Patent Application: US-201214002002-A

Abstract:
this invention provides a description of novel polypeptides and nucleotide sequences having gluco - oligosaccharide oxidase activity . the polypeptides of the invention can be used for enzymatic processes that modify carbohydrates from wood fiber . these polypeptides can be used in the oxidation of c 6 and c 5 mono - and oligomeric sugars . these polypeptides can also be used for the oxidation of glucose , xylose , galactose , nag , xylo - oligosaccharides , cello - oligosaccharides . the novel polypeptides of the invention can be used in a variety of pharmaceutical , agricultural and industrial contexts .

Description:
the inventors of the present application have demonstrated that goox with different substrate specificity were produced by different strains of a . strictum , widening the application of goox from a . strictum for the oxidation of mono - and oligo - saccharides . in addition to glucose , maltose and cello - oligosaccharides , the new goox - vn oxidized xylo - oligosaccharides , galactose , and n - acetylglucosamine . this was not detected in goox from previous studies . y300a and y300n substitutions increased the catalytic activity of goox - vn on all substrates , and gained low activity on mannose . rational engineering approaches are now being applied to decrease the k m of goox - vn and its mutant enzymes on oligomeric substrates . in particular , given the consistency between computational docking analyses and experimental data reported in the current study , docking analyses will be used to predict the effect of selected amino acid substitutions on the binding affinity , conformation , and orientation of substrates bound by goox - vn and variant enzymes . it is anticipated that resulting carbohydrate oxidases will constitute new tools for the quantitative detection and derivatization of carbohydrates . the goox gene cloned from a . strictum type strain cbs 346 . 70 encoded a mature protein containing 474 amino acids , which is the same length as a previously reported goox isolated from a . strictum strain t1 ( hereafter goox - t1 ) ( 13 , 15 ). however , there were 15 amino acid substitutions between the two proteins , 13 resulting from differences in corresponding wild - type gene sequences , and 2 ( a38v and s388n ) resulting from random mutations introduced during the construction of the expression system ( table 6 ). the new goox with v38 and n388 , hereafter goox - vn , shares 97 % sequence identity with the reported goox - t1 ( 13 ), and it has a similar fold to goox - t1 . the recombinant expression of goox - vn in p . pastoris gs115 was highest after three days of incubation with 0 . 5 % methanol . proteins were purified to more than 95 % homogeneity by affinity chromatography ; similar to previous reports of recombinant goox - t1 expression by p . pastoris ( 11 ). approximately 1 . 5 mg l − 1 of purified goox - vn was recovered , and after confirming that one freeze - thaw cycle did not affect enzyme activity , the purified enzyme was stored as 20 μl aliquots (˜ 4 μg ) at − 80 ° c . the enzyme remained active following pre - incubation at 37 ° c . for 60 min ( fig1 ). the deduced molecular mass of the mature protein with a 0 - mg epitope and a polyhistidine tag is approximately 56 kda ( protean , dnastar - lasergene ), which is less than the electrophoretic molecular weight of purified goox - vn (˜ 70 kda ) ( fig1 ). by comparison , the reported molecular weight of goox - t1 determined by size exclusion chromatography is approximately 61 kda ( 13 ). recombinant proteins expressed in p . pastoris g5115 can be n - glycosylated with high - mannose - type structures containing 8 to 14 man residues ( 2 , 9 ). and netnglyc predicted three n - glycosylation sites in goox - vn , including n305 , n341 , and n394 , which are all located in exposed loop regions . still , the molecular weight of deglycosylated goox - vn was ˜ 60 kda , suggesting that other post - translational modifications , including o - glycosylation and / or phosphorylation , probably occurred ( 3 , 4 , 14 ). notably , deglycosylation of goox - vn under native conditions did not cause a detectable loss in enzyme activity ( table 7 ). goox - vn oxidase activity was evaluated using glucose , xylose , galactose , n - acetylglucosamine ( nag ), mannose , and arabinose . glucose , xylose , galactose , and nag were oxidized by the recombinant goox - vn , and the highest catalytic efficiency was observed using glucose ( table 1 ). previous analyses of goox - t1 did not detect activity on xylose , galactose or nag , and activity was limited to glucose and oligosaccharides with reducing end - glucosyl residues ( 5 , 15 ). to check whether goox - vn can oxidize oligomers of c 5 sugars , the enzyme was then tested for oxidation of xylo - oligosaccharides . goox - vn oxidized xylo - oligosaccharides as efficiently as cello - oligosaccharides ( table 1 ), and the catalytic efficiency of goox - vn on these oligosaccharides was over two orders of magnitude higher than that of the corresponding monomers . these findings show that goox - vn has broader substrate specificity than goox - t1 , and goox - vn oxidizes c 6 and c 5 mono - and oligomeric sugars . the broader substrate range of goox - vn detected in the current study compared to previous reports using goox - t1 is unlikely the result of different assay conditions . while reactions for kinetic analyses of goox - vn proceeded for up to 15 min and included substrate concentrations over 500 mm , oxidation of xylose , galactose and nag by goox - vn was detected after 3 min using 10 mm of each sugar , which were the reaction conditions previously used to screen goox - t1 activity ( 13 ). furthermore , the k cat value of the recombinant goox - t1 on maltose is similar to that of goox - vn ( 361 min − 1 and 360 . 0 min − 1 , respectively ) ( 13 ), and goox - t1 oxidation of maltose was used by both lin et al . ( 15 ) and lee et al . ( 13 ) to calculate the relative activity of goox - t1 on other sugars . alternatively , novel substrate specificity of goox - vn is likely due to amino acid substitutions in this enzyme . most substitutions are located on the protein surface or far from the oxidation site ( table 6 ); however , n388 is positioned on the same β16 - sheet as conserved residues q384 and y386 , which are predicted to participate in substrate binding ( 11 ). the side chain of n388 is located near the predicted − 2 subsite , within 6 . 2 å from the substrate . when comparing the x - ray structures of precursor and mature galactose oxidase from fusarium spp ., firbank et al . ( 6 ) showed that the c α of tyr290 moved by 6 . 3 å and the loop containing this residue could shift up to 8 å ( 6 ). while general loop movement was not observed when comparing goox - t1 structures before and after inhibitor binding , the side chain of 5388 in goox - t1 turned significantly upon substrate binding to form a weak h - bond with the g349 backbone of the β15 - sheet ( 11 ). accordingly , the beneficial effect of the s388n substitution on goox - vn activity might be due to the potential of asn to stabilize substrates that contain fewer hydroxyl groups and / or to stabilize the β16 - sheet for substrate binding . docking analysis determined that the computational k d for xylose was two times higher than that for glucose , suggesting that low activity on xylose , which does not possess an exocyclic ch 2 oh , might be due to weak binding of this substrate by goox - vn ( table 8 ). the k m values for di - and tri - saccharides obtained experimentally , as well as the corresponding k d values derived from the docking models , are an order of magnitude lower than the k m and k d values for monosaccharides ( tables 1 , 8 ). these results support the presence of two glycosyl - binding subsites in the carbohydrate - binding groove of goox - vn , which was also predicted by the x - ray structure of goox - t1 ( 11 ). the catalytic activity of goox - vn on monosaccharides and oligosaccharides was further improved through site - directed mutagenesis . amino acids targeted for this analysis were chosen by : 1 ) referencing the published structure of goox - t1 ( 11 ), and 2 ) identifying amino acids in goox - vn that participate in substrate - binding , which consistently differ from corresponding residues in chito from f . graminearum and mnco from m . nivale . y300 and w351 are located at the − 2 glucosyl - binding subsite ( fig8 ), and likely stabilize oligosaccharide binding through stacking interactions . y300 is substituted by alanine in chito and asparagine in mnco while w351 is substituted by phenylalanine in mnco . since mnco is distinguished by its activity on galactose , xylose and to some extent on mannose ( 23 ), altering the polarity and / or size of y300 and w351 could increase the activity of goox on sugars with an axial oh 4 group or that lack an exocyclic ch 2 oh group . accordingly , y300n , y300a and w351f substitutions were generated in goox - vn , and 3 mg l − 1 , 4 mg l − 1 and 1 . 3 mg l − 1 of each purified protein was recovered , respectively . the mutant enzymes remained active after a one hour - pre - incubation at 37 ° c . ( fig1 ). the catalytic activity ( k cat ) of y300a ( table 2 ) and y300n ( table 3 ) mutant enzymes on all tested monosaccharides and oligosaccharides was approximately two times higher than that of goox - vn ( table 1 ). these two mutant enzymes also gained low activity on mannose . however , the loss in hydrophobic interactions at the − 2 subsite also increased the k m and k d values for oligosaccharides , reducing overall catalytic efficiency . these results suggest that y300 affects substrate positioning relative to the catalytic y429 residue and the fad cofactor , and that y300 contributes to stacking interactions with substrates containing more than two units . the w351f mutation slightly reduced the catalytic activity of goox - vn on all substrates . like y300a and y300n mutations , the w351f mutation also increased the k m values of goox - vn with oligomeric substrates ( table 4 ). these results are consistent with both y300 and w351 participating in stabilizing stacking interactions with penultimate reducing sugars of oligomeric substrates , which also explains why the impact of these mutations on k m is similar with di - and tri - saccharides ( table 4 ). notably , the w351f mutation also increased the k m values of goox - vn with glucose and xylose , but decreased the k m of goox - vn with galactose , resulting in higher catalytic efficiency with this substrate ( table 4 ). docking studies showed that while glucose and xylose binding at the active - site was not restricted , the axial oh 4 group of galactose points directly towards the benzene ring of tryptophan ( fig9 ), suggesting that the indole structure hinders goox - vn binding of sugars with axial oh 4 groups . acremonium strictum type strain cbs 346 . 70 was obtained from the american type culture collection ( atcc ) no . 34717 . a . strictum was grown on 1 g ml − 1 food grade wheat bran at 27 ° c . for 5 days , harvested by filtration through miracloth ( calbiochem ), and then flash - frozen using liquid nitrogen . total rna was extracted from the ground sample using the rneasy plant mini kit ( qiagen ). the full - length cdna encoding the goox protein was isolated using the long range 2step rt - pcr kit ( qiagen ), briefly , reverse transcription at 42 ° c . for 90 min was followed by pcr using pfu dna polymerase ( agilent technologies ), gene - specific primers ( 13 ), and 35 cycles of 93 ° c . for 30 s , annealing at 56 ° c . for 40 s , and extension at 72 ° c . for 2 min . the pcr product was purified using the qiaquick pcr purification kit ( qiagen ), and then sequenced at the centre of applied genomics ( tcag , the hospital for sick children ). the goox encoding gene was cloned into the ppiczαa expression vector ( invitrogen ), using ecori and xbai and t4 dna ligase ( invitrogen ). chito - oligosaccharide oxidase , chito , ( accession no . : xp — 391174 ) from fusarium graminearum and a carbohydrate oxidase from microdochium nivale , mnco , ( accession no . : cai94231 - 2 ) were aligned to goox ( accession no . : adi58761 ) using the megalign program ( dnastar - lasergene ) ( fig1 ). amino acids that were predicted to participate in substrate binding , and that varied between the enzymes analyzed , were selected for site - directed mutagenesis . mutations y300a , y300n and w351f were introduced using mutagenic primers ( table 5 ). pcr was performed for 14 cycles of 95 ° c . for 30 s ; 55 ° c . for 1 min ; and 68 ° c . for 5 min , using the quikchange method ( agilent technologies ). the mutations were confirmed by sequencing ( tcag , the hospital for sick children ). mutated plasmids were transformed into pichia pastoris gs115 according to the manufacturer &# 39 ; s instructions ( invitrogen , pichia expression version g ). transformants were selected on buffered minimal methanol medium containing histidine ( bmmh , 100 mm potassium phosphate , ph 6 . 0 ; 1 . 34 % yeast nitrogen base without amino acids ( ynb ); 4 × 10 5 % biotin ; 0 . 5 % methanol , 0 . 004 % histidine ), and then screened for protein expression by immuno - colony blot using nitrocellulose membranes ( 0 . 45 μm , bio - rad ), anti - myc antibodies ( sigma ), alkaline phosphatase - linked anti - rabbit igg conjugates ( sigma ), and 5 ′ bromo - 4 - chloro - 3 - indolyl phosphate nitroblue tetrazolium solution ( bcip / nbt , sigma ). positive transformants were grown overnight in 100 ml of buffered minimal glycerol medium containing histidine ( bmgh , 100 mm potassium phosphate , ph 6 . 0 ; 1 . 34 % ynb ; 4 × 10 5 % biotin ; 1 % glycerol , 0 . 004 % histidine ) at 30 ° c . with continuous shaking at 300 rpm . the cells were harvested by centrifugation at 1 , 500 × g for 10 min and suspended in 300 ml of bmmh medium in 1 l - flasks to od600 ˜ 1 . cultures were grown at 30 ° c . and 300 rpm for 3 days and 0 . 5 % methanol was added every 24 h to induce recombinant protein expression . levels of recombinant protein expression were monitored every 24 h by activity and sos - page . supernatants from methanol - induced cultures of p . pastoris expressing recombinant proteins were harvested by centrifugation at 6 , 000 × g for 10 min and filtration through 0 . 22 μm sterivex filter units ( millipore ). cleared supernatants were concentrated approximately 150 times using centricon concentration units ( millipore ). each recombinant protein was purified using a new ni - nta resin ( qiagen ). fractions were eluted with 250 mm imidazole and the buffer was replaced by 40 mm tris - hcl ( ph 8 . 0 ) using vivaspin6 concentration units ( ge healthcare ). protein concentration measurements were performed using the pierce bca assay ( thermo scientific ) and enzyme purity was verified by sds - page . in - gel trypsin digestion with sequencing - grade trypsin ( promega ), followed by tandem mass spectrometry was performed to confirm the identity of each protein sample . tryptic fragments were analyzed using the applied biosystems / mds sciex api qstar xl pulsar system coupled with an agilent nano hplc ( 1100 series ) ( the advanced protein technology centre , the hospital for sick children ). proteomic data were analyzed using scaffold viewer ( www . proteomesoftware . com ). a chromogenic assay was used to measure hydrogen peroxide production ( 15 ). reactions contained 0 . 1 mm 4aminoantipyrine ( 4aa ), 1 mm phenol , 0 . 5 u horseradish peroxidase , 40 mm tris - hcl ( ph 8 . 0 ), and different substrates were initiated by adding 0 . 2 μg of enzymes to the 250 μl reaction mixture . the production of h2o2 was coupled to the oxidation of 4aminoantipyrine by horseradish peroxidase and detected at 500 nm . reactions were incubated at 37 ° c . for 15 min to measure the specific activity of goox on 10 mm of monosaccharide or 1 mm of oligosaccharide . cello - oligosaccharides were purchased from sigma ( canada ) while xylo - oligosaccharides and manna - oligosaccharides were from megazyme . kinetic parameters were determined with a wide range of substrate concentrations : 0 . 1 mm to 300 mm glucose , 1 mm to 1500 mm xylose , 1 mm to 600 mm galactose , 1 mm to 600 mm n - acetyl - glucosamine ( nag ), 0 . 1 mm to 300 mm maltose , 5 μm to 1 . 5 mm cellobiose , 10 μm to 3 . 5 mm cellotriose , 20 μm to 40 mm xylobiose , and 20 μm to 50 mm xylotriose . at least 12 substrate concentrations were included to obtain kinetic parameters for each substrate . initial rates were obtained by measuring reaction products every 30 s for 15 min at 37 ° c . and ph 8 . 0 , and kinetic parameters were calculated using the michaelis - menten equation ( graphpad prism5 software ). the enzyme stability was evaluated in triplicate by incubating 0 . 6 μg of each enzyme preparation in 40 mm tris - hcl buffer ( ph 8 . 0 ) for 0 , 5 , 15 , 25 , 35 , and 60 min at 37 ° c . residual enzyme activity was measured at 37 ° c . for 15 min at ph 8 . 0 using 10 mm maltose and 0 . 2 μg of protein . approximately 2 μg of purified enzyme was treated with pngasef ( new england biolabs ) using denaturing and native conditions . samples that were deglycosylated using denaturing conditions were analysed by sds page , while samples deglycosylated using native conditions were used to evaluate the impact of glycosylation on enzyme activity . the activity of enzymes was measured on 10 mm maltose at 37 ° c . for 15 min . ( table 7 ). n - glycosylation was predicted by netnglyc ( http :// www . cbs . dtu . dk / services / netnglyc /) while o - glycosylation was predicted by ogpet ( http :// ogpet . utep . edu / ogpet /). the structural model of goox from a . strictum type strain cbs 346 . 70 expressed in pichia was built based on the x - ray structure of a . strictum strain t1 ( pdb id : 2axr ) using the swiss - model workspace ( 1 ). the structures of glucose , cellobiose , cellotriose , xylose , xylobiose , xylotriose and galactose were obtained from the protein database of the research collaboratory for structural bioinformatics ( pdb id : 2fvy , 3eng , 1uyy , x , 1b3w , 1ux7 and 2j1a , respectively ). the program autodocktools 1 . 5 . 2 ran on python 2 . 5 ( http :// autodock . scripps . edu /) was used to prepare the oligosaccharides and the enzyme for docking . all hydrogen atoms were added and the non - polar hydrogens were merged for all ligands and protein . a number of degrees of torsions of each oligosaccharide were set up to evaluate different thermodynamic properties . a lamarckian genetic algorithm ( 16 ) with different number of energy evaluations and a population size of 150 individuals were applied for docking . the program , autogrid 4 , which pre - calculates grip maps of interaction energies , was used to prepare the grid files , and then docking simulation was performed by autodock 4 ( http :// autodock . scripps . edu /). after docking , free energies of binding δgb and dissociation constants kd were reported . temperature stability was measured by incubating 0 . 2 μg of enzyme for 1 h at nine different temperatures ranging from 25 to 60 ° c . ( table 9 ). while goox - vn and the variant goox - v were stable at 45 ° c ., both lost more than 70 % activity after incubation for 1 h at 50 ° c . the residual activity was measured continuously for 15 min at 37 ° c . and ph 8 ( 50 mm tris - hcl ) using 1 mm cellobiose as the substrate , and 0 . 1 mm 4 - aminoantipyrine , 1 mm phenol and 0 . 5 u horseradish peroxidase to form the chromogenic product with absorbance at 500 nm . the ph stability of goox - vn was determined by incubating 0 . 2 μg of the enzyme for 1 h at ph values from ph 3 to 12 . after 1 h of incubation , goox - vn retained more than 80 % activity at ph 5 to ph 10 , 40 % activity at ph 4 , and less than 10 % activity at ph values below 3 or above 11 . finally , the optimum ph for goox - vn activity was determined by incubating 0 . 1 μg of enzyme at 37 ° c . for up to 5 min with 25 mm cellobiose in 25 mm britton - robinson universal buffer solutions at ph 5 to 12 . at regular time points , the chromogenic assay mix containing 400 mm potassium phosphate buffer ph 6 , 0 . 1 mm 4 - aminoantipyrine , 1 mm phenol , 3 u / ml horseradish peroxidase and 40 mm cellobiose was added to the reaction and was incubated for approximately 5 min at 37 ° c ., until the chromogenic compound was detected . this analysis revealed that the ph optimum of goox - vn is ph 10 , similar the optimal ph of goox - t1 ( 5 ). an additional eight mutations , identified as y72f , y72a , e247a , e314a , w351a , n388s , q353n , q384a , were created in goox - vn to assess the influence of these residues on the substrate selectivity of goox - vn . the specific amino acid substitutions were chosen based on substrate docking studies using model structures of the enzyme and substrate . the mutations were generated as previously described ; briefly , pcr with the mutagenic primers was performed for 14 cycles of 95 ° c . for 30 s ; 55 ° c . for 1 min ; and 68 ° c . for 5 min ( table 10 ). each variant was recombinantly expressed in p . pastoris as previously described , and purified to more than 95 % homogeneity by affinity chromatography . the specific activity of each mutant was then measured at 37 ° c . for 15 min using 10 mm mono - sugars and 1 mm oligosaccharides as performed for y300a , y300n , and w351f ( table 11 ). this analysis suggests that substituting the y300 residue may be sufficient to increase the activity of goox - vn on different monomeric sugars and linear oligosaccharides , and that e314 , q353 and 0384 are important to the activity of this enzyme . in several cases , mutations led to loss of activity on glucose and xylose , with retention of activity on corresponding oligomeric substrates . this result suggests that corresponding mutations affect the binding and positioning of sugars rather than catalytic mechanism of goox - vn . to assess the potential of goox - vn to direct subsequent chemical derivatization of cellulosic and hemicellulosic substrates , goox - vn was used to oxidize cellobiose to its acidic form , and then the carboxyl group of oxidized cellobiose was activated by a carbodimide ( n -( 3 - dimethylaminopropyl )- n ′- ethylcarbodiimide hydrochloride ( edac )) before it was derivatized by sulfanilic acid ( sa ) ( 24 ). the oxidation of 200 mm cellobiose by goox - vn ( 0 . 75 μm ) was performed in 50 mm tris - hcl ( ph 8 . 0 ) at 37 ° c . for 24 h to maximize the amount of the oxidized product . the enzyme was then removed from the reaction by centrifugation using nanosep 10k centrifuge filter tubes , and an equal amount of edac and sa ( 83 mm ) was added to the remaining reaction components . the consequent chemical derivatization proceeded in the dark for 2 h at room temperature before final reaction products were analyzed by mass spectrometry . the expected molecular weight of the derivatized product is 512 daltons , and the generation of the derivatized product only after goox - vn treatment was confirmed by mass spectrometry ( fig1 ). it is noted that the activated carboxyl group could also be coupled with other compounds containing other amino groups , including peptide or proteins . further , in addition to detecting the expected product , a new product with mass to charge ratio ( m / z ) of 699 was identified in derivatization reactions containing goox - vn ( fig1 ). based solely on its mass , it is anticipated that this product is a dimer of oxidized cellobiose , suggesting that goox - vn could be developed to increase the degree of polymerization of cellulosic and hemicellulosic compounds , as well as synthesize novel oligosaccharides and / or polysaccharides . the cloned gene encoding goox from acremonium strictum type strain cbs 346 . 70 ( goox - cbs ) has been deposited in the genbank database under accession number gu369974 ( http :// www . ncbi . nlm . nih . gov / nuccore / gu369974 ( submitted jun . 15 2010 )). the foregoing description illustrates only certain preferred embodiments of the invention . the invention is not limited to the foregoing examples . that is , persons skilled in the art will appreciate and understand that modifications and variations are , or will be , possible to utilize and carry out the teachings of the invention described herein . accordingly , all suitable modifications , variations and equivalents may be resorted to , and such modifications , variations and equivalents are intended to fall within the scope of the invention as described and within the scope of the claims . 1 . arnold , k ., l bordoli , j . kopp , and t . schwede . 2006 . the swiss - 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