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Nzira itsva dzekuongorora immune uye mass spectrometric dzekuongorora kwakaomarara kweoligosaccharides dzinogara dziri muchibage dzakagadziriswa neAFEX. Lignocellulosic biomass inzira inochengetedza mafuta ekare uye inoshandiswa zvakanyanya kugadzira biotechnologies yekugadzira zvigadzirwa zvakaita sechikafu, chikafu, mafuta uye makemikari. Chinhu chikuru chehunyanzvi uhu ndechekugadzira maitiro anokwikwidzana ekushandura makabhohaidhiretsi akaoma ari mumadziro emasero emiti kuita shuga dzakareruka dzakadai seglucose, xylose uye arabinose. Nekuti lignocellulosic biomass yakaoma kwazvo, inofanirwa kurapwa ne thermochemical (semuenzaniso, ammonia fiber exfoliation (AFEX), dilute acids (DA), ionic liquids (IL)) uye kurapwa kwe biological (semuenzaniso, enzymatic hydrolysis uye microbial fermentation) pamwe chete kuti uwane chigadzirwa chaunoda. Zvisinei, kana ma enzymes efungus ekutengeserana anoshandiswa mu hydrolysis process, 75-85% chete yeshuga inonyungudika inoumbwa ndiyo monosaccharides, uye 15-25% yasara ma oligosaccharides anonyungudika, asingapindiki, ayo asingawanzo kuwanikwa nezvipembenene. Kare, takakwanisa kubvisa nekuchenesa ma oligosaccharides anonyungudika anonyungudika tichishandisa musanganiswa we carbon ne diatomaceous earth separation uye size exclusion chromatography, uye takaongororawo maitiro avo ekudzivirira enzyme. Takaona kuti ma oligosaccharides ane higher degree of polymerization (DP) methylated uronic acid substitutions akaoma kugadzirisa ne commercial enzyme blends pane low DP ne neutral oligosaccharides. Pano tinoshuma kushandiswa kwemamwe maitiro akati wandei, kusanganisira glycan profiler uchishandisa monoclonal antibodies (mAbs) chaiyo kune plant biomass glycans kuratidza glycan bonds mu plant cell walls uye enzymatic hydrolysates, matrix-assisted laser desorption ionization, time-of-flight mass-spectrometry. MALDI-TOF-MS) inoshandisa structure-informative diagnostic peaks inowanikwa ne spectroscopy mushure mekuora kwechipiri kwema negative ions, gas chromatography uye mass spectrometry (GC-MS) kuratidza oligosaccharide bonds ine uye isina derivatization. Nekuda kwehukuru hudiki hwe oligosaccharides (DP 4–20), mamorekuru aya akaoma kushandisa pakubatanidza uye kuratidza hunhu hwe mAb. Kuti tikunde dambudziko iri, takashandisa nzira itsva ye biotin conjugation-based oligosaccharide immobilization iyo yakabudirira kunyora ruzhinji rwe low DP soluble oligosaccharides pamusoro pe microplate, iyo yakazoshandiswa mu high throughput mAb system yekuongorora kwakanangana ne ligation. Iyi nzira itsva ichaita kuti kuve nekuvandudzwa kwe high throughput glycome assays mune ramangwana inogona kushandiswa kupatsanura uye kuratidza oligosaccharides dziri mu biomarkers nekuda kwezvinangwa zvekuongorora.
Lignocellulosic biomass, inoumbwa nezvinhu zvekurima, zvemasango, zveuswa uye zvehuni, inzvimbo inogona kushandiswa kugadzira zvigadzirwa zvebio-based, zvinosanganisira chikafu, chikafu, mafuta uye makemikari ekugadzira zvigadzirwa zvine mutengo wakakwira1. Makabhohaidhiretsi (akadai secellulose nehemicellulose) ari mumadziro emasero ezvirimwa anobviswa mupolymer kuita monosaccharides nekugadzirwa kwemakemikari uye biotransformation (akadai se enzyme hydrolysis uye microbial fermentation). Kurapa kwakajairika kunosanganisira ammonia fiber expansion (AFEX), dilute acid (DA), ionic liquid (IL), uye steam explosion (SE), izvo zvinoshandisa musanganiswa wemakemikari nekupisa kuderedza kugadzirwa kwelignocellulose nekuvhura madziro emasero ezvirimwa3,4. kuomarara kwezvinhu, 5. Enzymatic hydrolysis inoitwa pahuwandu hwakawanda hwezvakasimba uchishandisa maenzymes ane carbohydrate (CAZymes) uye microbial fermentation uchishandisa yeast transgenic kana mabhakitiriya kugadzira mafuta nemakemikari akavakirwa pabio 6.
MaCAZymes mumaenzyme ekutengesa anoumbwa nemusanganiswa wakaomarara wemaenzymes anoparadzanisa zvisungo zve carbohydrate-shuga kuti zvigadzire monosaccharides2,7. Sezvatakambotaura, network yakaoma yemapolymers e aromatic e lignin nema carbohydrates inoita kuti asanyanya kugadzikana, izvo zvinotungamira mukushandurwa kweshuga kusina kukwana, zvichiunganidza 15-25% ye sex oligosaccharides isingagadzirwe panguva ye enzyme hydrolysis ye pretreated biomass. Iri idambudziko rinowanzoitika nenzira dzakasiyana-siyana dze biomass pretreatment. Zvimwe zvikonzero zveizvi zvinosanganisira kudzivirira enzyme panguva ye hydrolysis, kana kusavapo kana mazinga akaderera ema enzymes akakosha anodiwa kupwanya zvisungo zveshuga mu plant biomass. Kunzwisisa kuumbwa uye hunhu hweshuga, senge zvisungo zveshuga mu oligosaccharides, zvichatibatsira kuvandudza kushandurwa kweshuga panguva ye hydrolysis, zvichiita kuti maitiro e biotechnology ave nemutengo wakaderera nezvigadzirwa zvinobva mu petroleum.
Kuziva chimiro chemakabhohaidhiretsi kwakaoma uye kunoda musanganiswa wenzira dzakadai se liquid chromatography (LC)11,12, nuclear magnetic resonance spectroscopy (NMR)13, capillary electrophoresis (CE)14,15,16 uye mass spectrometry (MS)17. ,gumi nemasere. Nzira dzeMS dzakadai se time-of-flight mass spectrometry ne laser desorption uye ionization uchishandisa matrix (MALDI-TOF-MS) inzira inoshanda yekuziva maumbirwo emakabhohaidhiretsi. Munguva pfupi yapfuura, Collision-Induced Dissociation (CID) tandem MS ye sodium ion adducts yakashandiswa zvakanyanya kuona zvigunwe zvinoenderana nenzvimbo dzekunamatira dzeoligosaccharide, anomeric configurations, sequences, uye branching positions 20, 21.
Kuongorora kweGlycan chishandiso chakanaka chekuziva zvakadzama ma carbohydrate bonds22. Nzira iyi inoshandisa monoclonal antibodies (mAbs) yakanangana ne plant cell wall glycan se probes kuti inzwisise complex carbohydrate linkages. Anopfuura 250 mAbs anowanikwa pasi rese, akagadzirwa kurwisa akasiyana-siyana linear uye branched oligosaccharides achishandisa akasiyana-siyana saccharides24. Ma mAb akati wandei akashandiswa zvakanyanya kuratidza chimiro, kuumbwa, uye shanduko dze plant cell wall, sezvo paine misiyano yakakura zvichienderana nerudzi rwe plant cell, nhengo, zera, danho rekukura, uye nzvimbo yekukura25,26. Munguva pfupi yapfuura, nzira iyi yakashandiswa kunzwisisa vesicle populations mu plant and animals systems uye mabasa avo mu glycan transport sezvinotsanangurwa ne subcellular markers, development stages, kana Environmental stimulis, uye kuona enzyme activity. Mamwe maumbirwo akasiyana eglycans nemaxylans akawanikwa anosanganisira pectin (P), xylan (X), mannan (M), xyloglucans (XylG), mixed bond glucans (MLG), arabinoxylan (ArbX), galactomannan (GalG), glucuronic acid-arabinoxylan (GArbX) uye arabino-galactan (ArbG)29.
Zvisinei, pasinei nekuedza kwese uku kutsvagisa, zvidzidzo zvishoma chete zvakatarisa pamhando yekuunganidzwa kweoligosaccharide panguva yehydrolysis yemafuta akawanda (HSL), kusanganisira kuburitswa kweoligosaccharide, kuchinja kwehurefu hwecheni yeoligomeric panguva yehydrolysis, mapolymer akasiyana-siyana eDP akaderera, uye macurves awo. 30,31,32. Zvichakadaro, kunyangwe ongororo yeglycan yaratidza kuva chishandiso chinobatsira pakuongorora kwakazara kwechimiro cheglycan, zvakaoma kuongorora maoligosaccharide eDP akaderera anonyungudika mumvura uchishandisa nzira dzeantibody. Maoligosaccharide madiki eDP ane huremu hwemamorekuru ari pasi pe5-10 kDa haabatanidzi kumaplates eELISA 33, 34 uye anogezwa asati awedzerwa maantibody.
Pano, kekutanga, tinoratidza bvunzo yeELISA pamaplates akaputirwa neavidin tichishandisa monoclonal antibodies, tichibatanidza nzira imwe chete yebiotinylation ye soluble refractory oligosaccharides ne glycome analysis. Maitiro edu ekuongorora glycome akasimbiswa ne MALDI-TOF-MS ne GC-MS based analysis ye complementary oligosaccharide linkages tichishandisa trimethylsilyl (TMS) derivatization ye hydrolyzed sugar compositions. Iyi nzira itsva inogona kugadzirwa senzira yepamusoro-soro mune ramangwana uye kuwana kushandiswa kwakakura mu biomedical research35.
Kugadziriswa kwema enzymes nema antibodies mushure mekushandurwa, senge glycosylation,36 kunokanganisa mashandiro avo ehupenyu. Semuenzaniso, shanduko mu glycosylation yemapuroteni emuropa inoita basa rakakosha mukuputika kwearthritis, uye shanduko mu glycosylation inoshandiswa sezviratidzo zvekuongorora37. Ma glycans akasiyana-siyana akataurwa mumabhuku kuti anoonekwa nyore nyore muzvirwere zvakasiyana-siyana, kusanganisira zvirwere zvisingaperi zvemudumbu nechiropa, hutachiona hwehutachiona, kenza yemudumbu, yemazamu, uye prostate38,39,40. Kunzwisisa maumbirwo ema glycans uchishandisa nzira dze glycan ELISA dzinoshandisa antibody kuchapa chivimbo chakawedzerwa mukuongororwa kwechirwere pasina kushandisa nzira dzakaoma dzeMS.
Ongororo yedu yapfuura yakaratidza kuti maoligosaccharide akakombama akaramba asina hydrolyzed mushure mekurapwa kwekutanga uye enzyme hydrolysis (Mufananidzo 1). Mubasa redu rakaburitswa kare, takagadzira nzira yekubvisa charcoal solid-phase yakavhurwa kuti tiparadzanise maoligosaccharide kubva kuAFEX-pretreated corn stover hydrolyzate (ACSH)8. Mushure mekubviswa kwekutanga nekupatsanurwa, maoligosaccharide akapatsanurwa zvakare nehukuru hwekubviswa kwechromatography (SEC) uye akaunganidzwa zvichienderana nehuremu hwemamorekuru. Mamonomer eshuga nemaoligomer akaburitswa kubva mukurapa kwakasiyana-siyana akaongororwa nekuongorora kwehupfu. Pakuenzanisa zviri mumasugar oligomers akawanikwa nenzira dzakasiyana-siyana dzekurapa kwekutanga, kuvapo kwemasugar oligosaccharides idambudziko rinowanzoitika mukushandurwa kwebiomass kuita monosaccharides uye kunogona kutungamira mukuderedzwa kwegoho reshuga kweinenge 10-15% uye kunyange kusvika 18%. US. Nzira iyi inoshandiswa pakuwedzera kugadzirwa kukuru kwezvikamu zveoligosaccharide. ACH yakabuda pamwe chete nezvikamu zvayo zvinotevera zvine huremu hwakasiyana hwemamorekuru zvakashandiswa sezvinhu zvekuedza kutsanangura maoligosaccharides mubasa iri.
Mushure mekurapwa kwekutanga uye enzyme hydrolysis, maoligosaccharides anogara asina hydrolyzed. Pano (A) inzira yekuparadzanisa maoligosaccharides umo maoligosaccharides anoparadzaniswa kubva kuAFEX-pretreated corn stover hydrolysate (ACSH) achishandisa mubhedha wakazara we activated carbon uye diatomaceous earth; (B) Nzira yekuparadzanisa maoligosaccharides. Maoligosaccharides akaparadzaniswa zvakare nehukuru hwekubviswa kwechromatography (SEC); (C) Saccharide monomers uye oligomers dzakaburitswa kubva mukurapa kwakasiyana-siyana kwekutanga (diluted acid: DA, ionic liquid: IL uye AFEX). Mamiriro e enzyme hydrolysis: kuzara kwakasimba kwe25% (w/w) (inenge 8% glucan loading), hydrolysis kwemaawa 96, 20 mg/g commercial enzyme loading (Ctec2:Htec2:MP-2:1:1 ratio) uye (D) Mamonomer eshuga uye oligomers eglucose, xylose uye arabinose anoburitswa kubva kuAFEX pre-treated corn stover (ACS).
Kuongororwa kweGlycan kwaratidza kuti inzira inobatsira pakuongorora glycans muzvikamu zvakatorwa kubva muzvimedu zvebiomass. Zvisinei, masaccharides anonyungudika mumvura haana kuratidzwa zvakanaka achishandisa nzira iyi yechinyakare41 nekuti oligosaccharides ane huremu hwakaderera akaoma kudzimisa kufamba pamaplates eELISA uye anosukwa asati awedzerwa maantibody. Nokudaro, pakusunga uye kuratidza maantibody, nzira ye biotinylation yakashandiswa kuputira oligosaccharides anonyungudika, asingabvumirani nemaplates eELISA ane avidin. Nzira iyi yakaedzwa tichishandisa ACSH yedu yatakagadzira kare uye chidimbu chakavakirwa pahuremu hwayo hwemamorekuru (kana dhigirii repolymerization, DP). Biotinylation yeimwe nhanho yakashandiswa kuwedzera oligosaccharide binding affinity nekuwedzera biotin-LC-hydrazide kumucheto wekuderedza wecarbohydrate (Mufananidzo 2). Mukugadzirisa, boka rehemiacetal riri kumucheto wekuderedza rinoita hydrazide group yebiotin-LC-hydrazide kuti rigadzire hydrazone bond. Kana paine chinhu chinodzikisa NaCNBH3, chisungo chehydrazone chinoderedzwa kuita chigadzirwa chekupedzisira chakagadzikana chebiotinylated. Nekushandurwa kwechikamu chinodzikisa shuga, kubatana kweDP oligosaccharides shoma kumaplate eELISA kwakava kunogoneka, uye mukudzidza kwedu izvi zvakaitwa pamaplates akaputirwa neavidin tichishandisa glycan-targetted mAbs.
Kuongororwa kwema antibodies e monoclonal zvichibva pa ELISA kuti uone ma oligosaccharides ane biotinylated. Pano (A) kusanganiswa kwe biotinylation yema oligosaccharides uye kuongororwa kwe ELISA kunotevera ne glycan-targetted mAbs pama NeutrAvidin coated plates uye (B) kunoratidza nzira imwe chete ye biotinylation yezvigadzirwa zve reaction.
Maplate akaputirwa neAvidin ane ma antibodies akasanganiswa neoligosaccharide akabva awedzerwa kuma antibodies ekutanga neechipiri ndokugezwa nenzira ine chiedza uye inonzwa nguva. Mushure mekunge ma antibodies asungwa, wedzera TMB substrate kuti ufukidze ndiro. Mhinduro yacho yakazomiswa nesulfuric acid. Maplate akafukidzwa akaongororwa uchishandisa ELISA reader kuti aone simba rekusunga re antibody yega yega kuti aone antibody-specific cross-linking. Kuti uwane rumwe ruzivo uye parameters dzekuedza, ona chikamu chinoenderana "Zvishandiso uye Nzira".
Tinoratidza kushanda kwenzira iyi itsva yakagadzirwa pakushandisa kwakanangana nekutarisa maoligosaccharides anonyungudika ari muACSH pamwe chete nemaoligosaccharide fractions asina kuchena uye akacheneswa akaparadzaniswa kubva kumahydrolysates elignocellulosic. Sezvakaratidzwa muMufananidzo 3, maxylans anonyanya kushandiswa epitope akaonekwa muACSH achishandisa nzira dzekuongorora glycome assay assay anowanzo kuve uronic (U) kana methyluronic (MeU) uye pectic arabinogalactans. Mazhinji acho akawanikwawo muongororo yedu yapfuura pamusoro pekuongorora maglycans ezvisina kunyungudika (UHS)43.
Kuonekwa kwe recalcitrant oligosaccharide epitopes uchishandisa monoclonal antibody yakanongedzerwa kucell wall glycan. Chikamu "chisina kusimba" ndicho chikamu cheACN uye chikamu "chine acidic" ndicho chikamu cheFA. Matsvuku akajeka pa heatmap anoratidza huwandu hwakawanda hwe epitope, uye mabhuruu akajeka anoratidza kumashure kusina chinhu. Mavara ari pachikero akavakirwa pa raw OD values ​​​​yemafomula N=2. Ma epitopes makuru anozivikanwa nema antibodies anoratidzwa kurudyi.
Maumbirwo aya asiri e-cellulose haagone kupatsanurwa nema cellulases nema hemicellulases akajairika mumusanganiswa wema enzyme ekutengesa akaedzwa, unosanganisira ma enzymes ekutengesa anonyanya kushandiswa. Nokudaro, ma enzymes matsva ekubatsira anodiwa kuti aite hydrolysis. Pasina ma enzymes anodiwa asiri e-cellulose, aya ma bond asiri e-cellulose anodzivirira kushandurwa kwakazara kuita monosaccharides, kunyangwe kana ma polymers eshuga akanyanya kudirwa kuita zvidimbu zvipfupi uye akanyungudutswa achishandisa musanganiswa wema enzyme ekutengesa.
Kuenderera mberi nekudzidza nezvekugoverwa kwezviratidzo uye simba razvo rekubatanidza kwakaratidza kuti ma epitopes ekubatanidza aive akaderera muzvikamu zveshuga zveDP (A, B, C, DP kusvika 20+) pane muzvikamu zveDP zvakaderera (D, E, F, DP) muzvikamu zve dimers) (Mufananidzo 1). Zvidimbu zveasidhi zvakajairika muzvikamu zvisiri zve cellulose kupfuura zvikamu zvisina neutral. Izvi zvinoenderana nepatani yakaonekwa muchidzidzo chedu chekare, uko zvikamu zveDP neacid zvakawanda zvaive zvisingatsungiririke ne enzyme hydrolysis. Nokudaro, kuvapo kwe non-cellulose glycan epitopes uye U neMeU substitutions zvinogona kubatsira zvikuru mukugadzikana kweoligosaccharides. Zvinofanira kucherechedzwa kuti kubatana uye kugona kuona zvinogona kuva dambudziko kune low DP oligosaccharides, kunyanya kana epitope iri dimeric kana trimeric oligosaccharide. Izvi zvinogona kuyedzwa uchishandisa ma oligosaccharides ekutengeserana ane hurefu hwakasiyana, imwe neimwe ine epitope imwe chete inosunga kune mAb chaiyo.
Saka, kushandiswa kwema antibodies chaiwo echimiro kwakaratidza mamwe marudzi e recalcitrant bonds. Zvichienderana nerudzi rweantibody inoshandiswa, patani yakakodzera yekusunga, uye simba rechiratidzo chainoburitsa (chakawanda uye chisina kuwanda), ma enzyme matsva anogona kuonekwa uye kuwedzerwa semi-quantitatively kumusanganiswa we enzyme kuti glycoconversion iwedzere. Tichitora ongororo ye ACSH oligosaccharides semuenzaniso, tinogona kugadzira database yema glycan bonds echinhu chimwe nechimwe che biomass. Zvinofanira kucherechedzwa pano kuti hukama hwakasiyana hwema antibodies hunofanira kufungwa nezvahwo, uye kana hukama hwavo husingazivikanwe, izvi zvichagadzira mamwe matambudziko pakuenzanisa zviratidzo zvema antibodies akasiyana. Pamusoro pezvo, kuenzanisa ma glycan bonds kunogona kushanda zvakanaka pakati pemasampuro eantibody imwechete. Ma stubbonds aya anogona kubatanidzwa neCAZyme database, kwatinogona kuona ma enzymes, kusarudza ma enzymes akakodzera uye kuyedza ma enzymes anoputsa bond, kana kugadzira ma microbial systems ekuratidza ma enzymes aya ekushandisa mu biorefineries44.
Kuti tiongorore kuti nzira dzekudzivirira muviri dzinobatsirana sei nedzimwe nzira dzekutarisa maoligosaccharides ane huremu hwakaderera ari mu lignocellulosic hydrolysates, takaita MALDI (Mufananidzo 4, S1-S8) uye ongororo yeTMS-derived saccharides zvichibva paGC-MS pane imwe panel (Mufananidzo 5) oligosaccharide chikamu. MALDI inoshandiswa kuenzanisa kana kugoverwa kwehuwandu hwemamorekuru eoligosaccharide kuchienderana nechimiro chaunoda. Pamufananidzo 4 inoratidza MC yezvikamu zvisina kusimba ACN-A neACN-B. Ongororo yeACN-A yakasimbisa mhando dzakasiyana dzeshuga dzepentose kubva kuDP 4–8 (Mufananidzo 4) kusvika kuDP 22 (Mufananidzo S1), dzine huremu hunoenderana neMeU-xylan oligosaccharides. Ongororo yeACN-B yakasimbisa pentose neglucoxylan series neDP 8-15. Mune zvimwe zvinhu zvakaita seMufananidzo S3, mamepu ekugovera acidic moiety mass eFA-C anoratidza mhando dzakasiyana dzeshuga dzepentose dzakatsiviwa ne(Me)U dzine DP ye8-15 dzinoenderana ne xylans dzakatsiviwa dzinowanikwa mukuongorora kwemAb kwakavakirwa paELISA. Ma epitopes akafanana.
MALDI-MS spectrum ye oligosaccharides isingabvumirane nemakemikari inonyungudika iri muACS. Pano, (A) ACN-A low weight range fractions ine methylated uronic acid (DP 4-8) yakatsiva glucuroxylan oligosaccharides uye (B) ACN-B xylan uye methylated uronic acid oligosaccharides yakatsiva glucuroxylan (DP 8-15).
Kuongororwa kwechimiro cheglycan residue ye refractory oligosaccharides. Pano (A) TMS saccharide structure yezvikamu zvakasiyana-siyana zve oligosaccharide zvakawanikwa uchishandisa GC-MS analysis. (B) Maumbirwo eshuga dzakasiyana-siyana dzinobva kuTMS dziri mu oligosaccharides. ACN – acetonitrile fraction ine neutral oligosaccharides uye FA – ferulic acid fraction ine acid oligosaccharides.
Imwe mhedziso inonakidza yakawanikwa kubva muongororo yeLC-MS yechikamu cheoligosaccharide, sezvakaratidzwa muMufananidzo S9 (nzira dzinogona kuonekwa mune zvekuwedzera zvemagetsi). Zvidimbu zvemapoka ehexose ne -OAc zvakaonekwa kakawanda panguva yekusunga chikamu cheACN-B. Kuwanikwa uku hakungosimbisi chete kupatsanurwa kwakaonekwa muongororo yeglycome neMALDI-TOF, asiwo kunopa ruzivo rutsva nezvezvinogona kuwanikwa mumacarbohydrate mu biomass ye lignocellulosic yakagadziriswa kare.
Takaongororawo kuumbwa kweshuga kwechikamu cheoligosaccharide tichishandisa TMS shuga inobva mumasero. Tichishandisa GC-MS, takaona kuumbwa kweshuga dzetsinga dzeropa (dzisiri dzinobva mumasero) uye dzine acidic (GluA neGalA) muchikamu cheoligosaccharide (Mufananidzo 5). Glucuronic acid inowanikwa muzvikamu zvine acidic C naD, nepo galacturonic acid inowanikwa muzvikamu zvine acidic A naB, zvese zviri zvikamu zveDP zvine acidic. Mhedzisiro iyi haingosimbisi data redu reELISA neMALDI chete, asiwo inoenderana nezvidzidzo zvedu zvekare zvekuunganidzwa kweoligosaccharide. Nokudaro, tinotenda kuti nzira dzemazuva ano dzekudzivirira muviri dzinoshandisa biotinylation yeoligosaccharides uye kuongorora kweELISA kwakatevera kwakakwana kuona soluble recalcitrant oligosaccharides mumhando dzakasiyana dzebiological.
Sezvo nzira dzekuongorora mAb dzakavakirwa paELISA dzakasimbiswa nenzira dzakasiyana-siyana, taida kuongorora zvakanyanya kugona kwenzira iyi itsva yehuwandu. Maoligosaccharides maviri ekutengeserana, xylohexasaccharide oligosaccharide (XHE) uye 23-α-L-arabinofuranosyl-xylotriose (A2XX), akatengwa uye akaedzwa achishandisa nzira itsva ye mAb yakanangana neglycan yemadziro esero. Mufananidzo 6 unoratidza hukama huripo pakati pechiratidzo chekubatanidza biotinylated uye huwandu hwehuwandu hwe oligosaccharide, zvichiratidza kuti panogona kunge paine muenzaniso weLangmuir adsorption. Pakati pemamAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, uye CCRC-M151 zvakabatana neXHE, uye CCRC-M108, CCRC-M109, uye LM11 zvakabatana neA2XX pamusoro pe1 nm kusvika 100 nano. Nekuda kwekushaikwa kwema antibodies panguva yekuedza, kuyedza kushoma kwakaitwa ne oligosaccharide yega yega. Zvinofanira kucherechedzwa pano kuti mamwe ma antibodies anoita zvakasiyana zvikuru kune oligosaccharide imwechete se substrate, zvichida nekuti anobatana kune epitopes dzakasiyana zvishoma uye anogona kuva nehukama hwakasiyana kwazvo. Maitiro uye zvinorehwa nekuzivikanwa kwe epitope kwakarurama zvichave zvakaoma zvikuru kana nzira itsva ye mAb ikashandiswa kune chaiwo masampuli.
Maoligosaccharide maviri ekutengesa akashandiswa kuona huwandu hwema mAbs akasiyana-siyana anotarisa glycan. Pano, kuwirirana kwakanangana ne log concentration ye oligosaccharide kunoratidza mapatani eLangmuir adsorption e (A) XHE ne mAb uye (B) A2XX ne mAb. Ma epitopes anoenderana anoratidza maumbirwo ema oligosaccharide ekutengesa anoshandiswa se substrates mu assay.
Kushandiswa kwema antibodies e glycan-targetted monoclonal (glycocomic analysis kana ELISA-based mAb screening) chishandiso chine simba chekuongorora zvakadzama ma glycans makuru emasero anoumba biomass yemiti. Zvisinei, ongororo ye glycan yekare inongoratidza ma glycans makuru emasero, sezvo ma oligosaccharides mazhinji asingadzivirirwe zvakanaka pamaplate e ELISA. Muchidzidzo ichi, AFEX-pretreated corn stover yakanyungudutswa ne enzyme ine huwandu hwakawanda hwe solids. Kuongororwa kweshuga kwakashandiswa kuona kuumbwa kwema carbohydrates emasero e recalcitrant mu hydrolyzate. Zvisinei, kuongorora kwe mAb kwema oligosaccharides madiki muma hydrolysates hakukosheswi, uye zvimwe zvishandiso zvinodiwa kuti zvidzivise kufamba kwema oligosaccharides pamaplate e ELISA.
Pano tinopa nzira itsva uye inoshanda yekudzivirira oligosaccharide yekuongorora mAb nekubatanidza biotinylation yeoligosaccharide ichiteverwa nekuongorora ELISA pamaplates akaputirwa neNeutrAvidin™. Maoligosaccharides asina kufamba akaratidza hukama hwakakwana neantibody kuti zvikwanise kuona nekukurumidza uye zvinobudirira recalcitrant oligosaccharides. Kuongororwa kwekugadzirwa kweaya maoligosaccharides akaomarara kwakavakirwa pa mass spectrometry kwakasimbisa mhedzisiro yeiyi nzira itsva yekudzivirira muviri. Saka, zvidzidzo izvi zvinoratidza kuti kusanganiswa kweoligosaccharide biotinylation neELISA screening neglycan-targetted monoclonal antibodies kunogona kushandiswa kuona crosslinks muoligosaccharides uye kunogona kushandiswa zvakanyanya mune zvimwe zvidzidzo zve biochemical zvinoratidza chimiro cheoligosaccharides.
Iyi nzira yekuongorora glycan yakavakirwa pabiotin ndiyo mushumo wekutanga unokwanisa kuongorora ma recalcitrant carbohydrate bonds ema soluble oligosaccharides mu plant biomass. Izvi zvinobatsira kunzwisisa kuti nei zvimwe zvikamu zve biomass zvakaoma kudaro kana zvasvika pakugadzirwa kwe biofuel. Iyi nzira inozadza mukaha wakakosha munzira dzekuongorora glycome uye inowedzera kushandiswa kwayo kune akasiyana-siyana e substrates kupfuura ma plant oligosaccharides. Mune ramangwana, tinogona kushandisa robotics ye biotinylation uye kushandisa nzira yatakagadzira ye high-throughput ongororo yemasampuli tichishandisa ELISA.
Uswa hwechibage (CS) hwakarimwa kubva kumbeu dzakasanganiswa dzePioneer 33A14 hwakakohwewa muna 2010 kubva kuKramer Farms muRay, Colorado. Nemvumo yemuridzi wemunda, biomass iyi inogona kushandiswa pakutsvaga ruzivo. Mienzaniso yacho yakachengetwa yakaoma <6% yehunyoro mumabhegi e-zip-lock mutembiricha yemumba. Mienzaniso yacho yakachengetwa yakaoma <6% yehunyoro mumabhegi e-zip-lock mutembiricha yemumba. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Mienzaniso yakachengetwa yakaoma pahunyoro hwe<6% mumabhegi ane zipi patembiricha yemumba.樣品在室温下以干燥< 6% 的水分储存在自封袋中。样品在室温下以干燥< 6% Образцы хранят в пакетах с застежкой-молнией при комнатной температуре с влажностью < 6%. Mienzaniso inochengetwa mumabhegi ezipi patembiricha yemumba ine hunyoro hunosvika 6%.Chidzidzo ichi chakatevedzera gwara remunharaunda nerenyika. Kuongororwa kwemakemikari kwakaitwa uchishandisa nzira yeNREL. Zvakawanikwa kuti makemikari acho ane 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% protein uye 13.4% ash.
Cellic® CTec2 (138 mg protein/ml, lot VCNI 0001) musanganiswa wakaoma we cellulase, β-glucosidase uye Cellic® HTec2 (157 mg protein/ml, lot VHN00001) kubva kuNovozymes (Franklinton, NC, USA)). Multifect Pectinase® (72 mg protein/mL), musanganiswa wakaoma we pectin degenerative enzymes, wakapihwa neDuPont Industrial Biosciences (Palo Alto, CA, USA). Kuwanda kwema enzyme protein kwakatsanangurwa nekufungidzira huwandu hweprotein (uye kubvisa mupiro we non-protein nitrogen) uchishandisa Kjeldahl nitrogen analysis (AOAC method 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA). Diatomaceous earth 545 yakatengwa kubva kuEMD Millipore (Billerica, MA). Kabhoni yakakangwa (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, nemamwe makemikari ese akatengwa kubva kuSigma-Aldrich (St. Louis, MO).
Kurapa kweAFEX kwakaitwa paGLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). Kurapa kwakaitwa pa140°C kwemaminitsi gumi nemashanu. Nguva yekugara pa1:1 chiyero cheanhydrous ammonia kune biomass pa60% (w/w) loading mu stainless steel benchtop batch reactor (Parr Instruments Company). Zvakatora maminetsi makumi matatu. Reactor yakaunzwa pa140°C uye ammonia yakaburitswa nekukurumidza, zvichiita kuti biomass idzoke nekukurumidza patembiricha yemukamuri. Muumbirwo weAFEX pre-treated corn stover (ACS) wakafanana newe corn stover isina kurapwa (UT-CS).
Zvinhu zvakasimba zvakanyanya ACSH 25% (w/w) (inenge 8% dextran loading) zvakagadzirwa sechinhu chekutanga pakugadzirwa kwemaoligosaccharides akawanda. Enzymatic hydrolysis yeACS yakaitwa uchishandisa musanganiswa we enzyme unosanganisira Cellic® Ctec2 10 mg protein/g glucan (mu biomass yakarapwa kare), Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan, uye Multifect Pectinase (Genencor Inc, USA). ), 5 mg protein/g dextran. Enzymatic hydrolysis yakaitwa mu 5-liter bioreactor ine 3 litres inoshanda, pH 4.8, 50°C uye 250 rpm. Mushure me hydrolysis kwemaawa 96, hydrolyzate yakaunganidzwa ne centrifugation pa 6000 rpm kwemaminitsi 30 uye ipapo pa 14000 rpm kwemaminitsi 30 kubvisa zvinhu zvakasimba zvisina hydrolyzed. Hydrolyzate yakazoseferwa isina utachiona kuburikidza nebeaker re filter re 0.22 mm. Hydrolyzate yakaseferwa yakachengetwa mumabhodhoro asina utachiona pa4°C. yozobva yachekwa pa carbon.
Kuongororwa kwehuwandu hwemasampuli ebiomass akagadzirwa kubva muzvinhu zvakatorwa zvichienderana nemaitiro ekuongorora murabhoritari yeNREL: kugadzirira masampuli ekuongorora huwandu hwemasampuli (NREL/TP-510-42620) uye kuona ma structural carbohydrates uye lignin mu biomass (NREL/TP-510 - 42618)47.
Oligosaccharide ongororo ye hydrolyzate stream yakaitwa pachikero che 2 ml uchishandisa nzira ye autoclave-based acid hydrolysis. Sanganisa hydrolyzate sample ne 69.7 µl ye 72% sulfuric acid mu 10 ml screw cap culture tube woiisa kweawa imwe chete pa benchtop pa 121 °C, woitonhodza pachando woiisa mu high performance liquid chromatography (HPLC) vial. Kuwanda kwe oligosaccharides kwakawanikwa nekubvisa kuwanda kwe monosaccharides mu non-hydrolyzed sample kubva muhuwandu hweshuga mu acid-hydrolyzed sample.
Kuwanda kweglucose, xylose, uye arabinose mu acid hydrolysed biomass kwakaongororwa uchishandisa Shimadzu HPLC system ine autosampler, column heater, isocratic pump, uye refractive index detector pa Bio-Rad Aminex HPX-87H column. Column yakachengetedzwa pa50°C uye yakabviswa ne 0.6 ml/min 5 mM H2SO4 mumvura. flow.
Iyo hydrolyzate supernatant yakasanganiswa uye yakaongororwa kuti ione kana paine monomer neoligosaccharide. Monomeric shuga dzakawanikwa mushure mekunyungudika kweenzyme dzakaongororwa neHPLC dzakashongedzerwa neBio-Rad (Hercules, CA) Aminex HPX-87P column uye ash guard column. Tembiricha ye column yakachengetedzwa pa80°C, mvura yakashandiswa semobile phase ine flow rate ye0.6 ml/min. Oligosaccharides dzakaongororwa ne hydrolysis mu dilute acid pa121°C zvichienderana nenzira dzakatsanangurwa mu refs. 41, 48, 49.
Kuongororwa kweSaccharide kwakaitwa pane zvakasara zvebiomass zvisina kubikwa, zvakagadziriswa kare uye zvisina hydrolyzed (kusanganisira kugadzirwa kwezvakabviswa mumadziro emasero uye kuongorora kwavo mAb) uchishandisa nzira dzakatsanangurwa kare 27, 43, 50, 51. Pakuongorora glycome, zvakasara zvisinganyungudike zvemadziro emasero emiti zvinogadzirwa kubva mumarara ebiomass uye zvinobviswa mumasero zvine simba zvakanyanya senge ammonium oxalate (50 mM), sodium carbonate (50 mM uye 0.5% w/v), CON. (1M ne4M, zvese ne1% w/v sodium borohydride) uye acid chlorite sezvakatsanangurwa kare52,53. Zvakatorwa zvakazoiswa ELISA pakatarisana nepaneru yakaoma ye mAb50s yakanangidzirwa kumadziro emasero, uye mAb binding reactions dzakaratidzwa semepu yekupisa. mAbs inotarisa madziro emasero emiti glycan yakatengwa kubva muzvitoro zverabhoritari (CCRC, JIM neMAC series).
Kubatanidza maoligosaccharides ne biotin-LC-hydrazide kwakaitwa nenzira inotevera. Biotin-LC-hydrazide (4.6 mg/12 μmol) yakanyungudutswa mu dimethyl sulfoxide (DMSO, 70 μl) nekukurunga zvine simba uye kupisa pa 65° C. kwemaminitsi 1. Glacial acetic acid (30 µl) yakawedzerwa uye musanganiswa wacho wakadururwa pa sodium cyanoborohydride (6.4 mg/100 µmol) ndokunyungudutswa zvachose mushure mekudziya pa 65° C. kwemaminitsi angangoita 1. Zvadaro, kubva pa 5 kusvika 8 μl yemusanganiswa we reaction yakawedzerwa ku oligosaccharide yakaomeswa (1-100 nmol) kuti pave ne molar excess yakapetwa ka10 kana kupfuura ye label pamusoro pe reducing end. Reaction yakaitwa pa 65°C kwemaawa maviri, mushure mezvo sampuro dzakabva dzacheneswa ipapo ipapo. Hapana sodium cyanoborohydride yakashandiswa mukuyedza kunyora mazita pasina kuderedza, uye sampuro dzakaitwa pa65°C. kwemaawa maviri nehafu.
Kuputira nekugeza sampuli dzebiotinylated oligosaccharides neELISA. 25 μl yesampuli dzakasanganiswa (100 μl yemuenzaniso wega wega wakasanganiswa mu 5 ml ye 0.1 M Tris buffer solution (TBS)) yakawedzerwa kune rimwe nerimwe tsime replate rakaputirwa neavidin. Matsime ekudzora akaputirwa ne 50 μl yebiotin pahuwandu hwe 10 μg/ml mu 0.1 M TBS. Mvura yakabviswa ion yakashandiswa sejira rekuputira zviyero zvisina chinhu. Piritsi rakaputirwa kwemaawa maviri patembiricha yemumba murima. Geza ndiro katatu ne 0.1% mukaka wakabviswa mafuta mu 0.1 M TBS uchishandisa chirongwa nhamba 11 cheGrenier flat 3A.
Kuwedzera nekusuka ma antibodies ekutanga. Wedzera 40 µl yeantibody yekutanga mutsime rega rega. Isa microplate kweawa imwe chete patembiricha yemukamuri murima. Maplate acho akazosukwa katatu nemukaka we0.1% mu 0.1M TBS uchishandisa chirongwa chekushambidza #11 cheGrenier Flat 3A.
Wedzerai antibody yechipiri mogeza. Wedzerai 50 µl ye antibody yechipiri yembeva/gonzo (yakasanganiswa 1:5000 mumukaka we0.1% mu0.1 M TBS) mutsime rega rega. Isai microplate kweawa imwe chete patembiricha yemukamuri murima. Microplates dzakazogezwa kashanu nemukaka we0.1% mu0.1 M TBS uchishandisa chirongwa cheGrenier Flat 5A plate wash #12.
Kuwedzera substrate. Wedzera 50 µl ye3,3′,5,5′-tetramethylbenzidine (TMB) kune substrate yekutanga (nekuwedzera madonhwe maviri ebuffer, madonhwe matatu eTMB, madonhwe maviri ehydrogen peroxide ku15 ml yemvura yakabviswa ion). Gadzirira substrate yeTMB. uye vortex usati washandisa). Isa microplate patembiricha yemumba kwemaminitsi makumi matatu. Murima.
Pedzisa danho racho woverenga piritsi. Wedzera 50 µl ye1 N sulfuric acid mutsime rega rega wonyora kunyudzwa kwemvura kubva pa450 kusvika 655 nm uchishandisa ELISA reader.
Gadzirira mhinduro dze 1 mg/ml dze analytes idzi mumvura yakabviswa maion: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine. (glcNAc), N-acetylgalactosamine (galNAc), inositol (internal standard). Matanho maviri akagadzirwa nekuwedzera mhinduro dzeshuga dze 1 mg/mL dzakaratidzwa muTafura 1. Samples dzinoomeswa nechando uye dzinoiswa lyophilized pa -80° C. kusvika mvura yese yabviswa (kazhinji maawa angangoita 12-18).
Wedzera 100–500 µg yemuenzaniso kumachubhu ekuvhara akavharirwa pachiyero chekuongorora. Nyora huwandu hwakawedzerwa. Zvakanakisisa kunyungudutsa muenzaniso muhuwandu hwakati hwe solvent woiisa muchubhu semvura. Shandisa 20 µl ye 1 mg/ml inositol sechiyero chemukati chechubhu yega yega yemuenzaniso. Huwandu hwechiyero chemukati chakawedzerwa kumuenzaniso hunofanira kunge hwakafanana nehuwandu hwechiyero chemukati chakawedzerwa kuchubhu yakajairwa.
Wedzera 8 ml ye anhydrous methanol mubhodhoro re screw cap. Wobva waisa 4 ml ye 3 N. methanolic HCl solution, woivhara woizunza. Izvi hazvishandisi mvura.
Wedzera 500 µl ye 1 M HCl methanol solution kumasampuli e oligosaccharide nemachubhu eTMS akajairwa. Masampule akaiswa mu incubation husiku hwese (maawa 168) pa80° C. mu thermal block. Omesa chigadzirwa che methanolysis patembiricha yemumba uchishandisa manifold yekuomesa. Wedzera 200 µl MeOH woomesa zvakare. Maitiro aya anodzokororwa kaviri. Wedzera 200 µl ye methanol, 100 µl ye pyridine uye 100 µl ye acetic anhydride mu sample wosanganisa zvakanaka. Masampule akaiswa mu incubation patembiricha yemumba kwemaminitsi makumi matatu. woomesa. Wedzera 200 µl ye methanol woomesa zvakare.
Wedzera 200 µl yeTri-Sil wobva wapisa chubhu yakavharirwa kwemaminitsi makumi maviri. 80°C, wozoitonhodza kusvika patembiricha yemumba. Shandisa manifold yekuomesa kuti uomese sampuro kusvika pahuwandu hweinenge 50 µl. Zvakakosha kuziva kuti hatina kubvumira sampuro kuti dziome zvachose.
Wedzera 2 ml yehexane wobva wasanganisa zvakanaka nekushandisa vortex. Zadza mativi ePasteur pipettes (5-8 mm) nechidimbu chegirazi wool nekuisa girazi wool pamusoro pepipette ine dhayamita ye5-3/4 inch. Samples dzakaiswa centrifuge pa3000 g kwemaminitsi maviri. Chero zvasara zvisinganyunguduke zvinonyungudika. Omesa sampuli kusvika 100-150 µl. Vhoriyamu inosvika 1 μl yakapinzwa muGC-MS patembiricha yekutanga ye80 °C uye nguva yekutanga yemaminitsi 2.0 (Tafura 2).