Patent Application: US-201013380694-A

Abstract:
the present invention provides the field of enhancing proteins and in particular to that of proteins enhanced by molecular change . it provides a variant of a phytase that is termed enhanced in that is has better thermostability and / or activity than the original phytase . the invention also provides a nucleic acid coding for said variant , a cassette or an expression vector containing said variant , a host cell expressing said variant , a composition comprising said variant and uses thereof , principally in the preparation of food additives and animal feed .

Description:
producing enhanced phytase variants of the present invention required the construction of various plasmidic vectors that were capable of carrying out the directed mutagenesis experiments necessary in order to obtain libraries of variants or mutants as well as for the expression of said mutants in various screening or production hosts . using molecular biological techniques that are well known to the skilled person , the orf ( open reading frame ) zp — 00832361 corresponding to the sequence ncbi atcc 29909 and to the corresponding nucleotide sequence nz_aalf01000052 , region : 1889 . . . 3214 was cloned into the plasmidic vector pet25b . before cloning , the original signal sequence ( first 23 amino acids ) was deleted from the orf and replaced by the signal sequence for the phytase of escherichia coli . this signal sequence for the phytase of escherichia coli had been cloned into the vector pet25b in order to optimize expression of the phytase from yersinia intermedia in e . coli . the vector pet25b could also be used to express the protein of interest in the form of a fusion protein with a “ 6h is tag ” facilitating isolation and / or purification of the protein of interest using methods that are well known to the skilled person . this vector containing the phytase from yersinia intermedia fused with the signal sequence of the phytase from e . coli was transformed in a bl21 ( de3 ) e . coli strain from which the appa gene coding for the endogenous phytase of e . coli had been deleted . the orf of zp — 00832361 was cloned into the vector pnck using molecular biological techniques that are well known to the skilled person . this vector can be used to express , in a thermophilic microorganism thermus thermophilus , a fusion protein between the protein of interest and a thermostable kanamycin resistance gene using the method described in patent application wo 2006 / 134240 and corresponding to biométhodes &# 39 ; thr ™ technique . the orf of zp — 00832361 was cloned into the vector pyes2 using molecular biological techniques that are well known to the skilled person . this vector , containing the signal peptide for the phytase of aspergillus niger in the 5 ′ position of the orf zp — 00832361 , allowed expression of the phytase from yersinia intermedia by saccharomyces cerevisiae . the orf of zp — 00832361 was cloned into the vector ppic9 using molecular biological techniques that are well known to the skilled person . this vector , containing the signal peptide of the αfactor of pichia pastoris ( invitrogen ) in the 5 ′ position of the orf zp — 00832361 , allowed expression . of the phytase from yersinia intermedia by pichia pastoris . construction of libraries of mutants in pnck , screening and obtaining enhanced variants : several libraries of mutants of the phytase from yersinia intermedia were created using biométhodes &# 39 ; massive mutagenesis ® technique described in u . s . pat . no . 7 , 202 , 086 or in saboulard et si ( biotechniques , 2005 sep 39 ( 3 ): 363 - 8 ). briefly , mutagenic oligonucleotides were synthesized to produce phytase mutants on each of the amino acids constituting the enzyme . the libraries of mutants were constructed from the vector pnck containing the gene of the phytase using the massive mutagenesis ® protocol described in in u . s . pat . no . 7 , 202 , 086 or in saboulard et al ( biotechniques , 2005 sep 39 ( 3 ): 363 - 8 ), then transformed and amplified in the escherichia coli strain dh10b . the mutants of the phytase contained in these libraries were then screened using the thr ™ technique described in patent application wo 2006 / 134240 , the principle of which is summarized in fig2 . the possibility offered by the system for selecting , in a single step , a very large number of mutant molecules means that highly diverse libraries can be worked with . such a library , targeting all of the residues of the protein , approximately 10 8 clones , was constructed using massive mutagenesis ®. briefly , the libraries of mutants were transformed in cultures of thermus thermophilus that had been rendered competent . the transformants were set to grow in liquid medium at 70 ° c . to produce pre - cultures that were then spread onto a solid medium containing stringent concentrations of kanamycin of the order of 25 μg / ml . after incubating for 48 hours at 70 ° c ., only the mutants with a protein structure that resisted the selection temperature and that were folded correctly could allow functional folding of the thermostable kanamycin - resistant gene and thus could grow in the presence of kanamycin . using this approach , various mutants were isolated , and their plasmidic dna was isolated and sequenced . the various mutants revealed a total of 138 substitutions in the 89 positions shown in table 1 . the various substitutes were introduced individually into the phytase cloned into the plasmidic vector pet25b in order to be expressed in escherichia coli and characterized in more detail as regards their activity and their thermostability . example 4 details the protocols for measuring the activity used and example 5 details the protocols for characterizing the residual activity of the mutants as a function of temperature . a first series of experiments was able to identify at least 5 mutants with an enhanced thermostability relative to the wild type enzyme . of these , 3 mutants respectively contained substitutions on the amino acids k210 , y268 and q292 , particularly the substitutions k210s , y268e and q292p . these mutants had an 80 %- 20 % residual activity extinction coefficient of respectively 1 . 75 , 1 . 98 and 2 . 30 as can be seen in table 4a . these indices were calculated by the ratio between the differences in temperatures allowing firstly 80 % of residual activity to be retained and secondly 20 % of residual activity to be maintained for the various variants compared with the wild type enzyme . details of the calculation of this index are shown for the mutant k210s in table 4b . visualizing model structures in 3d using software such as the swiss - model ( http :// www . expasy . ch /) and spdbv v4 . 01 ( glaxosmithkline ) means that certain explanations can be advanced concerning the increases in thermostability obtained for the 3 mutants mentioned above . k210 is a potentially solvent - accessible residue located in a secondary “ sheet ”- like structure , possibly involved in bonding and / or interaction with the substrate ( phytate ) or the products derived from the reaction . k210 thus seems to be an important residue in particular because of its positive charge and its large volume , which could generate steric and electrostatic constraints as regards access of the substrate to or egress of products from the active site . the loss of this positive charge in the k210s substitution and modification of the associated spatial constraint could thus in general perturb the reaction ( access of substrate + egress of products + solvation of the active site cavity ) and modify the kinetic constants thereof , more particularly the apparent kms for the various substrates generated during the reaction . in some circumstances , it is known that the substrates / product could participate in stabilizing the 3d structure of the protein and provide increased thermostability . it might be envisaged that this substitution could , for example , facilitate positioning of the substrate by increasing the number of conformers tolerated in the active site and / or could also facilitate evacuation of the phosphates liberated during the reaction , preventing them from being too numerous in the cavity of the active site , which presence could clearly perturb the enzyme - substrate complex and destabilize the whole structure . y268 is a potentially solvent - accessible residue located in the secondary “ loop ” type structure . by modeling the y268e substitution using the software mentioned above , it can be established that the number of potential hydrogen bonds with structurally close residues ( with the co peptide function of the q143 residue for example ) are increased , but also that this substitution could produce an additional saline bridge , in particular with k146 . overall , the region in question becomes stiffer , which could explain the observed gain in thermostability . q292 is also a potentially solvent - accessible residue located in a secondary “ loop ” type structure . substitution by a proline residue is a relatively conventional engineering approach in this type of secondary structure with poor conservation . this type of residue generates few rotamers , and as a consequence stiffens the secondary structure in which it is located and thus can sometimes result in increased thermostability that could thus bring about the substitution q292p . certain post - translational modifications such as glycosylations have been described as being protein stabilizers . the signals for n - glycosylations in a protein sequence are nxt or nxs . such sites were introduced into the phytase of yersinia intermedia cloned into the vector pyes2 either using the protected biométhodes massive mutagenesis ® technique mentioned above or using a mutagenesis technique that is well known to the skilled person , such as overlapping pcr , by introducing a t residue at the + 2 position relative to a residue n present in the phytase of yersinia intermedia or by introducing a residue n at the − 2 position relative to an s or t residue . the positions into which the glycosylation sites were introduced are classified as a function of their percentage accessibility to solvents (% asa ) using the software available at : http :// mobyle . rpbs . univ - paris - diderot . fr / cgi - bin / portal . py ? from = asa ( t . j . richmond , solvent accessible surface area and excluded volume in proteins . j . mol . biol , 178 , 63 - 89 ( 1984 ). preferably , 22 substitutions on residues with a percentage accessibility to solvents of & gt ; 35 % were selected . more preferably , 10 substitutions on residues with a percentage accessibility to solvents of & gt ; 70 % were selected . these different variant constructs allowing the addition of glycosylation sites are summarized in table 2 as a function of their respective percentage accessibility to solvents . the mutant constructs were transformed in saccharomyces cerevisiae and characterized in more detail as regards their activity and thermostability . example 5 details the protocols for characterizing the residual activity of the mutants as a function of temperature . a first series of experiments was able to identify several mutants with an additional glycosylation site having enhanced thermostability compared with the wild type enzyme . these mutants contain substitutions on the amino acids t142 , a177 and q326 characterized by a percentage accessibility to solvents of & gt ; 70 %. more particularly , the substitutions on said amino acids are t142n , a177t and q326t . these mutants have a respective 80 %- 20 % residual activity extinction coefficient of 1 . 62 , 1 . 62 and 1 . 52 , as shown in table 4c . these indices were calculated from the ratio between the differences in temperatures allowing firstly 80 % of the residual activity and secondly 20 % of the residual activity to be retained for the various variants compared with the wild type enzyme . details of the calculations for this index are shown in table 4b for the mutant k210s . several pairs of residues were identified as regards their distance and orientation compatible with the formation of a disulfide bridge and replaced with cysteine residues . these pairs were identified by visualizing model or homologous structures of phytases in pdb format using a swisspdb viewer ( glaxosmithkline ) type program , taking into account optimized distances between residues and orientation of the side chains thereof . using this approach , 12 pairs of residues located at approximately 2 angstrom were selected and are listed in table 3 . the above residues were introduced into the phytase from yersinia intermedia cloned into the vector pyes2 , either using the protected biométhodes massive mutagenesis ® technique as mentioned above or using a mutagenesis technique that is well known to the skilled person such as overlapping pcr . the mutant constructs were transformed in saccharomyces cerevisiae and characterized in more detail as regards their activity and thermostability . example 5 details the protocols for characterizing the residual activity of mutants as a function of temperature . a first series of experiments was able to identify one mutant with an additional disulfide bridge site and enhanced thermostability . this mutant contains two substitutions on the residues g274 and n316 , more particularly the substitutions g274c and n316c . this mutant had an 80 %- 20 % residual activity extinction coefficient of 2 . 33 , as shown in table 4d . this index was calculated by the ratio between the differences in temperatures allowing firstly 80 % of the residual activity and secondly 20 % of the residual activity to be retained for the variant ss11 compared with the wild type enzyme . details of the calculations for this index for the mutant k210s are shown in table 4b . several residues were targeted in order to identify mutants having an enhanced activity . these residues were identified by visualizing model or homologous structures of phytases in the pdb format using a swisspdb viewer ( glaxosmithkline ) type program . the criterion for selection . that was selected was : residues located at a distance of 10 angstrom or less around the catalytic site of the enzyme . using this approach , 76 positions were selected and are listed in table 5a . complete diversity by using the oligonucleotides nns was introduced at these positions into the phytase of yersinia intermedia cloned into the pyes2 vector , either using the protected biométhodes massive mutagenesis ® technique as mentioned above or using a mutagenesis technique that is well known to the skilled person , such as overlapping pcr . thus , for each of the positions listed in table 5a , the mutants individually comprising one of the 19 possible substitutions from the list of 20 existing amino acids were constructed ; using the standard single letter code , these 20 amino acids are : a , r , n , d , c , e , q , g , h , i , l , k , m , f , p , s , t , w , y and v . the mutant constructs were transformed in saccharomyces cerevisiae and characterized in more detail as regards their respective activity and thermostability . example 5 details the protocols for characterizing the residual activity of the mutants as a function of temperature . a first series of experiments allowed 14 mutants containing the substitutions listed in table 5b to be characterized . the various approaches employed meant that the thermostability and / or activity and / or proteolysis resistance gains could be accumulated by screening and characterizing the various mutants in each of the approaches : thr ™, adding glycosylation sites , adding disulfide bridges and targeting activity . a first series of experiments was used to construct several mutants combining the thermostability enhancements obtained by screening libraries using thr ™, adding new glycosylation sites and adding disulfide bridges . these mutants were constructed using the protected biométhodes massive mutagenesis ® technique mentioned above . several mutants were constructed comprising combinations of substitutions : g274c + n316c , t142n + a177t + q326t , k210s + y268e + q292p , d140f + y268e + q292p , f167n + y268e + q292p , t142n + a177t + k210s + q326t , t142n + a177t + k210s + y268e + q292p + q326t , t142n + a177t + k210s + y268e + q292p + q326t + g274c + n316c . the mutant constructs were transformed in saccharomyces cerevisiae and characterized in more detail as regards their activity and their thermostability . example 5 details the protocols for characterizing the residual activity of mutants as a function of temperature . fig3 to 7 show the results of characterizations of some of these mutants , in particular the mutants phy - 98 - 4x , phy - 98 - 6x and phy - 98 - 6x - ss11 respectively containing combinations of mutations t142n + a177t + k210s + q326t , t142n + a177t + k210s + y268e + q292p + q326t and t142n + a177t + k210s + y268e + q292p + q326t + g274c + n316c , the positions being indicated in seq id no . 1 . three bacterial strains respectively containing the enzyme of origin phy - 98 and the mutants phy - 98 - 6x and phy - 98 - 6x - ss11 mentioned above , cloned into the plasmidic vector pyes2 as described above , form the subject matter of a deposit of biological material under respective references cncm i - 4172 , cncm i - 4173 and cncm i - 4174 , on jun . 24 , 2009 . these various mutants may act as a basis for adding one or more additional substitutions deriving from various selective approaches and show one / more enhancements in activity / thermostability in order to create novel combinations of mutations accumulating the enhancements or demonstrating synergistic effects in the enhancements due to these novel combinations of mutations . expression of enhanced variants of the phytase from yersinia intermedia in saccharomyces cerevisiae the plasmid pyes2 containing the enhanced phytase variant from yersinia intermedia as described above was transformed by electroporation in the yeast strain saccharomyces cerevisiae δpho4 and the transformants were selected on sd - u solid medium . several clones were pre - cultured in liquid sd - u medium overnight at 30 ° c ., with agitation . said pre - cultures allowed more culture to be seeded in 2 % yp galactose production medium . they were produced overnight at 30 ° c ., with agitation . expression of enhanced variants of the phytase from yersinia intermedia in pichia pastoris the plasmid ppic9 containing the enhanced phytase variant from yersinia intermedia as described above was transformed in cells of pichia pastoris that had been rendered competent . after selecting colonies containing the plasmid , one colony was grown for 16 hours at 28 ° c ., with agitation , in 50 ml of bmg medium to form a pre - culture . the production of variants was controlled by using different induction times in 0 . 5 % methanol depending on the desired quantity of said variant . before induction , the optical density ( od ) of the pre - cultures was measured at 600 nm ; for induction , optimized ods of 2 to 60d units were required . the pre - cultures were then centrifuged and re - suspended in bmm medium containing 0 . 5 % methanol at an initial od in the range 1 to 30 odu / ml depending on the envisaged induction time : 1 odu / ml , for 96 hours of induction , 6 odu / ml for an induction of 72 hours and 30 odu / ml , for an induction of 48 hours . 100 % methanol was added every 24 hours to give a final concentration of 0 . 5 %. these production procedures were employed for erlenmeyer flask production procedures adapted to volumes of 10 ml to 200 ml . for higher volumes , production was carried out in a 5 l to 50 l bioreactor adapted to production volumes of 2 l to 20 l . the type of bioreactor used ( applikon ) could automatically monitor yeast growth by carrying out regular measurements of the optical density at 600 nm and the oxygen pressure ( po 2 ), optimizing maintenance of the induction conditions with methanol . the procedure used was adapted from the protocol for fermentation in pichia pastoris from invitrogen (“ pichia expression kit ; a manual of methods of expression of recombinant proteins in pichia pastoris ” • version h dated 11 jan . 2002 ). fig8 shows a 12 % sds - page gel of production supernatants for various mutants expressed by pichia pastoris , digested or otherwise by the endoglycosidase hf ( new england biolabs p0703 ). the gel tracks denoted phy 98 , phy 98 6x and phy 98 6x ss11 correspond to 10 μg of production supernatants from the molecules phy - 98 , phy - 98 - 6x and phy - 98 - 6x - ss11 described in example 1 . the mutant phy 98 - 6x - ss5 is a mutant containing the substitutions l52c + l99c + t142n + a177t k2105 y268e + q292p + q326t . the phy98 tracks show the migration profile for wild type phytase of yersinia intermedia , not glycosylated , since it does not have the potential n - glycosylation sites nxs / t ; this is illustrated by the absence of differences in migration after digestion with the endoglycosidase hf . the tracks phy 98 6x , phy 98 6x ss5 and phy 98 6x ss11 , in the absence of digestion by the endoglycosidase hf , show the migration profiles of mutant phytases with high molecular masses due to respective glycosylation of the mutants . this glycosylation is shown , after digestion with the endoglycosidase hf ( tracks denoted “ digested endo hf ”), by a return of the migration profile for the various mutants towards that of the wild type phytase even if digestion by the endoglycosidase may be incomplete in some circumstances . measurement of the activity of enhanced phytase variants from yersinia intermedia the activity of variants of the phytase from yersinia intermedia as well as the activity of the protein of origin used as the control were measured using a colorimetric test measuring the phosphate liberated in the presence of a solution of phytate used as the substrate . in brief , 40 μl of supernatant from the test sample ( diluted or not in a sodium acetate buffer ) or 40 μl of a calibrating solution of a phosphate were mixed with 40 μl of phytate ( 20 g / l ). the reaction was incubated conventionally for 15 minutes at 37 ° c . the reaction was stopped with 80 μl of 0 . 5 m naoh or 20 % tca . 60 μl of the total volume of the reaction of 160 μl was transferred to be revealed with 60 μl of a fe — mo solution . the revealing reaction was left for 15 minutes in the dark before being read in a spectrophotometer at 620 nm . all of the reaction solutions were produced using wfi phosphate - free water . the reaction buffer was a sodium acetate buffer , 0 . 25 m , ph 4 . 5 . the substrate used was a phytate solution produced in the above reaction buffer from a 200 g / l stock solution . the revealing solution was formed extemporaneously using 4 volumes of mo solution mixed with 1 volume of fe solution . the mo solution was a solution of 0 . 012 m molybdate and the fe solution was a 0 . 38 m iron ii solution . in order to calculate the activity , a phosphate calibration curve was produced with a kh 2 po 4 range of 0 to 10 μmol / ml . by using the reaction conditions described above , the phytase activity of the test samples was calculated by applying the following formula : phytase activity in u / ml =[( od 620 nm )×( dilution factor )]/[( slope of calibration curve )×( reaction time in minutes )]. the protein concentrations were calculated using the conventional bradford method familiar to the skilled person . fig7 shows that the relative activities as a function of time ( 0 to 15 minutes ) were higher for the mutants phy - 98 - 4x and phy - 98 - 6x compared with the enzyme of origin phy - 98 in saccharomyces cerevisiae . the thermostability of variants isolated from the phytase of yersinia intermedia was determined by measuring a residual activity of various supernatants from production of the variants either after pre - heating to a constant temperature for varying times or after a fixed pre - heating time at varying temperatures . in the first alternative , the variants were pre - heated to 80 ° c . for times of 0 to 30 minutes . in the second alternative , two types of residual variant activity were measured either after pre - heating for 15 minutes to temperatures of 45 ° c . to 65 ° c ., or after pre - heating for 1 minute to temperatures of 60 ° c . to 80 ° c . the residual activities were determined in the first alternative as the % activity of the same sample without pre - heating or in the second alternative as the % of the activity of the same sample pre - heated to the first measured temperature , the two reference activities being considered as the 100 % points . the residual activity is shown as raw values for the optical density at 600 nm after pre - heating for 15 minutes in fig4 . fig3 , 4 and 5 show the results obtained for the variants phy - 98 - 4x and phy - 98 - 6x expressed by saccharomyces cerevisiae compared with the enzyme of origin phy - 98 . fig3 shows the higher residual activities of the two mutants phy - 98 - 4x and phy - 98 - 6x after pre - heating for 0 to 2 minutes at 80 ° c . compared with the enzyme of origin phy - 98 . fig4 shows the high residual activities of the two mutants phy - 98 - 4x and phy - 98 - 6x after pre - heating for 15 minutes at temperatures varying from 45 ° c . to 65 ° c . fig5 shows the high residual activities of the two mutants phy - 98 - 4x and phy - 98 - 6x beyond 60 ° c . using 1 minute of pre - heating . fig6 a ) and 6 b ) show the high residual activities of the mutants phy - 98 - 6x and phy - 98 - 6x - ss11 , expressed by pichia pastoris , after pre - heating times at 80 ° c . of respectively 0 to 30 minutes and 0 to 5 minutes . granulation tests could be carried out to determine the thermostability of the various mutants relative to the wild type and existing and / or commercially available enzymes . the various phytases could be incorporated into methods of forming and formulating granules intended to be added to animal feed , for example . these granules could be formed by mixing / kneading supernatants from the production of the mutants and reference enzymes that have to be compared , for example a matrix composed of corn starch and water , under the same conditions . the granulation matrix may contain different relative phytase / corn / water percentages . conventionally , after kneading , the matrix can be extruded with an extruder similar to the nica ™ e - 220 type and spheronized directly using a nica or fuji paudal ™ qj - 400g type spheronizer . the particles obtained are then dried in a glatt gpcg 1 . 1 type fluidized bed drier . the phytase activity in the granules is generally in the range 2500 to 3000 ftu / g . the granules formed may be mixed with food . depending on the volume of the tests , the quantity of food formed may vary . as an example , 250 g of granules may be mixed with 25 kg of food to form a pre - mix . just before the test , this pre - mix may be incorporated into 225 kg of food , for example , with the same composition . in non - limiting manner , a typical poultry feed may be composed of 45 % to 50 % corn , 0 to 5 % peas , 0 to 4 . 5 % rape flour , 0 to 4 . 5 % sunflower seed flour , 0 to 2 . 5 % corn flour gluten , 6 % to 10 % whole soya beans , approximately 25 % soya meal , approximately 4 % tapioca , 1 % to 3 . 5 % of soya oil , 0 to 4 % of animal fat , 0 . 5 % to 1 % of a cocktail of vitamins ( mervit 100 ), approximately 1 % of powdered chalk , 0 . 2 % to 1 . 3 % of monocalcium phosphate , 0 . 1 % to 0 . 4 % of salts , 0 to 0 . 3 % of sodium bicarbonate ( nahco 3 ), 0 . 05 % to 0 . 3 % of l - lysine , 0 . 15 % to 0 . 25 % of dl - methionine and 0 to 0 . 05 % of l - threonine . a pre - mix of approximately 25 kg can typically be mixed in a collete mp90 type planetary mixer for 10 minutes . a mixture of the order of 225 kg can be mixed in a nauta type 1200 liter mixer . samples of this mixture are taken at this stage to determine the activity and stability of the mutants and the reference phytases before forming the final granules . a mixture of the order of 250 kg is typically dosed into the mixer / conditioner using a dosing screw at a rate of approximately 600 kg / hour where it is heated by injecting steam at approximately 95 ° c . the total residence time is approximately 10 to 30 seconds , after which the hot mixture is directed towards a granulating press . for the tests , the sizes of the granules that could be produced were of the type 5 / 45 mm ( width / length ) or 3 / 65 mm . the temperature of the granules at the outlet from the press is typically 82 ° c . to 83 ° c . for the first type of granules and 91 ° c . to 93 ° c . for the second type . following this step , the granules are cooled on a cooling mat from which samples are taken in order to determine the activity and stability of the mutants and reference phytases after formation of the final granules . granulation yields in terms of activity may thus be obtained for each mutant , compared with the reference enzymes , by producing activity reports after and before the granulation step . a protocol for measuring phytase activity is given in example 4 . in addition , a standard protocol for measuring phytase activity adapted to these methods has been published with the following reference : van engelen et al ., journal of aoac international 1994 , 77 : 760 - 764 . several approaches could be used to measure the effectiveness of the mutants of the invention in liberating phosphate from phytate in vivo in order to contribute to animal growth compared with reference phytases . various animals such as pigs could be integrated into well - established protocols . these had free access to water and a typical diet constituted , for example , by 67 % corn , 28 % soya flour , 1 % powdered chalk , 0 . 1 % l - lysine , 1 % corn oil , 0 . 25 % of a conventional vitamin cocktail , 0 . 5 % salts , 0 . 5 % antibiotics . the feed waste was collected daily . the weight gain of the animals was measured weekly to calculate the mean gain per day , the mean daily food intake and the gain / intake ratio . the mutants with a particular advantage and the best performances were typically those which had an increased gain / intake ratio . in addition , in vitro models exist that can simulate digestion in the tract of a monogastric animal . as an example , feed samples composed of 30 % soya flour and 70 % corn flour may be supplemented with calcium phosphate in an amount of 5 g / kg of feed and preincubated at 40 ° c ., ph 3 for 30 minutes , followed by adding pepsin in an amount of 3000 u / g of feed and various dosages of phytase in the range 0 ( blank control ) to 1 u of phytase / g of feed . various phytase mutants could be tested and compared with reference phytases . the various samples were incubated at 40 ° c ., initially at a ph of 3 for 60 minutes then at a ph of 4 for 30 minutes . the reactions were then stopped and the phytate and inositol phosphates were extracted by adding hydrochloric acid in a final concentration of 0 . 5m , incubating for 2 hours at 40 ° c ., followed by a freeze - thaw cycle and one hour &# 39 ; s incubation at 40 ° c . the phytate and the inositol phosphates were separated by high performance ion exchange chromatography as described by q . c . chen , and b . w . li ( 2003 ), journal of chromatography a 1018 , 41 - 52 as well as by e . skoglund , n . g . carlson , and a . s . sandberg ( 1997 ), j . agric . food chem . 45 , 431 - 436 . the phosphate that was liberated was calculated from the difference between the phosphate bound to the inositol phosphates in the samples treated with phytase compared with the samples not treated with a phytase . the mutants of interest released a larger quantity of phosphate . three bacterial strains containing the constructs phy - 98 , phy - 98 - 6x and phy - 98 - 6x - ss11 used in the above examples were deposited with the collection nationale de cultures de microorganismes ( cncm ) at the institut pasteur , 25 , rue du flocteur roux , 75724 paris cedex france , under the terms of the treaty of budapest : d . j . cosgrove ( 1970 ) inositol phosphate phosphatases of microbiological origin , inositol phosphate intermediates in the dephosphorylation of the hexaphosphates of myo - inositol , scyllo - inositol , and d - chiro - inositol by a bacterial ( 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