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Usoro ọhụrụ nke immunological na mass spectrometric maka nyocha dị mgbagwoju anya nke oligosaccharides na-adịgide adịgide na stovu ọka e ji AFEX gwọọ. Lignocellulosic biomass bụ ihe ọzọ na-adịgide adịgide maka mmanụ ọkụ fosil ma ejiri ya mee ihe nke ukwuu iji mepụta teknụzụ biotechnology maka mmepụta ngwaahịa dịka nri, nri, mmanụ ọkụ na kemịkalụ. Isi ihe dị na teknụzụ ndị a bụ mmepe nke usoro asọmpi ọnụ ahịa maka ịtụgharị carbohydrates dị mgbagwoju anya dị na mgbidi sel osisi ka ọ bụrụ shuga dị mfe dịka glucose, xylose na arabinose. Ebe ọ bụ na lignocellulosic biomass siri ike nke ukwuu, a ga-etinye ya na ọgwụgwọ thermochemical (dịka ọmụmaatụ, ammonia fiber exfoliation (AFEX), diluted acids (DA), ionic liquids (IL)) na ọgwụgwọ bayọlọjik (dịka ọmụmaatụ, enzymatic hydrolysis na microbial fermentation) iji nweta ngwaahịa achọrọ. Agbanyeghị, mgbe ejiri enzymes fungal azụmahịa na usoro hydrolysis, naanị 75-85% nke shuga na-agbaze bụ monosaccharides, na 15-25% fọdụrụ bụ oligosaccharides na-agbaze, nke na-anaghị adịte aka na microorganisms mgbe niile. Na mbụ, anyị ekewapụla ma sachaa oligosaccharides siri ike nke na-agbaze site na iji ngwakọta nkewa carbon na diatomaceous earth na chromatography mwepụ nha, ma nyochaakwa ihe ndị na-egbochi enzyme ha. Anyị achọpụtala na oligosaccharides nwere ọkwa dị elu nke polymerization (DP) methylated uronic acid nnọchi anya na-esiri ike ịhazi ya na ngwakọta enzyme azụmahịa karịa obere DP na oligosaccharides na-anọpụ iche. N'ebe a, anyị na-akọ banyere ojiji nke ọtụtụ ụzọ ndị ọzọ, gụnyere profaịlụ glycan site na iji monoclonal antibodies (mAbs) nke akọwapụtara na osisi biomass glycans iji mara njikọ glycan na mgbidi sel osisi na enzymatic hydrolysates, matrix-assisted laser desorption ionization, time-of-flight mass-spectrometry. . MALDI-TOF-MS) na-eji elu nyocha ihe ọmụma nke nhazi nke enwetara site na spectroscopy mgbe ire ere nke abụọ nke ions na-adịghị mma, gas chromatography na mass spectrometry (GC-MS) iji mara njikọ oligosaccharides na na-enweghị derivatization. N'ihi obere nha nke oligosaccharides (DP 4–20), ọ na-esiri ike iji molekul ndị a maka njikọ na njirimara mAb. Iji merie nsogbu a, anyị tinyere usoro immobilization ọhụrụ nke dabere na biotin conjugation nke gosipụtara ọtụtụ oligosaccharides DP dị ala na elu microplate, nke ejiri ya mee ihe na sistemụ mAb dị elu maka nyocha njikọ kpọmkwem. Usoro ọhụrụ a ga-eme ka mmepe nke nnwale glycome dị elu dị elu n'ọdịnihu nke enwere ike iji kewapụ ma kọwaa oligosaccharides dị na biomarkers maka ebumnuche nchọpụta.
Lignocellulosic biomass, nke mejupụtara ọrụ ugbo, ọhịa, ahịhịa na ihe ndị dị n'osisi, bụ ihe nwere ike inye aka na mmepụta nke ngwaahịa ndị dabere na bio, gụnyere nri, nri, mmanụ ọkụ na ihe ndị na-ebute ụzọ kemịkalụ iji mepụta ngwaahịa bara uru karịa1. A na-agbanwe carbohydrates (dịka cellulose na hemicellulose) dị na mgbidi sel osisi ka ọ bụrụ monosaccharides site na nhazi kemịkalụ na biotransformation (dịka enzymatic hydrolysis na microbial fermentation). Ọgwụgwọ ndị a na-ejikarị eme ihe gụnyere mgbasa nke eriri ammonia (AFEX), diluted acid (DA), ionic liquid (IL), na steam explosive (SE), nke na-eji ngwakọta nke kemịkalụ na okpomọkụ iji belata mmepụta lignocellulose site na imepe mgbidi sel osisi3,4. isi ike nke ihe, 5. A na-eme hydrolysis enzymatic na ibu siri ike dị elu site na iji enzymes nwere carbohydrate na-arụ ọrụ azụmahịa (CAZymes) na fermentation microbial site na iji yist transgenic ma ọ bụ nje bacteria iji mepụta mmanụ na kemịkalụ bio-based 6.
CAZymes dị na enzymes azụmaahịa mejupụtara ngwakọta dị mgbagwoju anya nke enzymes nke na-ekewa njikọ carbohydrate na shuga dị mgbagwoju anya iji mepụta monosaccharides2,7. Dịka anyị kọrọ na mbụ, netwọk dị mgbagwoju anya nke polima aromatic nke lignin na carbohydrates na-eme ka ha sie ike nke ukwuu, nke na-eduga na mgbanwe shuga na-ezughị ezu, na-agbakọta 15-25% nke oligosaccharides mmekọahụ nke na-adịghị emepụta n'oge hydrolysis enzymatic nke biomass a gwọchara tupu oge eruo. Nke a bụ nsogbu a na-ahụkarị na ụzọ dị iche iche maka ọgwụgwọ biomass tupu oge eruo. Ụfọdụ ihe kpatara nsogbu a gụnyere mgbochi enzyme n'oge hydrolysis, ma ọ bụ enweghị ma ọ bụ obere ọkwa nke enzymes dị mkpa dị mkpa nke achọrọ iji mebie njikọ shuga na biomass osisi. Ịghọta ihe mejupụtara na njirimara nhazi nke shuga, dị ka njikọ shuga na oligosaccharides, ga-enyere anyị aka imeziwanye mgbanwe shuga n'oge hydrolysis, na-eme ka usoro biotechnological dị ọnụ ala na ngwaahịa sitere na mmanụ ala.
Ịchọpụta usoro nke carbohydrates siri ike ma chọọ njikọta nke ụzọ dị ka liquid chromatography (LC)11,12, nuclear magnetic resonance spectroscopy (NMR)13, capillary electrophoresis (CE)14,15,16 na mass spectrometry (MS)17. ,18. Usoro MS dị ka oge-n'ịgba ọsọ mass spectrometry na laser desorption na ionization site na iji matrix (MALDI-TOF-MS) bụ ụzọ dị iche iche maka ịchọpụta nhazi carbohydrate. N'oge na-adịbeghị anya, ejirila tandem Collision-Induced Dissociation (CID) tandem MS nke sodium ion adducts nke ukwuu iji chọpụta mkpịsị aka kwekọrọ na ọnọdụ njikọ oligosaccharide, nhazi anomeric, usoro, na ọnọdụ alaka 20, 21.
Nyocha Glycan bụ ngwa ọrụ dị mma maka ịchọpụta njikọ carbohydrate nke ọma22. Usoro a na-eji ọgwụ mgbochi monoclonal (mAbs) nke a na-eduzi na glycan mgbidi sel dị ka ihe nyocha iji ghọta njikọ carbohydrate dị mgbagwoju anya. Enwere ihe karịrị mAbs 250 n'ụwa niile, nke e mere megide ọtụtụ oligosaccharides dị larịị na nke alaka site na iji saccharides dị iche iche24. Ejirila ọtụtụ mAbs mee ihe nke ukwuu iji kọwaa nhazi, nhazi, na mgbanwe nke mgbidi sel osisi, ebe enwere nnukwu ọdịiche dabere na ụdị sel osisi, akụkụ ahụ, afọ, ọkwa mmepe, na gburugburu ebe obibi uto25,26. N'oge na-adịbeghị anya, ejirila usoro a ghọta ọnụọgụ vesicle na sistemụ osisi na anụmanụ na ọrụ ha na njem glycan dịka ihe nrịbama subcellular, ọkwa mmepe, ma ọ bụ ihe mkpali gburugburu ebe obibi si dị, yana ịchọpụta ọrụ enzymatic. Ụfọdụ n'ime ụdị dị iche iche nke glycans na xylans achọpụtala gụnyere pectin (P), xylan (X), mannan (M), xyloglucans (XylG), mixed bond glucans (MLG), arabinoxylan (ArbX), galactomannan (GalG), glucuronic acid-arabinoxylan (GArbX) na arabino-galactan (ArbG)29.
Agbanyeghị, n'agbanyeghị mbọ niile a na-agba n'ime nnyocha a, naanị nnyocha ole na ole lekwasịrị anya n'ụdị mkpokọta oligosaccharide n'oge hydrolysis dị elu (HSL), gụnyere ntọhapụ oligosaccharide, mgbanwe ogologo oligomeric n'oge hydrolysis, ọtụtụ polima DP dị ala, na usoro ha. nkesa 30,31,32. Ka ọ dị ugbu a, ọ bụ ezie na nyocha glycan egosila na ọ bụ ngwaọrụ bara uru maka nyocha zuru oke nke nhazi glycan, ọ siri ike inyocha obere oligosaccharides DP nke na-agbaze mmiri site na iji usoro antibody. Obere oligosaccharides DP nwere ibu molekul nke na-erughị 5-10 kDa anaghị ejikọta na efere ELISA 33, 34 ma a na-asacha ha tupu etinye antibody.
N'ebe a, maka oge mbụ, anyị na-egosi nnwale ELISA na efere ndị a kpuchiri avidin site na iji ọgwụ mgbochi monoclonal, na-ejikọta usoro biotinylation otu nzọụkwụ maka oligosaccharides na-agbaze agbaze na nyocha glycome. Usoro anyị maka nyocha glycome ka akwadoro site na nyocha MALDI-TOF-MS na GC-MS nke njikọ oligosaccharide mgbakwunye site na iji trimethylsilyl (TMS) nke ihe mejupụtara shuga hydrolyzed. Enwere ike ịmepụta usoro ọhụrụ a dị ka usoro mmepụta dị elu n'ọdịnihu ma chọta ojiji sara mbara na nyocha biomedical35.
Mgbanwe ndị e mere mgbe a sụgharịchara ya nke enzymes na ọgwụ mgbochi, dịka glycosylation,36, na-emetụta ọrụ ha na-arụ n'ime ahụ mmadụ. Dịka ọmụmaatụ, mgbanwe dị na glycosylation nke protein serum na-arụ ọrụ dị mkpa na ọrịa ogbu na nkwonkwo, a na-ejikwa mgbanwe dị na glycosylation dị ka ihe nrịbama nchọpụta37. Akọpụtala ọtụtụ glycans n'akwụkwọ ndị a ka ha pụta ìhè n'ọtụtụ ọrịa dị iche iche, gụnyere ọrịa mkpali na-adịghị ala ala nke eriri afọ na imeju, ọrịa nje, kansa ovarian, ara, na prostate38,39,40. Ịghọta usoro glycans site na iji usoro glycan dabere na antibody ga-enye ntụkwasị obi ọzọ na nchọpụta ọrịa na-enweghị iji usoro MS dị mgbagwoju anya.
Nnyocha anyị gara aga gosiri na oligosaccharides siri ike ka na-enweghị mmiri mmiri mgbe a gbasịrị ha tupu ọgwụgwọ na enzyme hydrolysis (Foto nke 1). N'ọrụ anyị bipụtara na mbụ, anyị mepụtara ụzọ mwepụta coal siri ike iji kewapụ oligosaccharides site na AFEX-pretreated corn stover hydrolyzate (ACSH)8. Mgbe ewepụtara na nkewa mbụ, e kewara oligosaccharides site na nha mwepụ chromatography (SEC) ma chịkọta ha n'usoro nke ibu molekul. E jiri nyocha nhazi shuga nyochaa monomers shuga na oligomers ewepụtara site na ọgwụgwọ dị iche iche. Mgbe a na-atụnyere ọdịnaya nke oligomers shuga enwetara site na ụzọ ọgwụgwọ dị iche iche, ọnụnọ nke oligosaccharides siri ike bụ nsogbu a na-ahụkarị na ntụgharị nke biomass ka ọ bụrụ monosaccharides ma nwee ike ibute mbelata na mmepụta shuga nke opekata mpe 10-15% na ọbụna ruo 18%. US. A na-eji usoro a maka mmepụta buru ibu nke oligosaccharides. Ejiri ACH na akụkụ ya ndị na-esote ya nwere ibu molekul dị iche iche mee ihe dị ka ihe nnwale maka njirimara nke oligosaccharides n'ọrụ a.
Mgbe a gbasịrị ọgwụgwọ tupu oge eruo na mgbe a gbasasịrị ya na enzyme hydrolysis, oligosaccharides na-adịgide adịgide ka na-enweghị hydrolyzed. Nke a bụ ụzọ nkewa oligosaccharides ebe a na-ekewapụ oligosaccharides na AFEX-pretreated corn stover hydrolysate (ACSH) site na iji akwa e tinyere n'ime carbon na diatomaceous earth; (B) Ụzọ maka nkewa nke oligosaccharides. E kewapụrụ oligosaccharides site na nha mwepụ chromatography (SEC); (C) Monomers na oligomers Saccharide ewepụtara site na ọgwụgwọ dị iche iche (diluted acid: DA, ionic liquid: IL na AFEX). Ọnọdụ hydrolysis enzymatic: ibu siri ike dị elu nke 25% (w/w) (ihe dị ka 8% glucan loading), hydrolysis awa 96, ibu 20 mg/g enzyme azụmahịa (Ctec2:Htec2:MP-2:1:1 ratio) na (D) Monomers shuga na oligomers nke glucose, xylose na arabinose ewepụtara na AFEX corn stover pre-treated (ACS).
Nnyocha Glycan egosila na ọ bụ ngwá ọrụ bara uru maka nyocha nhazi zuru oke nke glycans na ihe ndị ewepụtara na ihe fọdụrụ na biomass siri ike. Agbanyeghị, a naghị egosipụta saccharides ndị na-agbaze mmiri nke ọma site na iji usoro ọdịnala a41 n'ihi na oligosaccharides dị obere na-esi ike ịgbachi nkịtị na efere ELISA ma a na-asacha ha tupu etinye antibody. Ya mere, maka njikọ na njirimara antibody, ejiri usoro biotinylation otu nzọụkwụ kpuchie oligosaccharides ndị na-agbaze, ndị na-adịghị agbaso iwu na efere ELISA a na-ekpuchi avidin. A nwalere usoro a site na iji ACSH anyị mepụtara na obere akụkụ dabere na ibu molekul ya (ma ọ bụ ogo polymerization, DP). Ejiri otu nzọụkwụ biotinylation mee ihe iji mee ka njikọ oligosaccharide dịkwuo elu site na ịgbakwunye biotin-LC-hydrazide na njedebe mbelata nke carbohydrate (Foto 2). Na ngwọta, otu hemiacetal na njedebe mbelata na-emeghachi omume na otu hydrazide nke biotin-LC-hydrazide iji mepụta njikọ hydrazone. N'ihu ihe na-ebelata shuga NaCNBH3, njikọ hydrazone na-ebelata ka ọ bụrụ ngwaahịa njedebe biotinylated siri ike. Site na mgbanwe nke njedebe mbelata shuga, njikọ nke obere oligosaccharides DP na efere ELISA ghọrọ ihe kwere omume, na n'ọmụmụ ihe anyị, emere nke a na efere mkpuchi avidin site na iji mAbs glycan-targeted.
Nnyocha nke ọgwụ mgbochi monoclonal dabere na ELISA maka oligosaccharides biotinylated. N'ebe a (A) njikọta biotinylation nke oligosaccharides na nyocha ELISA sochirinụ na mAbs ezubere iche maka glycan na efere mkpuchi NeutrAvidin na (B) na-egosi usoro otu nzọụkwụ maka biotinylation nke ngwaahịa mmeghachi omume.
E tinyeziri efere ndị e ji Avidin kpuchie nwere ọgwụ mgbochi oligosaccharide jikọtara na ọgwụ mgbochi mbụ na nke abụọ ma saa ha n'ime ihe na-egosi ìhè na oge. Mgbe ejiri ọgwụ mgbochi mee ihe, tinye ihe TMB substrate iji mee ka efere ahụ dị ọkụ. Emesịa, e jiri sulfuric acid kwụsị mmeghachi omume ahụ. E jiri ihe na-agụ ELISA nyochaa efere ndị e ji ihe na-egosi ọkụ iji chọpụta ike njikọ nke ọgwụ mgbochi ọ bụla iji chọpụta njikọ njikọ nke antibody kpọmkwem. Maka nkọwa na paramita nke nnwale ahụ, lee ngalaba kwekọrọ na ya bụ "Ihe na Ụzọ".
Anyị na-egosi uru nke usoro a mepụtara ọhụrụ maka ojiji kpọmkwem site na ịkọwapụta oligosaccharides na-agbaze agbaze dị na ACSH yana na akụkụ oligosaccharides dị ọcha na nke e kewapụrụ site na lignocellulosic hydrolysates. Dịka egosiri na Foto nke 3, xylans epitope-substituted a na-ahụkarị na ACSH site na iji usoro bioacylated glycome assay bụkarị uronic (U) ma ọ bụ methyluronic (MeU) na pectic arabinogalactans. A hụkwara ọtụtụ n'ime ha na ọmụmụ anyị gara aga gbasara nyocha nke glycans nke ihe siri ike na-abụghị hydrolyzed (UHS)43.
Nchọpụta nke epitopes oligosaccharide recalcitrant site na iji monoclonal antibody nke e duziri na glycan mgbidi cell. Akụkụ "nọpụ iche" bụ akụkụ ACN na akụkụ "acidic" bụ akụkụ FA. Uhie na-egbuke egbuke na map okpomọkụ na-egosi ọdịnaya epitope dị elu, acha anụnụ anụnụ na-egbuke egbuke na-egosi ndabere efu. Ụkpụrụ agba dị na nha ahụ dabere na ụkpụrụ OD raw maka usoro N=2. E gosipụtara epitopes bụ isi nke ọgwụ mgbochi ahụ ghọtara n'aka nri.
A pụghị ikewapụ ihe owuwu ndị a na-abụghị cellulose site na cellulases na hemicellulases kachasị na ngwakọta enzyme azụmaahịa a nwalechara, nke gụnyere enzymes azụmaahịa a na-ejikarị eme ihe. Ya mere, a chọrọ enzymes enyemaka ọhụrụ maka hydrolysis ha. Na-enweghị enzymes mgbakwunye dị mkpa na-abụghị cellulose, njikọ ndị a na-abụghị cellulose na-egbochi mgbanwe zuru oke na monosaccharides, ọbụlagodi na a na-eme ka polymer shuga nne na nna ha dị obere ka ọ bụrụ iberibe dị mkpụmkpụ ma gbazee site na iji ngwakọta enzyme azụmahịa.
Nnyocha ọzọ gbasara nkesa mgbaàmà na ike njikọ ya gosiri na epitopes njikọ dị ala na nnukwu DP shuga (A, B, C, DP ruo 20+) karịa na obere DP (D, E, F, DP) na dimers) (Foto 1). Mpekere acid na-adịkarị na epitopes na-abụghị cellulose karịa iberibe na-anọpụ iche. Ihe ndị a kwekọrọ na ụkpụrụ a hụrụ na ọmụmụ ihe anyị gara aga, ebe nnukwu DP na acid moieties na-eguzogide hydrolysis enzymatic. Ya mere, ọnụnọ nke epitopes na-abụghị cellulose glycan na mgbanwe U na MeU nwere ike itinye aka nke ukwuu na nkwụsi ike nke oligosaccharides. E kwesịrị ịmara na arụmọrụ njikọ na nchọpụta nwere ike ịbụ nsogbu maka obere oligosaccharides DP, 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 nke na-ejikọ na mAb kpọmkwem.
Ya mere, ojiji nke ọgwụ mgbochi ndị a kapịrị ọnụ nke nhazi gosiri ụdị ụfọdụ nke njikọ recalcitrant. Dabere na ụdị ọgwụ mgbochi ejiri, usoro njikọ kwesịrị ekwesị, na ike nke mgbaàmà ọ na-emepụta (nke kachasị na nke kacha nta), enwere ike ịchọpụta enzymes ọhụrụ ma tinye ọkara ọnụọgụ na ngwakọta enzyme maka mgbanwe glycoconversion zuru oke. Site na nyocha nke ACSH oligosaccharides dị ka ihe atụ, anyị nwere ike ịmepụta nchekwa data nke njikọ glycan maka ihe biomass ọ bụla. A ga-ahụ ebe a na a ga-atụle njikọ dị iche iche nke ọgwụ mgbochi, ọ bụrụ na amaghị njikọ ha, nke a ga-emepụta ụfọdụ ihe isi ike mgbe a na-atụnyere akara nke ọgwụ mgbochi dị iche iche. Na mgbakwunye, ntụnyere nke njikọ glycan nwere ike ịrụ ọrụ kacha mma n'etiti ihe nlele maka otu ọgwụ mgbochi. Enwere ike ijikọ njikọ siri ike ndị a na nchekwa data CAZyme, nke anyị nwere ike ịchọpụta enzymes, họrọ enzymes ndị nwere ike ịbanye ma nwalee enzymes ndị na-agbaji njikọ, ma ọ bụ mepụta sistemụ nje iji gosipụta enzymes ndị a maka ojiji na biorefineries44.
Iji nyochaa otu ụzọ ndị na-egbochi ọrịa si ejikọta ụzọ ndị ọzọ maka ịchọpụta obere oligosaccharides dị na lignocellulosic hydrolysates, anyị mere MALDI (Foto 4, S1-S8) ma nyochaa saccharides sitere na TMS dabere na GC-MS na otu panel (Foto 5) oligosaccharide. A na-eji MALDI atụnyere ma nkesa oke nke molekul oligosaccharide kwekọrọ na nhazi ebumnuche. Na fig. 4 na-egosi MC nke ihe ndị na-anọpụ iche ACN-A na ACN-B. Nyocha ACN-A kwadoro ọtụtụ shuga pentose sitere na DP 4–8 (Foto 4) ruo DP 22 (Foto S1), nke ibu ya kwekọrọ na MeU-xylan oligosaccharides. Nyocha ACN-B kwadoro usoro pentose na glucoxylan na DP 8-15. N'ihe mgbakwunye dịka Foto S3, maapụ nkesa oke acidic nke FA-C na-egosi ọtụtụ shuga pentose (Me)U nke e tinyere n'ọnọdụ Me na DP nke 8-15 nke kwekọrọ na xylan ndị e tinyere n'ọnọdụ ha dị na nyocha mAb nke ELISA. Epitopes ndị ahụ na-agbanwe agbanwe.
Ụdị MALDI-MS nke oligosaccharides ndị na-agbaze agbaze nke na-adịghị agbaso iwu dị na ACS. N'ebe a, (A) ACN-A obere akụkụ dị arọ nke nwere methylated uronic acid (DP 4-8) dochie glucuroxylan oligosaccharides na (B) ACN-B xylan na methylated uronic acid oligosaccharides ejiri glucuroxylan dochie ya (DP 8-15).
Nyocha nke ihe mejupụtara glycan residue nke oligosaccharides na-adịghị ike. Lee (A) Ngwakọta TMS saccharide nke ọtụtụ akụkụ oligosaccharides e nwetara site na iji nyocha GC-MS. (B) Nhazi nke ụdị shuga dị iche iche sitere na TMS dị na oligosaccharides. ACN - akụkụ acetonitrile nke nwere oligosaccharides na-anọpụ iche na akụkụ FA - ferulic acid nke nwere acid oligosaccharides.
E nwetara nkwubi okwu ọzọ na-adọrọ mmasị site na nyocha LC-MS nke akụkụ oligosaccharide, dịka egosiri na Foto S9 (a na-ahụ usoro na ihe mgbakwunye eletrọniki). A hụrụ iberibe hexose na -OAC ugboro ugboro n'oge njikọta nke akụkụ ACN-B. Nchọpụta a abụghị naanị na ọ na-akwado nkewa a hụrụ na nyocha glycome na MALDI-TOF, kamakwa ọ na-enye ozi ọhụrụ gbasara ihe ndị nwere ike ịbụ carbohydrate na biomass lignocellulosic a gwọchara tupu a gwọchaa ya.
Anyị nyochakwara ihe mejupụtara shuga nke akụkụ oligosaccharide site na iji TMS generative shuga. Site na iji GC-MS, anyị chọpụtara ihe mejupụtara shuga neural (nke na-abụghị nke sitere) na shuga acidic (GluA na GalA) na akụkụ oligosaccharide (Foto 5). A na-ahụ Glucuronic acid na akụkụ acidic C na D, ebe a na-ahụ galacturonic acid na akụkụ acidic A na B, ha abụọ bụ nnukwu akụkụ DP nke shuga acidic. Nsonaazụ ndị a abụghị naanị na-akwado data ELISA na MALDI anyị, kamakwa ha kwekọrọ na ọmụmụ ihe anyị gara aga gbasara mkpokọta oligosaccharide. Ya mere, anyị kwenyere na usoro immunological nke oge a na-eji biotinylation nke oligosaccharides na nyocha ELISA na-esote zuru oke iji chọpụta oligosaccharides recalcitrant na-agbaze na ọtụtụ ihe atụ bayọlọjik.
Ebe ọ bụ na e jiri ọtụtụ ụzọ dị iche iche kwado usoro nyocha mAb nke dabeere na ELISA, anyị chọrọ inyochakwu ike nke usoro ọnụọgụgụ ọhụrụ a. E zụtara ma nwalee oligosaccharides azụmaahịa abụọ, xylohexasaccharide oligosaccharide (XHE) na 23-α-L-arabinofuranosyl-xylotriose (A2XX), site na iji usoro mAb ọhụrụ na-elekwasị anya na glycan mgbidi sel. Foto nke 6 na-egosi njikọ kwụ ọtọ n'etiti akara njikọ biotinylated na ntinye log nke ntinye oligosaccharide, na-atụ aro ụdị nnabata Langmuir nwere ike. 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 ya na A2XX n'ofe 1 nm ruo 100 nano. N'ihi obere nnweta nke ọgwụ mgbochi n'oge nnwale ahụ, emere nnwale pere mpe na ntinye oligosaccharide ọ bụla. A ga-amarịrị ebe a na ụfọdụ ọgwụ mgbochi na-emeghachi omume n'ụzọ dị iche na otu oligosaccharide dị ka substrate, ikekwe n'ihi na ha na-ejikọ na epitopes dịtụ iche ma nwee ike inwe njikọ dị iche iche. Usoro na ihe ndị metụtara njirimara epitope ziri ezi ga-adị mgbagwoju anya karị mgbe etinyere usoro mAb ọhụrụ na ezigbo ihe nlele.
E jiri oligosaccharides azụmaahịa abụọ chọpụta oke nchọpụta nke mAbs dị iche iche na-elekwasị anya na glycan. N'ebe a, njikọ kwụ ọtọ na mkpokọta ndekọ nke mkpokọta oligosaccharides na-egosi ụkpụrụ nnabata Langmuir maka (A) XHE na mAb na (B) A2XX na mAb. Epitopes ndị kwekọrọ na ha na-egosi nhazi nke oligosaccharides azụmaahịa ejiri dị ka ihe ndị dị n'ime nnwale ahụ.
Ojiji nke ọgwụ mgbochi monoclonal nke glycan lekwasịrị anya (nyocha glycocomic ma ọ bụ nyocha mAb nke ELISA) bụ ngwa dị ike maka njirimara miri emi nke ọtụtụ nnukwu glycans mgbidi sel nke mejupụtara biomass osisi. Agbanyeghị, nyocha glycan oge ochie na-egosipụta naanị glycans mgbidi sel buru ibu, ebe ọ bụ na ọtụtụ oligosaccharides anaghị arụ ọrụ nke ọma na efere ELISA. N'ọmụmụ ihe a, e tinyere stovu ọka AFEX nke a na-agwọ tupu emezi ya na enzyme hydrolyzed na ọdịnaya siri ike dị elu. Ejiri nyocha shuga chọpụta ihe mejupụtara carbohydrates mgbidi sel recalcitrant na hydrolyzate. Agbanyeghị, a naghị eleda nyocha mAb nke obere oligosaccharides na hydrolysates anya, a chọkwara ngwaọrụ ndị ọzọ iji gbochie oligosaccharides na efere ELISA nke ọma.
Anyị na-akọ ebe a ụzọ ọhụrụ na nke dị irè maka nyocha mAb site na ijikọta biotinylation oligosaccharide nke e tinyere nyocha ELISA na efere mkpuchi NeutrAvidin™. Oligosaccharides biotinylated ndị a na-anaghị agbanwe agbanwe gosipụtara ike zuru oke maka antibody iji mee ka nchọpụta ngwa ngwa na nke dị irè nke oligosaccharides recalcitrant. Nyocha nke ihe mejupụtara oligosaccharides ndị a siri ike dabere na mass spectrometry kwadoro nsonaazụ nke ụzọ ọhụrụ a maka nyocha immunoscreening. Ya mere, ọmụmụ ihe ndị a na-egosi na enwere ike iji njikọta nke biotinylation oligosaccharide na nyocha ELISA na mgbochi monoclonal nke glycan-targeted iji chọpụta njikọ crosslinks na oligosaccharides ma nwee ike itinye ya n'ọrụ nke ukwuu na ọmụmụ ihe ndị ọzọ na-akọwapụta nhazi nke oligosaccharides.
Usoro a nke e ji biotin mepụta glycan profiling bụ akụkọ mbụ nwere ike inyocha njikọ carbohydrate recalcitrant nke oligosaccharides na-agbaze na biomass osisi. Nke a na-enyere aka ịghọta ihe mere ụfọdụ akụkụ nke biomass ji sie ike nke ukwuu ma a bịa n'ihe gbasara mmepụta biofuel. Usoro a na-emeju oghere dị mkpa na usoro nyocha glycome ma gbasaa ojiji ya na ọtụtụ ihe ndị ọzọ karịrị oligosaccharides osisi. 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.
E si Kramer Farms dị na Ray, Colorado nweta ahịhịa ọka (CS) e si na mkpụrụ Pioneer 33A14 kpụọ n'afọ 2010. Site n'ikike nke onye nwe ala, enwere ike iji biomass a mee nnyocha. E debere ihe nlele ndị a n'ime akpa mkpọchi zip nke ruru ihe dịka pasentị 6 n'ime okpomọkụ ụlọ. E debere ihe nlele ndị a n'ime akpa mkpọchi zip nke ruru ihe dịka pasentị 6 n'ime okpomọkụ ụlọ. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. E debere ihe nlele ndị a kpọrọ nkụ na oke iru mmiri nke ihe na-erughị pasentị isii n'ime akpa e ji zipa kpuchie n'ọnọdụ 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 n'ime akpa zip na okpomọkụ ụlọ na iru mmiri na-erughị 6%.Nnyocha ahụ mere ihe kwekọrọ na ntuziaka mpaghara na nke mba. Ejiri usoro NREL mee nyocha nhazi ya. A chọpụtara na ihe mejupụtara ya nwere 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% protein na 13.4% ntụ.
Cellic® CTec2 (138 mg protein/ml, lot VCNI 0001) bụ ngwakọta dị mgbagwoju anya nke cellulase, β-glucosidase na Cellic® HTec2 (157 mg protein/ml, lot VHN00001) sitere na Novozymes (Franklinton, NC, USA)). DuPont Industrial Biosciences (Palo Alto, CA, USA) nyere onyinye nke Multifect Pectinase® (72 mg protein/mL), ngwakọta dị mgbagwoju anya nke enzymes na-emebi pectin. E kpebiri oke protein enzyme site na 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). E zụtara ala diatomaceous 545 site na EMD Millipore (Billerica, MA). E si Sigma-Aldrich (St. Louis, MO) zụta kabọn a na-arụ ọrụ (DARCO, granules mesh 100), Avicel (PH-101), beech xylan, na kemịkalụ ndị ọzọ niile.
E mere ọgwụgwọ AFEX tupu oge eruo na GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). Emere ọgwụgwọ tupu oge eruo na 140°C. maka nkeji iri na ise. Oge obibi 46 na oke 1:1 nke ammonia anhydrous na biomass na 60% (w/w) na-ebunye na ihe nrụpụta benchtop ígwè anaghị agba nchara (Parr Instruments Company). O were nkeji iri atọ. E wetara ihe nrụpụta ahụ na 140°C ma wepụta ammonia ngwa ngwa, na-ekwe ka biomass ahụ laghachi ngwa ngwa na okpomọkụ ụlọ. Ihe mejupụtara AFEX stover ọka e gwọrọ tupu oge eruo (ACS) yiri nke stover ọka a na-agwọghị (UT-CS).
E kwadebere ACSH siri ike dị elu 25% (w/w) (ihe dị ka 8% dextran loading) dị ka ihe mmalite maka mmepụta nnukwu oligosaccharides. E jiri ngwakọta enzyme azụmaahịa gụnyere Cellic® Ctec2 10 mg protein/g glucan (na biomass a gwọchara tupu oge eruo), Htec2 (Novozymes, Franklinton, NC), protein 5 mg/g glucan, na Multifect Pectinase (Genencor Inc, USA). ), protein 5 mg/g dextran. E mere hydrolysis enzymatic na bioreactor lita 5 nke nwere olu ọrụ nke lita 3, pH 4.8, 50°C na 250 rpm. Mgbe hydrolysis gasịrị ruo 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 hydrolyzed. E jiri ihe nzacha nke dị milimita 0.22 mee ka hydrolyzate ahụ dị ọcha. E tinyere hydrolyzate ahụ n'ime karama ndị dị ọcha na okpomọkụ dị ogo 4 Celsius wee kewaa ya na carbon.
Nyocha nke ihe mejupụtara ihe atụ biomass nke sitere na ihe ewepụtara dịka usoro nyocha ụlọ nyocha NREL si dị: nkwadebe nke ihe atụ maka nyocha nhazi (NREL/TP-510-42620) na mkpebi nke carbohydrates nhazi na lignin na biomass (NREL/TP-510 - 42618)47.
E mere nyocha oligosaccharide nke iyi hydrolyzate na nha 2 ml site na iji usoro hydrolysis acid nke autoclave-based. Gwakọta ihe nlele hydrolyzate na 69.7 µl nke 72% sulfuric acid n'ime tube ọdịbendị mkpuchi skru 10 ml wee tinye ya na benchtop na 121 °C, jụọ oyi na ice ma tinye ya na karama chromatography mmiri nwere arụmọrụ dị elu (HPLC). E kpebiri mkpokọta oligosaccharides site na iwepụ mkpokọta monosaccharides na ihe nlele na-abụghị hydrolyzed site na mkpokọta mkpokọta shuga na ihe nlele acid-hydrolyzed.
E jiri sistemụ Shimadzu HPLC nke nwere ihe nlele onwe, ihe na-ekpo ọkụ kọlụm, ọkpọkọ isocratic, na ihe nchọpụta ihe na-eme ka ihe dị n'ime kọlụm Bio-Rad Aminex HPX-87H nyochaa mkpokọta glucose, xylose, na arabinose dị na biomass acid hydrolyzed.
A gwakọtara ma nyochaa ihe na-eme ka mmiri dị n'ime ya dị ka mmiri hydrolyzate maka ọdịnaya monomer na oligosaccharide. HPLC nke nwere kọlụm Bio-Rad (Hercules, CA) Aminex HPX-87P na kọlụm nche ntụ ka e ji mee ya nyocha shuga monomeric e nwetara mgbe e mechara mmiri enzymatic hydrolysis. A nọgidere na-enwe okpomọkụ kọlụm ahụ na 80°C, ejiri mmiri mee ihe dị ka usoro mmịpụta nke 0.6 ml/min. A chọpụtara oligosaccharides site na hydrolysis na acid diluted na 121°C dịka usoro akọwara na refs. 41, 48, 49 si dị.
E mere nyocha Saccharide na ihe fọdụrụ n'ime AFEX nke a gwọrọ tupu oge eruo na ihe niile a na-anaghị amịpụta mmiri (gụnyere mmepụta nke ihe ewepụtara na mgbidi sel na nyocha mAb ha) site na iji usoro ndị a kọwara na mbụ 27, 43, 50, 51. Maka nyocha glycome, a na-akwadebe ihe fọdụrụ n'ime ihe osisi site na ihe fọdụrụ n'ime biomass ma na-ewepụta ya site na ihe ndị na-eme ka ihe dị ike 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 dịka akọwara na mbụ52,53. E mesịa tinye ihe ewepụtara na ELISA megide otu panel dị mgbagwoju anya nke mAb50s nke a na-eduzi na mgbidi sel glycan, e gosikwara mmeghachi omume njikọ mAb dị ka maapụ okpomọkụ. A zụtara mAbs na-elekwasị anya na mgbidi sel sel site na ngwaahịa ụlọ nyocha (CCRC, JIM na usoro MAC).
Otu usoro biotinylation nke oligosaccharides. Ejiri usoro a mee njikọ nke carbohydrates na biotin-LC-hydrazide. A gbazere Biotin-LC-hydrazide (4.6 mg/12 μmol) na dimethyl sulfoxide (DMSO, 70 μl) site na ịkpalite ike ma kpoo ọkụ na 65°C. maka nkeji 1. A gbakwunyere glacial acetic acid (30 µl) ma wụsa ngwakọta ahụ na sodium cyanoborohydride (6.4 mg/100 µmol) ma gbazee kpamkpam mgbe ọ kpochara ọkụ na 65°C. maka ihe dị ka nkeji 1. Mgbe ahụ, site na 5 ruo 8 μl nke ngwakọta mmeghachi omume ahụ na oligosaccharide kpọrọ nkụ (1-100 nmol) iji nweta oke molar okpukpu iri ma ọ bụ karịa nke akara ahụ n'elu njedebe mbelata. Emere mmeghachi omume ahụ na 65°C maka awa 2, mgbe nke ahụ gasịrị, a sachara ihe nlele ahụ ozugbo. E jirighị sodium cyanoborohydride mee ihe n'ule ndị e ji mee akara n'enweghị mbelata, e mekwara ihe nlele ndị ahụ na okpomọkụ 65°C ruo awa 2.5.
Mkpuchi ELISA na ịsacha ihe atụ nke oligosaccharides biotinylated. Agbakwunyere 25 μl nke ihe atụ biotinylated (100 μl nke ihe nlele ọ bụla a gwakọtara na 5 ml nke 0.1 M Tris buffer solution (TBS)) na olulu mmiri ọ bụla nke efere avidin kpuchiri. E ji 50 μl nke biotin tee olulu mmiri njikwa na mkpokọta 10 μg/ml na 0.1 M TBS. E jiri mmiri e tinyere n'ọkụ mee ihe dị ka mkpuchi maka nha efu. E tinyere mbadamba ahụ n'ime ụlọ ruo awa abụọ na okpomọkụ ụlọ n'ọchịchịrị. Jiri mmiri ara ehi a kpụchara akpụcha 0.1% saa efere ahụ ugboro atọ na 0.1 M TBS site na iji mmemme nke 11 maka Grenier flat 3A.
Mgbakwunye na ịsacha ọgwụ mgbochi mbụ. Tinye 40 µl nke ọgwụ mgbochi mbụ n'olulu nke ọ bụla. Kpoo ihe mkpuchi ahụ ruo otu awa n'ọnọdụ okpomọkụ ụlọ n'ọchịchịrị. E mesịa, a sachara efere ndị ahụ ugboro atọ na mmiri ara ehi 0.1% na 0.1M TBS site na iji usoro ịsa ahụ #11 maka Grenier Flat 3A.
Tinye ihe mgbochi nke abụọ ma saa ya. Tinye ihe mgbochi nke abụọ nke òké/oke (a gwakọtara ya 1:5000 na mmiri ara ehi 0.1% na 0.1 M TBS) n'olulu nke ọ bụla. Tinye ihe mgbochi nke abụọ ahụ ruo otu awa n'ime okpomọkụ ụlọ n'ọchịchịrị. E jiri usoro ịsa efere Grenier Flat 5A nke e ji asa efere #12 sachaa ihe mgbochi nke abụọ ahụ ugboro ise.
Ịtinye ihe mkpuchi. Tinye 50 µl nke 3,3′, 5,5′-tetramethylbenzidine (TMB) na ihe mkpuchi ala (site na ịgbakwunye ntapụ abụọ nke buffer, ntapụ atọ nke TMB, ntapụ abụọ nke hydrogen peroxide na 15 ml nke mmiri deionized). Kwadebe ihe mkpuchi TMB. ma kpụọ ya tupu e jiri ya). Tinye ihe mkpuchi ahụ na okpomọkụ ụlọ maka nkeji iri atọ. N'ọchịchịrị.
Mezue usoro a ma gụọ mbadamba ahụ. Tinye 50 µl nke 1 N sulfuric acid na olulu mmiri ọ bụla ma dekọọ mmiri mmiri site na 450 ruo 655 nm site na iji ihe na-agụ ELISA.
Kwadebe ihe ngwọta 1 mg/ml nke ihe ndị a na-enyocha n'ime mmiri e tinyere ion: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine. (glcNAc), N-acetylgalactosamine (galNAc), inositol (ọkọlọtọ dị n'ime). E tinyere ihe ngwọta shuga 1 mg/mL egosiri na Tebụl 1. A na-akpọnwụ ihe nlele ma gbanye ha na -80°C. ruo mgbe ewepụrụ mmiri niile (ọ na-abụkarị ihe dị ka awa 12-18).
Tinye 100–500 µg nke ihe nlele ahụ iji kpụọ ọkpọkọ mkpuchi ahụ na nguzozi nyocha. Dekọọ ego etinyere. Ọ kacha mma ịgbaze ihe nlele ahụ na nguzozi pụrụ iche nke ihe mgbaze ma tinye ya na ọkpọkọ ahụ dị ka ihe mmiri mmiri. Jiri 20 µl nke 1 mg/ml inositol dị ka ọkọlọtọ dị n'ime maka ọkpọkọ nlele ọ bụla. Ọnụọgụ ọkọlọtọ dị n'ime agbakwunyere na ihe nlele ahụ ga-abụ otu ihe ahụ na ọnụọgụ ọkọlọtọ dị n'ime agbakwunyere na ọkpọkọ ọkọlọtọ.
Tinye 8 ml nke methanol anhydrous na karama mkpuchi skru. Wee tinye 4 ml nke 3 N. methanol HCl mmiri, kpuchie ya ma maa jijiji. Usoro a anaghị eji mmiri eme ihe.
Tinye 500 µl nke 1 M HCl methanol solution na ihe nlele oligosaccharide na tube TMS ọkọlọtọ. E tinyere ihe nlele n'ime akpa n'abalị (awa 168) na okpomọkụ 80°C. n'ime ihe mkpuchi okpomọkụ. Kpoo ngwaahịa methanolysis na okpomọkụ ụlọ site na iji ihe na-akpọ nkụ. Tinye 200 µl MeOH ma kpọọ nkụ ọzọ. A na-emeghachi usoro a ugboro abụọ. Tinye 200 µl nke methanol, 100 µl nke pyridine na 100 µl nke acetic anhydride na ihe nlele ahụ ma gwakọta nke ọma. E tinyere ihe nlele n'ime akpa n'ime ụlọ maka nkeji iri atọ ma kpọọ nkụ. Tinye 200 µl nke methanol ma kpọọ nkụ ọzọ.
Tinye 200 µl nke Tri-Sil ma kpoo tube ahụ e ji mkpuchi kpuchie maka nkeji iri abụọ. 80°C, wee jụọ oyi ruo na okpomọkụ ụlọ. Jiri ihe na-akpọ nkụ mee ka ihe nlele ahụ kpọọ nkụ ruo ihe dị ka 50 µl. Ọ dị mkpa ịmara na anyị ekweghị ka ihe nlele ahụ kpọọ nkụ kpamkpam.
Tinye 2 ml nke hexane ma gwakọta ya nke ọma site na ịchagharị ya. Jupụta nsọtụ Pasteur pipettes (5-8 mm) na obere ajị iko site na itinye ajị iko n'elu pipette nke dị sentimita 5-3/4. E tinyere ihe nlele na centrifuge na 3000 g maka nkeji abụọ. A na-eme ka ihe fọdụrụ na-anaghị agbaze agbaze. Kpoo ihe nlele ahụ ruo 100-150 µl. A gbanyere ihe dị ka 1 μl n'ime GC-MS na okpomọkụ mbụ nke 80 °C na oge mbụ nke nkeji 2.0 (Tebụl 2).