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Mitsva yeimmunological uye mass spectrometric nzira dzekuongorora kwakaoma kweanoramba ari oligosaccharides mune chibage stover pretreated neAFEX.Lignocellulosic biomass inzira inogadzika kune zvisaririra uye inoshandiswa zvakanyanya kugadzira biotechnologies yekugadzira zvigadzirwa zvakaita sechikafu, chikafu, mafuta uye makemikari.Chinokosha kune aya matekinoroji kuvandudzwa kwemitengo-yemakwikwi maitiro ekushandura makabhohaidhiretsi akaomesesa aripo muchirimwa masero madziro kuita shuga iri nyore senge glucose, xylose uye arabinose.Nekuti lignocellulosic biomass yakasindimara zvakanyanya, inofanirwa kuve pasi pekurapwa kwe thermochemical (semuenzaniso, ammonia fiber exfoliation (AFEX), dilute acids (DA), ionic zvinwiwa (IL)) uye kurapwa kwebiological (semuenzaniso, enzymatic hydrolysis uye microbial fermentation) musanganiswa kuwana chigadzirwa chaunoda..Zvisinei, kana ma enzymes ekutengesa fungal anoshandiswa mu hydrolysis process, chete 75-85% yeshuga yakanyunguduka inoumbwa monosaccharides, uye iyo yakasara 15-25% inonyunguduka, isingagadziriswi oligosaccharides, iyo isingawanikwi nguva dzose kune microorganisms.Kare, takabudirira kutsaura nekunatsa soluble yakasindimara oligosaccharides tichishandisa musanganiswa wekabhoni uye diatomaceous pasi kupatsanurwa uye saizi yekusabatanidzwa chromatography, uye zvakare kuongorora yavo enzyme inhibitory properties.Takaona kuti oligosaccharides ine yepamusoro dhigirii yepolymerization (DP) methylated uronic acid substitutions inonyanya kuoma kugadzirisa nekutengeserana enzyme blends pane yakaderera DP uye neutera oligosaccharides.Pano tinoshuma kushandiswa kwedzimwe nzira dzakati wandei, kusanganisira glycan profiling uchishandisa monoclonal antibodies (mAbs) yakananga kudyara biomass glycans kuratidza glycan bond muplant cell wall uye enzymatic hydrolysates, matrix-assisted laser desorption ionization, nguva-ye-ndege mass- spectrometry..MALDI-TOF-MS) inoshandisa dhizaini-inozivisa diagnostic peaks inowanikwa nespectroscopy mushure mekuora kwechipiri kweiyoni isina kunaka, gasi chromatography uye mass spectrometry (GC-MS) kuratidza oligosaccharide zvisungo zvine uye pasina kubuda.Nekuda kwehukuru hudiki hwe oligosaccharides (DP 4-20), mamorekuru aya anonetsa kushandisa mAb kusunga uye hunhu.Kuti tikunde dambudziko iri, takashandisa nzira itsva yebiotin conjugation-based oligosaccharide immobilization iyo yakabudirira kunyora ruzhinji rweyakaderera DP soluble oligosaccharides pane microplate pamusoro, iyo yakazoshandiswa mune yakakwira throughput mAb system yekunyatsoongorora ligation.Iyi nzira itsva ichaita kuti kuvandudzwa kweimwe yepamusoro yepamusoro ye glycome assays mune ramangwana inogona kushandiswa kuparadzanisa uye kuratidza oligosaccharides iripo mune biomarkers nekuda kwekuongorora.
Lignocellulosic biomass, inoumbwa nekurima, masango, huswa uye zvinhu zvehuni, inogona kugadzirwa yezvigadzirwa zvebio-based, zvinosanganisira chikafu, chikafu, mafuta uye makemikari precursors kugadzira yakakwira kukosha zvigadzirwa1.Makabhohaidhiretsi (akadai seselulosi uye hemicellulose) ari muchirimwa sero madziro anoiswa mumonosaccharides nekugadziriswa kwemakemikari uye biotransformation (senge enzymatic hydrolysis uye microbial fermentation).Kazhinji pre-kurapwa kunosanganisira ammonia fiber kuwedzera (AFEX), dilute acid (DA), ionic liquid (IL), uye steam kuputika (SE), iyo inoshandisa musanganiswa wemakemikari uye kupisa kuderedza kugadzirwa kwe lignocellulose nekuvhura plant cell wall3,4.kusindimara kwenyaya, 5. Enzymatic hydrolysis inoitwa pamutoro wakasimba uchishandisa bhizimusi rinoshanda carbohydrate-ine enzymes (CAZymes) uye microbial fermentation uchishandisa mbiriso ye transgenic kana mabhakitiriya kugadzira bio-based mafuta uye makemikari 6.
MaCAZymes muma enzymes ekutengesa anoumbwa nemusanganiswa wakaoma wema enzymes kuti synergistically cleave yakaoma carbohydrate-shuga zvisungo kuita monosaccharides2,7.Sezvatakambotaura, iyo yakaoma network yeanonhuhwirira mapolymers e lignin ane makabhohaidhiretsi anoita kuti ive yakanyanya kusagadzikana, izvo zvinotungamira kune isina kukwana kutendeuka kweshuga, kuunganidza 15-25% yebonde oligosaccharides isingabudiswe panguva ye enzymatic hydrolysis ye pretreated biomass.Iri idambudziko rakajairika nenzira dzakasiyana dzebiomass pretreatment.Zvimwe zvikonzero zvebhodhoro iri zvinosanganisira inhibition ye enzyme panguva yehydrolysis, kana kusavapo kana kuderera kwemazinga akakosha ma enzymes anodiwa kutyora shuga zvisungo muchirimwa biomass.Kunzwisisa kuumbwa uye maitiro ehuga hwehuga, zvakadai seshuga zvisungo mu oligosaccharides, zvichatibatsira kuvandudza kushanduka kweshuga panguva ye hydrolysis, zvichiita kuti biotechnological process inodhura-kukwikwidzana ne petroleum-derived products.
Kuona maumbirwo emakabhohaidhiretsi kunonetsa uye kunoda musanganiswa wenzira dzakadai semvura chromatography (LC)11,12, nuclear magnetic resonance spectroscopy (NMR)13, capillary electrophoresis (CE)14,15,16 uye mass spectrometry (MS)17.,gumi nesere.Nzira dzeMS dzakadai senguva-ye-ndege mass spectrometry ine laser desorption uye ionization uchishandisa matrix (MALDI-TOF-MS) inzira inosiyana-siyana yekuziva zvimiro zvecarbohydrate.Munguva ichangopfuura, Collision-Induced Dissociation (CID) tandem MS ye sodium ion adducts yakashandiswa zvakanyanya kuona zvigunwe zvinowirirana ne oligosaccharide attachment positions, anomeric configurations, sequences, uye branching positions 20, 21.
Glycan ongororo chishandiso chakanakisa chekuzivikanwa kwakadzama kwecarbohydrate bonds22.Iyi nzira inoshandisa monoclonal antibodies (mAbs) inonangwa kudyara sero madziro glycan se probes kunzwisisa yakaoma carbohydrate linkages.Anopfuura 250 mAbs anowanikwa pasi rese, akagadzirirwa kupesana neakasiyana mutsara uye branched oligosaccharides achishandisa akasiyana saccharides24.Akawanda maAbs akashandiswa zvakanyanya kuratidza chimiro, kuumbwa, uye kugadziridzwa kwechirimwa cell wall, sezvo paine misiyano yakakura zvichienderana nechirimwa sero rudzi, nhengo, zera, nhanho yekukura, uye nharaunda yekukura25,26.Nguva pfupi yadarika, nzira iyi yakashandiswa kunzwisisa huwandu hwevanhu muzvirimwa nemhuka masisitimu uye mabasa adzo mukutakura glycan sekutsanangurwa kwazvinoitwa ne subcellular marker, nhanho dzebudiriro, kana kusimudzira kwezvakatipoteredza, uye kuona enzymatic chiitiko.Zvimwe zvezvakasiyana zvimiro zve glycans uye xylans zvakaonekwa zvinosanganisira pectin (P), xylan (X), mannan (M), xyloglucans (XylG), yakasanganiswa bond glucans (MLG), arabinoxylan (ArbX), galactomannan (GalG) , glucuronic acid-arabinoxy-XbGGGGN2 uye2Arbinoxylan (9Arbinoxylan).
Zvisinei, pasinei nekuedza kwese uku kwekutsvaga, zvidzidzo zvishomanana chete zvakatarisana nemhando ye oligosaccharide kuunganidza panguva yakakwirira solids load (HSL) hydrolysis, kusanganisira oligosaccharide kusunungurwa, oligomeric chain urefu hunoshanduka panguva ye hydrolysis, yakasiyana-siyana yakaderera DP polymers, uye ma curves avo.kugoverwa 30,31,32.Zvichakadaro, kunyange zvazvo kuongorora kweglycan kwakaratidza kuva chinhu chinobatsira chekuongorora kwakazara kwechimiro cheglycan, zvakaoma kuongorora mvura-inonyunguduka yakaderera DP oligosaccharides uchishandisa nzira dzeantibody.Diki DP oligosaccharides ine molecular uremu isingasviki 5-10 kDa haisungiri kune ELISA mahwendefa 33, 34 uye inoshambidzwa isati yawedzerwa antibody.
Pano, kekutanga, tinoratidza ELISA assay paavidin-coated plates vachishandisa monoclonal antibodies, tichibatanidza imwe-nhanho biotinylation nzira ye soluble refractory oligosaccharides ne glycome analysis.Maitiro edu ekuongorora glycome akasimbiswa neMALDI-TOF-MS uye GC-MS yakavakirwa ongororo yeino wedzera oligosaccharide linkages uchishandisa trimethylsilyl (TMS) inotorwa kubva kune hydrolyzed shuga zvinyorwa.Iyi nzira yekuvandudza inogona kuvandudzwa senzira yepamusoro-soro mune ramangwana uye kuwana kushandiswa kwakakura mukutsvakurudza kwebiomedical35.
Post-translational modifications ye enzymes uye antibodies, zvakadai se glycosylation,36 inokanganisa basa ravo rehupenyu.Semuenzaniso, shanduko mu glycosylation yemapuroteni e serum inoita basa rinokosha mukuputika kweArthritis, uye kuchinja kweglycosylation kunoshandiswa sekuongorora zviratidzo37.Maglycans akasiyana-siyana akave akashumwa mumabhuku kuti aonekwe nyore nyore muzvirwere zvakasiyana-siyana, kusanganisira zvirwere zvisingaperi zvinoputika zvegastrointestinal turakiti uye chiropa, zvirwere zvehutachiona, ovarian, mazamu, uye kenza yeprostate38,39,40.Kunzwisisa chimiro cheglycans uchishandisa antibody-based glycan ELISA nzira ichapa humwe chivimbo mukuongororwa kwechirwere pasina kushandisa nzira dzakaoma dzeMS.
Chidzidzo chedu chekare chakaratidza kuti oligosaccharides yakasindimara yakaramba isina hydrolyzed mushure mekutanga uye enzymatic hydrolysis (Mufananidzo 1).Mubasa redu rakamboburitswa, takagadzira yakabatiswa marasha akasimba-chikamu chekubvisa nzira yekuparadzanisa oligosaccharides kubva kuAFEX-pretreated corn stover hydrolyzate (ACSH)8.Mushure mekutanga kubviswa uye kupatsanurwa, oligosaccharides yakawedzera kukamurwa nehukuru kusarudzika chromatography (SEC) uye yakaunganidzwa mukurongeka kwemamorekuru.Shuga monomers uye oligomers akaburitswa kubva kwakasiyana pretreatment akaongororwa neshuga kuumbwa kwekuongorora.Kana tichienzanisa zvinyorwa zveshuga oligomers inowanikwa nenzira dzakasiyana-siyana dzekugadzirira, kuvapo kweoligosaccharides yakasindimara idambudziko rinowanzoitika mukushandurwa kwebiomass kune monosaccharides uye rinogona kutungamirira kuderedzwa kwegoho reshuga ye10-15% uye kunyange kusvika ku18%.US.Iyi nzira inoshandiswa pakuwedzera kugadzirwa kwakakura kwezvikamu zve oligosaccharide.Izvo zvinokonzerwa neACH uye zvikamu zvaro zvakatevera zvine uremu hwakasiyana hwema molecular zvakashandiswa sechinhu chekuedza chechimiro che oligosaccharides mubasa iri.
Mushure mekutanga uye enzymatic hydrolysis, kuramba oligosaccharides yakaramba isina hydrolysed.Pano (A) inonzi oligosaccharide separation method iyo oligosaccharides inoparadzaniswa kubva kuAFEX-pretreated corn stover hydrolyzate (ACSH) uchishandisa mubhedha wakazara we activated carbon uye diatomaceous pasi;(B) Nzira yekuparadzanisa oligosaccharides.Iyo oligosaccharides yakawedzera kuparadzaniswa nehukuru kusarudzika chromatography (SEC);(C) Saccharide monomers uye oligomers akaburitswa kubva kwakasiyana-siyana pretreatments (diluted acid: DA, ionic liquid: IL uye AFEX).Enzymatic hydrolysis situations: high solids loading ye25% (w/w) (inenge 8% glucan loading), 96 hours hydrolysis, 20 mg/g commercial enzyme loading (Ctec2:Htec2:MP-2:1:1 ratio) uye (D) Sugar monomers uye oligomers yeglucose, xyraafCSA yakabudiswa yeglucose, xyraAFCSA yechibage pre-shinose yakabudiswa.
Ongororo yeGlycan yakaratidza kuve chishandiso chinobatsira chekuongorora kwakadzama kwemaitiro eglycans mune zvakatorwa zvakaparadzaniswa kubva kune yakasimba biomass masara.Nekudaro, saccharides inonyungudika nemvura inomiririrwa nekushandisa iyi nzira yechinyakare41 nekuti yakaderera molecular uremu oligosaccharides inonetsa kusamira pamahwendefa eELISA uye inogezwa kunze pamberi pekuwedzera antibody.Nokudaro, nokuda kwekusungirirwa kweantibody uye maitiro, imwe-nhanho biotinylation nzira yakashandiswa kupfeka oligosaccharides yakanyungudika, isingaenderani pane avidin-coated ELISA mahwendefa.Iyi nzira yakaedzwa tichishandisa yedu yakambogadzirwa ACSH uye chidimbu chinoenderana nehuremu hwayo hwemamorekuru (kana dhigirii repolymerization, DP).Imwe-nhanho biotinylation yakashandiswa kuwedzera oligosaccharide kusunga hukama nekuwedzera biotin-LC-hydrazide kusvika kumagumo ekuderedza carbohydrate (Fig. 2).Mukugadzirisa, boka rehemiacetal pamagumo ekuderedza rinopindirana neboka re hydrazide re biotin-LC-hydrazide kuti rigadzire hydrazone bond.Muhupo hwekudzikisa mumiririri NaCNBH3, hydrazone bond inoderedzwa kusvika kune yakagadzikana biotinylated end product.Nekugadziriswa kwekupedzisira kuderedza shuga, kusungirirwa kweDP oligosaccharides yakaderera kune ELISA mahwendefa zvakave zvichigoneka, uye mukudzidza kwedu izvi zvakaitwa paavidin-coated plates vachishandisa glycan-targeted mAbs.
Kuongororwa kwemonoclonal antibodies yakavakirwa paELISA yebiotinylated oligosaccharides.Pano (A) yakasanganiswa biotinylation ye oligosaccharides uye yakatevera ELISA kuongorora ne glycan-targeted mAbs paNeutrAvidin coated plates uye (B) inoratidza imwe-nhanho nzira ye biotinylation yezvinoita zvigadzirwa.
Avidin-coated plates ane oligosaccharide-conjugated antibodies akabva awedzerwa kune ma antibodies ekutanga uye echipiri uye akashambidzwa muchiedza-uye-nguva-sensitive medium.Mushure mekunge kusungirirwa kwemasoja ekudzivirira chirwere kwapera, wedzera TMB substrate kuti ubaye ndiro.Mhedzisiro yacho pakupedzisira yakamiswa ne sulfuric acid.Mahwendefa akaiswa akaongororwa pachishandiswa ELISA muverengi kuona simba rekusunga remasoja ega ega kuti aone antibody-specific cross-linking.Kuti uwane ruzivo uye ma paramita ekuedza, ona chikamu chinoenderana "Zvishandiso uye Nzira".
Isu tinoratidza kushandiswa kweiyi nzira ichangobva kugadzirwa yezvikumbiro zvakananga kuburikidza nekuratidzira maoligosaccharides anonyungudika aripo muACSH pamwe neakacheneswa uye akacheneswa oligosaccharide zvikamu zvakaparadzaniswa kubva kune lignocellulosic hydrolysates.Sezvinoratidzwa muMufananidzo 3, epitope-inotsiva xylans inowanikwa muACSH vachishandisa bioacylated glycome assay nzira dzinowanzoita uronic (U) kana methyluronic (MeU) uye pectic arabinogalactans.Mazhinji acho akawanikwawo mune yedu yapfuura kudzidza pamusoro pekuongororwa kweglycans yeasina-hydrolyzed solids (UHS) 43.
Kuonekwa kwe recalcitrant oligosaccharide epitopes uchishandisa monoclonal antibody inotungamirirwa kune cell wall glycan.Chikamu "chisina kwazvakarerekera" chikamu cheACN uye chikamu che "acidic" chikamu cheFA.Zvitsvuku zvinopenya pamepu yekupisa zvinoratidza zvemukati epitope, uye mabhururu anopenya anoratidza kuseri kusina chinhu.Rudzi rwemavara pachiyero chakavakirwa pane yakasvibirira OD tsika dzemaumbirwo N = 2.Maepitopes makuru anozivikanwa nemasoja ekudzivirira chirwere anoratidzwa kurudyi.
Aya maitiro asiri e-cellulose haaigona kutsemurwa nema cellulases akajairika uye hemicellulases mumusanganiswa wekutengesa enzyme musanganiswa, unosanganisira anonyanya kushandiswa ekutengesa enzymes.Naizvozvo, ma enzyme matsva ekubatsira anodiwa kune yavo hydrolysis.Pasina anodiwa asiri-cellulose accessory enzymes, aya asiri-cellulose mabhondi anodzivirira kutendeuka kwakazara kune monosaccharides, kunyangwe kana mubereki wavo mapolymers eshuga akaiswa hydrolyzed kuita zvimedu zvipfupi uye akanyungudutswa vachishandisa musanganiswa weenzayimu yekutengeserana.
Kuwedzera kudzidza kwekugovera kwechiratidzo uye simba rayo rekusunga kwakaratidza kuti kusunga epitopes kwaive kwakaderera muzvikamu zvepamusoro zveDP shuga (A, B, C, DP kusvika ku20 +) kupfuura muzvikamu zvakaderera zveDP (D, E, F, DP) mu dimers) (Fig. 1).Zvimedu zveAcid zvinowanzoonekwa mune zvisiri-cellulose epitopes pane zvimedu zvisina kwazvakarerekera.Izvi zviitiko zvinopindirana nemaitiro akaonekwa muchidzidzo chedu chekare, uko yakakwira DP uye acid moieties yainyanya kurwisa enzymatic hydrolysis.Nokudaro, kuvapo kwe-non-cellulose glycan epitopes uye U neMeU zvinotsiva zvinogona kubatsira zvikuru kugadzikana kwe oligosaccharides.Zvinofanira kucherechedzwa kuti kusunga uye kuona kushanda zvakanaka kunogona kunetsa kune yakaderera DP oligosaccharides, kunyanya kana epitope iri dimeric kana trimeric oligosaccharide.Izvi zvinogona kuongororwa uchishandisa maoligosaccharides ekutengeserana ehurefu hwakasiyana, imwe neimwe iine epitope imwe chete inosunga kune chaiyo mAb.
Saka, kushandiswa kwemaitiro-chaiwo masoja ekudzivirira chirwere akaburitsa mamwe marudzi erecalcitrant bond.Zvichienderana nemhando yeantibody inoshandiswa, iyo yakakodzera ligation pateni, uye simba rechiratidzo chainoburitsa (chakawanda uye chishoma chakawanda), ma enzymes matsva anogona kuzivikanwa uye kuwedzerwa semi-quantitatively kumusanganiswa we enzyme kuti iwedzere kuzere glycoconversion.Tichitora kuongororwa kweACSH oligosaccharides semuenzaniso, tinogona kugadzira dhatabhesi yeglycan bond kune yega biomass zvinhu.Zvinofanira kucherechedzwa pano kuti hukama hwakasiyana hwemasoja ekudzivirira chirwere hunofanirwa kuverengerwa, uye kana hukama hwavo husingazivikanwe, izvi zvinogadzira mamwe matambudziko kana uchienzanisa masaini emasoja akasiyana.Pamusoro pezvo, kuenzanisa kweglycan bond kunogona kushanda zvakanyanya pakati pemasampuli eiyo antibody yakafanana.Izvi zvisungo zvakasindimara zvinokwanisa kubatanidzwa nedatabase yeCAZyme, kubva kwatinogona kuziva ma enzymes, sarudza ma enzymes uye kuyedza ma enzymes anopwanya chisungo, kana kugadzira ma microbial system kuratidza ma enzymes aya kuti ashandiswe mu biorefineries44.
Kuti tiongorore kuti nzira dzekudzivirira zvirwere dzinopindirana sei nedzimwe nzira dzekuratidza pasi molecular weight oligosaccharides iripo mu lignocellulosic hydrolysates, takaita MALDI (Fig. 4, S1-S8) uye kuongorora kwe-TMS-yakagadzirwa saccharides inobva kuGC-MS pane imwechete panel (Fig. 5) oligosaccharide chikamu.MALDI inoshandiswa kuenzanisa kana kuwanda kweiyo oligosaccharide mamorekuru inofanana neyakarongwa chimiro.Pamusoro pemuonde.4 inoratidza MC yezvakarerekera zvikamu ACN-A uye ACN-B.ACN-A kuongororwa kwakasimbisa huwandu hwehuga hwepentose kubva kuDP 4-8 (Fig. 4) kusvika kuDP 22 (Fig. S1), iyo inorema inofanana neMeU-xylan oligosaccharides.ACN-B ongororo yakasimbisa pentose uye glucoxylan akatevedzana ane DP 8-15.Mune zvekuwedzera senge Figure S3, FA-C acidic moiety mass kugovera mepu inoratidza huwandu hwe (Me) U inotsiva pentose shuga ine DP ye8-15 inowirirana neinotsiviwa xylans inowanikwa muELISA-based mAb kuongorora.Epitopes inowirirana.
MALDI-MS spectrum ye soluble non-compliant oligosaccharides iripo mu ACS.Pano, (A) ACN-A low weight range zvidimbu zvine methylated uronic acid (DP 4-8) inotsiva glucuroxylan oligosaccharides uye (B) ACN-B xylan uye methylated uronic acid oligosaccharides inotsiviwa neglucuroxylan (DP 8-15).
Kuongororwa kwekuumbwa kweglycan yakasara ye refractory oligosaccharides.Pano (A) TMS saccharide kuumbwa kwezvikamu zvakasiyana-siyana zveoligosaccharide zvakawanikwa uchishandisa GC-MS kuongorora.(B) Zvimiro zvemhando dzakasiyana-siyana dzeTMS-yakatorwa shuga dziripo mune oligosaccharides.ACN - acetonitrile chikamu chine neutral oligosaccharides uye FA - ferulic acid chikamu chine acid oligosaccharides.
Imwe mhedziso inofadza yakatorwa kubva kuLC-MS kuongororwa kwechikamu che oligosaccharide, sezvinoratidzwa muMufananidzo S9 (nzira dzinogona kuonekwa mumagetsi ekuwedzera).Zvimedu zvehexose uye -OAc mapoka zvakadzokororwa zvakaonekwa panguva yekubatanidza chikamu che ACN-B.Kuwana uku hakungotsigiri kupatsanurwa kunoonekwa mukuongorora kweglycome uye MALDI-TOF, asi kunopawo ruzivo rutsva pamusoro pezvingangove zvinobva kune kabhohaidhiretsi mu pretreated lignocellulosic biomass.
Isu takaongorora zvakare kuumbwa kweshuga kwechikamu che oligosaccharide tichishandisa TMS shuga derivatization.Tichishandisa GC-MS, takasarudza kuumbwa kweneural (isina-derivative) uye acidic shuga (GluA uye GalA) muchikamu che oligosaccharide (Fig. 5).Glucuronic acid inowanikwa mu acidic components C uye D, nepo galacturonic acid inowanikwa mu acidic components A ne B, zvese zviri high DP zvikamu zve acidic sugar.Iyi mhedzisiro haingosimbisi yedu ELISA neMALDI data, asi zvakare inopindirana nezvidzidzo zvedu zvekare zvekuunganidza oligosaccharide.Nokudaro, isu tinotenda kuti nzira dzemazuva ano dzekudzivirira zvirwere dzinoshandisa biotinylation ye oligosaccharides uye inotevera ELISA kuongorora inokwana kuona soluble recalcitrant oligosaccharides mune zvakasiyana-siyana zvehupenyu sampuli.
Sezvo ELISA-yakavakirwa mAb nzira dzekuongorora dzakasimbiswa nenzira dzinoverengeka dzakasiyana, isu taida kuenderera mberi nekuongorora kugona kweiyi nzira itsva yehuwandu.Oligosaccharides maviri ekutengeserana, xylohexasaccharide oligosaccharide (XHE) uye 23-α-L-arabinofuranosyl-xylotriose (A2XX), akatengwa uye akaedzwa achishandisa nzira itsva mAb inotarisa sero wall glycan.Mufananidzo 6 unoratidza kuwirirana kwemutsara pakati peiyo biotinylated inosunga chiratidzo uye iyo yerogi yekumisikidza oligosaccharide concentration, ichikurudzira inogona kuitika yeLangmuir adsorption modhi.Pakati pemAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, uye CCRC-M151 yakabatana neXHE, uye CCRC-M108, CCRC-M109, uye LM11 yakabatana neA2XX pamusoro pehuwandu hwe100 nmno.Nekuda kwekuwanikwa kushoma kwemasoja ekudzivirira chirwere panguva yekuedza, zvishoma zviedzo zvakaitwa nega yega oligosaccharide concentration.Zvinofanira kucherechedzwa pano kuti mamwe masoja ekudzivirira chirwere anoita zvakasiyana zvakanyanya kune imwechete oligosaccharide se substrate, pamwe nekuti anosunga kune zvishoma zvakasiyana epitopes uye anogona kuve akasiyana zvakanyanya anosunga affinities.Maitiro uye zvinorehwa neiyo epitope yekuzivikanwa ichave yakaoma zvakanyanya kana iyo itsva mAb nzira inoshandiswa kune chaiyo sampuli.
Maoligosaccharides maviri ekutengesa akashandiswa kuona huwandu hwekuonekwa kweakasiyana glycan-targeting mAbs.Pano, kuwirirana kwemutsara ne log concentration ye oligosaccharide concentration inoratidza Langmuir adsorption maitiro e (A) XHE ine mAb uye (B) A2XX ine mAb.Iwo epitopes anowirirana anoratidza zvimiro zvekutengesa oligosaccharides anoshandiswa se substrates muyedzo.
Kushandiswa kweglycan-targeted monoclonal antibodies (glycocomic analysis kana ELISA-based mAb screening) chinhu chine simba chekudzika-dzika kwemaitiro akawanda emasero makuru emadziro glycans anoumba biomass yemiti.Nekudaro, classical glycan kuongororwa inongoratidzira hombe sero madziro glycans, sezvo mazhinji oligosaccharides asina kunyatso gadziriswa pamahwendefa eELISA.Muchidzidzo ichi, AFEX-pretreated corn stover yaive enzymatically hydrolyzed pane yakakwira solids yemukati.Kuongororwa kweshuga kwakashandiswa kuona kuumbwa kwerecalcitrant cell wall carbohydrates mu hydrolyzate.Nekudaro, mAb kuongororwa kwediki oligosaccharides mu hydrolysates inotarisirwa, uye mamwe maturusi anodiwa kuti anyatso immobilize oligosaccharides pamahwendefa eELISA.
Isu tinoshuma pano inoveli uye inoshanda oligosaccharide immobilization nzira yemAb yekuongorora nekubatanidza oligosaccharide biotinylation inoteverwa neELISA kuongorora paNeutrAvidin™ yakavharwa mahwendefa.Iyo immobilized biotinylated oligosaccharides yakaratidza hukama hwakakwana kune antibody kuti igone kukurumidza uye inobudirira kuona kwe recalcitrant oligosaccharides.Ongororo yekuumbwa kweaya akasindimara oligosaccharides akavakirwa pamisa spectrometry yakasimbisa mhedzisiro yeiyi nzira nyowani yekudzivirira muviri.Nokudaro, zvidzidzo izvi zvinoratidza kuti kusanganiswa kwe oligosaccharide biotinylation uye ELISA kuongorora ne glycan-targeted monoclonal antibodies inogona kushandiswa kuona crosslinks mu oligosaccharides uye inogona kushandiswa zvakanyanya mune zvimwe zvidzidzo zvebiochemical zvinoratidza chimiro che oligosaccharides.
Iyi biotin-based glycan profiling nzira ndiyo yekutanga rondedzero inokwanisa kuongorora recalcitrant carbohydrate zvisungo zve soluble oligosaccharides muchirimwa biomass.Izvi zvinobatsira kunzwisisa kuti sei zvimwe zvikamu zvebiomass zvakasindimara kana zvasvika pakugadzirwa kwebiofuel.Iyi nzira inozadza gaka rinokosha mune nzira dzekuongorora glycome uye inowedzera kushandiswa kwayo kune yakawanda yakawanda ye substrates kupfuura chirimwa oligosaccharides.Mune ramangwana, tinogona kushandisa marobhoti ebiotinylation uye toshandisa nzira yatakagadzira yekuongororwa kwepamusoro-soro yemasampuli tichishandisa ELISA.
Chibage mashanga (CS) akarimwa kubva kuPioneer 33A14 mbeu dzakauchikwa zvakakohwewa muna 2010 kubva kuKramer Farms kuRay, Colorado.Nemvumo yemuridzi wevhu, iyi biomass inogona kushandiswa kutsvaga. Iwo masampuli akachengetwa akaoma <6% mwando mumabhegi e-zip-lock mune tembiricha yekamuri. Iwo masampuli akachengetwa akaoma <6% mwando mumabhegi e-zip-lock mune tembiricha yekamuri. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Samples dzakachengetwa dzakaoma pa <6% humidity mumabhegi akaiswa zippered pakamuri tembiricha.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%. Samples inochengetwa mumabhegi ezipi patembiricha yekamuri ine hunyoro <6%.Chidzidzo ichi chaitevedzera gwara renzvimbo nemunyika.Compositional analysis yakaitwa uchishandisa NREL protocol.Kuumbwa kwacho kwakawanikwa kuine 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% protein uye 13. 4% madota.
Cellic® CTec2 (138 mg protein/ml, lot VCNI 0001) isanganiswa yakaoma ye 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 inoshatisa enzymes, yakapihwa neDuPont Industrial Biosciences (Palo Alto, CA, USA).Enzyme protein concentrations yakatemwa nekufungidzira maprotein content (uye kubvisa mupiro wenon-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).Activated carbon (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, nemamwe makemikoro ese akatengwa kubva kuSigma-Aldrich (St. Louis, MO).
AFEX pretreatment yakaitwa kuGLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA).Pre-kurapwa kwakaitwa pa 140 ° C. kwemaminitsi gumi nemashanu.46 nguva yekugara pa1:1 reshiyo yeanhydrous ammonia kune biomass pa60% (w/w) ichitakura musimbi isina tsvina yebhenji rebhenji reactor (Parr Instruments Company).Zvakatora maminitsi makumi matatu.Iyo reactor yakaunzwa ku140 ° C uye ammonia yakakurumidza kuburitswa, ichibvumira iyo biomass kuti ikurumidze kudzoka kune tembiricha yekamuri.Kuumbwa kweAFEX pre-treated corn stover (ACS) yakanga yakafanana neyeiyo isina kubatwa corn stover (UT-CS).
High solids ACSH 25% (w/w) (inenge 8% dextran loading) yakagadzirirwa sechinhu chekutanga chekugadzirwa kukuru kweoligosaccharides.Enzymatic hydrolysis ye ACS yakaitwa uchishandisa musanganiswa we enzyme yekutengeserana kusanganisira Cellic® Ctec2 10 mg protein / g glucan (mu pretreated biomass), Htec2 (Novozymes, Franklinton, NC), 5 mg protein / g glucan, uye Multifect Pectinase (Genencor Inc, USA).)), 5 mg protein/g dextran.Enzymatic hydrolysis yakaitwa mu5-litre bioreactor ine vhoriyamu yekushanda ye3 litres, pH 4.8, 50 ° C uye 250 rpm.Mushure me hydrolysis kwemaawa 96, iyo hydrolyzate yakaunganidzwa ne centrifugation pa 6000 rpm kwemaminitsi makumi matatu uye ipapo pa 14000 rpm kwemaminitsi makumi matatu kubvisa unhydrolysed solids.Iyo hydrolyzate yakabva yaiswa mukusefa isina kuchena kuburikidza ne 0.22 mm sefa beaker.Iyo hydrolyzate yakasefa yaichengetwa mumabhodhoro asina utachiona pa4° C. yobva yapatsanurwa pacarbon.
Ongororo yekuumbwa kweakatorwa-based biomass samples zvinoenderana neNREL laboratory analysis maitiro: kugadzirira kwemasamples ekuongorora kuumbwa (NREL/TP-510-42620) uye kutsunga kwemaitiro carbohydrates uye lignin mubiomass (NREL/TP-510 - 42618)47.
Oligosaccharide kuongororwa kwemvura yehydrolyzate yakaitwa pachiyero che 2 ml uchishandisa autoclave-based acid hydrolysis nzira.Sanganisa sampu yehydrolyzate ne 69.7 µl ye72% sulfuric acid mu10 ml screw cap culture chubhu uye incuite kweawa imwe pabhenji pa121 °C, inotonhorera paaizi uye kusefa mubhodhoro remvura rinoshanda kwazvo (HPLC) .Iko kusanganiswa kwe oligosaccharides kwakagadziriswa nekubvisa huwandu hwemonosaccharides mumuenzaniso usina-hydrolyzed kubva kuhuwandu hwehuga hwehuga mumuenzaniso we-acid-hydrolyzed.
Glucose, xylose, uye arabinose concentrations mu acid hydrolysed biomass yakaongororwa pachishandiswa Shimadzu HPLC system ine autosampler, column heater, isocratic pump, uye refractive index detector pane Bio-Rad Aminex HPX-87H column.Iyo column yakachengetedzwa pa 50 ° C uye yakasvibiswa ne 0.6 ml / min 5 mM H2SO4 mumvura.kuyerera.
Iyo hydrolyzate supernatant yakaderedzwa uye yakaongororwa kune monomer uye oligosaccharide zvemukati.Monomeric shuga yakawanikwa mushure me enzymatic hydrolysis yakaongororwa neHPLC ine Bio-Rad (Hercules, CA) Aminex HPX-87P column uye ash guard column.Iyo tembiricha yekoramu yakachengetedzwa pa80 ° C, mvura yakashandiswa sechikamu chekufambisa ine kuyerera kwe 0.6 ml / min.Oligosaccharides yakagadziriswa ne hydrolysis mu dilute acid pa 121 ° C maererano nenzira dzinotsanangurwa mu refs.41, 48, 49.
Kuongororwa kweSaccharide kwakaitwa pane mbishi, AFEX pre-yakagadziriswa uye yese isina-hydrolysed biomass residues (kusanganisira kugadzirwa kwe serial cell wall extracts uye yavo mAb kuongorora) vachishandisa zvakambotsanangurwa maitiro 27, 43, 50, 51.Kuongorora glycome, doro-isina kunyungudika zvechirimwa cell wall zvinhu zvinogadzirwa kubva kune biomass zvisaririra uye zvichiiswa pasiri kudhirowa nekuwedzera hukasha reagents senge ammonium oxalate (50 mM), sodium carbonate (50 mM uye 0.5% w/v), CON.(1M uye 4M, zvose zvine 1% w/v sodium borohydride) uye acid chlorite sezvakatsanangurwa kare52,53.Zvinyorwa zvakabva zvaiswa pasi peELISA pakatarisana nepaneru yakaoma yemAb50s yakanangidzirwa kune cell wall glycan, uye iyo mAb inosunga maitiro yakaunzwa semepu yekupisa.mAbs yakanangana nechirimwa sero madziro glycan yakatengwa kubva murabhoritari stocks (CCRC, JIM uye MAC akatevedzana).
Imwe-nhanho biotinylation ye oligosaccharides.Iko kusanganiswa kwemakhahydrates ne biotin-LC-hydrazide yakaitwa uchishandisa nzira inotevera.Biotin-LC-hydrazide (4.6 mg / 12 μmol) yakanyungudutswa mu dimethyl sulfoxide (DMSO, 70 μl) nekukurudzira kwakasimba uye kupisa pa65 ° C. kwe1 min.Glacial acetic acid (30 µl) yakawedzerwa uye musanganiswa wakadirwa pasodium cyanoborohydride (6.4 mg/100 µmol) uye yakanyungudika zvachose mushure mekupisa pa65 ° C. kweinenge 1 miniti.Zvadaro, kubva pa5 kusvika ku8 μl yemusanganiswa wekuita yakawedzerwa kune oligosaccharide yakaomeswa (1-100 nmol) kuti uwane 10-fold or more molar over the label pamusoro pekuderedza kupera.Mhedzisiro yacho yakaitwa pa65 ° C ye2 h, mushure mezvo masampuli akacheneswa pakarepo.Hapana sodium cyanoborohydride yakashandiswa mukuedza kwekunyora pasina kuderedza, uye sampuli dzakaitwa pa 65 ° C. kwemaawa 2.5.
ELISA kupfekedza uye kugezwa kwemasampuli ebiotinylated oligosaccharides.25 μl yebiotinylated samples (100 μl yega yega yakanyungudutswa sampu yakanyungudutswa mu5 ml ye0.1 M Tris buffer solution (TBS)) yakawedzerwa kune rimwe nerimwe tsime reavidin-coated plate.Kudzora matsime akaputirwa ne50 μl yebiotin pachikamu che10 μg/ml mu 0.1 M TBS.Mvura yakasvibiswa yakashandiswa sechifukidzo chezviyero zvisina chinhu.Iyo piritsi yakavharwa kwemaawa maviri pakamuri tembiricha murima.Geza ndiro katatu ne 0.1% mukaka wakapfava mu 0.1 M TBS uchishandisa chirongwa che.11 yeGrenier flat 3A.
Kuwedzera uye kugezwa kwekutanga masoja ekudzivirira chirwere.Wedzera 40 µl yemasoja ekutanga ekudzivirira chirwere patsime rega rega.Isa iyo microplate kweawa imwe pane tembiricha murima.Mahwendefa akazogezwa katatu nemukaka 0.1% mu 0.1M TBS uchishandisa wash program #11 yeGrenier Flat 3A.
Wedzera masoja echipiri wogeza.Wedzera 50 µl yembeva/gonzo yechipiri antibody (yakaderedzwa 1:5000 mu0.1% mukaka mu0.1 M TBS) kutsime rega rega.Isa iyo microplate kweawa imwe pane tembiricha murima.Iwo ma microplates akazogezwa kashanu ne 0.1% mukaka mu 0.1 M TBS uchishandisa Grenier Flat 5A ndiro yekuwacha chirongwa #12.
Kuwedzera substrate.Wedzera 50 µl ye3,3′,5,5'-tetramethylbenzidine (TMB) kune base substrate (nekuwedzera madonhwe maviri e-buffer, madonhwe matatu eTMB, madonhwe maviri ehydrogen peroxide kusvika 15 ml yemvura yakasvibiswa).Gadzirira iyo TMB substrate.uye vortex isati yashandiswa).Isa iyo microplate panzvimbo yekushisa kwemaminetsi makumi matatu.Murima.
Pedzisa danho uye uverenge piritsi.Wedzera 50 µl ye1 N sulfuric acid kutsime rega rega uye rekodha kunyura kubva pa450 kusvika 655 nm uchishandisa ELISA muverengi.
Gadzirira 1 mg/ml mhinduro dzevaongorori ava mumvura yakasvibiswa: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc) N-coamine .(glcNAc), N-acetylgalactosamine (galNAc), inositol (yemukati chiyero).Zviyero zviviri zvakagadzirirwa nekuwedzera 1 mg / mL mishonga yeshuga inoratidzwa muTebhu 1. Mienzaniso yakaomeswa nechando uye lyophilized pa -80 ° C. kusvikira mvura yose yabviswa (kazhinji inenge maawa 12-18).
Wedzera 100–500 µg yesample kuscrew cap machubhu pachiyero chekuongorora.Rekodha mari yakawedzerwa.Zvakanakisisa kunyungudutsa sampuli mune chaiyo yakasanganiswa ye solvent uye kuiwedzera kune chubhu seyemvura aliquot.Shandisa 20 µl ye1 mg/ml inositol seyero yemukati yemuenzaniso wechubhu imwe neimwe.Kuwanda kwechiyero chemukati chakawedzerwa kumuenzaniso chinofanira kunge chakafanana nehuwandu hwemukati wemukati wakawedzerwa kune yakajairwa chubhu.
Wedzera 8 ml yeanhydrous methanol kune screw cap vial.Zvadaro 4 ml ye3 N. methanolic HCl solution, yakavharwa uye yakazununguswa.Iyi nzira haishandisi mvura.
Wedzera 500 µl ye1 M HCl methanol mhinduro kune oligosaccharide samples uye yakajairwa TMS machubhu.Mienzaniso yakavharwa usiku hwose (maawa 168) pa80 ° C. mune imwe nzvimbo inopisa.Pukuta chigadzirwa chemethanolysis pane tembiricha yekamuri uchishandisa manifold ekuomesa.Wedzera 200 µl MeOH woomesa zvakare.Iyi nzira inodzokororwa kaviri.Wedzera 200 µl yemethanol, 100 µl yepyridine uye 100 µl yeacetic anhydride kumumuenzaniso uye sanganisa zvakanaka.Samples akaiswa pakamuri tembiricha kwemaminitsi makumi matatu.uye akaomeswa.Wedzera 200 µl yemethanol woomesa zvakare.
Wedzera 200 µl yeTri-Sil uye inopisa capped chubhu kwemaminitsi makumi maviri.80 ° C, yobva yatonhodzwa kusvika kune tembiricha yekamuri.Shandisa manifold yekuomesa kuti uwedzere kuomesa sampuli kusvika kuhuwandu hunosvika 50 µl.Zvakakosha kuziva kuti hatina kubvumira masampuli kuti aome zvachose.
Wedzera 2 ml yehexane uye sanganisa zvakanaka nevortexing.Zadzai matipi ePasteur pipettes (5-8 mm) nechidimbu chegirazi shinda nekuisa girazi makushe pamusoro pepipette ye5-3/4 inch diayamita.Samples dzakaiswa centrifuged pa3000 g kwemaminetsi maviri.Chero inseluble residues inonaya.Pukuta sampuli kusvika 100-150 µl.Vhoriyamu ingangoita 1 μl yakaiswa muGC-MS pane yekutanga tembiricha ye80 ° C uye yekutanga nguva ye2.0 maminetsi (Tafura 2).