Patent Application: US-63550400-A

Abstract:
the present invention relates to heat tolerant phytases and dna sequences which code therefor . the phytases are useful in hydrolyzing phytate to inositol and inorganic phosphates . the phytases are valuable feed additives .

Description:
the aspergillus terreus cbs 116 . 46 strain was deposited under the terms of the budapest treaty on mar . 3 , 1995 at the centralbureau voor schimmel - cultures , oosterstraat 1 , p . o . box 273 , 3740 ag baarn , the netherlands , and it was given the deposit no : cbs 220 . 95 . the aspergillus terreus 9a1 strain was deposited under the terms of the budapest treaty on mar . 17 , 1994 at the dsm - deutsche sammlung von , mikroorganismen und zellkulturen , gmbh , maacheroder weg 1b , d - 38124 braunschweig , and it was given the deposit no . : dsm 9076 . m3 medium - phosphate with the addition of 5 g / l of na 12 phytate fungi were screened on a three plate system , using the following three media : “ m3 ” a defined medium containing phosphate ); “ m3 - p ” ( m3 medium lacking phosphate ); and “ m3 p + phytate ” ( m3 medium lacking phosphate but containing phytate as a sole phosphorus source ). plates were made with agarose to decrease the background level of phosphate . fungi were grown on the medium and at the temperature recommended by the supplier . either spores or mycelium were transferred to the test plates and incubated at the recommended temperature until growth was observed . the following thermotolerant strains were found to exhibit growth consistent with the production of an extracellular phytase : strains of myceliophthora thermophila , talaromyces thermophilus , aspergillus fumigatus , aspergillus nidulans and aspergillus terreus 9a - 1 were grown in potato dextrose broth ( difco lab ., detroit , mich ., usa ) or complete medium ( clutterbuck ). aspergillus terreus 9a - 1 and aspergillus nidulans have been deposited under the budapest treaty for patent purposes at the dsm in braunschweig , brd at mar . 17 , 1994 under accession number dsm 9076 and at feb . 17 , 1995 under accession number dsm 9743 , respectively . medium was innoculated at a high density with spores and grown overnight ( o / n ) with shaking . this produced a thick culture of small fungal pellets . the mycelium was recovered by filtration blotted dry and weighed . up to 2 . 0 g was used per preparation . the mycelium was ground to a fine powder in liquid nitrogen and immediately added to 10 mls of extraction buffer ( 200 mm tris / hcl , 250 mm nacl , 25 mm edta , 0 . 5 % sds , ph 8 . 5 ) and mixed well . phenol ( 7 mls ) was added to the slurry and mixed and then chloroform ( 3 mls ) was also added and mixed well . the mixture was centrifuged ( 20 , 000 g ) and the aqueous phase recovered . rnase a was added to a final concentration of 250 μ / ml and incubated at 37 ° c . for 15 minutes . the mixture was then extracted with 1 volume of chloroform and centrifuged ( 10 , 000 g , 10 minutes ). the aqueous phase was recovered and the dna precipitated with 0 . 54 volumes of rt isopropanol for 1 hour at room temperature ( rt ). the dna was recovered by spooling and resuspended in water . a portion of the dna was digested with proteinase k for 2 hrs at 37 ° c . and then extracted repeatedly ( twice to three times ) with an equal volume of phenol / chloroform and then ethanol precipitated prior to resuspension in water to a concentration of approximately 1 μg / μl . pcr was performed essentially according to the protocol of perkin elmer cetus [( pec ); norwalk , conn ., usa ]. the following two primers were used ( bases indicated in brackets are either / or ): phyt 8 : 5 ′ atg ga ( c / t ) atg tg ( c / t ) tc ( n ) tt ( c / t ) ga 3 ′ [ seq id no : 15 ] all components with the exception of the taq polymerase were incubated at 95 ° c . for 10 minutes and then 50 ° c . for 10 minutes and then the reaction placed on ice . the taq polymerase ( amplitaq , f . hoffmann - la roche , basel , ch ) was then added and 35 cycles of pcr performed in a triothermoblock ( biometra , göttingen , de ) according to the following cycle profile : an aliquot of the reaction was analyzed on 1 . 5 % agarose gel . pcr products of the expected size ( approximately 146 bp predicted from the aspergillus niger dna - sequence ) were excised from low melting point agarose and purified from a nacs — prepac — column ( brl life technologies inc ., gaithersburg , md ., usa ) essentially according to the manufacturer &# 39 ; s protocol . the fragment was polyadenylated in 50 μl 100 mm sodiumcacodylate ph6 . 6 , 12 . 5 mm tris / hci ph 7 . 0 , 0 . 1 mm dithiothreitol , 125 μg / ml bovine serum albumin , 1 mm cocl 2 , 20 μmdatp , 10 units terminal deoxytransferase ( boehringer mannheim , mannheim , de ) for 5 minutes at 37 ° c . and cloned into the p123t vector [ mitchell et al ., pcr meth . app . 2 , 81 - 82 ( 1992 )]. alternatively , pcr fragments were purified and cloned using the “ sure clone ” ligation kit ( pharmacia ) following the manufacturers instructions . sequencing was performed on dsdna purified on a quiagen - column ( diagen gmbh , hilden , de ) using the dideoxy method and the pharmacia t7 kit ( pharmacia , lkb biotechnology ab , uppsala , se ) according to the protocol supplied by the manufacturer . the fragments from aspergillus terreus strain 9a - 1 and myceliophthora thermophila were used to probe bamhi and bglii southerns to determine the suitable restriction enzyme to use to construct genomic libraries in the lambda fix ii vector ( strategene , la jolla , calif ., usa ). lambda fix ii can only accept inserts from 9 - 23 kb . southerns were performed according to the following protocol . genomic dna ( 10 gg ) was digested in a final volume of 200 μl . the reaction without enzyme was prepared and incubated on ice for 2 hours . the enzyme ( 50 units ) was added and the reaction incubated at the appropriate temperature for 3 hours . the reaction was then extracted with an equal volume of phenol / chloroform and ethanol precipitated . the resuspended dna in loading buffer was heated to 65 ° c . for 15 minutes prior to separation on a 0 . 7 % agarose gel ( o / n 30 v ). prior to transfer the gel was washed twice in 0 . 2 m hcl / 10 ′/ room temperature ( rt ) and then twice in 1 m nacl / 0 . 4 m naoh for 15 ′ at rt . the dna was transferred in 0 . 4 m naoh in a capillary transfer for 4 hours to nytran 13 n nylon membrane ( schleicher and schuell ag , feldbach , zürich , ch ). following transfer the membrane was exposed to uv . [ auto cross - link , uv stratalinker 2400 , stratagene ( la jolla , calif ., usa )]. the membrane was prehybridized in hybridization buffer [ 50 % formamide , 1 % sodium dodecylsulfate ( sds ), 10 % dextransulfate , 4 × sspe ( 180 mm nacl , 10 mm nah 2 po 4 , 1 mm edta , ph 7 . 4 )] for 4 hours at 42 ° c . and following addition of the denatured probe o / n at 42 ° c . the blot was washed : results indicate that aspergillus terreus strain 9a - 1 genomic dna digested with bamhi and myceliophthora thermophila genomic dna digested with bglii produce fragments suitable for cloning into the lambda fix ii vector . the construction of genomic libraries of aspergillus terreus strain 9a - 1 and myceliophthora thermophila in lambda fix ii was performed according to the manufacturer &# 39 ; s protocols ( stratagene ). the lambda libraries were plated out on 10 137 mm plates for each library . the plaques were lifted to nytran 13n round filters and treated for 1 minute in 0 . 5 m naoh / 1 . 5 m nacl followed by 5 minutes in 0 . 5 m tris - hcl ph 8 . 0 / 1 . 5 m nacl . the filters were then treated in 2 × ssc for 5 minutes and air dried . they were then fixed with uv ( 1 minute , uv stratalinker 2400 , stratagene ). the filters were hybridized and washed as above . putative positive plaques were cored and the phage soaked out in sm buffer ( 180 mm nacl , 8 mm mgso 4 . 7h 2 o , 20mm tris / hcl ph 7 . 5 , 0 . 01 % gelatin ). this stock was diluted and plated out on 137 mm plates . duplicate filters were lifted and treated as above . a clear single positive plaque from each plate was picked and diluted in sm buffer . three positive plaques were picked . two from aspergillus terreus strain 9a - 1 ( 9a1λ17 and 9a1λ22 ) and one from myceliophthora thezmophila ( mtλ27 ). lambda dna was prepared from the positive plaques . this was done using the “ magic lambda prep ” system ( promega corp ., madison , wis ., usa ) and was according to the manufactures specifications . to confirm the identity of the clones , the lambda dna was digested with psti and sali and the resultant blot probed with the pcr products . in all cases this confirmed the clones as containing sequences complementary to the probe . dna from 9a1λ17 was digested with psti and the resultant mixture of fragments ligated into pbluescript ii sk + ( stratagene ) cut with psti and treated with shrimp alkaline phosphatase ( united states biochemical corp ., cleveland , ohio , usa ). the ligation was o / n at 16 ° c . the ligation mixture was transformed into xl - 1 blue supercompetent cells ( stratagene ) and plated on lb plates containing 0 . 5 mm isopropyl - β - d - thiogalactopyranoside ( iptg ), 40 μg / ml 5 - bromo - 4 - chloro - 3 - indoyl - β - d - galactopyranoside ( xgal ), 50 μg / ml ampicillin . dna from 9aλ17 was digested with bglii and xbai and the resultant mixture ligated into pbluescript ii sk + digested with bamhi / xbai . ligation , transformation and screening were performed as described above . dna from mtλ27 was digested with sali and the resultant mixture of fragments ligated into pbluescript ii sk + cut with sali and treated with shrimp alkaline phosphatase . the ligation was o / n at 16 ° c . the ligation mixture was transformed into xl1 blue supercompetent cells and plated on lb plates containing xgal / iptg and ampicillin . colonies from the above transformations were picked and “ gridded ” approximately 75 to a single plate . following o / n incubation at 37 ° c . the colonies were lifted to a nylon filter (“ hybond - n ”, amersham corp ., arlington heights , ill ., usa ) and the filters treated with 0 . 5 m naoh for 3 minutes , 1 m tris / hcl ph7 . 5 twice for 1 minute , then 0 . 5 m tris / hcl ph7 . 5 / 1 . 5 m nacl for 5 minutes . the filters were air dried and then fixed with uv ( 2 minutes , uv stratalinker 2400 , stratagene ). the filters were hybridized with the pcr products of example 5 . positive colonies were selected and dna prepared . the subclones were sequenced as previously described in example 4 . sequences determined are shown in fig1 ( fig1 ) for the phytase from aspergillus terreus strain 9a1 and its encoding dna sequence , fig2 for the phytase from myceliophthora thermophila and its encoding dna - sequence , fig3 a shows a restriction map for the dna of aspergillus terreus ( wherein the arrow indicates the coding region , and the strips the regions sequenced in addition to the coding region ) and 3b for m . thermophila , and fig4 for the phytase from talaromyces thermophilus and its encoding dna sequence , fig5 for the phytase from aspergillus fumigatus and its encoding dna - sequence and fig6 for the phytase from aspergillus nidulans and its encoding dna - sequence . the sequences for the phytases and its encoding dna - sequences from talaromyces thermophilus , aspergillus fumigatus and aspergillus nidulans were obtained in the same way as described for those of aspergillus terreus strain 9a1 and myceliophthora thermophila in examples 2 - 7 . bases are given for both strands in small letters by the typically used one letter code abbreviations . derived amino acid sequences of the phytase are given in capital letters by the typically used one letter code below the corresponding dna - sequence . construction of a chimeric construct between a . niger and a . terreus phytase dna - sequences all constructions were made using standard molecular biological procedures as described by sambrook et al ., ( 1989 ) ( molecular cloning , a laboratory manual , cold spring harbor laboratory press , ny ). the first 146 amino acids ( aa ) of the aspergillus niger phytase , as described in ep 420 358 , were fused to the 320 cterminal aa of the aspergillus terreus 9a1 gene . a ncoi site was introduced at the atg start codon when the a . niger phytase gene was cloned by pcr . the intron found in the a . niger phytase was removed by site directed mutagenesis ( bio - rad kit , cat nr 170 - 3581 ; bio - rad , richmond , calif ., usa ) using the following primer ( wherein the vertical dash indicates that the sequence to its left hybridizes to the 3 ′ end of the first exon and the sequence to its right hybridizes to the 5 ′ end of the second exon ): to construct the chimeric construct of phytases from a . niger and a . terreus an eco 47iii site was introduced into the a . niger coding sequence to aid cloning . pcr with a mutagenic primer ( 5 ′ cga ttc gta gcg ctg gta g 3 ′) in conjunction with the t3 primer was used to produce a dna fragment that was cleaved with bamhi and eco 47iii . the bamhi / eco 47iii fragment was inserted into bamhi / eco 47iii cut p9a1pst ( example 7 ). fig7 shows the amino acid sequence of the fusion construct and its encoding dna - sequence . for expression of the fusion construct in a . niger an expression cassette was chosen where the fusion gene was under control of the inducible a . niger glucoamylase ( glaa ) promoter . for the complete a . terreus 9a1 gene , expression cassettes with the constitutive a . nidulans glyceraldehyde - 3 - phosphate dehydrogenase ( gpda ) promoter were made . all genes used for expression in a . niger carried their own signal sequence for secretion . the a . niger glucoamylase ( glaa ) promoter was isolated as a 1960 bp xhoi / clai fragment from plasmid pdh33 [ smith et al . ( 1990 ), gene 88 : 259 - 262 ] and cloned into pbluescriptsk + - vector ( pbs ) [ stratagene , la jolla , calif ., usa ] containing the 710 bp bamhi / xbai fragment of the a . nidulans trpc terminator . the plasmid with the cassette was named pglac . the fusion gene , as described in example 8 , was put under control of the a . niger glaa promoter by ligating the blunt ended ncoi / ecori fragment to the blunt ended clai site and the ecorv site of plasmid pglac . the correct orientation was verified by restriction enzyme digests . the entire cassette was transferred as a kpni / xbai fragment to puc19 ( new england biolabs , gmbh , schwalbach , brd ), that carried the neurospora crassa pyr4 gene ( puc19 - pyr4 ), a selection marker in uridine auxotrophic aspergilli , resulting in vector pfpan1 ( see fig8 with restriction sites and coding regions as indicated ; crossed out restriction sites indicate sites with blunt end ligation ). the a . nidulans glyceraldehyd - 3 - phosphate dehydrogenase ( gpda ) promoter was isolated as a ˜ 2 . 3 kb ecori / ncoi fragment from plasmid pan52 - 1 [ punt et al . ( 1987 ), gene 56 : 117 - 124 ], cloned into puc19 - ncoi ( puc19 having a smai - site replaced by a ncoi - site ), reisolated as ecori / bamhi fragment and cloned into pbs with the trpc terminator as described above . the obtained cassette was named pgpdn . the a . terreus gene was isolated as a ncoi / ecori fragment , where the ecori site was filled in to create blunt ends . plasmid pgpdn was cut with bamhi and ncoi . the bamhi site was filled in to create blunt ends . the ncoi / ecori ( blunt ) fragment of the a . terreus gene was cloned between the gpda promoter and trpc terminator . the expression cassette was isolated as kpni / xbai fragment and cloned into puc19 - pyr4 resulting in plasmid ppat1 ( see fig9 ; for explanation of abbreviations see legend to fig8 ). the plasmid pfpan1 was used to transform a . niger by using the transformation protocol as described by ballance et al . [( 1983 ), biochem . biophys . res . commun 112 , 284 - 289 ] with some modifications : ypd medium ( 1 % yeast extract , 2 % peptone , 2 % dextrose ) was inoculated with 10 6 spores per ml and grown for 24 hours at 30 ° c . and 250 rpm cells were harvested using wero - lene n tissue ( no . 8011 . 0600 wernli ag verbandstoffabrik , 4852 rothrist , ch ) and once washed with buffer ( 0 . 8 m kcl , 0 . 05 m cacl 2 , in 0 . 01 m succinate buffer ; ph 5 . 5 ) for protoplast preparation only lysing enzymes ( sigma l2265 , st . louis , mo ., usa ) were used the cells were incubated for 90 min at 30 ° c . and 100 rpm , and the protoplasts were separated by filtration ( wero - lene n tissue ) the protoplasts were once washed with stc ( 1 m sorbitol , 0 . 05 m cacl 2 , 0 . 01 m tris / hcl ph 7 . 5 ) and resuspended in the same buffer 150 μl protoplasts (˜ 10 8 / ml ) were gently mixed with 10 - 15 μg plasmid dna and incubated at room temperature ( rt ) for 25 min polyethylene glycol ( 60 % peg 4000 , 50 mm cacl 2 , 10 mm tris / hcl ph 7 . 5 ) was added in three steps , 150 μl , 200 μl and 900 μl , and the sample was further incubated at room temperature ( rt ) for 25 min 5 ml stc were added , centrifuged and the protoplasts were resuspended in 2 . 5 ml ygs ( 0 . 5 % yeast extract , 2 % glucose , 1 . 2 m sorbitol ) the sample was incubated for 2 hours at 30 ° c . ( 100 rpm ) centrifuged and the protoplasts were resuspended in 1 ml 1 . 2 m sorbitol the transformed protoplasts were mixed with 20 ml minimal regeneration medium ( 0 . 7 % yeast nitrogen base without amino acids , 2 % glucose , 1 m sorbitol , 1 . 5 % agar , 20 mm tris / hcl ph 7 . 5 supplemented with 0 . 2 g arginine and 10 mg nicotinamide per liter ) single transformants were isolated , purified and tested for overproduction of the fusion protein . 100 ml m25 medium ( 70 g maltodextrin ( glucidex 17 d , sugro basel , ch ), 12 . 5 g yeast extract , 25 g casein - hydrolysate , 2 g kh 2 po 4 , 2 g k 2 so 4 , 0 . 5 g mgso 4 . 7h 2 o , 0 . 03 g zncl 2 , 0 . 02g cacl 2 , 0 . 05 g mnso 4 . 4h 2 o , 0 . 05 g feso 4 per liter ph 5 . 6 ) were inoculated with 10 6 spores per ml from transformants fpan1 # 11 , # 13 , # 16 , # e25 , # e30 respectively # e31 and incubated for 5 days at 30 ° c . and 270 rpm . supernatant was collected and the activity determined . the fusion protein showed the highest activity with phytic acid as substrate at ph 2 . 5 , whereas with 4 - nitrophenyl phosphate as substrate it showed two activity optima at ph 2 . 5 and 5 . 0 ( table 1 ). a 1 ml enzyme reaction contained 0 . 5 ml dialyzed supernatant ( diluted if necessary ) and 5 . 4 mm phytic acid ( sigma p - 3168 ). the enzyme reactions were made in 0 . 2 m sodium acetate buffer ph 5 . 0 , respectively 0 . 2 m glycine buffer ph 2 . 5 . the samples were incubated for 15 min at 37 ° c . the reactions were stopped by adding 1 ml 15 % tca ( trichloroacetic acid ). for the colour reaction 0 . 1 ml of the stopped sample was diluted with 0 . 9 ml distilled water and mixed with 1 ml reagent solution ( 3 volumes 1 m h 2 so 4 , 1 volume 2 . 5 % ( nh 4 ) 6 mo 7 o 24 , 1 volume 10 % ascorbic acid ). the samples were incubated fot 20 min at 50 ° c . and the blue color was measured spetrophotometrically at 820 nm . since the assay is based on the release of phosphate a phosphate standard curve , 11 - 45 nmol per ml , was used to determine the activity of the samples . a 1 ml enzyme reaction contained 100 μl dialyzed supernatant ( diluted if necessary ) and 1 . 7 mm 4 - nitrophenyl phosphate ( merck , 6850 , darmstadt , brd ). the enzyme reactions were made in 0 . 2 m sodium acetate buffer ph 5 . 0 , respectively 0 . 2 m glycine buffer ph 2 . 5 . the samples were incubated for 15 min at 37 ° c . the reactions were stopped by adding 1 ml 15 % tca . for the determination of the enzyme activity the protocol described above was used . a . niger nw205 was transformed with plasmid ppat1 as described above . single transformants were isolated , purified and screened for overproduction of the a . terreus protein . 50 ml ypd medium were inoculated with 106 spores per ml from transformants pat1 # 3 , # 10 , # 11 , # 13 and # 16 and incubated for 3 days at 30 ° c . and 270 rpm . supernatant was collected and the activity determined as described above except that the ph for the enzyme reactions were different . the enzyme showed its main activity at ph 5 . 5 with phytic acid as substrate and at ph 3 . 5 with 4 - nitrophenyl phosphate as substrate ( table 2 ). preculture medium [ 30 g maltodextrin ( glucidex 17 d ), 5 g yeast extract , 10 g casein - hydrolysate , 1 g kh 2 po 4 , 0 . 5 g mgso 4 . 7h 2 o , 3 g tween 80 per liter ; ph 5 . 5 ] was inoculated with 10 6 spores per ml in a shake flask and incubated for 24 hours at 34 ° c . and 250 rpm . a 10 liter fermenter was inoculated with the pre - culture to a final dilution of the pre - culture of 1 : 100 . the batch fermentation was run at 30 ° c . with an automatically controlled dissolved oxygen concentration of minimum 25 % ( po 2 ≧ 25 %). the ph was kept at 3 . 0 by automatic titration with 5 m naoh . the medium used for the fermentation was : 35 g maltodextrin , 9 . 4 g yeast extract , 18 . 7 g casein - hydrolysate , 2 g kh 2 po 4 , 0 . 5 g mgso 4 . 7h 2 o , 2 g k 2 so 4 , 0 . 03 g zncl 2 , 0 . 02 g cacl 2 , 0 . 05 g mnso 4 . 4h 2 o , 0 . 05 g feso 4 per liter ; ph 5 . 6 . enzyme activities reached after 3 days under these conditions were 35 units / ml respectively 16 units / ml at ph 2 . 5 respectively ph 5 . 0 with phytic acid as substrate and 295 units / ml respectively 90 units / ml at ph 2 . 5 respectively ph 5 . 0 and 4 - nitrophenyl phosphate as substrate . preculture , inoculation of the fermenter and the fermentation medium were as described above , except that the ph was kept at 4 . 5 by automatic titration with 5 m naoh . enzyme activities reached after 4 days under these conditions were 17 . 5 units / ml at ph 5 . 5 with phytic acid as substrate and 2 units / ml at ph 3 . 5 with 4 - nitrophenyl phosphate as substrate . isolation of pcr fragments of a phytase gene of asperaillus terreus ( cbs 116 . 46 ) two different primer pairs were used for pcr amplification of fragments using dna of aspergillus terreus [ cbs 116 . 46 ]. the primers used are shown in the table below . dna sequences in bold show the sense primer and in italics the antisense primer . the primers correspond to the indicated part of the coding sequence of the aspergillus niger gene . the combinations used are primers 8 plus 9 and 10 plus 11 . the taq - start antibody kit from clontech ( palo alto , calif ., usa ) was used according to the manufacturer &# 39 ; s protocol . primer concentrations for 8 plus 9 were 0 . 2 mm and for primers 10 plus 11 one mm . touch - down pcr was - used for amplification [ don , r . h . et al . ( 1991 ), nucleic acids res . 19 , 4008 ]. first the dna was denatured for 3 min at 95 ° c . then two cycles were done at each of the following annealing temperatures : 60 ° c ., 59 ° c ., 58 ° c ., 57 ° c ., 56 ° c ., 55 ° c ., 54 ° c ., 53 ° c ., 52 ° c . and 51 ° c ., with an annealing time of one min . each . prior to annealing the incubation was heated to 95 ° c . for one min and after annealing elongation was performed for 30 sec at 72 ° c . cycles 21 to 35 were performed as follows : denaturation one min at 95 ° c ., annealing one min at 50 ° c . and elongation for 30 sec at 72 ° c . two different pcr fragments were obtained . the dna sequences obtained and their comparison to relevant parts of the phytase gene of aspergillus terreus 9a1 are shown in fig1 [ relevant parts of the phytase gene of aspergillus terreus 9a1 “ 9a1 ”( top lines ) ( 1 ) and the pcr fragments of aspergillus terreus cbs 116 . 46 “ aterr2 ” ( bottom lines ). panel a : fragment obtained with primer pair 8 plus . 9 ( aterr21 ). panel b : fragment obtained with primer pair 10 plus 11 ( aterr58 ). dna sequences of aspergillus terreus cbs 116 . 46 ( top lines ) are compared with those of aspergillus terreus 9a1 ( 1 ) ( bottom lines ). pcr amplifications were performed as described in the legend to table 4 . panel a : the bold gc sequence ( bases 16 plus 17 ) in the aterr21 fragment could possibly be cg ( dna sequencing uncertainty ). panel b : the n at position 26 of the aterr58 pcr fragment could possibly represent any of the four nucleotides ]. five mg &# 39 ; s of genomic dna of each strain listed in table 3 were incubated with 4 units of hindiii or psti , respectively , per mg of dna at 37 ° c . for 4 hours . after digestion , the mixtures were extracted with phenol and dnas were precipitated with ethanol . samples were then analyzed on 0 . 8 % agarose gels . dna - s were transferred to nytran membranes ( schleicher & amp ; schuell , keene , n . h ., usa ) using 0 . 4m naoh containing 1m nacl as transfer solution . hybridizations were performed for 18 hours at 42 ° c . the hybridization solution contained 50 % formamide , 1 % sds , 10 % dextran sulphate , 4 × sspe ( 1 × sspe = 0 . 18m nacl , 1 mm edta , 10 mm nah2 po 4 , ph 7 . 4 ), 0 . 5 % blotto ( dried milk powder in h 2 o ) and 0 . 5 mg salmon sperm dna per ml . the membranes were washed under non - stringent conditions using as last and most - stringent washing condition incubation for 30 min at room temperature in 0 . 1 × sspe containing 0 . 1 % sds . the probes ( labeled at a specific activity of around 10 9 dpm / mg dna ) used were the pcr fragments generated with primers 8 plus 9 ( see example 11 ) using genomic dna of myceliophthora thermophila ; mycelio . thermo . ; aspergillus nidulans , asperg . nidul . ; aspergillus fumigatus , asperg . fumig .,; aspergillus terreus 9a1 , asperg . torrous 9a1 . talaromyces thermophilus , talarom . thermo . the mt2 genomic probe was obtained by random priming ( according to the protocol given by pharmacia , uppsala , sweden ) and spans 1410 bp , from the bspei site upstream of the n - terminus of the mycelo . thezmo . phytase gen to the pvuii site in the c - terminus ( positions 2068 is to 3478 ). the at2 genomic probe was obtained by random priming and spans 1365 bp , from the apai site to the ndei site of the auperg . terreus 9a1 phytase gene ( positions 491 to 1856 ). the an2 dna probe was obtained by random priming and spans the complete coding sequence ( 1404 bp ) of the asperg . niger gene ( ep 420 358 ). results are given in table 3 . [“*” except for weak signal corresponding to a non - specific 20 kb fragment ; in case of the very weak cross - hybridization signal at 20 kb seen with dna from aspergillus niger using the pcr fragment from talaromyces thermophilus this signal is unspecific , since it differs significantly from the expected 10 kb hindiii - fragment , containing the phytase gene ; “**” signal due to only particle digest of dna ]. for cross - hybridizations with stringent washing conditions membranes were further washed for 30 min . at 65 ° c . in 0 . 1 × sspe containing 0 . 1 % sds . results are shown in table 4 [ ( 1 ) only the 10 . 5 - kb hindiii fragment is still detected , the . 6 . 5 - kb hindiii fragment disappeared ( see table 3 )]. organisms and growth conditions : aspergillus nidulans ( dsm 9743 ), aspergillus fumigatus ( atcc 34625 ), aspergillus terreus ( cbs 116 . 46 ) and talaromyces thermophilus ( atcc 20186 ) were grown on potato dextrose broth ( difco lab ., detroit , mich ., usa ) at 28 ° c . except for t . thermophilus , a thermotolerant fungus , which was grown at 45 ° c . transformed e . coli ( tg - 1 ) were grown in luria broth ( lb ) at 37 ° c . with 100 μg / ml ampicillin for selection . gencmic dna : fungal mycelium was obtained by incubating potato dextrose medium at a high density with spores o / n ( 200 rpm ) at the temperatures indicated above . up to 2 grams of the mycelium , obtained by filtration through a whatmann filter , were used for the isolation of genomic dna as described in the present application . dna amplification : genomic dna of the coding regions of the different phytase genes was amplified using pcr on a gene amp kit ( perkin elmer cetus ) according to the manufacturer &# 39 ; s instructions using degenerate primers : for t . thermophilus all components of the reaction , including the primers 8 [ seq id no : 15 ] and 9 [ seq id no : 16 ] at a final concentration of 10 mm , but with the exception of the taq polymerase , were incubated at 95 ° c . for 10 min and 50 ° c . for 1 min before the reaction was placed on ice . the taq polymerase was then added and 35 cycles of pcr performed according to the following cycle profiles : 60 sec , 95 ° c ./ 60 sec , 50 ° c ./ 90 sec , 72 ° c ./ 120 sec . for a . nidulans , a . terreus cbs116 . 46 and a . fumigatus a “ touch - down ” pcr with a final primer concentration of 0 . 2 mm for primers 8 [ seq id no : 151 and 9 [ seq id no : 16 ] was performed as described in don , r . h ., cox , p . t ., wainwright , b . j ., baker , k ., mattick , j . s ., “ touchdown ” pcr to circumvent spurious priming during gene amplification . nucleic acids res ., 19 : 4008 ( 1991 ).]. in the case of a . terreus cbs116 . 46 additional amplifications with primers 10 [ seq id no : 17 ] and 11 [ seq id no : 18 ] at a final concentration of 1 mm were also done . in all “ touch - down ” reactions first the dna was denatured for 3 min at 95 ° c ., followed by two cycles at each of the following annealing temperatures : 60 ° c ., 59 ° c ., 58 ° c ., 57 ° c ., 56 ° c ., 55 ° c ., 54 ° c ., 53 ° c ., 52 and 51 ° c ., with an annealing time of one min each . prior to annealing the incubation was heated to 95 ° c . for one min and after annealing elongation was performed for 30 sec at 72 ° c . cycles 21 to 35 were performed as follows : denaturation one min at 95 ° c ., annealing one min at 50 ° c . and elongation for 30 sec at 72 ° c . as template , 1 μg of the genomic dna was used in a total reaction mixture of 50 μl . primers aterr21 , aterr58 , terr380 and terr1810 were used to isolate the complete a . terreus cbs 116 . 46 phytase gene by pcr ( see results ). the a . terreus cbs 116 . 46 specific primer , aterr21 ( 5 ′- ctgtcgccgttctgcgacc tc - 3 ′) [ seq id no : 20 ] and aterr 58 ( 5 ′ cggtgccgttgtacagacccagc - 3 ′) [ seq id no : 21 ] were designed using the nucleotide sequence of the two pcr fragments obtained with primers 8 [ seq id no : 15 ] and 9 [ seq id no : 16 ] and primers 10 [ seq id no : 17 ] and 11 [ seq id no : 18 ] on genomic dna of a . terreus cbs116 . 46 ( see fig1 ). primer terr380 ( 5 ′ atgggctttcttgccattgt - 3 ′) [ seq id no : 22 ] and terrl8 lo ( 5 ′ tcagaaacaatccgcccaagtt - 3 ′) [ seq id no : 23 ] are specific for the 5 ′ and 3 ′ of the coding sequence of the phytase gene of a . terreus 9a1 . in all cases an aliquot of the reaction was analysed on 1 . 5 % agarose gel . pcr products of the expected size were excised from the agarose and isolated by centrifugation of the gel slices through siliconized glass wool as described by heery , d . m ., gannon , f . and powell , r ., a simple method for subcloning dna fragments from gel slices . trends . genet ., 6 : 173 ( 1990 ) or using a geneclean kit ( bio101 . inc .) essentially according to the manufacturer &# 39 ; s protocol . the fragment was subsequently cloned 25 into puc 18 using the sure - clone ligation kit ( pharmacia ). southern blot analysis : southern hybridization experiments were performed to construct genomic maps to find appropriate dna fragments carrying the phytase gene . genomic dna ( 3 μg ) was digested with the different restriction enzymes as indicated in the legends of the figures and electrophoresed on a 0 . 75 % agarose gel . the transfer to zeta - probe blotting membranes ( bio - rad ) was done as described in southern , e . m ., detection of specific sequences among dna fraaments separated by gel electrophoresis . j . mol . biol ., 98 : 503 ( 1975 ). prehybridization and hybridization was in 7 % sds , 1 % bsa ( fraction v ; boehringer ), 0 . 5m na 2 hpo 4 , ph 7 . 2 at 65 ° c . probes derived from pcr products of the respective phytase genes ( see fig1 ) were labeled with ( α - 32 p )- dgtp ( amersham ) by random - priming according to sambrook , j ., fritsch , e . f ., maniatis , t ., molecular cloning : a laboratory manual . second edition ed . 1989 , cold spring harbor n . y ., cold spring harbor laboratory press , u . s . a . and used in the hybridization experiments . after hybridization the membranes were washed twice for 5 minutes in 2 × ssc ., 1 % sds at room temperature and twice for 15 minutes in 0 . 1 % ssc ., 0 . 1 % sds at 65 ° c . before exposure o / n on kodak x - omat ar film . library construction : prior to the partial library construction , southern blot analysis with a given probe was done in order to identify a specific restriction fragment of interest . subsequently 10 - 20 μg of genomic dna was digested with the appropriate restriction enzymes and electrophoresed on an agarose gel . according to comigrating dna markers , the region of interest was cut out of the gel , the dna isolated and subcloned into the pbluescriptii —( ks ) vector . transformation of the ligation mixture into e . coli tg - 1 cells resulted in partial genomic libraries carrying the fragment of interest . the genomic a . fumigatus ( nih stock # 5233 ) lambda fixii library was obtained from stratagene ( cat . nr . 946055 ). the size of the cloned fragments , generated by partial sau3ai digestion of genomic dna were in the range of 9 - 22 kb , according to the manufacturer . screening of genomic libraries : e . coli transformants of the partial genomic libraries of a . nidulans , a . terreus cbs 116 . 46 and t . thermophilus were screened using the colony lift assay described in sambrook , j ., fritsch , e . f ., maniatis , t ., molecular cloning : a laboratory manual . second edition ed . 1989 , cold spring harbor n . y ., cold spring harbor laboratory press , u . s . a . and the appropriate probe ( see fig1 ). the a . fumigatus lambda fixii library was screened according to the manufacturer &# 39 ; s instructions using the dna fragment pcrafu as probe . putative positive plaques were cored and subjected to a second round of purification . a clear single positive plaque was picked and used to make a large scale phage preparation as described in sambrook , j ., fritsch , e . f ., maniatis , t ., molecular cloning : a laboratory manual . second edition ed . 1989 , cold spring harbor n . y ., cold spring harbor laboratory press , u . s . a . the analysis of the dna insert and further subcloning steps are outlined herein . dna sequencing : the sequence was determined by the dideoxy chain termination technique described in sanger , f ., nicklen , s . and coulson , a . r ., dna sequencing with chain - terminating inhibitors . proc . natl . acad . sci . usa ., 74 : 5463 - 5467 ( 1977 ) using the sequenase kit ( united states biochemical ). both strands were completely sequenced and the sequence analyzed using the gcg sequence analysis software package ( version 8 . 0 ) by genetics computer , inc . see devereux , j ., haeberli , p . and smithies , o ., a comprehensive set of sequence analysis programs for the vax . nucleic acids res ., 12 : 387 - 395 ( 1984 ). cloning fragments of phytase genes by pcr : to have suitable probes for the library screening , specific dna fragments of the different phytases were obtained by performing pcr on . genomic dna of the individual fungi using degenerate primers as described above . these degenerate pcr amplifications on genomic dna with primers 8 [ seq id no : 15 ] and 9 [ seq id no : 16 ] gave discrete bands of about 120 to 130 bp for a . nidulans , a . fumigatus , t . thermophilus and about 150 bp for a . terreus cbs 116 . 46 . furthermore amplification with primers 10 [ seq id no : 17 ] and 11 [ seq id no : 18 ] on genomic dna of a . terreus cbs116 . 46 gave a amplification product of 220 bp . the sequences of these fragments are shown in fig1 . the a . nidulans phytase : to identify and isolate dna fragments carrying the putative phytase gene the pcr fragment pcrani ( fig1 ) was used to probe a southern blot carrying chromosomal dna of a . nidulans digested with different restriction enzymes ( fig1 ). the 6 kb hindiii / kpni fragment hybridizing to the probe seemed the most suitable for cloning genomic a . nidulans dna was digested with hindiii and kpni and electrophoresed on an agarose gel . according to comigrating dna markers , the region of about 5 - 7 kb was cut out of the gel and the dna isolated . one positive transformant , clone 1 , was chosen and the sequence determined . fig1 shows 1931 nts of the insert [ seq id no : 28 ] carrying the complete phytase gene . the encoded protein [ seq id no : 29 ] consists of 463 amino acids with a theoretical mw of 51785 da and is interrupted by a single intron with a predicted length of 54 nts and positioned close to the 5 ′ of the gene . in the open reading frame ( orf ) upstream of the intron there is one additional potential initiation codon at position 125 - 127 followed by a putative signal peptide , however when the amino acid sequences of all known phytases are aligned the atg at position 158 - 160 is the most likely translation start site . the t . themchiwus phytase : a partial library with bamhi / xbai fragments of about 4 . 7 kb was constructed based on the southern blot analysis results with genomic dna of t . thermophilus ( fig1 ). one positive transformant , clone tt29 , was chosen and sequencing reactions with primer and reverse primer performed . sequencing data showed that the bamhi restriction site was present within the phytase gene and therefore only part of the complete gene had been isolated . comparison of the amino acid sequence to known phytase proteins predicted that we had cloned the c - terminus of the putative phytase gene . to get the missing n - terminus a chromosome walking approach was taken using 5 ′ end of clone tt29 ( a 370 bp bamhi - bsteii fragment ) as probe , to hybridize to t . thermophilus genomic dna double digested with bsteii and a number of other restriction enzymes ( fig1 ). the 4 . 5 kb ecori / bsteii dna fragment identified by the probe was the most appropriate for cloning . genomic dna digested with ecori and bsteii and having a size between 4 and 5 kb was isolated and subcloned into the ecori and bsteii sites of plasmid tt29 - 1 . plasmid tt29 - 1 is a shorter variant of construct tt29 , and was obtained by deleting a 3 . 6 kb . saci fragment . this reduction of the plasmid size was done to avoid instability problems which potentially could arise when cloning the 4 . 5 kb ecori - bsteii fragment into the plasmid tt29 , already containing a 4 . 7 kb dna insert . transformant carrying the tt29 - 132 construct were identified by hybridization to the bamhi - bsteii dna probe . one positive clone carrying the ecori - saci insert of approx . 5 . 5 kb was chosen and the phytase sequence determined . fig1 shows a linear map of the position of the insert of the plasmid clones tt29 , tt29 - 1 and tt29 - 132 . the 1845 nts of the insert tt29 - 132 [ seq id no : 30 ] are shown in fig1 . the phytase gene of t . thermophilus encodes , interrupted by a single intron located similar to the above mentioned phytase genes , a protein [ seq id no : 31 ] of 466 amino acids . the theoretical molecular weight is 51450 da . one additional potential initiation codon is present upstream at position 236 - 238 followed by a putative signal sequence . however based on amino acid homology comparisons to the other phytases the atg at position 288 - 290 is the most likely translation initiation site . the a . fumigatus phytase : screening of 5 . 3 × 10 6 plaques of the a . fumigatus fixii library with the probe pcrafu gave 115 hybridizing plaques . two plaques were picked and subjected to a second round of purification . bacteriophage dna of the two candidates was isolated and digested with noti . the lambda clone having the largest insert ( approx . 15 kb ) was further mapped by restriction analysis and genomic southern . fig1 shows the position of the bamhi fragment within the insert of the bacteriophage lambda clone . the 6 kb bamhi fragment giving a strong signal with the above mentioned probe was isolated , subcloned and part of the sequence encoding the phytase gene was determined . fig1 shows 1571 nts of the insert [ seq id no : 32 ] carrying the complete phytase gene . one single intron of 56 nts is found close to the 5 ′ of the gene . this is in accordance with the aforementioned phytase genes . the enzyme . consists of 465 amino acids [ seq id no : 33 ] with a theoretical mw of 50704 da . the a . terres cbs 116 . 46 phytase : based on the high sequence identity seen between both strains of a . terreus , 9a1 and cbs 116 . 46 , we tried to isolate the phytase gene of the latter strain by pcr , using primers derived from the 5 ′ and 3 ′ of the a . terreus 9a1 sequence ( terr 380 [ seq id no : 22 ] and terr 1810 [ seq id no : 23 ] ) and two internal primers ( aterr 21 [ seq id no : 20 ] and aterr 58 [ seq id no : 21 ]) derived from the a . terreus cbs 116 . 46 dna fragments described in fig1 . fig2 outlines the position of the primers on the phytase gene of a . terreus strain 9a1 and the expected amplification products . only primer atterr 21 [ seq id no : 20 ] and terr 1810 [ seq id no : 23 ] gave a product with an expected size of about 570 bp . the pcr fragment was cloned into the smai site of puc18 , resulting in plasmid puc18 - 569 . sequencing of the insert confirmed that we had cloned the c - terminus of the phytase gene . the missing n - terminus of the gene was cloned basically as described for the other phytases . southern blot analysis of genomic dna of a . terreus cbs - 116 . 46 , with the 570 bp a . terreus cbs116 . 46 dna piece as probe , identified a kpni / kpni fragment of 2 kb carrying the complete phytase gene . the region of about 2 kb was isolated and used to construct a partial genomic library . one e . coli transformant , clone 227 , hybridizing to the probe was then used for further analysis . fig2 shows 1567 nts of the insert [ seq id no : 34 ]. the encoded phytase has 466 amino acids [ seq id no : 35 ] and a theoretical molecular weight of 51054 da . the phytases from aspergillus fumigatus , a . nidulans , a . terreus 9a1 , a . terreus cbs , and myceliophthora thermophila were overexpressed in a . niger or hansenula polymozpha and purified to apparent homogeneity . after removal of the cells , the clear culture supernatant was concentrated by ultrafiltration and subjected to buffer exchange on a fast desalting column hr 10 / 10 ( pharmacia ). final purification was achieved by cation exchange chromatography on either a mono s hr 5 / 5 ( pharmacia ) or a poros hs / m column ( perseptive biosystems ) in the case of a . fumigatus phytase ( fig2 ). the sample was loaded in a buffer containing 10 mm sodium acetate , ph 5 . 0 . a . fumigatus phytase was eluted ith a linear gradient from 10 mm sodium acetate , ph 5 . 0 to 10 mm sodium acetate , 1 m nacl , ph 5 . 0 . anion exchange chromatography on a poros hq / m column ( perseptive biosystems ) was used in the case of the other phytases . in order to corroborate the identities of the purified proteins , samples were separated by sds - page and blotted onto pvdf membranes ( immobilon - p sq , millipore ). n - terminal sequencing of the proteins was done by automated edman degradation on an applied biosystems 494a sequencer with on - line microbore phenylthiohydantoin detection . the results of the n - terminal sequencing are set forth in table 5 . for the determination of the specific activity of the respective proteins , protein concentration was calculated from od 280 according to the theoretical absorption calculated from the amino acid sequences . phytase activity was measured in an assay mixture containing 0 . 5 % phytic acid (˜ 5 mm ), 200 mm sodium acetate , ph 5 . 0 . after a 15 min - incubation period at 37 ° c ., the reaction was stopped by addition of an equal volume of 15 % tca . the liberated phosphate ions were quantified by mixing 100 μl of the assay mixture with 900 μl h 2 o and 1 ml of 0 . 6 m h 2 so 4 , 2 % ascorbic acid and 0 . 5 % ammonium molybdate . standard solutions of potassium phosphate were used as reference . one unit ( u ) is defined as the amount of protein liberating 1 μmol of inorganic phosphate per minute at 37 ° c . for the investigation of the substrate specificities of a . fumigatus , a . nidulans and a . terreus cbs phytase , phytic acid was replaced in the activity assay by 5 mm - concentrations of pnitrophehyl phosphate , phenyl phosphate , fructose 1 , 6 - diphosphate , fructose 6 - phosphate , glucose 6 - phosphate , ribose 5 - phosphate , α - glycerophosphate , β - glycerophosphate , 3 - phosphoglycerate , amp , adp , atp , or nadh . the activities found with these substrates were expressed relative ( in %) to the activity found with phytic acid . the results of . the substrate specificities are set forth in fig2 . cgtgatggct acc atg ggc ttt ctt gcc att gtg ctc tcc gtc gcc ttg 409 cggcgcag c aca tcg ggc acc ccg ttg ggc ccc cgg ggc aaa cat agc 508 gac tgc aac tca gtc gat cac ggc tat caa tgc ttt cct gaa ctc tct 556 asp cys asn ser val asp his gly tyr gln cys phe pro glu leu ser cat aaa tgg gga ctc tac gcg ccc tac ttc tcc ctc cag gac gag tct 604 his lys trp gly leu tyr ala pro tyr phe ser leu gln asp glu ser ccg ttt cct ctg gac gtc cca gag gac tgt cac atc acc ttc gtg cag 652 pro phe pro leu asp val pro glu asp cys his ile thr phe val gln gtg ctg gcc cgc cac ggc gcg cgg agc cca acc cat agc aag acc aag 700 gcg tac gcg gcg acc att gcg gcc atc cag aag agt gcc act gcg ttt 748 ccg ggc aaa tac gcg ttc ctg cag tca tat aac tac tcc ttg gac tct 796 pro gly lys tyr ala phe leu gln ser tyr asn tyr ser leu asp ser gag gag ctg act ccc ttc ggg cgg aac cag ctg cga gat ctg ggc gcc 844 glu glu leu thr pro phe gly arg asn gln leu arg asp leu gly ala cag ttc tac gag cgc tac aac gcc ctc acc cga cac atc aac ccc ttc 892 gln phe tyr glu arg tyr asn ala leu thr arg his ile asn pro phe gtc cgc gcc acc gat gca tcc cgc gtc cac gaa tcc gcc gag aag ttc 940 gtc gag ggc ttc caa acc gct cga cag gac gat cat cac gcc aat ccc 988 val glu gly phe gln thr ala arg gln asp asp his his ala asn pro cac cag cct tcg cct cgc gtg gac gtg gcc atc ccc gaa ggc agc gcc 1036 his gln pro ser pro arg val asp val ala ile pro glu gly ser ala tac aac aac acg ctg gag cac agc ctc tgc acc gcc ttc gaa tcc agc 1084 acc gtc ggc gac gac gcg gtc gcc aac ttc acc gcc gtg ttc gcg ccg 1132 gcg atc gcc cag cgc ctg gag gcc gat ctt ccc ggc gtg cag ctg tcc 1180 ala ile ala gln arg leu glu ala asp leu pro gly val gln leu ser acc gac gac gtg gtc aac ctg atg gcc atg tgt ccg ttc gag acg gtc 1228 thr asp asp val val asn leu met ala met cys pro phe glu thr val agc ctg acc gac gac gcg cac acg ctg tcg ccg ttc tgc gac ctc ttc 1276 acg gcc act gag tgg acg cag tac aac tac ctg ctc tcg ctg gac aag 1324 thr ala thr glu trp thr gln tyr asn tyr leu leu ser leu asp lys tac tac ggc tac ggc ggg ggc aat ccg ctg ggt ccg gtg cag ggg gtc 1372 ggc tgg gcg aac gag ctg atg gcg cgg cta acg cgc gcc ccc gtg cac 1420 gly trp ala asn glu leu met ala arg leu thr arg ala pro val his gac cac acc tgc gtc aac aac acc ctc gac gcg agt ccg gcc acc ttc 1468 asp his thr cys val asn asn thr leu asp ala ser pro ala thr phe ccg ctg aac gcc acc ctc tac gcc gac ttc tcc cac gac agc aac ctg 1516 gtg tcg atc ttc tgg gcg ctg ggc ctg tac aac ggc acc gcg ccg ctg 1564 val ser ile phe trp ala leu gly leu tyr asn gly thr ala pro leu tcg cag acc tcc gtc gag agc gtc tcc cag acg gac ggg tac gcc gcc 1612 gcc tgg acg gtg ccg ttc gcc gct cgc gcg tac gtc gag atg atg cag 1660 ala trp thr val pro phe ala ala arg ala tyr val glu met met gln tgt cgc gcc gag aag gag ccg ctg gtg cgc gtg ctg gtc aac gac cgg 1708 gtc atg ccg ctg cat ggc tgc cct acg gac aag ctg ggg cgg tgc aag 1756 val met pro leu his gly cys pro thr asp lys leu gly arg cys lys cgg gac gct ttc gtc gcg ggg ctg agc ttt gcg cag gcg ggc ggg aac 1804 tgg gcg gat tgt ttc tgatgttgag aagaaaggta gatagatagg tagtacatat 1859 thr ser gly thr pro leu gly pro arg gly lys his ser asp cys asn ser val asp his gly tyr gln cys phe pro glu leu ser his lys trp leu asp val pro glu asp cys his ile thr phe val gln val leu ala thr pro phe gly arg asn gln leu arg asp leu gly ala gln phe tyr glu arg tyr asn ala leu thr arg his ile asn pro phe val arg ala thr asp ala ser arg val his glu ser ala glu lys phe val glu gly ser pro arg val asp val ala ile pro glu gly ser ala tyr asn asn gln arg leu glu ala asp leu pro gly val gln leu ser thr asp asp val val asn leu met ala met cys pro phe glu thr val ser leu thr glu trp thr gln tyr asn tyr leu leu ser leu asp lys tyr tyr gly asn glu leu met ala arg leu thr arg ala pro val his asp his thr ala thr leu tyr ala asp phe ser his asp ser asn leu val ser ile phe trp ala leu gly leu tyr asn gly thr ala pro leu ser gln thr ctc gga gtg atg gtg gtg atg gtc ggc ttc ctg gcg atc gcc tct ct 2263 caa tcc gag tcc cgg cca tgc gac acc cca gac ttg ggc ttc cag tgt 2369 gln ser glu ser arg pro cys asp thr pro asp leu gly phe gln cys ggt acg gcc att tcc cac ttc tgg ggc cag tac tcg ccc tac ttc tcc 2417 gly thr ala ile ser his phe trp gly gln tyr ser pro tyr phe ser gtg ccc tcg gag ctg gat gct tcg atc ccc gac gac tgc gag gtg acg 2465 ttt gcc caa gtc ctc tcc cgc cac ggc gcg agg gcg ccg acg ctc aaa 2513 phe ala gln val leu ser arg his gly ala arg ala pro thr leu lys cgg gcc gcg agc tac gtc gat ctc atc gac agg atc cac cat ggc gcc 2561 atc tcc tac ggg ccg ggc tac gag ttc ctc agg acg tat gac tac acc 2609 ile ser tyr gly pro gly tyr glu phe leu arg thr tyr asp tyr thr ctg ggc gcc gac gag ctc acc cgg acg ggc cag cag cag atg gtc aac 2657 leu gly ala asp glu leu thr arg thr gly gln gln gln met val asn tcg ggc atc aag ttt tac cgc cgc tac cgc gct ctc gcc cgc aag tcg 2705 atc ccc ttc gtc cgc acc gcc ggc cag gac cgc gtc gtc cac tcg gcc 2753 ile pro phe val arg thr ala gly gln asp arg val val his ser ala gag aac ttc acc cag ggc ttc cac tct gcc ctg ctc gcc gac cgc ggg 2801 glu asn phe thr gln gly phe his ser ala leu leu ala asp arg gly tcc acc gtc cgg ccc acc ctc ccc tat gac atg gtc gtc atc ccg gaa 2849 ser thr val arg pro thr leu pro tyr asp met val val ile pro glu acc gcc ggc gcc aac aac acg ctc cac aac gac ctc tgc acc gcc ttc 2897 gag gaa ggc ccg tac tcg acc atc ggc gac gac gcc caa gac acc tac 2945 ctc tcc acc ttc gcc gga ccc atc acc gcc cgg gtc aac gcc aac ctg 2993 leu ser thr phe ala gly pro ile thr ala arg val asn ala asn leu ccg ggc gcc aac ctg acc gac gcc gac acg gtc gcg ctg atg gac ctc 3041 tgc ccc ttc gag acg gtc gcc tcc tcc tcc tcc gac ccg gca acg gcg 3089 gac gcg ggg ggc ggc aac ggg cgg ccg ctg tcg ccc ttc tgc cgc ctg 3137 ttc agc gag tcc gag tgg cgc gcg tac gac tac ctg cag tcg gtg ggc 3185 phe ser glu ser glu trp arg ala tyr asp tyr leu gln ser val gly aag tgg tac ggg tac ggg ccg ggc aac ccg ctg ggg ccg acg cag ggg 3233 gtc ggg ttc gtc aac gag ctg ctg gcg cgg ctg gcc ggg gtc ccc gtg 3281 ttc ccg ctc ggc cgg ccc ctc tac gcc gac ttc agc cac gac aac gac 3377 phe pro leu gly arg pro leu tyr ala asp phe ser his asp asn asp atg atg ggc gtc ctc ggc gcc ctc ggc gcc tac gac ggc gtc ccg ccc 3425 ctc gac aag acc gcc cgc cgc gac ccg gaa gag ctc ggc ggg tac gcg 3473 gcc agc tgg gcc gtc ccg ttc gcc gcc agg atc tac gtc gag aag atg 3521 ala ser trp ala val pro phe ala ala arg ile tyr val glu lys met gag aag gat gag gag atg gtc agg gtg ctg gtg aac gac cgg gtg atg 3617 acg ctg aag ggg tgc ggc gcc gac gag agg ggg atg tgt acg cta gaa 3665 cgg ttc atc gaa agc atg gcg ttt gcg agg ggg aac ggc aag tgg gat 3713 arg phe ile glu ser met ala phe ala arg gly asn gly lys trp asp ala ser leu gln ser glu ser arg pro cys asp thr pro asp leu gly phe gln cys gly thr ala ile ser his phe trp gly gln tyr ser pro tyr phe ser val pro ser glu leu asp ala ser ile pro asp asp cys glu val thr phe ala gln val leu ser arg his gly ala arg ala pro thr leu lys arg ala ala ser tyr val asp leu ile asp arg ile his his gly ala ile ser tyr gly pro gly tyr glu phe leu arg thr tyr met val asn ser gly ile lys phe tyr arg arg tyr arg ala leu ala arg lys ser ile pro phe val arg thr ala gly gln asp arg val val ile pro glu thr ala gly ala asn asn thr leu his asn asp leu cys thr ala phe glu glu gly pro tyr ser thr ile gly asp asp ala gln asp thr tyr leu ser thr phe ala gly pro ile thr ala arg val asn met asp leu cys pro phe glu thr val ala ser ser ser ser asp pro cys arg leu phe ser glu ser glu trp arg ala tyr asp tyr leu gln pro arg thr phe pro leu gly arg pro leu tyr ala asp phe ser his arg val met thr leu lys gly cys gly ala asp glu arg gly met cys thr leu glu arg phe ile glu ser met ala phe ala arg gly asn gly g acc ttg gct cgc aac cac aca gac acg ctg tct ccg ttc tgc gct 46 thr leu ala arg asn his thr asp thr leu ser pro phe cys ala ctt tcc acg caa gag gag tgg caa gca tat gac tac tac caa agt ctg 94 thr leu ala arg asn his thr asp thr leu ser pro phe cys ala leu t acg gta gcg cgc acc agc gac gca agt cag ctg tca ccg ttc tgt 46 thr val ala arg thr ser asp ala ser gln leu ser pro phe cys caa ctc ttc act cac aat gag tgg aag aag tac aac tac ctt cag tcc 94 gln leu phe thr his asn glu trp lys lys tyr asn tyr leu gln ser thr val ala arg thr ser asp ala ser gln leu ser pro phe cys gln leu phe thr his asn glu trp lys lys tyr asn tyr leu gln ser leu c acc atg gcg cgc acc gcc act cgg aac cgt agt ctg tct cca ttt 46 tgt gcc atc ttc act gaa aag gag tgg ctg cag tac gac tac ctt caa 94 cys ala ile phe thr glu lys glu trp leu gln tyr asp tyr leu gln ala ile phe thr glu lys glu trp leu gln tyr asp tyr leu gln ser atg ggc gtc tct gct gtt cta ctt cct ttg tat ctc cta gct gga gtc 48 acc tcc gga ctg gca gtc ccc gcc tcg aga aat caa tcc act tgc gat 96 thr ser gly leu ala val pro ala ser arg asn gln ser thr cys asp acg gtc gat caa ggg tat caa tgc ttc tcc gag act tcg cat ctt tgg 144 thr val asp gln gly tyr gln cys phe ser glu thr ser his leu trp ggt caa tac gcg ccg ttc ttc tct ctg gca aac gaa tcg gtc atc tcc 192 gly gln tyr ala pro phe phe ser leu ala asn glu ser val ile ser cct gat gtg ccc gcc ggt tgc aga gtc act ttc gct cag gtc ctc tcc 240 pro asp val pro ala gly cys arg val thr phe ala gln val leu ser cgt cat gga gcg cgg tat ccg acc gag tcc aag ggc aag aaa tac tcc 288 gct ctc att gag gag atc cag cag aac gtg acc acc ttt gat gga aaa 336 ala leu ile glu glu ile gln gln asn val thr thr phe asp gly lys tat gcc ttc ctg aag aca tac aac tac agc ttg ggt gca gat gac ctg 384 act ccc ttc gga gag cag gag cta gtc aac tcc ggc atc aag ttc tac 432 thr pro phe gly glu gln glu leu val asn ser gly ile lys phe tyr cag cgc tac aac gcc ctc acc cga cac atc aac ccc ttc gtc cgc gcc 480 gln arg tyr asn ala leu thr arg his ile asn pro phe val arg ala acc gat gca tcc cgc gtc cac gaa tcc gcc gag aag ttc gtc gag ggc 528 thr asp ala ser arg val his glu ser ala glu lys phe val glu gly ttc caa acc gct cga cag gac gat cat cac gcc aat ccc cac cag cct 576 tcg cct cgc gtg gac gtg gcc atc ccc gaa ggc agc gcc tac aac aac 624 ser pro arg val asp val ala ile pro glu gly ser ala tyr asn asn acg ctg gag cac agc ctc tgc acc gcc ttc gaa tcc agc acc gtc ggc 672 gac gac gcg gtc gcc aac ttc acc gcc gtg ttc gcg ccg gcg atc gcc 720 cag cgc ctg gag gcc gat ctt ccc ggc gtg cag ctg tcc acc gac 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gln tyr asn tyr leu leu ser leu asp lys tyr tyr gly asn glu leu met ala arg leu thr arg ala pro val his asp his thr ala thr leu tyr ala asp phe ser his asp ser asn leu val ser ile phe trp ala leu gly leu tyr asn gly thr ala pro leu ser gln thr g acg gtc agc ctg acc gac gac gcg cac acg ctg tcg ccg ttc tgc 46 gac ctc ttc acc gcc gcc gag tgg acg cag tac aac tac ctg ctc tcg 94 asp leu phe thr ala ala glu trp thr gln tyr asn tyr leu leu ser c agt aac ctg gtg tcg atc ttc tgg gcg ctg ggt ctg tac aac ggc 46 acc aag ccc ctg tcg cag acc acc gtg gag gat atc acc cgg acg gac 94 thr lys pro leu ser gln thr thr val glu asp ile thr arg thr asp ggg tac gcg gcc gcc tgg acg gtg ccg ttt gcc gcc cgc gcc tac atc 142 gag atg atg cag tgt cgc gcg gag aag cag ccg ctg gta 181 ser asn leu val ser ile phe trp ala leu gly leu tyr asn gly thr lys pro leu ser gln thr thr val glu asp ile thr arg thr asp gly ggggatgagg aggagcaggc tcgtgttcat ttcggtc atg gct ttt ttc acg gtc 175 gct ctt tcg ctt tat tac ttg cta tcg ag gtgagatctc tacaatatct 224 gtctgcttag ttgaattggt acttatctgt acag a gtc tct gct cag gcc cca 277 gtg gtc cag aat cat tca tgc aat acg gcg gac ggt gga tat caa tgc 325 val val gln asn his ser cys asn thr ala asp gly gly tyr gln cys ttc ccc aat gtc tct cat gtt tgg ggt cag tac tcg ccg tac ttc tcc 373 atc gag cag gag tca gct atc tct gag gac gtg cct cat ggc tgt gag 421 ile glu gln glu ser ala ile ser glu asp val pro his gly cys glu gtt acc ttt gtg cag gtg ctc tcg cgg cat ggg gct agg tat ccg aca 469 val thr phe val gln val leu ser arg his gly ala arg tyr pro thr gag tcg aag agt aag gcg tac tcg ggg ttg att gaa gca atc cag aag 517 aat gct acc tct ttt tgg gga cag tat gct ttt ctg gag agt tat aac 565 asn ala thr ser phe trp gly gln tyr ala phe leu glu ser tyr asn tat acc ctc ggc gcg gat gac ttg act atc ttc ggc gag aac cag atg 613 tyr thr leu gly ala asp asp leu thr ile phe gly glu asn gln met gtt gat tcg ggt gcc aag ttc tac cga cgg tat aag aat ctc gcc agg 661 val asp ser gly ala lys phe tyr arg arg tyr lys asn leu ala arg aaa aat act cct ttt atc cgt gca tca ggg tct gac cgt gtc gtt gcg 709 lys asn thr pro phe ile arg ala ser gly ser asp arg val val ala tct gcg gag aag ttc att aat gga ttt cgc aag gct cag ctc cac gac 757 ser ala glu lys phe ile asn gly phe arg lys ala gln leu his asp cat ggc tcc aaa cgt gct acg cca gtt gtc aat gtg att atc cct gaa 805 his gly ser lys arg ala thr pro val val asn val ile ile pro glu atc gat ggg ttt aac aac acc ctg gac cat agc acg tgc gta tct ttt 853 ile asp gly phe asn asn thr leu asp his ser thr cys val ser phe gag aat gat gag cgg gcg gat gaa att gaa gcc aat ttc acg gca att 901 atg gga cct ccg atc cgc aaa cgt ctg gaa aat gac ctc cct ggc atc 949 aaa ctt aca aac gag aat gta ata tat ttg atg gat atg tgc tct ttc 997 lys leu thr asn glu asn val ile tyr leu met asp met cys ser phe gac acc atg gcg cgc acc gcc cac gga acc gag ctg tct cca ttt tgt 1045 asp thr met ala arg thr ala his gly thr glu leu ser pro phe cys gcc atc ttc act gaa aag gag tgg ctg cag tac gac tac ctt caa tct 1093 ala ile phe thr glu lys glu trp leu gln tyr asp tyr leu gln ser cta tca aag tac tac ggc tac ggt gcc gga agc ccc ctt ggc cca gct 1141 cag gga att ggc ttc acc aac gag ctg att gcc cga cta acg caa tcg 1189 gln gly ile gly phe thr asn glu leu ile ala arg leu thr gln ser ccc gtc cag gac aac aca agc acc aac cac act cta gac tcg aac cca 1237 gcc aca ttt ccg ctc gac agg aag ctc tac gcc gac ttc tcc cac gac 1285 ala thr phe pro leu asp arg lys leu tyr ala asp phe ser his asp aat agc atg ata tcg ata ttc ttc gcc atg ggt ctg tac aac ggc acc 1333 cag ccg ctg tca atg gat tcc gtg gag tcg atc cag gag atg gac ggt 1381 tac gcg gcg tct tgg act gtt ccg ttt ggt gcg agg gct tac ttt gag 1429 tyr ala ala ser trp thr val pro phe gly ala arg ala tyr phe glu ctc atg cag tgc gag aag aag gag ccg ctt gtg cgg gta tta gtg aat 1477 gat cgc gtt gtt cct ctt cat ggc tgc gca gtt gac aag ttt gga cgg 1525 asp arg val val pro leu his gly cys ala val asp lys phe gly arg tgc act ttg gac gat tgg gta gag ggc 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leu gly pro ala gln gly ile gly phe thr asn glu leu ile ala arg leu thr gln ser pro val gln asp asn thr ser thr asn his thr leu asp ser asn pro ala thr phe pro leu asp arg lys leu tyr gly leu tyr asn gly thr gln pro leu ser met asp ser val glu ser ile gln glu met asp gly tyr ala ala ser trp thr val pro phe gly ala arg ala tyr phe glu leu met gln cys glu lys lys glu pro leu val asp lys phe gly arg cys thr leu asp asp trp val glu gly leu asn phe ala arg ser gly gly asn trp lys thr cys phe thr leu ttg ttg ctg gtg ctg tcc ggc ggg ttg gtc gcg tta ta gtatgctcct 344 tctctctggt catattgttt tctgctaacg ttctcataat tgaag t gtc tca aga 399 aat ccg cat gtt gat agc cac tct tgc aat aca gtg gaa gga ggg tat 447 asn pro his val asp ser his ser cys asn thr val glu gly gly tyr cag tgt cgt cca gaa atc tcc cac tcc tgg ggc cag tat tct cca ttc 495 gln cys arg pro glu ile ser his ser trp gly gln tyr ser pro phe ttc tcc ctg gca gac cag tcg gag atc tcg cca gat gtc cca cag aac 543 phe ser leu ala asp gln ser glu 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gga cat gac gca cag gaa aag 1023 cys pro val phe glu asp ser ser gly gly his asp ala gln glu lys ttc gca aag caa ttc gca cca gct atc ctg gaa aag atc aag gac cat 1071 ctt ccc ggc gtg gac ctg gcc gtg tcg gat gta ccg tac ttg atg gac 1119 ttg tgt ccg ttt gag acc ttg gct cgc aac cac aca gac acg ctg tct 1167 leu cys pro phe glu thr leu ala arg asn his thr asp thr leu ser ccg ttc tgc gct ctt tcc acg caa gag gag tgg caa gca tat gac tac 1215 pro phe cys ala leu ser thr gln glu glu trp gln ala tyr asp tyr tac caa agt ctg ggg aaa tac tat ggc aat ggc ggg ggt aac ccg ttg 1263 ggg cca gcc caa ggc gtg ggg ttt gtc aac gag ttg att gct cgc atg 1311 gly pro ala gln gly val gly phe val asn glu leu ile ala arg met acc cat agc cct gtc cag gac tac acc acg gtc aac cac act ctt gac 1359 tcg aat ccg gcg aca ttc cct ttg aac gcg acg ctg tac gca gat ttc 1407 agc cac gac aac aca atg acg tca att ttc gcg gcc ttg ggc ctg tac 1455 ser his asp asn thr met thr ser ile phe ala ala leu gly leu tyr aac ggg acc gcg aag ctg 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