a
gradient of MeOH (30% to 90%) in 1 mM of H.sub.2SO.sub.4 at 1
mL/min. Injection was 20 .mu.L of sample. TA, MHET and BHET were
measured according to standard curves prepared from commercial TA
and BHET and in house synthesized MHET in the same conditions than
samples. The specific degrading activity of PET (mg of equivalent
TA/hour/mg of enzyme) was determined in the linear part of the
hydrolysis curve.
[0179] The results for both specific degrading activity and
thermostability of the esterase variants of the invention are shown
in Table 3.
[0180] The specific degrading activity of the esterase of SEQ ID No
1 is used as a reference and considered as 100% degrading activity.
The degrading activity is measured as exposed in example 3 (mg of
equivalent TA/hour/mg of enzyme). Equivalent TA corresponds to the
sum of TA measured and of TA contained in measured MHET and BHET.
The thermostability is expressed in Tm values (measured according
to example 2.6) and the gain of Tm as compared to the Tm of the
esterase of SEQ ID No 1 is noted in brackets.
TABLE-US-00003 TABLE 3 Specific activity and Tm of the esterases of
the invention Specific Variants of the degrading Tm of the variant
of the invention activity invention F208W 143% 85.90.degree. C. +/-
0.17.degree. C. (+1.20.degree. C.) Y92P 177% 85.80.degree. C. +/-
0.00.degree. C. (+1.10.degree. C.) T61M 121% 87.40.degree. C. +/-
0.17.degree. C. (+2.70.degree. C.) Y92G 120% 87.00.degree. C. +/-
0.00.degree. C. (+2.30.degree. C.) Y92P + F208W 116% 86.60.degree.
C. +/- 0.17.degree. C. (+1.90.degree. C.) F208W + V170I 128%
85.80.degree. C. +/- 0.00.degree. C. (+1.10.degree. C.) F208W +
D203C + 123% 94.80.degree. C. +/- 0.00.degree. C. (+10.10.degree.
C.) S248C F208I + D203C + 133% 90.90.degree. C. +/- 0.00.degree. C.
(+6.20.degree. C.) S248C
Sequence CWU 1
1
11258PRTartificial sequenceesterase 1Ser Asn Pro Tyr Gln Arg Gly
Pro Asn Pro Thr Arg Ser Ala Leu Thr1 5 10 15Ala Asp Gly Pro Phe Ser
Val Ala Thr Tyr Thr Val Ser Arg Leu Ser 20 25 30Val Ser Gly Phe Gly
Gly Gly Val Ile Tyr Tyr Pro Thr Gly Thr Ser 35 40 45Leu Thr Phe Gly
Gly Ile Ala Met Ser Pro Gly Tyr Thr Ala Asp Ala 50 55 60Ser Ser Leu
Ala Trp Leu Gly Arg Arg Leu Ala Ser His Gly Phe Val65 70 75 80Val
Leu Val Ile Asn Thr Asn Ser Arg Phe Asp Tyr Pro Asp Ser Arg 85 90
95Ala Ser Gln Leu Ser Ala Ala Leu Asn Tyr Leu Arg Thr Ser Ser Pro
100 105 110Ser Ala Val Arg Ala Arg Leu Asp Ala Asn Arg Leu Ala Val
Ala Gly 115 120 125His Ser Met Gly Gly Gly Gly Thr Leu Arg Ile Ala
Glu Gln Asn Pro 130 135 140Ser Leu Lys Ala Ala Val Pro Leu Thr Pro
Trp His Thr Asp Lys Thr145 150 155 160Phe Asn Thr Ser Val Pro Val
Leu Ile Val Gly Ala Glu Ala Asp Thr 165 170 175Val Ala Pro Val Ser
Gln His Ala Ile Pro Phe Tyr Gln Asn Leu Pro 180 185 190Ser Thr Thr
Pro Lys Val Tyr Val Glu Leu Asp Asn Ala Ser His Phe 195 200 205Ala
Pro Asn Ser Asn Asn Ala Ala Ile Ser Val Tyr Thr Ile Ser Trp 210 215
220Met Lys Leu Trp Val Asp Asn Asp Thr Arg Tyr Arg Gln Phe Leu
Cys225 230 235 240Asn Val Asn Asp Pro Ala Leu Ser Asp Phe Arg Thr
Asn Asn Arg His 245 250 255Cys Gln
* * * * *
References