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Ụzọ ọhụrụ immunological na mass spectrometric maka nyocha dị mgbagwoju anya nke oligosaccharides na-adịgide adịgide na stovu ọka ejiri AFEX mee ya.Lignocellulosic biomass bụ ihe na-adigide na mmanụ ọkụ ma na-ejikarị emepụta teknụzụ biotechnology maka mmepụta nke ngwaahịa dịka nri, nri, mmanụ ọkụ na kemịkalụ.Isi ihe dị na teknụzụ ndị a bụ mmepe nke usoro asọmpi na-efu maka ịtụgharị carbohydrates dị mgbagwoju anya dị na mgbidi cell osisi ka ọ bụrụ shuga dị mfe dị ka glucose, xylose na arabinose.N'ihi na lignocellulosic biomass dị nnọọ isi ike, a ga-edozi ya na ọgwụgwọ thermochemical (dịka ọmụmaatụ, amonia fiber exfoliation (AFEX), dilute acid (DA), ionic liquids (IL)) na ọgwụgwọ ndu (dịka ọmụmaatụ, enzymatic hydrolysis na microbial gbaa ụka) na ngwakọta iji nweta ngwaahịa a chọrọ..Otú ọ dị, mgbe a na-eji enzymes fungal azụmahịa eme ihe na usoro hydrolysis, naanị 75-85% nke sugars soluble guzobere bụ monosaccharides, na 15-25% fọdụrụnụ bụ soluble, oligosaccharides na-adịghị ike, nke na-adịghị adị mgbe niile maka microorganisms.N'oge gara aga, anyị ejirila ngwakọta nke carbon na diatomaceous ụwa na oke chromatography wepụrụ ma sachaa oligosaccharides soluble soluble, wee nyochaa njirimara mgbochi enzyme ha.Anyị achọpụtala na oligosaccharides nwere ogo dị elu nke polymerization (DP) methylated uronic acid nnọchi na-esiri ike ịhazi ya na ngwakọta enzyme azụmahịa karịa DP dị ala na oligosaccharides na-anọpụ iche.N'ebe a, anyị na-akọ na iji ọtụtụ ụzọ ndị ọzọ, gụnyere glycan profiling site na iji monoclonal antibodies (mAbs) kpọmkwem ka osisi biomass glycans ji mara glycan bonds na osisi cell mgbidi na enzymatic hydrolysates, matrix-enyere laser desorption ionization, oge-of-flight mass-spectrometry..MALDI-TOF-MS) na-eji ọnụ ọgụgụ nyocha ihe nhazi-enye nkọwa nke enwetara site na spectroscopy mgbe mmebi nke abụọ nke ion na-adịghị mma, chromatography gas na spectrometry mass (GC-MS) iji mara njikọ oligosaccharides na enweghị mwepu.N'ihi obere oligosaccharides (DP 4-20), ụmụ irighiri ihe ndị a siri ike iji maka njikọ mAb na njirimara.Iji merie nsogbu a, anyị na-etinye usoro biotin conjugation nke dabeere na oligosaccharides immobilisation nke ọma na-edepụta ihe ka ọtụtụ n'ime DP soluble oligosaccharides dị ala n'elu microplate, bụ nke a na-eji eme ihe na usoro mAb dị elu maka nyocha ligation kpọmkwem.Usoro ọhụrụ a ga-eme ka mmepe nke nyocha glycome dị elu dị elu dị elu na-eme n'ọdịnihu nke a ga-eji kewapụ ma kọwaa oligosaccharides dị na biomarkers maka nchọpụta nchọpụta.
Lignocellulosic biomass, nke mejupụtara ọrụ ugbo, oke ohia, ahihia na ihe osisi, bụ nri nwere ike imepụta ngwaahịa sitere na bio, gụnyere nri, nri, mmanụ ọkụ na ihe ndị na-ebu ụzọ kemịkalụ iji mepụta ngwaahịa bara uru1.Carbohydrates (dị ka cellulose na hemicellulose) dị na mgbidi cell osisi na-depolymerized n'ime monosaccharides site chemical nhazi na biotransformation (dị ka enzymatic hydrolysis na microbial gbaa ụka).Usoro ọgwụgwọ ndị a na-emekarị na-agụnye mgbasawanye fiber amonia (AFEX), dilute acid (DA), mmiri mmiri ionic (IL), na mgbawa uzuoku (SE), nke na-eji ngwakọta nke kemịkal na okpomọkụ iji belata mmepụta lignocellulose site na imeghe mgbidi cell3,4.isi ike nke ihe, 5. Enzymatic hydrolysis na-rụrụ na a elu solids ibu na-eji azụmahịa na-arụ ọrụ carbohydrate-nwere enzymes (CAZymes) na microbial gbaa ụka site na iji transgenic yists ma ọ bụ nje bacteria na-emepụta bio-based fuels na chemicals 6.
CAZymes na enzymes azụmahịa mejupụtara ngwakọta mgbagwoju anya nke enzymes nke na-ejikọta njikọ carbohydrate-sugar siri ike iji mepụta monosaccharides2,7.Dị ka anyị kwuru na mbụ, netwọk mgbagwoju anya nke polymers aromatic nke lignin na carbohydrates na-eme ka ha bụrụ ihe na-adịghị ike, nke na-eduga na ntụgharị shuga na-ezughị ezu, na-agbakọta 15-25% nke mmekọahụ oligosaccharides na-emepụtaghị n'oge enzymatic hydrolysis nke pretreated biomass.Nke a bụ nsogbu a na-enwekarị na usoro ọgwụgwọ biomass dị iche iche.Ụfọdụ ihe kpatara ụgbụ a gụnyere mgbochi enzyme n'oge hydrolysis, ma ọ bụ enweghị ma ọ bụ ọkwa dị ala nke enzymes dị mkpa dị mkpa achọrọ iji mebie njikọ shuga na biomass osisi.Ịghọta ihe mejupụtara na njirimara nhazi nke shuga, dị ka njikọ shuga dị na oligosaccharides, ga-enyere anyị aka imeziwanye mgbanwe shuga n'oge hydrolysis, na-eme ka usoro biotechnological bụrụ ọnụ ahịa na ngwaahịa mmanụ mmanụ.
Ịchọpụta nhazi nke carbohydrates dị mgbagwoju anya ma na-achọ nchikota ụzọ dị ka mmiri chromatography (LC) 11,12, nuklia magnetik resonance spectroscopy (NMR) 13, capillary electrophoresis (CE) 14,15,16 na mass spectrometry (MS)17.,iri na asatọ.Ụzọ MS dị ka oge nke ụgbọ elu mass spectrometry nwere laser desorption na ionization site na iji matrix (MALDI-TOF-MS) bụ usoro dị iche iche maka ịchọpụta ihe owuwu carbohydrate.N'oge na-adịbeghị anya, Mkpakọrịta-Induced Dissociation (CID) tandem MS nke sodium ion adducts ka ejirila mee ihe iji chọpụta akara mkpịsị aka kwekọrọ na ọnọdụ ntinye oligosaccharides, nhazi nke anomeric, usoro, na ọnọdụ alaka 20, 21.
Nyocha Glycan bụ ngwá ọrụ magburu onwe ya maka ịmata omimi nke bonds carbohydrate22.Usoro a na-eji ọgwụ mgbochi monoclonal (mAbs) na-eduzi glycan cell mgbidi dị ka nyocha iji ghọta njikọ carbohydrate dị mgbagwoju anya.Ihe karịrị 250 mAbs dị n'ụwa niile, emebere megide oligosaccharides dị iche iche na alaka na-eji saccharide24 dị iche iche.A na-eji ọtụtụ mAbs mee ihe n'ọtụtụ ebe iji mara nhazi, nhazi, na mgbanwe nke mgbidi cell osisi, n'ihi na enwere ọdịiche dị ịrịba ama dabere na ụdị mkpụrụ ndụ ihe ọkụkụ, akụkụ ahụ, afọ, ọkwa mmepe, na gburugburu ebe obibi25,26.N'oge na-adịbeghị anya, a na-eji usoro a iji ghọta ọnụọgụ vesicle na usoro ihe ọkụkụ na anụmanụ na ọrụ ha dị iche iche na njem glycan dị ka akara akara subcellular, ọkwa mmepe, ma ọ bụ mkpali gburugburu ebe obibi, na iji chọpụta ọrụ enzymatic.Ụfọdụ n'ime usoro dị iche iche nke glycans na xylan ndị a chọpụtarala gụnyere pectin (P), xylan (X), mannan (M), xyloglucans (XylG), mix bond glucans (MLG), arabinoxylan (ArbX), galactomannan (GalG) , glucuronic acid-arabinoxylan (GarbX) na 2 (arabinoxylan).
Otú ọ dị, n'agbanyeghị mgbalị nyocha ndị a nile, ọ bụ nanị nnyocha ole na ole lekwasịrị anya na ọdịdị nke nchịkọta oligosaccharides n'oge nnukwu ihe siri ike (HSL) hydrolysis, gụnyere ntọhapụ oligosaccharides, ogologo ogologo ogologo oligomeric na-agbanwe n'oge hydrolysis, dị iche iche DP polymers, na akụkụ ha.nkesa 30,31,32.Ka ọ dị ugbu a, ọ bụ ezie na nyocha glycan egosila na ọ bụ ngwá ọrụ bara uru maka nyocha zuru ezu nke usoro glycan, ọ na-esiri ike nyochaa mmiri-soluble DP oligosaccharides dị ala site na iji ụzọ mgbochi.Oligosaccharides DP pere mpe nwere ibu ihe na-erughị 5-10 kDa anaghị ejikọta ya na efere ELISA 33, 34 ma na-asachapụ ya tupu mgbakwunye mgbochi.
N'ebe a, maka oge mbụ, anyị na-egosipụta nyocha ELISA na efere avidin nwere mkpuchi site na iji ọgwụ mgbochi monoclonal, na-ejikọta usoro biotinylation nke otu nzọụkwụ maka oligosaccharides refractory soluble na glycome analysis.Usoro anyị na nyocha glycome kwadoro site na MALDI-TOF-MS na GC-MS nyocha dabere na njikọ oligosaccharides na-emeju ya site na iji trimethylsilyl (TMS) nke mejupụtara shuga hydrolyzed.Enwere ike ịmepụta ụzọ ọhụrụ a dị ka usoro dị elu n'ọdịnihu wee chọta ngwa ka ukwuu na nchọpụta biomedical35.
Mgbanwe ntụgharị nke enzymes na ọgwụ mgbochi, dị ka glycosylation,36 na-emetụta ọrụ ndụ ha.Dịka ọmụmaatụ, mgbanwe na glycosylation nke protein serum na-ekere òkè dị mkpa na ọrịa ogbu na nkwonkwo na-egbuke egbuke, na mgbanwe na glycosylation na-eji dị ka nchọpụta nchọpụta37.A kọwo glycans dị iche iche n'akwụkwọ ahụ ka ha pụta ngwa ngwa n'ọrịa dị iche iche, gụnyere ọrịa na-adịghị ala ala nke na-adịghị ala ala nke tract gastrointestinal na imeju, nje virus, ovarian, ara, na prostate cancer38,39,40.Ịghọta usoro nke glycans site n'iji ụzọ ELISA glycan dabeere na-emegide mmadụ ga-enyekwu obi ike na nchọpụta ọrịa na-ejighị usoro MS mgbagwoju anya.
Ọmụmụ ihe anyị gara aga gosiri na oligosaccharides isi ike nọgidere na-enweghị mmiri mgbe emechara ya na enzymatic hydrolysis (Nyocha 1).N'ime ọrụ anyị ebipụtara na mbụ, anyị mepụtara usoro mmepụta unyi siri ike na-arụ ọrụ iji kewapụ oligosaccharides na AFEX-pretreated corn stover hydrolyzate (ACSH) 8.Mgbe mmịpụta mbụ na nkewa gasịrị, a na-ekewa oligosaccharides site na nha chromatography (SEC) ma chịkọta ya n'usoro nha molekụla.A na-enyocha sugar monomers na oligomers ewepụtara site na usoro ọgwụgwọ dị iche iche site na nyocha ihe mejupụtara shuga.Mgbe ị na-atụnyere ọdịnaya nke oligomers shuga nwetara site na ụzọ ọgwụgwọ dị iche iche, ọnụnọ oligosaccharides isi ike bụ nsogbu a na-ahụkarị na ntụgharị nke biomass na monosaccharides ma nwee ike ibute mbelata mkpụrụ shuga ma ọ dịkarịa ala 10-15% na ọbụna ruo 18%.USA na-eji usoro a eme ihe maka mmepụta ihe dị ukwuu nke oligosaccharides.ACH nke si na ya pụta na ụmụ irighiri ihe ndị na-esote ya nwere nha molekụla dị iche iche ka ejiri mee ihe nyocha maka njirimara oligosaccharides na ọrụ a.
Mgbe ọgwụgwọ mbụ na enzymatic hydrolysis gasịrị, oligosaccharides na-adịgide adịgide nọgidere na-enweghị mmiri.Ebe a (A) bụ usoro nkewa nke oligosaccharides nke oligosaccharides dịpụrụ adịpụ site na AFEX-pretreated corn stover hydrolyzate (ACSH) na-eji akwa akwa nke carbon na-arụ ọrụ na ala diatomaceous;(B) Usoro maka nkewa nke oligosaccharides.A na-ekewakwa oligosaccharides site na nha chromatography (SEC);(C) Saccharide monomers na oligomers wepụtara site na usoro ọgwụgwọ dị iche iche (acid diluted: DA, ionic liquid: IL na AFEX).Enzymatic hydrolysis ọnọdụ: elu solids loading nke 25% (w / w) (ihe dị ka 8% glucan loading), 96 awa hydrolysis, 20 mg / g azụmahịa enzyme loading (Ctec2: Htec2: MP-2: 1: 1 ratio) na (D) Sugar monomers na oligomers nke glucose, xylose wepụtara na CS stovu (Arabin).
Nyocha Glycan egosila na ọ bụ ngwá ọrụ bara uru maka nyocha nhazi zuru oke nke glycans na ihe ndị dịpụrụ adịpụ site na residues biomass siri ike.Agbanyeghị, saccharides nwere mmiri soluble na-egosighi ya site na iji usoro ọdịnala a41 n'ihi na oligosaccharides dị obere dị obere na-esiri ike imegharị na efere ELISA ma na-asachapụ ya tupu mgbakwunye mgbochi.Ya mere, maka njide mgbochi na njirimara, a na-eji usoro biotinylation otu nzọụkwụ na-ekpuchi oligosaccharides na-akwadoghị na efere ELISA nke avidin.A nwalere usoro a site na iji ACSH anyị mepụtara na mbụ yana akụkụ dị nta dabere na ịdị arọ ya (ma ọ bụ ogo nke polymerization, DP).A na-eji biotinylation otu-nzọụkwụ mee ka njikọ nke oligosaccharides dịkwuo elu site n'ịgbakwunye biotin-LC-hydrazide na njedebe mbelata nke carbohydrate (Fig 2).Na ngwọta, otu hemiacetal na njedebe mbelata na-emeghachi omume na ìgwè hydrazide nke biotin-LC-hydrazide iji mepụta njikọ hydrazone.N'ebe ọnụnọ nke NaCNBH3 na-ebelata, njikọ hydrazone na-ebelata ka ọ bụrụ ngwaahịa njedebe biotinylated kwụsiri ike.Site na ngbanwe nke njedebe na-ebelata shuga, ijikọ DP oligosaccharides dị ala na efere ELISA bịara kwe omume, na n'ọmụmụ ihe anyị, a na-eme nke a na efere avidin na-ekpuchi site na iji glycan-ezubere iche mAbs.
Nyochaa ọgwụ mgbochi monoclonal dabere na ELISA maka oligosaccharides biotinylated.N'ebe a (A) jikọtara biotinylation nke oligosaccharides na nyocha ELISA na-esote ya na mAbs glycan ezubere iche na efere mkpuchi NeutrAvidin na (B) na-egosi usoro otu nzọụkwụ maka biotinylation nke ngwaahịa mmeghachi omume.
A na-agbakwunye efere ndị nwere Avidin nwere ọgwụ mgbochi oligosaccharides na ọgwụ mgbochi nke mbụ na nke abụọ ma sachaa ya na ọkụ na oge.Ka emechara ihe mgbochi mmadụ, tinye mkpụrụ osisi TMB ka ọ gbanye efere ahụ.Emechara kwụsị mmeghachi omume ahụ na sulfuric acid.Ejiri onye na-agụ ELISA nyochara efere ndị ahụ agbakwunyere iji chọpụta ike njide nke ọgwụ mgbochi ọ bụla iji chọpụta njikọ njikọ mgbochi-kpọmkwem.Maka nkọwa na paramita nke nnwale ahụ, lee ngalaba kwekọrọ "Ihe na ụzọ".
Anyị na-egosipụta uru nke usoro a emepụtara ọhụrụ maka ngwa dị iche iche site n'ịkọwapụta oligosaccharides soluble dị na ACSH yana n'ụdị oligosaccharides crude na sachara nke dịpụrụ adịpụ na lignocellulosic hydrolysates.Dị ka egosiri na eserese 3, xylan ndị a na-ahụkarị na-anọchi anya epitope na ACSH na-eji usoro nyocha nke bioacylated glycome na-abụkarị uronic (U) ma ọ bụ methyluronic (MeU) na pectic arabinogalactans.Achọpụtakwara ọtụtụ n'ime ha n'ọmụmụ ihe anyị gara aga na nyocha nke glycans nke ihe ndị na-abụghị hydrolyzed solids (UHS)43.
Nchọpụta nke recalcitrant oligosaccharides epitopes site na iji ọgwụ mgbochi monoclonal na-eduga na glycan cell mgbidi.Akụkụ "nọpụ iche" bụ akụkụ ACN na "acid" bụ akụkụ FA.Uhie na-egbuke egbuke na map okpomọkụ na-egosi ọdịnaya epitope dị elu, na blues na-egbuke egbuke na-egosi ndabere oghere.Ụkpụrụ agba na nha dabere na ụkpụrụ OD raw maka usoro N = 2.E gosipụtara epitopes bụ isi nke ọgwụ mgbochi ahụ ghọtara n'aka nri.
Ihe ndị a na-abụghị cellulose enweghị ike ịkewaa site na cellulases na hemicellulases ndị a na-ahụkarị na ngwakọta enzyme azụmahịa a nwalere, nke gụnyere enzymes azụmahịa a na-ejikarị eme ihe.Ya mere, a na-achọ enzymes inyeaka ọhụrụ maka hydrolysis ha.Na-enweghị ihe ndị dị mkpa na-abụghị cellulose ngwa enzymes, ndị a na-abụghị cellulose agbụ na-egbochi ntughari zuru ezu na monosaccharides, ọbụna ma ọ bụrụ na ha nne na nna sugar polymers na-ukwuu hydrolyzed n'ime mkpụmkpụ mkpụmkpụ ma gbazee na-eji azụmahịa enzyme ngwakọta.
Ọmụmụ ihe ọzọ gbasara nkesa mgbaàmà na ike njide ya gosiri na epitopes na-ejikọta ọnụ dị ala na nnukwu DP sugar fractions (A, B, C, DP ruo 20+) karịa na obere DP DP (D, E, F, DP) na dimers) (Fig 1).Iberibe acid na-adịkarị na epitopes na-abụghị cellulose karịa iberi na-anọpụ iche.Ihe omume ndị a kwekọrọ n'ụkpụrụ ahụ a na-ahụ n'ọmụmụ ihe anyị gara aga, ebe nnukwu DP na acid moieties na-eguzogide ọgwụ enzymatic hydrolysis.Ya mere, ọnụnọ nke na-abụghị cellulose glycan epitopes na U na MeU ngbanwe nwere ike inye aka dị ukwuu na nkwụsi ike nke oligosaccharides.Ekwesiri ighota na njikota na nghota nke oma nwere ike ibu nsogbu maka DP oligosaccharides di ala, karịsịa ma ọ bụrụ na epitope bụ dimeric ma ọ bụ trimeric oligosaccharides.Enwere ike ịnwale nke a site na iji oligosaccharides azụmahịa nke ogologo dị iche iche, nke ọ bụla nwere naanị otu epitope na-ejikọta na mAb kpọmkwem.
N'ihi ya, iji ọgwụ mgbochi ndị a kapịrị ọnụ ekpughere ụfọdụ ụdị nkekọ na-atụgharị uche.Dabere n'ụdị mgbochi a na-eji, ụkpụrụ ligation kwesịrị ekwesị, na ike nke mgbaàmà ọ na-emepụta (ọtụtụ na nke kacha nta karịa), enwere ike ịchọpụta enzymes ọhụrụ ma tinye ya na ọkara na ngwakọta enzyme maka ntụgharị glycoconversion zuru oke.N'iji nyocha nke ACSH oligosaccharides dịka ọmụmaatụ, anyị nwere ike ịmepụta nchekwa data nke glycan bond maka ihe ọ bụla biomass.Ekwesiri iburu n'uche ebe a na ekwesiri iburu n'uche mmekorita di iche iche nke ogwu, ma oburu na amaghi ihe ha na-eme, nke a ga-eme ka ihe isi ike di iche iche tulee ihe mgbaàmà nke ogwu di iche iche.Na mgbakwunye, ntụnyere njikọ glycan nwere ike ịrụ ọrụ kacha mma n'etiti ihe nlele maka otu mgbochi ahụ.Enwere ike jikọta njikọ ndị a siri ike na nchekwa data CAZyme, nke anyị nwere ike isi mata enzymes, họrọ enzymes nwa akwukwo ma nwalee maka enzymes na-agbaji, ma ọ bụ mepụta usoro microbial iji gosipụta enzymes ndị a maka ojiji na biorefineries44.
Iji nyochaa otú usoro immunological si akwado usoro ndị ọzọ maka ịkọwapụta oligosaccharides dị ala dị ala dị na lignocellulosic hydrolysates, anyị mere MALDI (Fig 4, S1-S8) na nyocha nke saccharides sitere na TMS sitere na GC-MS na otu panel (Fig 5) akụkụ oligosaccharide.A na-eji MALDI atụnyere ma oke nkesa ụmụ irighiri oligosaccharides dabara n'usoro a chọrọ.Na fig.4 na-egosi MC nke ihe ndị na-anọpụ iche ACN-A na ACN-B.Nyocha ACN-A kwadoro ọtụtụ pentose sugars sitere na DP 4-8 (Fig 4) ruo DP 22 (Fig. S1), nke nha ya kwekọrọ na MeU-xylan oligosaccharides.Nyocha ACN-B kwadoro usoro pentose na glucoxylan na DP 8-15.N'ihe mgbakwunye dị ka eserese S3, maapụ nkesa oke acidic nke FA-C na-egosi ọtụtụ (Me) U ngbanwe pentose sugars nwere DP nke 8-15 dabara na xylans nọchiri anya achọtara na nyocha mAb dabere na ELISA.Epitopes na-agbanwe agbanwe.
MALDI-MS ụdịdị oligosaccharides soluble na-akwadoghị dị na ACS.N'ebe a, (A) ACN-A obere nha nha nke nwere methylated uronic acid (DP 4-8) dochie glucuroxylan oligosaccharides na (B) ACN-B xylan na methylated uronic acid oligosaccharides dochie ya na glucuroxylan (DP 8-15).
Nyocha nke ihe mejupụtara glycan residue nke refractory oligosaccharides.N'ebe a (A) Ngwakọta TMS saccharide dị iche iche nke oligosaccharides dị iche iche nwetara site na iji nyocha GC-MS.(B) Ọdịdị nke ụdị shuga dị iche iche sitere na TMS dị na oligosaccharides.ACN - akụkụ nke acetonitrile nwere oligosaccharides na-anọpụ iche na FA - akụkụ ferulic acid nwere oligosaccharides acid.
Nkwubi okwu ọzọ na-adọrọ mmasị sitere na nyocha LC-MS nke akụkụ nke oligosaccharides, dị ka egosiri na Figure S9 (usoro nwere ike ịhụ na ihe mgbakwunye electronic).A na-ahụ akụkụ nke hexose na -OAc ugboro ugboro n'oge a na-ejikọta akụkụ nke ACN-B.Nchọpụta a abụghị naanị na-akwado nkewa nke a hụrụ na glycome na nyocha MALDI-TOF, kamakwa na-enye ozi ọhụrụ gbasara ihe ndị nwere ike ịnweta carbohydrate na lignocellulosic biomass pretreated.
Anyị tụlekwara ihe mejupụtara shuga nke ntakiri oligosaccharides site na iji mwepu shuga TMS.N'iji GC-MS, anyị kpebiri nhazi nke neural (na-abụghị nke mmepụta) na acidic sugars (GluA na GalA) na oligosaccharides fraction (Fig 5).A na-achọta Glucuronic acid na ihe acidic C na D, ebe a na-ahụ galacturonic acid na acidic components A na B, nke abụọ bụ akụkụ DP dị elu nke sugar acidic.Nsonaazụ ndị a abụghị naanị na-akwado data ELISA na MALDI anyị, mana ọ dabara na ọmụmụ ihe gara aga gbasara mkpokọta oligosaccharides.Ya mere, anyị kwenyere na usoro mgbochi nke ọgbara ọhụrụ site na iji biotinylation nke oligosaccharides na nyocha ELISA na-esote ezuola iji chọpụta oligosaccharides soluble recalcitrant n'ụdị ụdị dị iche iche.
Ebe ọ bụ na ejiri ụzọ dị iche iche akwado usoro nyocha mAb dabere na ELISA, anyị chọrọ inyochakwu ikike nke usoro ọnụọgụ ọhụrụ a.Azụtara oligosaccharides abụọ azụmahịa, xylohexasaccharide oligosaccharide (XHE) na 23-α-L-arabinofuranosyl-xylotriose (A2XX), zụrụ ma nwalee site na iji ụzọ mAb ọhụrụ na-elekwasị anya na mgbidi glycan cell.Ọgụgụ 6 na-egosi njikọ ahịrị n'etiti mgbama njikọ biotinylated na ntinye uche nke oligosaccharides, na-atụ aro ụdị mgbasa ozi Langmuir ga-ekwe omume.N'ime mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, na CCRC-M151 jikọtara ya na XHE, na CCRC-M108, CCRC-M109, na LM11 jikọtara na A2XX karịa 10 namno.N'ihi na enwere obere ọgwụ mgbochi n'oge nnwale ahụ, a na-eme nnwale ole na ole na ntinye oligosaccharides ọ bụla.Okwesiri iburu n'uche ebe a na ufodu ogwu mgbochi na-emeghachi omume n'ụzọ di iche na otu oligosaccharides dị ka mkpụrụ, ma eleghị anya n'ihi na ha na-ejikọta na epitopes dịtụ iche ma nwee ike inwe njikọ dị iche iche.Usoro na mmetụta nke njirimara epitope ziri ezi ga-adị mgbagwoju anya karị mgbe a na-etinye usoro mAb ọhụrụ na ezigbo sample.
A na-eji oligosaccharides azụmahịa abụọ eme ihe iji chọpụta ọnụọgụ nke mAbs dị iche iche na-eche glycan.N'ebe a, njikọta ahịrị na ntinye uche nke oligosaccharides na-egosi usoro mgbasa ozi Langmuir maka (A) XHE na mAb na (B) A2XX na mAb.Epitopes kwekọrọ ekwekọ na-egosi ihe owuwu nke oligosaccharides azụmahịa nke ejiri mee ihe dị ka ihe eji eme ihe na nyocha.
Ojiji nke glycan-ezubere iche maka monoclonal antibodies (glycocomic analysis or ELISA based mAb screening) bụ ngwá ọrụ dị ike maka njiri mara omimi nke ọtụtụ n'ime glycans cell cell bụ isi nke mejupụtara biomass osisi.Agbanyeghị, nyocha glycan oge gboo na-akọwapụta glycans cell buru ibu, n'ihi na ọtụtụ oligosaccharides anaghị emegharị nke ọma na efere ELISA.N'ime ọmụmụ ihe a, AFEX-pretreated ọka stover bụ enzymatically hydrolyzed na nnukwu ihe siri ike.A na-eji nyocha shuga chọpụta ihe mejupụtara carbohydrates mgbidi cell recalcitrant na hydrolyzate.Agbanyeghị, nyocha mAb nke obere oligosaccharides na hydrolysates adịghị eleda anya, yana ngwaọrụ ndị ọzọ achọrọ iji mebie oligosaccharides nke ọma na efere ELISA.
Anyị na-akọ ebe a akwụkwọ akụkọ na usoro oligosaccharides immobilization nke ọma maka nyocha mAb site na ijikọta oligosaccharides biotinylation na-esote ELISA screening na NeutrAvidin™ plates mkpuchi.Oligosaccharides biotinylated a na-adịghị ahụ anya gosipụtara mmekọrịta zuru oke maka mgbochi ahụ iji mee ka nchọpụta ngwa ngwa na nke ọma nke oligosaccharides recalcitrant.Nyocha nke ihe mejupụtara oligosaccharides ndị a siri ike dabere na spectrometry mass spectrometry kwadoro nsonaazụ nke ụzọ ọhụrụ a maka nyocha immunoscreening.Ya mere, ọmụmụ ihe ndị a na-egosi na nchikota oligosaccharides biotinylation na ELISA screening na glycan-ezubere iche monoclonal antibodies nwere ike iji chọpụta crosslinks na oligosaccharides na enwere ike tinye ya n'ọtụtụ ebe na nchọpụta biochemical ndị ọzọ na-akọwa nhazi nke oligosaccharides.
Usoro ịkọwapụta glycan dabere na biotin bụ akụkọ izizi nwere ike nyocha njikọ nke carbohydrate nke soluble oligosaccharides na biomass osisi.Nke a na-enyere aka ịghọta ihe kpatara akụkụ ụfọdụ nke biomass ji enwe isi ike ma a bịa n'ichepụta biofuel.Usoro a na-ejupụta oghere dị mkpa na usoro nyocha nke glycome ma gbasaa ngwa ya n'ọtụtụ ihe dị iche iche karịa osisi oligosaccharides.N'ọdịnihu, anyị nwere ike iji robotics maka biotinylation ma jiri usoro anyị mepụtara maka nyocha dị elu nke ihe nlele site na iji ELISA.
Ahịhịa ọka (CS) nke sitere na mkpụrụ osisi Pioneer 33A14 bụ nke a na-egbute na 2010 site na Kramer Farms na Ray, Colorado.Site na ikike nke onye nwe ala, enwere ike iji biomass a mee nyocha. A na-echekwa ihe nlele ahụ kpọrọ nkụ <6% mmiri na akpa mkpọchi zip n'ime ụlọ okpomọkụ. A na-echekwa ihe nlele ahụ kpọrọ nkụ <6% mmiri na akpa mkpọchi zip n'ime ụlọ okpomọkụ. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. A na-echekwa ihe nlele akọrọ na <6% iru mmiri na akpa zipper na okpomọkụ ụlọ.样品在室温下以干燥< 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%. A na-echekwa ihe nlele na akpa zipa na ụlọ okpomọkụ na iru mmiri <6%.Ọmụmụ ihe ahụ kwekọrọ na ntuziaka mpaghara na nke mba.Emere nyocha nhazi site na iji usoro NREL.Achọpụtara ihe mejupụtara ya nwere 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, protein 1.7% na 13. 4% ash.
Cellic® CTec2 (138 mg protein / ml, otutu VCNI 0001) bụ ngwakọta mgbagwoju anya nke cellulase, β-glucosidase na Cellic® HTec2 (157 mg protein / ml, otutu VHN00001) sitere na Novozymes (Franklinton, NC, USA)).Multifect Pectinase® (72 mg protein/mL), ngwakọta mgbagwoju anya nke pectin na-emebi enzymes, bụ DuPont Industrial Biosciences (Palo Alto, CA, USA) nyere onyinye.Ekpebiri mkpokọta protein nke Enzyme site n'ịtụle ọdịnaya protein (na iwepụ onyinye nke nitrogen na-abụghị protein) site na iji nyocha Kjeldahl nitrogen (usoro AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA).Azụtara Diatomaceous Earth 545 n'aka EMD Millipore (Billerica, MA).Carbon arụ ọrụ (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, na kemịkalụ ndị ọzọ ka zụtara n'aka Sigma-Aldrich (St. Louis, MO).
Emere AFEX pretreatment na GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA).A na-eme ọgwụgwọ tupu oge eruo na 140 ° C. maka nkeji 15.Oge obibi 46 na 1: 1 nke amonia anhydrous na biomass na 60% (w / w) na-ebunye n'ime igwe anaghị agba nchara benchtop reactor (Parr Instruments Company).O were nkeji iri atọ.A na-ebute reactor na 140 ° C na amonia na-ahapụ ngwa ngwa, na-ekwe ka biomass laghachi ngwa ngwa na okpomọkụ.Ngwakọta nke AFEX pre-treated corn stover (ACS) yiri nke ọka ọka na-adịghị edozi (UT-CS).
Akwa siri ike ACSH 25% (w / w) (ihe dị ka 8% dextran loading) kwadebere dị ka ihe mmalite maka mmepụta nnukwu oligosaccharides.Emere enzyme hydrolysis nke ACS site na iji ngwakọta enzyme azụmahịa gụnyere Cellic® Ctec2 10 mg protein / g glucan (na pretreated biomass), Htec2 (Novozymes, Franklinton, NC), 5 mg protein / g glucan, na Multifect Pectinase (Genencor Inc, USA).).), 5 mg protein / g dextran.A na-eme hydrolysis enzymatic na bioreactor 5-lita nwere olu na-arụ ọrụ nke 3 lita, pH 4.8, 50 ° C na 250 rpm.Mgbe hydrolysis maka awa 96, a na-anakọta hydrolyzate site na centrifugation na 6000 rpm maka nkeji 30 wee na 14000 rpm maka nkeji 30 iji wepụ ihe siri ike na-enweghị mmiri.A na-edozi hydrolyzate ahụ na nzacha na-adịghị mma site na beaker 0.22 mm.A na-echekwa hydrolyzate nzacha n'ime karama na-adịghị ọcha na 4 Celsius C. wee kewaa ya na carbon.
Nyocha nke ihe mejupụtara nke ihe nleba anya biomass nke biomass na-ewepụta dị ka usoro nyocha ụlọ nyocha NREL: nkwadebe nke nlele maka nyocha ihe mejupụtara (NREL / TP-510-42620) na mkpebi nke carbohydrates structural na lignin na biomass (NREL / TP-510 - 42618) 47.
A na-eme nyocha nke oligosaccharides nke iyi hydrolyzate na nha 2 ml site na iji usoro hydrolysis acid nke dabeere na autoclave.Gwakọta ihe nlele hydrolyzate na 69.7 µl nke 72% sulfuric acid na 10 ml screw cap Culture tube na incubate maka 1 h na benchtop na 121 Celsius C, dị jụụ na ice na nyochaa n'ime nnukwu chromatography mmiri (HPLC) vial.Achọpụtara mkpokọta oligosaccharides site n'iwepụ mkpokọta nke monosaccharides na nlele na-abụghị nke hydrolyzed site na mkpokọta shuga dị na nlele acid-hydrolyzed.
A na-enyocha ọkwa glucose, xylose, na arabinose na acid hydrolysed biomass site na iji sistemu Shimadzu HPLC nke nwere ihe autosampler, ikpo ọkụ kọlụm, mgbapụta isocratic, na onye na-achọpụta ihe nrịba ama na kọlụm Bio-Rad Aminex HPX-87H.A na-edobe kọlụm ahụ na 50 Celsius ma jiri 0.6 ml / min 5 mM H2SO4 na mmiri.eruba.
A na-agbaze ma nyochaa hydrolyzate supernatant maka ọdịnaya monomer na oligosaccharides.Monomeric sugars enwetara mgbe enzymatic hydrolysis ka HPLC kwadoro ya na kọlụm Bio-Rad (Hercules, CA) Aminex HPX-87P na kọlụm nche ash.A na-edobe okpomoku kọlụm na 80 ° C, a na-eji mmiri mee ihe dị ka usoro mkpanaka na-agba ọsọ nke 0.6 ml / min.Oligosaccharides kpebisiri ike site na hydrolysis na dilute acid na 121 Celsius dịka usoro akọwara na refs.41, 48, 49 .
A na-eme nyocha nke saccharide na raw, AFEX pre-treated na ihe niile na-abụghị hydrolysed biomass residues (gụnyere mmepụta nke serial cell cell extracts na ha mAb screening) site na iji usoro 27, 43, 50, 51 akọwapụtara na mbụ.Maka nyocha nke glycome, a na-akwadebe ihe mkpofu ihe ọṅụṅụ na-aba n'anya nke ihe ọkụkụ cell site na residues biomass ma tinye ya na mmịpụta usoro ya na ndị na-eme ihe ike na-arịwanye elu dị ka ammonium oxalate (50 mM), sodium carbonate (50 mM na 0.5% w / v), CON.(1M na 4M, ha abụọ nwere 1% w/v sodium borohydride) na acid chlorite dị ka akọwara na mbụ52,53.A na-edobe ihe ndị ahụ na ELISA megide otu mgbagwoju anya nke mAb50s na-eduzi na mgbidi cell glycan, na mAb na-ejikọta mmeghachi omume dịka map okpomọkụ.A zụtara mAbs ezubere iche maka glycan cell cell site na ebuka ụlọ nyocha (CCRC, JIM na MAC usoro).
Otu nzọụkwụ biotinylation nke oligosaccharides.A na-eme njikọ nke carbohydrates na biotin-LC-hydrazide site na iji usoro ndị a.Biotin-LC-hydrazide (4.6 mg/12 μmol) gbazere na dimethyl sulfoxide (DMSO, 70 μl) site na mkpali siri ike na kpo oku na 65 ° C. maka 1 min.Glacial acetic acid (30 µl) gbakwụnye na ngwakọta a na-awụsa na sodium cyanoborohydride (6.4 mg/100 µmol) na agbaze kpamkpam mgbe kpochara na 65 ° C. maka ihe dị ka 1 nkeji.Mgbe ahụ, site na 5 ruo 8 μl nke ngwakọta mmeghachi omume agbakwunyere na oligosaccharides a mịrị amị (1-100 nmol) iji nweta 10-fold ma ọ bụ karịa molar ngafe nke akara n'elu mbelata njedebe.Emere mmeghachi omume ahụ na 65 Celsius C maka awa 2, mgbe nke ahụ gasịrị, a na-ehichapụ ihe nlele ahụ ozugbo.A naghị eji sodium cyanoborohydride mee ihe na nyocha nke ịde aha na-enweghị mbelata, a na-emeghachikwa ihe nlele ahụ na 65 ° C. maka awa 2.5.
Mkpuchi ELISA na ịsacha ihe atụ nke oligosaccharides biotinylated.25 μl nke ihe nlele biotinylated (100 μl nke ihe nlele ọ bụla etinyere na 5 ml nke 0.1 M Tris buffer solution (TBS)) ka agbakwunyere n'olulu mmiri ọ bụla nke efere avidin.A na-ekpuchi olulu mmiri nchịkwa na 50 μl nke biotin na ntinye nke 10 μg / ml na 0.1 M TBS.A na-eji mmiri deionized mee ihe mkpuchi maka nha oghere.A na-etinye mbadamba nkume ahụ maka awa 2 na ụlọ okpomọkụ n'ime ọchịchịrị.Jiri mmiri ara ehi 0.1% sachaa efere ugboro 3 na 0.1 M TBS site na iji mmemme no.11 maka Grenier flat 3A.
Mgbakwunye na ịsacha ọgwụ mgbochi mbụ.Tinye 40 µl nke ọgwụ mgbochi mbụ n'olulu mmiri ọ bụla.Tinye microplate maka awa 1 n'ime ụlọ okpomọkụ n'ime ọchịchịrị.A na-asacha efere ndị ahụ ugboro atọ na mmiri ara ehi 0.1% na 0.1M TBS site na iji mmemme ịsacha #11 maka Grenier Flat 3A.
Tinye ọgwụ mgbochi nke abụọ wee sachaa.Tinye 50 µl nke òké/oke mgbochi nke abụọ (gbasara 1:5000 na mmiri ara ehi 0.1% na 0.1 M TBS) na olulu mmiri ọ bụla.Tinye microplate maka awa 1 n'ime ụlọ okpomọkụ n'ime ọchịchịrị.A na-asacha microplates ahụ ugboro 5 na mmiri ara ehi 0.1% na 0.1 M TBS site na iji Grenier Flat 5A plate wash program #12.
Na-agbakwụnye mkpụrụ.Tinye 50 µl nke 3,3′,5,5′-tetramethylbenzidine (TMB) na mkpụrụ nke isi (site na itinye 2 tụlee nke ihe nchekwa, 3 tụlee nke TMB, 2 tụlee nke hydrogen peroxide na 15 ml nke mmiri deionized).Kwadebe mkpụrụ osisi TMB.na vortex tupu eji).Tinye microplate n'ime ụlọ okpomọkụ maka nkeji 30.N'ọchịchịrị.
Mezue nzọụkwụ ma gụọ mbadamba nkume ahụ.Tinye 50 µl nke 1 N sulfuric acid na olulu mmiri ọ bụla wee dekọọ nnabata site na 450 ruo 655 nm site na iji onye na-agụ ELISA.
Kwadebe 1 mg / ml ngwọta nke nyocha ndị a na mmiri deionized: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine.(glcNAc), N-acetylgalactosamine (galNAc), inositol (ụkpụrụ ime).A kwadebere ụkpụrụ abụọ site n'ịgbakwụnye ihe ngwọta shuga nke 1 mg / mL nke egosiri na tebụl 1. A na-echekwa ihe nlele na lyophilized na -80 Celsius C. ruo mgbe ewepụrụ mmiri niile (na-emekarị ihe dịka awa 12-18).
Tinye 100-500 µg nke ihe nlele iji kpochie tubes okpu na nguzozi nyocha.Dekọọ ego agbakwunyere.Ọ kacha mma igbari ihe nlele ahụ na ntinye ihe mgbaze ma tinye ya na tube dị ka mmiri mmiri.Jiri 20 µl nke 1 mg/ml inositol dị ka ọkọlọtọ ime maka tube ọ bụla.Ọnụ ọgụgụ nke ọkọlọtọ dị n'ime agbakwunyere na nlele ahụ ga-abụrịrị otu ihe dị n'ime ya agbakwunyere na tube ọkọlọtọ.
Tinye 8 ml nke methanol anhydrous na vial okpu ịghasa.Mgbe ahụ, 4 ml nke 3 N. methanol HCl ngwọta, kpuchie ma maa jijiji.Usoro a anaghị eji mmiri eme ihe.
Tinye 500 µl nke 1 M HCl methanol ngwọta na oligosaccharides samples na ọkọlọtọ TMS tubes.A na-etinye ihe nlele n'otu abalị (awa 168) na 80 Celsius C. na ngọngọ okpomọkụ.Ghichaa ngwaahịa methanolysis na okpomọkụ nke ụlọ site na iji nnukwu ihicha.Tinye 200 µl MeOH wee kpochaa ọzọ.A na-emeghachi usoro a ugboro abụọ.Tinye 200 µl nke methanol, 100 μl nke pyridine na 100 μl acetic anhydride na nlele wee gwakọta nke ọma.A na-etinye ihe nlele na ụlọ okpomọkụ maka nkeji iri atọ.na akpọnwụwo.Tinye 200 µl nke methanol wee kpochaa ọzọ.
Tinye 200 µl nke Tri-Sil na kpoo tube mkpuchi maka nkeji 20.80 Celsius C, wee mee ka ọ dị jụụ na okpomọkụ.Jiri nnukwu ihicha ihicha iji mee ka nlele ahụ kụọ ya ka ọ dị ihe dị ka 50 µl.Ọ dị mkpa iburu n'obi na anyị ekweghị ka ihe nlele ahụ kpoo kpamkpam.
Tinye 2 ml nke hexane ma gwakọta nke ọma site na vortexing.Jupụta ntụ ntụ nke Pasteur pipettes (5-8 mm) na mpempe ajị anụ site na ịtinye ajị anụ iko n'elu pipette 5-3 / 4 inch dayameta.A na-etinye ihe nlele na 3000 g maka nkeji 2.A na-ebuli ihe mkpofu ọ bụla a na-apụghị imeri emeri.Kpochapụ ihe nlele ahụ ruo 100-150 µl.A gbanyere olu ihe dịka 1 μl n'ime GC-MS na okpomọkụ mbụ nke 80 Celsius C yana oge mbụ nke nkeji 2.0 (Table 2).