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EP2449100A2 - Variants ameliores d'une phytase - Google Patents

Variants ameliores d'une phytase

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Publication number
EP2449100A2
EP2449100A2 EP10737804A EP10737804A EP2449100A2 EP 2449100 A2 EP2449100 A2 EP 2449100A2 EP 10737804 A EP10737804 A EP 10737804A EP 10737804 A EP10737804 A EP 10737804A EP 2449100 A2 EP2449100 A2 EP 2449100A2
Authority
EP
European Patent Office
Prior art keywords
phytase
variant
improved
functional derivative
substitutions
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10737804A
Other languages
German (de)
English (en)
French (fr)
Inventor
Stéphane BLESA
Hélène CHAUTARD
Marc Delcourt
Laurent Mesta
Bruno Winter
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biomethodes SA
Original Assignee
Biomethodes SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biomethodes SA filed Critical Biomethodes SA
Publication of EP2449100A2 publication Critical patent/EP2449100A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03008—3-Phytase (3.1.3.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase

Definitions

  • the present invention relates to the field of protein improvement and in particular that of proteins improved by molecular evolution.
  • HIIe relates to a variant of a phytase, said improved, in that it has a greater thermostability and / or activity than the original phytase.
  • the invention also relates to a nucleic acid encoding the variant, a cassette or expression vector containing this variant, a host cell expressing this variant, a composition comprising said variant as well as uses thereof, mainly in the preparation of food additives and animal feed.
  • Phytate is the main phosphorus storage compound in the plant. This molecule, also called phytic acid or inositol hexa-phosphate (I ⁇ sl'6 or mvo-inositol hexakisphosphatc), consists of a cyclohexane on which are connected six phosphate groups. Phytate represents about 70% of the vegetable phosphate, the remaining 30% being present in free form. On the other hand, phosphate residues of phytate chelate divalent and trivalent cations such as metal ions calcium, iron, zinc, magnesium, copper, manganese and mohbdenum essential for nutrition.
  • Phytases are enzymes that hydrolyze phytules: these enzymes naturally release one or two phosphates, rarely three, then adsorbable in the digestive system: the other products of the reaction being rinositol-triphosphate reactivated, mainly of the iositol tetra- and penta-phosphate ( Figure 1).
  • Phylascs are a family of enzymes that are widely represented in nature: many organisms, from bacteria to plants to fungi and some animals, express one or more. Mammals, however, express none; ruminants or polygastric animals such as cows and horses have endogenous microorganisms specific to their gastrointestinal tract which can degrade the plntatc.
  • the objective of the present invention to overcome the current limits by generating a phytase that is sufficiently active to significantly affect the need for supplementation. phosphates, and enough power for £ thermnstahlc be added directly to food animals without the use of sprayage technique.
  • Phyuises isolated from fungi are numerous and come mainly from the ⁇ . ⁇ ergi / lus family but also from Absiili ⁇ , Ivn ⁇ hi ⁇ lophor ⁇ .
  • Some phytase from other fungi have shown interesting properties including highierii ⁇ qnes activities such as phyta.se of Tr ⁇ mcws piihesct-ns or Ia phytuse of Pcniophor ⁇ lycii (respectively 1200 l. Mg-1 and 1-Kiso U.mg in WO W28408).
  • Itttfosporiuiti ' shows an interesting phytase with a specific activity of 1 MO l '.my-l and a Km Je 1 5.2 ⁇ M but an optimal temperature busse at 40' - "C. Kgaleme ⁇ l the 6-ph ⁇ tase of Ccripnri ⁇ ⁇ .
  • the phytase of the application WO2007I 28 I60 has a high specific activity greater than 3000 I / mg. of the same order as the specific activity recorded for Yersinia pestis in WO2002048332.
  • WO2007128160 the intrinsic biochemical characteristics of the protein are claimed namely a molecular weight of 45.5 kl) a. an optimal pH of 4.0-5.0. an optimum temperature of 50-6 (PC. a theoretical pi of 7.7. a specific activ ity superior to 3000 l. " 'mg and high resistance to pepsin and your trypsin.
  • the present invention makes it possible to exceed current limits by proposing an improved variant by molecular evolution of the Yersinki intenncilia phtase.
  • improved variant means a variant having a greater degree of thermophilicity and / or activity than the phyiasis of origin of Yersinia irrigatoria. allowing it to be used in industrial processes and in particular as a food additive.
  • the present application discloses an improved variant of a phylase Ia whose sequence is SKQ ID NO: 1 or a functional derivative thereof, wherein it comprises at least one substitution on one of the amino acids of the group consisting of 1 * 3, V4. ⁇ 5. P8. T ' ) . GI O. VIo. V 17. LI 9.
  • the improved variant of the phytase whose sequence is SIiQ ID N 0 I or a functional derivative thereof comprises at least one substitution selected from the group consisting of P3L, P3V. V4G, ⁇ 5I ⁇ P8N. P8V. HI. '1 "9Q, T9S. P9Y. CilO ⁇ . GMLP. V16M Vl. 7W.
  • PI55N PI55T. L156N. F158N. IM58N • S 1601 “, Flh7N, ⁇ 177N, ⁇ 177S, AI 771 " .
  • the improved variant of phytaso whose sequence is SHQ II ) N r 1 or a functional derivative thereof.
  • ci comprises at least one combination of substitutions selected from the substitutions of the preceding group.
  • SHQ ID N -1 1 is the sequence appearing in the NC Bl database filed on October 20, 2005, only the entry number / P U0832361. however, not including the 23 amino acid signal sequence at the 5 'end of the protein.
  • SIiQ ID No. I corresponds to residues 24-4-41 in the previously cited entry number.
  • SKQ ID No. 11 also contains the nucleic sequence coding for the preceding pioleic sequence, referenced under the number NZ ⁇ I. FO 1000052 in the NCBI database.
  • a nucleic acid encoding a variant according to the present invention can easily be prepared on the basis of this sequence by techniques well known to those skilled in the art, for example by "directed mutagenesis of the eodon to be modified, to obtain the substitution of
  • the sequence of the improved variant of the phytasc according to the present invention corresponds to S1, JD N "1 including the selected substitution (s).
  • the improved variant of the present invention comprises a single substitution.
  • the improved variant of the present invention or a functional derivative thereof comprises at least one substitution on one of the amino acids of the group consisting of K20. Q30. Y51. 1 52. ( 1775) 1. VTv Q. 5. IN8 .9 ( K 1-129. H 130. D 140, T142. P155. F 167, ⁇ 177.0189. O ) I. K2I0, L219.1250. S251.1.252, L255, M263, Y2G8.0274, Q292, G293. P297, G3O8. N3I6, Q326. D349. cl 1-391. the positions being indicated in SHQ ID N 0 I.
  • the improved variant Ia present invention or a functional derivative thereof comprises substitutions on a back amino acids of the group 'consisting of K29. Q30. Y51.1,52. 075. C81, V93, Q95. R98, L99, F 129.11130. D 140. Tl 42. Pl 55, F167, ⁇ 177.0189, K201. K210. L2I9.1250. S251. L252. 1.255, M263. Y268.0274. Q292.0293.1 * 2 ⁇ 7. G308, N3I6. Q326, D349, and F.39I. the positions being indicated in SKQ ID N 0 I.
  • the improved variant of the present invention or a functional derivative thereof includes substitutions on one of the amino acids of the group consisting of K29. QM). Y51, L52. O75. V93. R08, 1.99. ⁇ > 9.11130.
  • substitutions on the foregoing amino acids are selected from the group consisting of K29N. Q30D. Y51O. Y51Q. Y51W.1.520 (" 75R. V93 (i.
  • the improved variant of the present invention or a functional derivative thereof comprises a combination of substitutions selected from the group consisting of G274C ⁇ - N3I6C-. F142N ' - ⁇ 177T * Q326T.
  • K210S ⁇ Y268I- r ( ⁇ D 292P 1401- ⁇ - Y2 (.> 8H - Q292P FI67N * Y2 ⁇ 8l- t ⁇ > 292P I I42N.. + ⁇ 177T + K210S + Q326T.
  • the improved variant of the present invention or a functional derivative thereof comprises a combination of substitutions consisting of T142N * - ⁇ 177T + K210S + Y268L ⁇ Q292P +
  • the improved variant of the present invention or a functional derivative thereof comprises a combination of I42N * ⁇ substitutions. 177T ⁇ K2i 0S + G274C Y2681- f * t Q292P N316C -.. Q326T positions being indicated in SKQ ID NO: 1 in another preferred embodiment of this particular embodiment I variant improved in present invention or a functional derivative it comprises a combination of substitutions consisting of TI42N + ⁇ I 77T-K2 I0S • Q32o T. the positions being indicated in SIiQ ID N 0 1.
  • the improved variant of the present invention or a functional derivative thereof comprises a combination of substitutions consisting of (f J274C N3 Î 6C, the positions being indicated in IF-Q ID NO: I.
  • the improved variant of the present invention or a functional derivative thereof comprises a combination of substitutions consisting of 1 142N- ⁇ I 77T f Q326T, the positions being indicated in SEQ ID NO: i.
  • the improved variant of the present invention or a functional derivative thereof comprises a combination of substitutions consisting of K.210S + Y268M f -Q2 ⁇ ⁇ 2P. the positions being indicated in SI-; Q ID NO 1 î.
  • the improved variant of the present invention or a functional derivative thereof comprises a combination of substitutions consisting of D 140 F + Y 268 I-! + Q2O2P. In another preferred embodiment of this particular embodiment, the improved variant of the present invention or a functional derivative thereof comprises a combination of substitutions consisting of F167N-YIhHIl 'Q292P.
  • the present invention relates to an improved variant of a phyta.se whose sequence is SIiQ ID N 0 I or a functional derivative thereof comprising the at least one substituted substituent.
  • the present invention also relates to a nucleic acid encoding an improved variant of the phytase according to the present invention or a functional derivative thereof, an expression cassette comprising a nucleic acid according to the present invention, and a vector comprising a nucleic acid or an expression cassette according to the present invention.
  • the vector may be preferably selected from a plasmid. a phage. a phagemid and a viral vector.
  • the present invention also relates to a composition comprising at least one improved variant of the phytase whose sequence is SMQ ID N ⁇ 1 or a functional derivative thereof with the one or more substitutions selected according to the present invention. It also concerns any solid mixture. liquid or gas comprising a certain percentage of at least one improved variant of the phytase according to the present invention. It also relates to mixtures preferably containing one, two, three, four, five or ten improved variants of the phylase according to the present invention or functional derivatives thereof.
  • the present invention also relates to compositions or preparations of phytase containing a certain percentage of at least one v Ariant improved ph) tasc according to the present invention or a functional derivative thereof and one or more other enzymes having properties av antagates.
  • Lu present invention relates to the use of an improved variant of the phytase of the present invention or a functional derivative thereof, for the preparation of a food additive.
  • IA presents invention relates to the use of a nucleic acid, an expression cassette or a vector encoding and / or containing at least one variant of the improved phvtase whose sequence is Sl] Q II) the NP or a functional derivative thereof with the one or more substitutions selected according to the present invention for transforming or trafecting a host cell ; It further relates to a host cell comprising a nucleic acid, an expression cassette or a vector encoding an improved variant of the phylase according to present invention or a functional drift thereof.
  • the present invention also relates to the use of such a nucleic acid, such an expression cassette, such a vector or host cell to produce an improved variant of the phytase according to the present invention.
  • lillc also relates to a method for producing an improved variant of the phytase of the present invention comprising transforming or transiccti ⁇ n a host cell with a nucleic acid, expression cassette or a vector according to the present invention, culturing the transformed or truncated transfected cell and harvesting the improved variant of the phytase or a functional derivative thereof, produced by the host cell, the host cell may be procaryolc or cucaryote.
  • the hole cell may be a microorganism, preferably a bacterium, a yeast or a fungus.
  • the host cell may also be a mammalian cell such as a COS7 or CUO cell.
  • “functional derivative” is meant any compound derived from the phvlusc variant of the present invention comprising structural modifications while preserving phytase activity. These modifications can be, for example, an extension of the enzyme by addition of new domains, partial or complete substitutions of domains such as replacements of "siretches” of amino acids by amino acids of other enzymes that can confer other functions / properties.
  • “functional derivative” is also inclusive diinérisée form of the variant of the enzyme of the present invention, homo- or hommerodime tablette, or polymeric. having improved properties, such as thcnnostubilitû for example, by the multiplication of domains.
  • “functional derivative” is also understood a chimeric form of the phytase derivative of the present invention, fused with a protein / molecule of interest or with one or more domains of that enzyme of interest.
  • “functional derivative” is also meant a functional fragment of the phytase variant of the present invention preserving the activity of the phytase. This activity can be measured according to "one of the protocols described in Examples 4 and 5. The fragment may comprise 250. 275. 300, 325. 350. 375. 380. 385, WO. 395. 400. 405. 410 or 415 consecutive amino acids of the phytase according to the present invention This functional moiety may also be dimerized or polyniérisé and;..
  • variant or “mutant” is meant a nucleotide sequence exhibiting mutations with respect to a reference nucleotide sequence. These mutations can be silent due to the degeneracy of the genetic code: in this case the protein encoded by the variant is identical to the protein encoded by the reference nucleotide sequence. These mutations may also result in amino acid substitutions in read by Ic encoded protein variant as compared to the protein encoded by the nucleotide sequence of reference.
  • Variant includes sequences containing mutations obtained by directed muiagenesis. The term “variant” is assigned to the sequences nuciéotidique.s well as protein sequences encoded by iesdites nuclcotidiquus sequences. presenting said mutations.
  • the improved variant of the phyiase according to the present invention or a functional derivative thereof may comprise other substitutions on one of the amino acids of the group consisting of P3. V4. ⁇ 5. PX.19. CiK), V16. V17.1.19. S20. R21, 1122, C23. V24. R25. S26. I J 27. T28. K29, QV ) . T31. Q32, 133. M34.1) 36. P39. K41. W45. ⁇ 49. G50, Y5I.
  • substitutions may be so-called “conservative” substitutions, that is, substitutions within a group of amino acids with similar or equivalent characteristics, such as amino acids having a small footprint of acidic, basic, polar, hydrophobic and aromatic amino acids according to the table below:
  • the improved variant of the present invention or a derivative works! of it may include substitutions equivalent to the P276I substitution. described in the preceding group, such as 1 * 2761 substitutions. P276M or P276V according to the classification of the previous table. The above interpretation also applies to combinations of substitutions.
  • the improved variant of the plasmid according to the present invention or a functional derivative thereof may comprise other mutations not described in this urology. preferably substitutions, including some known in the art.
  • II * improved variant of ptiytase or a functional derivative thereof comprises at most 40. 35, 30, 25. 20. 15. 14. 13. 12. 1 1. IU. 9. 8, 7. 6. 5. 4.
  • the J ar variant improved on emend a variant having improved properties and particularly a et ⁇ the ⁇ nostrise> u a specific activity and / or increased expression compared to the parent phytasc
  • the variant improved by the present invention may have greater resistance to proteolysis. by proteases or other.
  • the increase in one or more properties of the improved variant of the phytase according to the present invention is at least 5%. preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or a factor of 2.5, K) or 100 relative to the properties of the parent phytase, measured under the same conditions experimental.
  • thermostabiiInstitut phytase can be measured by following the procedures in Example 5.
  • the specific activity of phytase read can be measured by following the procedures in Example 4.
  • I the expression of phytase can be measured following the procedures detailed in Examples 2 CL 3.
  • Such visualizations also allow, in a predictive approach, to target certain residues for mutage ⁇ ippoe experiments.
  • the targeted residues may be those which allow stiffening of the secondary structure. I ; such rigidificalion can be obtained in different ways; for example, the targeted residues may be substituted with a proline residue which.
  • the expression vector may be any type of recombinant vector (in particular, plasmid, virus, etc.) that makes it possible to express the nucleotide sequence of the improved variant of the present invention.
  • the choice of expression vector depends on its compatibility with the H ⁇ ie targeted expression in which it is processed or traasleclé.
  • This vector can be linear or circular closed. He can * e replicate autonomously, that is to say be a cxtruchrom ⁇ s ⁇ miquc entity! Has involvement is independent of the chromosome of the host that contains a plasmidc an extrachromosomal clement. a minichromosome or a .irtilkiel chromosome.
  • the vector can, when introduced into the host cell, s integrate into the host genome to replicate the same time as him. Also, several vectors may be required for the expression of the improved variant of the present invention and may be used simultaneously, as well as a transposon.
  • the vectors for expression of the improved variant of the present invention may contain one or more markers that allow easy selection of transformed or transfected host cells.
  • These selection markers are typically genes whose product confers an advantage on the host and allows, for example, bacterial resistance to an antibiotic, prototrophy for auxotrophs. resistance to heavy metals etc.
  • Examples of bacterial selection markers are genes that confer resistance to antibiotics IcIs as ampicillin, ka ⁇ amycin. Tetracyline and chloramphenol especially.
  • Markers suitable for selection in yeasts are, for example, the ⁇ DF ⁇ 2 genes. HIS3. I .I-U2. LYS 2. MIi Yi, TRJM and 1 : R ⁇ 3 in particular. Examples of markers used in the filamentous fungi were: AmdS (uelamidase).
  • argH tornithino earbamo ⁇ ltransferase bar phosphinolhricinicketyltransferase). liph (hygromycin phosphotransferase). niai ) (nitrate reductant), pyr (orotidine 5 '-phos ⁇ hate deearhoxyla.se), sC (adcnyltransicru.su sulfate) and trpC (anthranilate synthase). especially.
  • the vectors for expression of the improved variant of the present invention may not contain selection markers.
  • the vector, dnns Ie case of an autonomous réplieation must contain an origin of r ⁇ plication suitable host cell Ia.
  • bacterial origin of replication are, in particular, pBR322 plasmids. pl ,! C19, p ⁇ CYC 177 and p ⁇ CYC I 84 allowing replication in Esdiehchia coll. and pL ' BHO, pi-! l c > 4, pT ⁇ I 060 and p. ⁇ MjbetaJ allowing replication in Bacillus.
  • Examples of replication origin in yeasts are, without this list being exhaustive, the origins of 2 micron replieation. ⁇ RSJ. ⁇ RS4.
  • I vector e in the case of integration into Ic genome of the host cell should allow its integration with the coding sequence of the variant of the present invention improves or n 'any other suitable sequence into the vector. in a homologous or non-homologous recinbinization. It may also contain additional nucleic acid sequences to direct its integration into the genome of the host cell. To maximize the chances of integration into the host genome, the sequences of integration must be of sufficient length, such as 100 to 10,000 base pairs, 400 independentlyentielleme ⁇ l ⁇ 10,000, even more preferably 800-10000 base pairs.
  • the integration sequences may be coding or non-coding.
  • this sequence is also a parameter well known to those skilled in the art when an expression of the gene in question is desired in another organism or in another cellular compartment via a plasmid vector or AWRE chosen in line with the body of expression desired. So, this sequence can wax replaced by the signal sequence of other genes such as that of PeIIi, Pho ⁇ , OmpA or ⁇ -luctamasc in particular, for its expression in a prokaryotic host.
  • I .ors of expression in yeast such as Ichia pavions signal sequences present at P 5 'end of the phytase gene of Yersiniti inwnnedia may be the PI signal sequences K) ⁇ làctor I below respectively of a gene phnsphatase and ⁇ faet ⁇ r of this organism.
  • Sacchar ⁇ myccs ecrevisiae the same signal sequence can be used.
  • the signal sequence present at the 5 'end of the Yersinia hueniwdia phytase gene may be the XPR2 signal sequence of the same XPR2 gene. a protease of this organism.
  • the cell may be prokaryotic or cucalandotu: it may be a gram positive bacterium such as liacillus alkulmphil. liacillus ⁇ nyloliquefaciens, liaciilu.v hrevis. liacillus circulam. Bacillus clauxii. liacilhts coagulate. BtK he read liwtux. Iacilus Icntu *. liacillus tichvnifhrmis, Bacillus me ⁇ uie ⁇ um. liactllus swuroiherrm ⁇ hilus.
  • Ht here Uns .whtilis. liacillus tlmringk'tisis, Sireptomyccs lividam or Slrepiamyces murmusque. in particular, or a gram negative bacterium such as ' b ' scherichia cols or Psciuhmionas sp. for example, without this list being exhaustive.
  • the present invention also relates to a method of producing a soluble and active phytase or variant thereof in a bacterium, preferably Escherichia coli. comprising expressing a nucleic acid encoding the phytase or Ie variant thereof in a bacterium, preferably do Hscherichia coli. optionally recovering the phytase thus expressed.
  • the host cell may be a yeast of the genus ( 'anJiih. Llamen ⁇ la. Kkiyveromyccs. The Ichia, Siiccharomyces. SchKosacchar ⁇ mycws. Yarr ⁇ wia or, in particular. LEMENT the host cell can be Sucdiarotmrc.s Preferred carlshcrgemis. Saccharomyccs cvn'vi. ⁇ Iae.
  • the host cell may be a filamentous fungus of the genus Acrcmanium. Aspergillus. Aui T ohasidium. Hji-rkanderu. This is the case, the oprimts. ( 'Nriolus. The ryptococcus, the ilihasidium. ⁇ -u.suri ⁇ m. IlitmicoUt. Magnaporlhi. Yiiicor. ⁇ Lycclinphllmra., ⁇ C ⁇ c ⁇ illi ⁇ mtslix. ⁇ Ewnspora, Ptmciloinycvs. Pvmcillitun. P / uincmcluieie. Phlehiu.
  • Pir ⁇ myccs The Icin niiis. Schiiuphytlum. Talon mnves. Thcnuoascus. the hiulavia. the olyp ⁇ cladium. ⁇ mmctes. or Trichoi / erma.
  • the host cell may be activated by awamori. Aspergillus fumigatus. Aspergillus fungi ⁇ A ⁇ pergillus japonicus. Aspergillus nidukms. Aspergillus et / vr. Aspergillus oryzae, Culdariomyces jumago. Fusarium bactridiaiiies.
  • Fusarium cerealis Fusarhim crniikwi'ilense. Fusai htm culmorum. Fusarium ⁇ rc ⁇ minearum. Fusuhum hetymuspulum, Fusarium negwuti. Fusarium oxyspnrum. Fusarium reliadatum. Fusarium rosvttm. Fusarium sumhuumum, Fusarium sarcochrumum, Fusarium sparotrichioides. Fusarium sulphureum. Fusarium torulosum. Fusarium irichoihecioides.
  • the host cell may be a mammalian cell such as COS7 or CIK) (US 4.8iW.803: 1JS 5.047..V> 5).
  • Pure nucleic acid is understood to mean DNA (cDNA or gDNA), RNA or a mixture of both.
  • the nucleic acid may comprise modified nucleotides, including, for example, a modified linkage, a purified or modified pyrimidine bose, or a modified sugar. It can be prepared by any method known to those skilled in the art, including chemical synthesis, recombination, mulching. eic ...
  • An improved variant variant nucleic acid The phyiase according to the present invention can be optimized in terms of the codons constituting it to maximize its expression in a particular host different from its original organism. Since the universal genetic code is degenerate, there are several codons (codon-triplet of nucleotides) which encode a given amino acid. These homonymous codons are not used at random because the corresponding ⁇ UN ' t (transfer ⁇ RN) do not exist in all cells at the same concentrations. So that certain codons are less likely to be expressed in tissues where the corresponding ⁇ RNi is rare.
  • nucleic acids encoding the improved variant of the phytase of the present invention may require optimization to promote expression in the selected production host.
  • percent identity or identity between two nucleic acid sequences or amino acids within the meaning of In present invention, is intended to denote a percentage of nuciéotides or amino acid residues identical between two sequences to be compared ies, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length, the best alignment or alignment is the alignment for which the percentage identity between the two sequences compare, as calculated below, is the highest.
  • Sequence comparisons between two nucleic acid sequences or amino ac ⁇ dcs are traditionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out by segment or by conipuraîMon window to identify and compare local regions of sequence similarity.
  • the optimal alignment of the sequences for the comparison can be realized, besides manually, with the nxne ⁇ of the algorithm the homologue.
  • Smith and Waterman's locality > 81) ( ⁇ dpp Maih 2: 482). using the local homology algorithm of Neddleman and Wunsch (l ⁇ > 70) (J. Mol, Hiol 48: 443). using the similarity search method of Pearso ⁇ and Lipman (1 ⁇ > 88) (Rock Natl. adcad. Sci. USA 85: 2444). by means of computer software using these algorithms ( ⁇ i> P.HhSTI-TI.I- ' AST ⁇ and 1! " AS IA in the Wisconsin Cicnutics Software Package, Genets of the Computer Group, 575 Science Dr ..
  • percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences optimally aligned for comparison window in which the region of the nucleic acid or amino acid sequence to be compared can comprise additions or deletions relative to the reference sequence for optimal alignment between these two sequences.
  • I e percentage identity is calculated by determining Ic number of identical positions for which the nueléoude or the amino acid residue is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window and multiplying the result obtained by I OD in. to get the percentage of identity between these two sequences.
  • the enhancement conferred by the selected substitution (s) in the improved variant of the present invention can be accomplished by performing an equivalent substitution in a phytase of another organism than that from which the phytase of the present invention originates. understood that these equivalent substitutions are within the scope of the present invention.
  • the improved variant of the phytase according to the present invention can be used in the aforementioned reactions and methods in a purified or partially purified form.
  • the purification of the variant of the phytase according to the present invention may be basic and carried out in particular by lysis and llltration of the content of the flasks or production containers and / or by centrifugation steps. and / or by selective latter successive precipitations ammonium sulfate and / or evaporation. These basic procedures make it possible to obtain fractions of the improved variant of the phytase according to the invention exhibiting a strong increase in specific activity.
  • the purified or partially purified fraction of the improved variant of the phytase according to the present invention can be used in the aforementioned reactions and methods in immobilized or non immobilized form.
  • Methods for immobilizing the improved variant of the present invention on organic or inorganic carriers are well known to those skilled in the art. These last may be especially polyacrylamides, agaroscs. celluloses, sephadex or dextrans. porous glass beads *, hydroxides of aluminum or tilanium.
  • composition is generally meant a composition comprising at least one improved variant of the phytase whose sequence is SLiQ ID N 0 I with or selected substitutions according to the present invention.
  • Kilo also relates to any solid, liquid or titer mixture comprising a certain percentage of at least one improved variant of the phytase according to the present invention.
  • LIIe also relates to mixtures eonte ⁇ .uu a. two, three, four, five or ten improved variants of the phytase according to the present invention.
  • the compositions containing phytases are liquid or so-called dry compositions.
  • the liquid compositions need not contain anything other than the highly purified phytase. However stabilizers such as glycerol. Sulfitol or monopropylene glycol can be added. Said composition may also contain other additives such as salts, sugars, preservatives, buffering agents opposite the fold, protein and phytate. Typical liquid compositions are aqueous compositions or oil-based suspensions. The liquid compositions may be added to the feed before or after an optional granulation step.
  • the so-called dry compositions can be dry compositions by freezing, spraying or extraded dry compositions: in these cases said composition does not need to contain anything other than the enzyme in its dry form.
  • the dry compositions can also be granules that may be mixed or ready to be with a food component, or form a component of a premix.
  • the size of the enzyme granules is preferably ,, compatible with the other components of chronic intestinal disease. This is a safe and convenient way to incorporate enzyme (s) into foods.
  • a stable formulation of enzyme may be prepared by spraying a liquid mixture of phytase on ⁇ m component Here soybean meal and then drying the assembly.
  • the decrease in the percentage of moisture and binding interactions of the phytase ⁇ DC component protect the enzyme external environmental factors such as extreme temperatures used during manufacture of the food component.
  • presentation as a dry preparation of Ia phyiase may increase stability by decreasing the activity of potential proteolytic enzymes may be present ii trace at the end of the liquid fermentation stages during the production process.
  • the dry phytase preparation can, for example, be used as a dietary supplement in the poultry and hog production industry.
  • granules are pre-prepared using agglomeration techniques well known to those skilled in the art, in a mixer inside which a filling material and the enzyme are co- agglomcrés to form granules, f es granules are prepared from the templates on which the enzyme can come s absorb or on which an enzyme layer can be applied.
  • Typical materials that can serve as a matrix are soils such as disodium sulfate.
  • Other potential matrices may be, or based on, clay, magnesium silicate, aluminum silicate or cellulose fibers.
  • binding agents such as dextrins may be included in the granules.
  • Trainers may be included in any of the following components: starch, cassava, potato, rice. corn. corn ... Salts can also be added.
  • the granules may be covered by specifically dedicated blends, and in particular hydrophilic mixtures, based on palm oil, beef tallow and, if necessary, other additives such as calcium carbonate and clay.
  • the phylaxis preparation may contain other agents such as coloring agents, aromatic compounds, stabilizers, vitamins, minerals as well as other enzymes or enzyme mixtures having advantageous properties. This is especially true for premixes.
  • a food additive is meant a substantially pure component or a composition containing several components for wax added to a food.
  • this additive is intended to become a full-fledged component of the food and ot aims to affect, modify, increase one / the properties of said food.
  • a phylase preparation used as a food additive means a pbytase which is not a natural component of the diet in which it is added, or which is not present in this food at its natural concentration, or which is added separately. mature components of the food, alone or in combination with other food additives. T> piqtiemont.
  • a food additive contains several components such as vitamins, minerals, entrainers, excipients, other enzymes or enzyme mixtures with advantageous properties.
  • a food additive is understood that is not produced in situ in food or animal feed.
  • a phytase or preparation can be given directly as food to humans or animals and preferably directly after mixing with other constituents of said food.
  • a food additive according to this aspect of the present invention is combined with other 23 compounds to produce a food. It is included in these other compounds one or more other enzymes, preferably thcrmosiables. vitamins food additives, mineral food additives, amino acids as food additives. Ie result of this mixture or this combination of compounds can be mixed in an appropriate proportion with other compounds such as protein supplements or cereal to form the final food.
  • phytase preparation or phyiase composition of the present invention are meant preparations or compositions which contain a significant amount of at least one improved variant of the phytase according to the present invention and one or more other enzymes having advantageous properties. for the preparation of food.
  • Such enzymes can belong to the following list without this being exhaustive: alpl ⁇ ugaiaclosid ⁇ ses. beta-galactosidases, in particular lactases, other phytases.
  • beta-lluea ⁇ ases in particular endo-beta-1,4-gli ⁇ canuscs and end ⁇ -bcta-).
  • -glucanascs celluloses, xyiosiduses, yalactanases. in the context of arabiuliogulactan-eiidu- 1.4-hetgalalaetosidus and arabino-ulactan-endo-1.3-beta-galutfosida.se:. cndoghicunases. especially e ⁇ do-! 2-betu glucanase. cnd ⁇ -l .3-alpha- ⁇ lucanasc.
  • pectin degrading enzymes especially peetinases. peciincsterases. pectin I vases. polygalacturonase. u ⁇ thinanu ⁇ es. rhuninogalacfuronases. ihamno ⁇ alacîiironan-sceiyl-esteiases. rhamnogalacluronan-alpha-rhamnosidase. peelate lyases. alpha-galaeturoni.sida.ses. mannanasos. beta- i ⁇ annosidases. mannan-acetyl-esierascs. xyian-acetyl-esteiases. proteases. xylanases, arabinoxylanases and lipolytic enzymes such as lipases, phospholipases and eutinases.
  • the addition of the feed additive to the animals according to the present invention can be done before or simultaneously with the meal. Preferably, the supplementation is done at the same time as the meal.
  • 1.1 no effective amount of phyta.se can be added in foods is from about 10 to 20000 I'U- 'kji food; preferably between 10 and 15000 PPU.'kg; more preferably between 10 and 10,000 PI 1 I '/ kg. In particular from 100 to 50 IW ) IMM. ks ". particularly from 100 to 2000 PPl ; ⁇ ky of uliment. 24
  • a variant improves the phytase according to the present invention in the manufacture of foods intended for human consumption or for animal feed.
  • Grains or meals for human consumption may be treated with phytase to reduce their phytate content.
  • a here phyiasis treatment can increase the efficiency of the production of this food.
  • the addition of phytase to white soy mocons during the soy protein extraction process can increase the yield and quality of the extracted proteins.
  • Phytase is active during food production but not in the final product.
  • soybean or col / a grains may be pretreated with phytase prior to manufacture and / or final packaging. Such pretreatment makes it possible to degrade ani-nutritional elements such as phytate and to increase the quality and the nutritional value of the foodstuffs in question. In that case. phytase may or may not always be active in the digestive tract of animals after they have been ingested.
  • L 'sl also included in the scope of the invention, the use of an improved variant of the phytase of the present invention as facilitaleur agent for food processing.
  • the phytase according to the present invention can be used as a supplement in the human diet to facilitate digestion.
  • a cachet (s) containing an adequate amount of phytase may be ingested by a person before consuming food in order to provide an active enzyme to his digestive tract.
  • the benefit of this phthalase ingestion is particularly remarkable in the case of consuming a food which can not be treated with phytase during its manufacture.
  • the phytase according to the present invention can be advantageously used with mono- or polygastric animal islands. especially young calves. Diets for fish and crustaceans can also be treated with phytase to improve conversion efficiencies between Iburnie Ia food and animal growth and 25 reduce the amount of phosphate excreted in intensive production systems.
  • Food treated according to the present invention may be supplied to poultry (turkeys, ducks, owls, partridges, chickens, broilers) to pigs, horses, cattle, goats, canines and retines.
  • KIIe is particularly suitable for poultry and pigs, including but not limited to hens, broilers, turkeys, ducks and geese.
  • I. phyta.se according to the present invention is used to produce novel combinations of food ingredients or foods with advantageous qualities. For example, it can be used to produce foods with a lowered inorganic phosphate content. This content is adjusted depending on the amount and activity of the added phytase, present in the final food or active in a food ingredient in the composition of the final food. In a preferred manner, such a food may contain ingredients which are microbiolitic. vitamins, amino acids and optimized and effective amounts of phytase and inorganic phosphate canvases that the amount of phytase is between 50 and 200 (JO units of phytase per kilogram of food and the amount of inorganic phosphate is less than 0.45%.
  • these two amounts are between 100 and 1000 phytase units per kilogram of feed and less than 0.225% inorganic phosphate; more preferably between 150 and 10,000 units of phytase per kilogram of feed and less than 5% of inorganic phosphate; even more preferably between 250 and 20000 phytase units per kilo of feed and no added inorganic phosphate.
  • These new combinations have various interests such as reducing the release of phosphate into the environment and optimizing the conversion efficiencies between the food supplied and the growth of the animals, which is particularly sought after in intensive livestock production.
  • Table 2 List of mutants having an additional glycosylation site classified according to their percentages of accessibility to the respective solvents.
  • fablcau 3 couples list of mutations permitting the addition of disulfuros additional bridges
  • Table 4 ⁇ extinction coefficients of activity for 80-20 K.2 I0S mutants. Y268: and Q292P.
  • Table 4C Coef cients of extinguishing activity for 80-20 T142-N mutants.
  • Block 3B I wastes substitutions targeting an improvement in activity characterized in a first series of experiments.
  • Figure 1 Degradation of phytate by a phytase
  • Mgurc ⁇ Measurement of ACLI ⁇ ities residual mutants Pl-I Y 1 Y JM 98-4X and 98-6X-products by Succhar ⁇ myccs cerevisiac after preheating from 0 to 2 minutes 80''C *.
  • Figure 4 Measurement of residual activity nants Phy- 1 MMX and J J HY- ⁇ > 8-6X produced by Sitccfuimtnive.s cerevisiue nréchaut ⁇ age after 15 minutes at temperatures of 45 ⁇ ca ⁇ C to 65 "C.
  • I- 'igure 5 Measurement of residual activities of mutants IM IY-98-4X IM and IY-98- ⁇ X produced by Sticcharnmy ⁇ L's vi'revistac crosschaul ⁇ age after 1 minute at temperatures from 60 0 C to 80''C.
  • Figure f> A Measurement of the residual activities of mutants PHY-W-6X and lM lY-W-6X-ssl I produced by Pidiia panions after a precaution tic 0 to 30 minuies at 80 1 C.
  • Figure 8 SDS-PACiI sky-12% of pn> deciding supernatants for different mutants expressed by Pichia pastoris. digested or not by Tendoglycosidase Hf.
  • the production of the improved variants of the phytase according to the present invention required the construction of plasmid vectors which make it possible to carry out the directed-oriented experiments required to obtain variant or mutant libraries as well as the expression of these mutants in different hosts. screening or production.
  • ORF Open Reading the train / P 00,832,561 corresponding to the sequence NCBI ⁇ TCC 2990Q cl Ia corresponding sequence micléotidique NZ ⁇ LI 01000052 region: 188 '
  • ORF Open Reading the train / P 00,832,561 corresponding to the sequence NCBI ⁇ TCC 2990Q cl Ia corresponding sequence micléotidique NZ ⁇ LI 01000052 region: 188 '
  • ORF ZP! 083 236 the cloned by molecular biology techniques well known to the skilled gifts pNCK the vector.
  • This vector makes it possible to express a fusion protein between a protein of interest and a thermostable resistance gene with kanamyein in a thermophilic Thriminus thurmapuHus miemorganism according to the method described in the patent application WO2006134240 and corresponding to the proprietary technique of Hiirmélhodes II. IR 'JM .
  • LORr from ZP 00832361 was cloned according to molecular biology techniques well known to those skilled in the art pYHS2. This ector, containing the peptide Pheniasis phyiasis was isolated at the 5 'position of IORF ZP 00832361, allowing expression of Yeninia phytase intcrmviliu by Saccharomyces cerevixiae.
  • ORF ZP 0083236 was clo ⁇ ée according to molecular biology techniques well known in humans of the melting in the vector pPIC9.
  • This vector containing the signal peptide of Pichia positron P ⁇ laetor (Tnvitr ⁇ gen) 5 'position of 1 "ORF ZP 00832361, allowed the expression of the phytase of Ycrsmia inwrmetiia by Pichia postons. Construction of Mutant Banks in p.NCK. screening and obtaining an improvement in:
  • mutant libraries of your phytase ⁇ erùnhi intermvdia were created by technology owner Miomélhodes Massive Mutagene.MS'R 'described in t' S 7,202,086 cm in Saboulard ei al (Bioteehniques 2005 September 39 (3): 363 -8). Briefly, mulagèn ⁇ s olmonueléotides were synthesized to achieve the phytabc nants on each of the amino acids constituting the enzyme.
  • the mutant libraries were constructed from a pNCK vector containing the phytase gene. according to the Masske Mutagenesis-K 1 protocol described in 7 : 222,086 or in Salmonella (2005). (Sep.
  • the mutant banks are transformed into cultures of Thcrmus ihenm ⁇ hilus made competent. Transformants were grown in liquid medium at 70 ° C to achieve precultures which are then plated on solid medium containing kanamycin stringent concentrations of the order of 25 mcg / ml. After incubation for 48 hours at 70 ° C., only those mutants whose protein structure is resistant to the selection temperature and whose folding is correct allow a functional folding of the resistance gene thcrm ⁇ stahlc to the kananiveinc and therefore can push in the presence of the kanamyc. In this study, isolated key mutants were isolated, their ⁇ DN plasminidic isolated and sequenced, and the different mutants revealed a total of 13S.
  • K.210 therefore seems to be an important residue, in particular because of its positive charge and its large volume, which can generate steric and electrostatic stresses with respect to the entry of the substrate or the exit of the products from the active site.
  • the door of Celtic positive charge in the K.210S substitution and the change in the spatial constraint associated generally may therefore disturb the reaction (entry ⁇ r output products solvataiion substrate of the cavity of the active site) and alter the kinetic constants of the and more particularly the apparent Km for the different substrates generated during the reaction. It is known in some cases. that the produced substrates can participate in the stabilization of the structure 31) of the protein and impart increased stability.
  • Y268 is a potentially solvent accessible residue located in a "loop" secondary strand.
  • I. are signals I N-glyeosy lalions in a protein sequence are Nx 1 "or N ⁇ S Such sites were introduced into the phytasc of Yersinn 't i.'iurmi'iiia cloned djiw the ⁇ ecieur Pyi. S2.
  • Example 5 details the characterization protocols of the residual activity of mutants as a function of temperature.
  • ⁇ 'Is first set of experiments has identified several mutants with .supplpiraire site glycosylalion having a th ⁇ rmoslahilitc increased relative to the wild-type enzyme r. These mutants contain substitutions of amino acids ⁇ 142. ⁇ 177 and Q326 characterized by a solvent accessibility percentage> 70%. More particularly, the substitutions on these amino acids are TI 42N. ⁇ 177F and Q326T. These mutants have an activity extinction coefficient between 80% and 20% of residual activity respectively of 1.62. 1, 62 and 1.52 as presented in the table -IC.
  • the preceding residues have been introduced into the plasmid enzyme inserted in the vector ⁇ YLS2, or by the proprietary technique of Ithomethodes Massive Mutaye ⁇ esistt. as mentioned above or by a technique of mutayenèse well known to those skilled in the art, such that a PCIR overlay.
  • the mutant constructs have been transformed in Succession and Criteria and characterized in more detail in their activity and thermosilability.
  • Example 5 details the protocols for characterization of the residual activity of mutants as a function of temperature.
  • a first series of experiments has identified a mutant with an additional disulfide bridge and improved thermostability.
  • Cc mutant contains two .substitutions on IE- * residues (J274 and N3 l o.
  • This mutant has a coefficient of extinction of activity between 80% and 20% 32 of residual activity of 2.33 as shown in Table 4D Ie.
  • This index is calculated by Io ratio between temperatures dilTércnccs allowing on one hand the maintenance of 80% residual activity and the other 20% of the residual activity for the variant SS I 1 compared to the enzyme wild. The detail of the calculation of this index is shown for the mutant K21 OS in Table 4 B.
  • Example 5 details the protocols for characterizing the residual activity of mutants as a function of temperature.
  • PHY-98-6X and PHY-98-6X-SS 1 1 respectively containing the combinations of imitations T142N * ⁇ I 77T-K2 I0S t O3261 " , ⁇ 42N * - 77 I 77T f-K210S * Y2 ⁇ 8F.
  • - K2I0S T "- * Y268 ⁇ - Q292P> Q '3261” r (J274C. "- I N3 6C positions being indicated in SLQ ID!.
  • Plasmid pY1-! S2 containing the improved variant of the Yemniu intermc ⁇ a phytase as previously described was transformed by electroporation into the yeast strain Succfiarumomyces cerevisiae ⁇ Pho4 strain and the transformants were selected on solid medium Sl) -I '.
  • Several clones were grown in a liquid medium for 30 hours at 30 ° C. under shaking, and these seedlings sown as much cooked rice in YP Galactose production medium. * -a were performed overnight at 30 '1 C. with agitation.
  • Example 3 Expression of Enhanced Variants of the Yersinia intermedia phytase in Pichia Pastom
  • the plasmid pPK ' 9 containing the improved variant of the Yersinia iniermediu phytase as previously described was transformed into competent Pichia pastoris cells. After selection of colonies containing the plasmid, a colony was allowed to grow for 16 hours at 28 'C with stirring in 1 50 ml of FiM medium (J to form a preculture.
  • the production of variants is controlled by induction time Different amounts of 0.5% methanol, depending on the desired amount of the variant dilter, were used to determine the Optical Density (ViO) of the prccultures at oOOnm: for induction, optimal OD values of 2 to 6 uDO were required. then centrifuged and resuspended in a BMVI medium containing 0.5% methanol at a starting I) o between i and .K) uDO ml according to the induction times envisaged: luDO / ml for an induction of 06 flowers. 6 u I XVmI for a 72 hour induction and M ) u IX VmI for a 48 hour induction. 100% methanol was added every hour 24 final concentration 0.5 "• '.” These pmducii ⁇ n procedures were used for productions in L' rlcn ⁇ e ⁇ cr suitable for volumes ranging from IO ml to 200 ml.
  • bioreactor For higher volumes, the productions were carried out in bioreactor from 5 to 50 I. adapted to production volumes ranging from 2 to 20 I.
  • the type of bioreactor used ( ⁇ pplikon) allows automated control of yeast growth by regular measurements of Optical Density at 600 nm and Oxygen pressure (p ⁇ >) optimizing the maintenance of induction conditions by methanol.
  • the procedure used was adapted from the Pichia paxtnris fermentation protocol of Invitrogyn ("Pichia expression kit: a manu ⁇ l de methodes de expression de reeomhina ⁇ i proteins en Pichia pastnris" - Version M of January 1, 2002 *).
  • the Phy 98-6X-SS5 mutant is a mutant containing the substitutions I.52C • I .WC ( T142N '- ⁇ 177T r K210S * Y268I- t ⁇ 2') 2P 'O326T.
  • Phy tracks ⁇ ⁇ S show the profile of i ⁇ igration ⁇ t the phytase healthy age of ⁇ vrtinia inwrmvtliu, not ⁇ lycosveal since not having potential sites of N-giycosN lalions N ⁇ S / T: this is illustrated by the absence of migration difference after Digestion with Pendoglycosidase Wf.
  • nO ⁇ i of the total reaction volume of 160 ⁇ l was transferred for the Union dream with 60 ⁇ l of a Fe-Mo solution.
  • the revelation reaction was left for 1 5 minutes in the absence of light before being iu in a spectrophot ⁇ mèlre to 620 ntn. All the reaction solutions were carried out with the water ppi phosphate.
  • the rcaelionnel buffer is a 0.25 M nH 4.5 sodium acetate buffer.
  • the substrate used was a phytate solution achieved in the previous reaetionnel buffer from a stock solution to 200 g / l.
  • the solution of the solution was formed by four microliters of Mo solution mixed with one volume of Fe solution.
  • the solution of Mo is a solution of Molyhdutc at 0.012M and the solution of I ' is a solution of 1st to O. SM.
  • Figure 7 shows levels of relative activity vs. time (O to 15 minutes) higher for PHY-mutants 98-4X and 98-6X PI-IY compared to the enzyme from PHY-98 origin in Sacchur ⁇ myces cerevisiue .
  • thermostability of the improved variants of the Yersiiriu phytase intermedia i.a thermostability of isolated variants of the phyiasc of Ycrsima Inwrnnulia was determined by measuring a residual activity of the different supernatants of production of the variants either after a pre-harvesting at a constant temperature for various times, either after a fixed time railch.mJ 'wise at varying temperatures, in the first ca.s. the variants are preheated to 80 ° C. for periods ranging from 0 to 30 minutes. In the second case.
  • Figures 3, 4 and 5 show the results obtained for the varimils Phy- 1 JJMX and Phy- *) 8-f "expressed by X Sarcharoinyics ccrevisiue with respect to the original enzyme iY Pi-08.
  • Figure 3 shows the higher residual activities of the two mutants PHY- 4) 8-4X and Pl IY-Wi-GX after 0-2 min pre-chase at 80 ° C relative to the enzyme of PHY-V8 origin.
  • Figure 4 shows the upper residual activities of two mutants PHY- ⁇ 8-4X and PHY-JS-oX after 15 minutes of preheating at room temperature.
  • Ligure 5 shows the higher residual activities of the two PHY-98-4X mutants and PHY-M- ⁇ X beyond 6OT using I minute chargeehaulfage.
  • Figures 6a) and 6b) provided the upper residual activity of mutants PI IY-98- 6X L M PH Y-y8-6X-SS I. expressed by Pichia pusloris. after preheating times at SO 1 C of respectively 0 to M) minutes and 0 to 5 minutes.
  • Example 6 Hermosiabilit ⁇ Mutants in Granulation Assays
  • Granulation tests can be small to determine the thermostability of the different mutants with respect to the wild-type enzyme and existing and / or commercial enzymes, i.
  • Various phytases can be incorporated into the processes for forming and formulating wax granules added to animal feed, for example.
  • the granules formed can be mixed with the food. According! "Largest volume of testing, as island of formed food may vary. For example. 250 g of granules can be mixed with 25 kg feed to form a pr ⁇ mclange. This 53 ⁇ télangc can be incorporated just before the test with 225 kg of
  • food of the same composition, but not limited to, typical poultry feed can be composed as a percentage of 45 to 50% corn 0 to 5% pea 0 to 4.5 0 0 eol flour 0 to 4.5% of sunflower seed meal, 0 to 2.5% of corn flower gluten.
  • the mixture of the order of 250 kg is typically dosed in a mixer / conditioner by v is a dosage in a ⁇ iiesse approximately (> 0U 3X kg / h. where it is heated by steam injection to about 95 ° C.
  • the total residence time is about 10 to 30 seconds after which the hot mixture is directed to a granulation press.
  • the sizes of granules that can be manufactured are 5/45 mm (width / length) or 3/65 mm.
  • the temperature of the pellets at the outlet of the press is typically 82 to 83 ° C for the first type of granules and 91 to 93 ° C for the second type.
  • the granules are cooled on a carpet of cooling, where samples are recovered to determine the activity and stability of the mutants and reference phytases after the formation of the final granules, and granulation yields in terms of activity can be obtained for each mutant, compared to Reference is made to the activity reports after and before the granulation step
  • a protocol for measuring the phytase activity is given in Example 4. Also, a standard protocol for measuring the phytase activity adapted to these methods is published as: van.hngelen et al .. Journal oi " ⁇ O ⁇ C International 1994. 77: 760-764.
  • Dil ⁇ rcnis phytase mutants can be tested compared to phytase retro 'erent.
  • the different samples are incubated at 40 ° C., firstly at pH 3 for 60 minutes and then at 40 ° C. for 30 minutes.
  • the reactions are then stopped and the phytate and the innsilol phosphate are extracted by addition of hydrochloric acid to a final concentration of 0.5M, incubation for 2 hours at 40 ° C. followed by a freeze-thaw cycle and one hour of incubation at 40 ° C.
  • Phytaia and inositus phosphates are separated by high-performance ion exchange chromalography here as described in Chen. Q.C. and Li. B ⁇ V. (2003) and Journal of Chromatography (K) 18. 41-52 as well as in Skoglund. 1- .. Carlson. N. (i.and Snndherg, ⁇ .S. ⁇ J * W7) and J. ⁇ gric. I-ood Chem. 45. 431-436.
  • Rcla ⁇ ic phosphate is calculated as the difference between inositol phosphates-bound phosphate in phytase-treated samples compared to untreated samples with phthalc. Interesting mutants result in a greater amount of phosphate.

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