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Uburyo bushya bwo gupima ubudahangarwa bw'umubiri n'ubwinshi bw'ibipimo mu gusesengura neza oligosaccharides zihoraho mu gikoni cy'ibigori byavuwe mbere na AFEX. Lignocellulosic biomass ni ubundi buryo burambye bwo gusimbura ibikomoka kuri peteroli kandi bukoreshwa cyane mu guteza imbere ikoranabuhanga rya biotechnologies mu gukora ibicuruzwa nk'ibiribwa, ibiryo, ibikomoka kuri peteroli n'imiti. Urufunguzo rw'ubu buryo ni iterambere ry'inzira zihendutse zo guhindura karubohidrati zikomeye ziri mu nkuta z'uturemangingo tw'ibimera mo isukari yoroshye nka glucose, xylose na arabinose. Kubera ko lignocellulosic biomass ikomera cyane, igomba gukorerwa ubuvuzi bwa thermochemical (urugero, exfoliation ya ammonia fibre (AFEX), dilute aside (DA), ionic liquids (IL)) n'ubuvuzi bwa biyoloji (urugero, hydrolysis ya enzymatic na mikorobe fermentation) hamwe kugira ngo haboneke umusaruro wifuza. Ariko, iyo enzymes zo mu bucuruzi zikoreshwa muri hydrolysis, 75-85% by'isukari ishongeshwa yakozwe ni monosaccharides, naho 15-25% bisigaye ni oligosaccharides zishobora gushonga, zidakunze kuboneka ku dukoko. Mbere, twashoboye gutandukanya no gutunganya oligosaccharides zishongeshwa hakoreshejwe uruvange rwa karuboni na diatomaceous earth separation na chromatography yo gukuraho ingano, ndetse tunasuzuma imiterere yazo yo gukumira enzyme. Twasanze oligosaccharides zifite methylated uronic aside substitutions nyinshi zo mu bucuruzi zigoye cyane gutunganya hamwe n'imvange za enzymes zo mu bucuruzi kurusha DP nke na oligosaccharides zitagira aho zibogamiye. Aha turatangaza ikoreshwa ry'ubundi buryo bwinshi, harimo no gusesengura glycan hakoreshejwe antibodies za monoclonal (mAbs) zihariye kuri glycans za biomass plant kugira ngo hamenyekane glycan bonds mu nkuta z'uturemangingo tw'ibimera na hydrolysates za enzymatic, matrix-assisted laser desorption ionization, time-of-flight mass-spectrometry. MALDI-TOF-MS) ikoresha imiterere y’ibipimo by’imiterere n’amakuru atangwa na spectroscopy nyuma yo kwangirika kwa kabiri kwa iyoni mbi, chromatography ya gas na mass spectrometry (GC-MS) kugira ngo igaragaze imigozi ya oligosaccharide hamwe n’ibidafite iyo bikomokaho. Bitewe n’uko ingano nto ya oligosaccharide (DP 4–20) itoroshye kuyikoresha mu guhuza no kugaragaza mAb. Kugira ngo dukemure iki kibazo, twakoresheje uburyo bushya bwo guhagarika mobilisation bwa biotin bushingiye kuri oligosaccharide, bwagaragaje neza ko inyinshi muri DP soluble oligosaccharides nkeya ku buso bwa microplate, hanyuma bukoreshwa muri sisitemu ya mAb yo hejuru kugira ngo habeho isesengura ryihariye rya ligation. Ubu buryo bushya buzoroshya iterambere ry’ibizamini bya glycome byo hejuru mu gihe kizaza bishobora gukoreshwa mu gutandukanya no kugaragaza oligosaccharides ziri mu bipimo by’isuzuma.
Lignocellulosic biomass, igizwe n'ibikomoka ku buhinzi, amashyamba, ibyatsi n'ibiti, ni ububiko bw'ibiribwa bushobora gukoreshwa mu gukora ibikomoka ku binyabuzima, harimo ibiryo, ibiryo, ibikomoka kuri peteroli n'ibikomoka kuri shimi kugira ngo bikore ibicuruzwa bifite agaciro kanini1. Karubohidrati (nka selulose na hemicellulose) ziri mu nkuta z'uturemangingo tw'ibimera zikurwaho polymeri mu buryo bwa monosaccharides binyuze mu gutunganya imiti no guhindura imiterere y'ibinyabuzima (nk'amazi ya enzyme na fermentation ya mikorobe). Uburyo busanzwe bwo kuvura burimo kwagura fibre ya ammonia (AFEX), aside ivanze (DA), ionic liquid (IL), n'ibyuka by'umwuka (SE), bikoresha uruvange rw'imiti n'ubushyuhe kugira ngo bigabanye umusaruro wa lignocellulose binyuze mu gufungura inkuta z'uturemangingo tw'ibimera3,4. Kudakomera kw'ibintu, 5. Hydrolysis ya enzime ikorwa ku mutwaro mwinshi w'ibintu bikomeye hakoreshejwe enzymes zirimo karubohidrati (CAZymes) n'imisemburo ya mikorobe hakoreshejwe imyembe ya transgenic cyangwa bagiteri kugira ngo hakorwe ibikomoka kuri shimi n'imiti 6.
CAZymes mu enzymes zicuruzwa zigizwe n'uruvange rw'imisemburo ivangavanga imiyoboro ikomeye ya karubohidrati n'isukari kugira ngo habeho monosaccharides2,7. Nkuko twabitangaje mbere, urusobe rw'imisemburo ihumura ya lignin hamwe na karubohidrati rutuma idakira cyane, bigatuma isukari ihinduka idatunganyije, bigatuma 15-25% bya oligosaccharides z'igitsina zidakorwa mu gihe cy'amazi ya enzyme yavuwe mbere. Iki ni ikibazo gikunze kugaragara mu buryo butandukanye bwo kuvura biomass. Zimwe mu mpamvu zitera iki kibazo zirimo gukumira enzymes mu gihe cy'amazi ya hydrolysis, cyangwa kubura cyangwa kugabanuka kwa enzymes z'ingenzi zikenewe kugira ngo zice imiyoboro y'isukari mu bimera. Gusobanukirwa imiterere n'imiterere y'isukari, nk'imiyoboro y'isukari muri oligosaccharides, bizadufasha kunoza ihinduka ry'isukari mu gihe cy'amazi ya hydrolysis, bigatuma inzira z'ikoranabuhanga mu binyabuzima zihenda cyane n'ibikomoka kuri peteroli.
Kumenya imiterere ya karubohidrati biragoye kandi bisaba uburyo butandukanye nka chromatography y'amazi (LC)11,12, spectroscopy ya magnetic resonance nuclear (NMR)13, electrophoresis ya capillary (CE)14,15,16 na spectrometry ya mass (MS)17. ,18. Uburyo bwa MS nka spectrometry ya mass ya time-of-flight hamwe na laser desorption na ionization hakoreshejwe matrix (MALDI-TOF-MS) ni uburyo bukoreshwa cyane mu kumenya imiterere ya karubohidrati. Vuba aha, Collision-Induced Dissociation (CID) tandem MS ya sodium ion adducts yakoreshejwe cyane mu kumenya ibikumwe bijyanye n'aho oligosaccharide ihurira, imiterere ya anomeric, urukurikirane, n'aho amashami ahurira 20, 21.
Isesengura rya Glycan ni igikoresho cyiza cyane cyo kumenya byimbitse imigozi ya karubohidrati22. Ubu buryo bukoresha antibodies za monoclonal (mAbs) zigenewe glycan y'urukuta rw'uturemangingo tw'ibimera nk'ibikoresho byo gupima kugira ngo bumve isano ikomeye ya karubohidrati. Haraboneka mAbs zirenga 250 ku isi yose, zakozwe ku miterere itandukanye ya oligosaccharide n'amashami hakoreshejwe saccharides zitandukanye24. MAbs nyinshi zakoreshejwe cyane mu kugena imiterere, imiterere, n'impinduka z'urukuta rw'uturemangingo tw'ibimera, kuko hari itandukaniro rinini bitewe n'ubwoko bw'uturemangingo tw'ibimera, ingingo, imyaka, intambwe y'iterambere, n'aho bikura25,26. Vuba aha, ubu buryo bwakoreshejwe mu gusobanukirwa ubwinshi bw'uturemangingo mu bimera n'inyamaswa ndetse n'uruhare rwabyo mu gutwara glycan nk'uko bigenwa n'ibimenyetso by'uturemangingo, intambwe z'iterambere, cyangwa ibintu bikurura ibidukikije, no kumenya imikorere ya enzyme. Zimwe mu miterere itandukanye ya glycans na xylan zagaragaye harimo 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.
Ariko, nubwo hari ubushakashatsi bwose bwakozwe, ubushakashatsi buke ni bwo bwibanze ku miterere y’ubwiyongere bwa oligosaccharide mu gihe cy’amazi menshi (HSL), harimo no kurekurwa kwa oligosaccharide, impinduka z’uburebure bw’umunyururu wa oligomeric mu gihe cy’amazi, polimeri zitandukanye za DP nke, n’imirongo yazo. Hagati aho, nubwo isesengura rya glycan ryagaragaye ko ari igikoresho cy’ingirakamaro mu isesengura ryimbitse ry’imiterere ya glycan, biragoye gusuzuma oligosaccharide nke za DP zishobora gushonga mu mazi hakoreshejwe uburyo bwa antibody. Oligosaccharide nto za DP zifite uburemere bwa molekile buri munsi ya 5-10 kDa ntizifatana ku ma plate ya ELISA 33, 34 kandi zirogezwa mbere yo kongeramo antibody.
Aha, ku nshuro ya mbere, tugaragaza isuzuma rya ELISA ku masahani ya avidin hakoreshejwe antibodiste za monoclonal, duhuza uburyo bwo gukora biotinylation bw'intambwe imwe kuri oligosaccharides zishonga hamwe n'isesengura rya glycome. Uburyo bwacu bwo gusesengura glycome bwemejwe n'isesengura rishingiye kuri MALDI-TOF-MS na GC-MS ry'imibanire ya oligosaccharide yuzuzanya hakoreshejwe trimethylsilyl (TMS) ikomoka ku isukari ya hydrolyzed. Ubu buryo bushya bushobora guterwa nk'uburyo bwo gutanga umusaruro mwinshi mu gihe kizaza kandi bugakoreshwa cyane mu bushakashatsi bwa siyansi35.
Impinduka mu mikorere ya enzymes na antibody nyuma yo guhindura imikorere, nka glycosylation,36 zigira ingaruka ku mikorere yabyo mu mubiri. Urugero, impinduka mu glycosylation ya poroteyine zo mu mara zigira uruhare runini mu ndwara ziterwa n’ububyimbirwe, kandi impinduka mu glycosylation zikoreshwa nk'ibimenyetso byo gusuzuma37. Ibinyabutabire bitandukanye byavuzwe mu nyandiko ko bigaragara mu ndwara zitandukanye, harimo indwara zidakira zo mu gifu no mu mwijima, indwara za virusi, kanseri y'intanga ngore, iy'ibere, na prostate38,39,40. Gusobanukirwa imiterere ya glycans hakoreshejwe uburyo bwa glycan ELISA bushingiye ku antibody bizatanga icyizere cyinshi mu gusuzuma indwara hatabayeho gukoresha uburyo bugoye bwa MS.
Ubushakashatsi bwacu bwa mbere bwagaragaje ko oligosaccharide zitagira amazi nyuma yo kuvurwa mbere no gushonga hakoreshejwe enzyme (Ishusho ya 1). Mu kazi kacu kasohotse mbere, twakoze uburyo bwo gukuramo amakara bukoresha uburyo bwo gutandukanya oligosaccharide na AFEX-pretreated corn stover hydrolyzate (ACSH)8. Nyuma yo gukuramo no gutandukanya, oligosaccharide zongeye gucibwamo ibice hakoreshejwe ingano ya chromatography (SEC) maze zikusanywa hakurikijwe uburemere bwa molekile. Monomer na oligomer zakuwe mu buryo butandukanye bwo kuvurwa mbere byasesenguwe hakoreshejwe isesengura ry’imiterere y’isukari. Mu kugereranya ingano ya oligosaccharide zabonetse hakoreshejwe uburyo butandukanye bwo kuvurwa mbere, kuba hariho oligosaccharide zitagira amazi ni ikibazo gikunze kubaho mu guhindura biomass muri monosaccharide kandi bishobora gutuma umusaruro w’isukari ugabanuka nibura 10-15% ndetse no kugeza kuri 18%. US. Ubu buryo bukoreshwa mu kongera gukora ibice bya oligosaccharide ku rugero runini. Muri iki gikorwa, ACH n'ibice byayo byakurikiyeho bifite uburemere butandukanye bwa molekile byakoreshejwe nk'ibikoresho byo kugerageza mu kugaragaza oligosaccharides.
Nyuma yo kuvurwa mbere no gukurwaho enzyme, oligosaccharides zihoraho zagumye zitarimo amazi. Aha (A) hari uburyo bwo gutandukanya oligosaccharides aho oligosaccharides zikurwa muri AFEX-pretreated corn stover hydrolysate (ACSH) hakoreshejwe AFEX-activated corn stover na diatomaceous earth; (B) Uburyo bwo gutandukanya oligosaccharides. Oligosaccharides zakomeje gutandukana hakoreshejwe chromatography y'ingano (SEC); (C) Saccharide monomers na oligomers zakuwe mu buryo butandukanye bwo kuvurwa mbere (aside yavuyemo: DA, ionic liquid: IL na AFEX). Imiterere ya hydrolysis ya enzyme: kwiyongera kw'ibintu bikomeye bya 25% (w/w) (hafi 8% by'umutwaro wa glucan), hydrolysis mu masaha 96, 20 mg/g y'inzoga zicuruzwa (Ctec2:Htec2:MP-2:1:1 ratio) na (D) Monomer z'isukari na oligomer za glucose, xylose na arabinose biva muri AFEX pre-treated corn stover (ACS).
Isesengura rya Glycan ryagaragaye ko ari igikoresho cy'ingirakamaro mu isesengura ryimbitse ry’imiterere ya glycans mu binyabutabire byakuwe mu bisigazwa by’ibinyabuzima bikomeye. Ariko, saccharides zishongeshwa mu mazi ntizigaragara neza hakoreshejwe ubu buryo gakondo41 kuko oligosaccharides zifite uburemere buke bwa molekile zigorwa no kuzihagarika ku masahani ya ELISA kandi zigakurwaho mbere yo kongeramo antibody. Kubwibyo, kugira ngo antibody ifatanye kandi imenyekane, hakoreshejwe uburyo bwa biotinylation bw’intambwe imwe kugira ngo hapfukwe oligosaccharides zishongeshwa kandi zidakurikiza amategeko ku masahani ya ELISA apfutswe na avidin. Ubu buryo bwageragejwe hakoreshejwe ACSH yacu yakozwe mbere n’igice gishingiye ku buremere bwayo bwa molekile (cyangwa urugero rwa polymerization, DP). Biotinylation y’intambwe imwe yakoreshejwe mu kongera ubushobozi bwo guhuza oligosaccharide hiyongereyeho biotin-LC-hydrazide ku mpera igabanya karubohidrati (Ishusho ya 2). Mu gusesengura, itsinda rya hemiacetal ku mpera igabanya rikorana n’itsinda rya hydrazide rya biotin-LC-hydrazide kugira ngo rikore umugozi wa hydrazone. Iyo hari ikintu kigabanya NaCNBH3, umugozi wa hydrazone ugabanuka ukagera ku musozo uhamye wa biotinylated. Hamwe no guhindura umusozo ugabanya isukari, guhuza oligosaccharides nke za DP na ELISA byarashobotse, kandi mu bushakashatsi bwacu ibi byakozwe ku masahani ya avidin hakoreshejwe mAbs zigamije glycan.
Gusuzuma utunyangingo tw’umubiri dushingiye kuri ELISA kugira ngo harebwe oligosaccharide za biotinyle. Aha (A) guhuza biotinyle ya oligosaccharide n’isuzuma rya ELISA nyuma yabyo hakoreshejwe mAbs zigamije glycan ku masahani ya NeutrAvidin atwikiriwe na (B) bigaragaza inzira imwe yo guhuza biotinyle y’ibikomoka ku bisubizo.
Hanyuma ibyuma bya Avidin bifite antibodies zifatanye na oligosaccharide byongewe kuri antibodies z’ibanze n’iza kabiri hanyuma byozwa mu buryo bworoheje kandi bukoresha igihe. Nyuma yo guhuza antibodies, shyiramo substrate ya TMB kugira ngo ushyire kuri plate. Amaherezo igisubizo cyahagaritswe na aside sulfurike. Ibikoresho byashyizwe muri plate byasesenguwe hakoreshejwe ELISA reader kugira ngo hamenyekane imbaraga zo guhuza za buri antibodies kugira ngo hamenyekane aho antibodies zihurira. Kubindi bisobanuro n'ibipimo by'igerageza, reba igice kijyanye nacyo "Ibikoresho n'Uburyo".
Tugaragaza akamaro k'ubu buryo bushya bwo gukoresha mu buryo bwihariye, dusobanura oligosaccharides zishongeshwa ziba muri ACSH ndetse no mu duce twa oligosaccharide twakuwe muri hydrolysates za lignocellulosic. Nkuko bigaragara ku Ishusho ya 3, xylans zikunze gusimburwa na epitope zabonetse muri ACSH hakoreshejwe uburyo bwa bioacylated glycome assay akenshi ni uronic (U) cyangwa methyluronic (MeU) na pectic arabinogalactans. Inyinshi muri zo zabonetse mu bushakashatsi bwacu bwa mbere ku isesengura rya glycans z'ibintu bidafite hydrolyzed (UHS)43.
Gutahura epitopes za oligosaccharide zisubira inyuma hakoreshejwe antibody ya monoclonal yerekeza kuri glycan yo ku rukuta rw'uturemangingo. Igice "kidahuza" ni igice cya ACN naho igice "kidahuza" ni igice cya FA. Umutuku utukura cyane ku gipimo cy'ubushyuhe ugaragaza ingano nini ya epitope, naho ubururu butangaje bugaragaza inyuma y'ubusa. Ingano y'amabara ku gipimo ishingiye ku gaciro ka OD gasanzwe ku miterere N = 2. Epitopes z'ingenzi zigaragazwa n'abasirikari b'umubiri zigaragara iburyo.
Izi ngingo zitari selulosi ntizashoboraga gucibwamo ibice na selulosi zisanzwe cyane mu mvange ya enzymes zo mu bucuruzi zageragejwe, zirimo na enzymes zikoreshwa cyane mu bucuruzi. Kubwibyo, hakenewe enzymes nshya z'inyongera kugira ngo zihingurwe. Hatabayeho enzymes zikenewe zitari selulosi, izi mvange zitari selulosi zibuza guhinduka burundu muri monosaccharides, nubwo polymers z'isukari zazo zavangwa cyane mu bice bigufi kandi zigashonga hakoreshejwe uruvange rwa enzymes zo mu bucuruzi.
Inyigo y’inyongera ku ikwirakwizwa ry’ibimenyetso n’imbaraga zabyo zo gufatana byagaragaje ko epitopes zo gufatana zari nke mu bice by’isukari bya DP byinshi (A, B, C, DP kugeza kuri 20+) kurusha mu bice by’isukari bya DP bike (D, E, F, DP) mu bipimo (Ishusho ya 1). Ibice bya aside bikunze kugaragara mu bice by’isukari bitari cellulose kurusha ibice bidafite aho bibogamiye. Ibi bintu bihuye n’icyitegererezo cyagaragaye mu bushakashatsi bwacu bwabanje, aho ibice byinshi bya DP na aside byari birwanya cyane hydrolysis ya enzymatic. Kubwibyo, kuba hariho epitopes za glycan zitari cellulose hamwe n’izindi nsimburangingo za U na MeU bishobora kugira uruhare runini mu gutuma oligosaccharide ziguma neza. Tugomba kumenya ko gufatana no kumenya neza bishobora guteza ikibazo kuri oligosaccharide nke za DP, cyane cyane niba epitope ari oligosaccharide ya dimeric cyangwa trimeric. Ibi bishobora kugeragezwa hakoreshejwe oligosaccharide zo mu bucuruzi zifite uburebure butandukanye, buri kimwe kirimo epitope imwe gusa ifatana na mAb runaka.
Bityo, ikoreshwa ry’uturemangingo tw’umubiri twihariye ryagaragaje ubwoko bumwe na bumwe bw’imigozi isubira inyuma. Bitewe n’ubwoko bw’uturemangingo twakoreshejwe, imiterere ikwiye yo kwihuza, n’imbaraga z’ikimenyetso gikora (cyane cyane kandi gito cyane), enzymes nshya zishobora kumenyekana no kongerwamo mu buryo bungana ku mvange y’uturemangingo kugira ngo habeho glycoconversion yuzuye. Dufashe urugero rw’isesengura rya oligosaccharides za ACSH, dushobora gukora ububiko bw’imigozi ya glycan kuri buri kintu cy’ibinyabuzima. Aha ni ngombwa kuzirikana ko ubwinshi butandukanye bw’uturemangingo bugomba kwitabwaho, kandi niba ubwinshi bwatwo butazwi, ibi bizatera ingorane zimwe na zimwe mu kugereranya ibimenyetso by’uturemangingo dutandukanye. Byongeye kandi, kugereranya imigozi ya glycan bishobora gukora neza hagati y’ingero z’uturemangingo tumwe. Izi migozi zikomeye zishobora guhuzwa na database ya CAZyme, aho dushobora kumenya enzymes, guhitamo enzymes zigomba gukoreshwa no gupima enzymes zica imigozi, cyangwa guteza imbere sisitemu za mikorobe kugira ngo zigaragaze izi enzymes zo gukoreshwa muri biorefineries44.
Kugira ngo dusuzume uburyo uburyo bwo kurwanya indwara bwuzuzanya n’ubundi buryo bwo kugaragaza oligosaccharide zifite uburemere buke bwa molekile ziba muri hydrolysates za lignocellulosic, twakoze MALDI (Ishusho ya 4, S1-S8) no gusesengura saccharide zikomoka kuri TMS zishingiye kuri GC-MS ku gice kimwe cya oligosaccharide (Ishusho ya 5). MALDI ikoreshwa mu kugereranya niba ikwirakwizwa ry’ibice bya oligosaccharide rihuye n’imiterere yagenwe. Ku ishusho ya 4 hagaragaramo MS y’ibice bidafite aho bibogamiye bya ACN-A na ACN-B. Isesengura rya ACN-A ryemeje ubwoko butandukanye bw’isukari ya pentose kuva kuri DP 4–8 (Ishusho ya 4) kugeza kuri DP 22 (Ishusho ya S1), uburemere bwayo buhuye na MeU-xylan oligosaccharides. Isesengura rya ACN-B ryemeje uruhererekane rwa pentose na glucoxylan hamwe na DP 8-15. Mu bikoresho by'inyongera nka Figure S3, amakarita yo gukwirakwiza aside ya FA-C agaragaza ubwoko butandukanye bwa (Me)U isukari ya pentose yasimbuwe na (DP) ifite 8-15 ihuye na xylan zasimbuwe ziboneka mu gusuzuma mAb bishingiye kuri ELISA. Epitopes zirahuye.
MALDI-MS spectrum y'ibinure bitarahuza n'ibinure biboneka muri ACS. Aha, (A) Ibice bya ACN-A bifite uburemere buke birimo aside uronic ya methyl (DP 4-8) byasimbuje glucuroxylan oligosaccharides na (B) ACN-B xylan na methylated uronic acid oligosaccharides byasimbujwe glucuroxylan (DP 8-15).
Isesengura ry'imiterere y'ibisigazwa bya glycan bya oligosaccharides zidashobora guhumeka. Hano (A) Imiterere ya TMS ya saccharides z'ibice bitandukanye bya oligosaccharides byabonetse hakoreshejwe isesengura rya GC-MS. (B) Imiterere y'isukari itandukanye ikomoka kuri TMS iboneka muri oligosaccharides. ACN - igice cya acetonitrile kirimo oligosaccharides zidafite aho zibogamiye na FA - igice cya aside ferulic kirimo oligosaccharides zidafite aho zibogamiye.
Undi mwanzuro ushimishije wavuye mu isesengura rya LC-MS ry'igice cya oligosaccharide, nk'uko bigaragara ku Ishusho S9 (uburyo buboneka mu bikoresho by'inyongera bya elegitoroniki). Ibice by'amatsinda ya hexose na -OAc byagaragaye kenshi mu gihe cyo gushyira igice cya ACN-B. Iki cyavumbuwe nticyemeza gusa icikamo ibice ryagaragaye mu isesengura rya glycome na MALDI-TOF, ahubwo gitanga amakuru mashya ku bikomoka kuri karubohidrati bishobora kuba mu bimera bya lignocellulosic byavuwe mbere.
Twakoze kandi isesengura ry’imiterere ya glycan ku bice bya oligosaccharide dukoresheje TMS glycan derivatization. Dukoresheje GC-MS, twamenye imiterere y’isukari y’imitsi (idafite ikomoka kuri yo) n’isukari ishaje (GluA na GalA) mu gice cya oligosaccharide (Ishusho ya 5). Aside Glucuronic iboneka mu bice bya aside C na D, mu gihe aside galacturonic iboneka mu bice bya aside A na B, byombi bikaba ari ibice byinshi bya DP by’isukari ishaje. Ibi bisubizo ntibishimangira gusa amakuru yacu ya ELISA na MALDI, ahubwo binahuye n’ubushakashatsi twakoze mbere ku ikusanyirizo rya oligosaccharide. Kubwibyo, twizera ko uburyo bugezweho bwo kurwanya indwara bukoresha biotinylation ya oligosaccharide no gusuzuma ELISA nyuma yaho bihagije kugira ngo harebwe oligosaccharide zisubira inyuma mu buryo busanzwe mu bipimo bitandukanye by’ibinyabuzima.
Kubera ko uburyo bwo gusuzuma mAb bushingiye kuri ELISA bwemejwe hakoreshejwe uburyo butandukanye, twifuje gukomeza gusuzuma ubushobozi bw'ubu buryo bushya bwo gupima. Oligosaccharides ebyiri z'ubucuruzi, xylohexasaccharide oligosaccharide (XHE) na 23-α-L-arabinofuranosyl-xylotriose (A2XX), zaguzwe kandi zigeragezwa hakoreshejwe uburyo bushya bwa mAb bugamije glycan yo ku rukuta rw'uturemangingo. Ishusho ya 6 igaragaza isano iri hagati y'ikimenyetso cyo gufatana kwa biotinylated n'ubwinshi bwa oligosaccharide, bigaragaza ko Langmuir adsorption mode ishoboka. Muri mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, na CCRC-M151 bifitanye isano na XHE, na CCRC-M108, CCRC-M109, na LM11 bifitanye isano na A2XX ku ntera iri hagati ya nano 1 nano 100. Bitewe n’uko antibodies zitaboneka neza mu gihe cy’igerageza, hakozwe igerageza rito kuri buri gipimo cya oligosaccharide. Aha ngaha, ni ngombwa kumenya ko antibodies zimwe na zimwe zigira ingaruka zitandukanye cyane kuri oligosaccharide imwe nk’iy’ishingiro, bishoboka ko biterwa nuko zigira epitopes zitandukanye gato kandi zishobora kugira isano zitandukanye cyane. Uburyo n’ingaruka zo kumenya epitopes neza bizaba bigoye cyane iyo uburyo bushya bwa mAb bukoreshejwe ku bipimo nyabyo.
Hakoreshejwe oligosaccharide ebyiri zo mu bucuruzi kugira ngo hamenyekane urwego rwo gupima mAbs zitandukanye zigamije glycan. Aha, isano iri hagati y’umurongo n’ingano ya oligosaccharide igaragaza imiterere ya Langmuir yo guterwa (A) XHE hamwe na mAb na (B) A2XX hamwe na mAb. Epitopes zijyanye nazo zigaragaza imiterere ya oligosaccharide zo mu bucuruzi zikoreshwa nk'ibipimo mu isuzuma.
Gukoresha antibodies za monoclonal zigamije glycan (isesengura rya glycocomic cyangwa isuzuma rya mAb rishingiye kuri ELISA) ni igikoresho gikomeye cyo gusobanura byimbitse inyinshi mu nkuta z’uturemangingo zigize biomass y’ibimera. Ariko, isesengura rya glycan rya kera rigaragaza gusa insengero nini za glycans, kuko inyinshi muri oligosaccharides zidashyirwa mu buryo bunoze ku masahani ya ELISA. Muri ubu bushakashatsi, ikigori cya AFEX cyatezwe mbere cyashyizwe mu buryo bwa enzyme ku gipimo kinini cy’ibintu bikomeye. Isesengura ry’isukari ryakoreshejwe kugira ngo hamenyekane imiterere ya karubohidrati y’uturemangingo isubira inyuma muri hydrolyzate. Ariko, isesengura rya mAb rya oligosaccharides nto muri hydrolyzate ntirifatwa nk’iry’ingenzi, kandi hakenewe izindi mfashanyigisho kugira ngo oligosaccharides zihagarike neza ku masahani ya ELISA.
Aha turatangaza uburyo bushya kandi bwiza bwo gupima oligosaccharide mu buryo bwo gupima mAb binyuze mu guhuza biotinylation ya oligosaccharide hagakurikiraho gusuzuma ELISA ku masahani ya NeutrAvidin™. Oligosaccharide za biotinylated zitagenda zagaragaje ubushobozi buhagije kuri antibody kugira ngo habeho gupima vuba kandi neza oligosaccharide zisubira inyuma. Isesengura ry’imiterere y’izi oligosaccharide zikomeye hashingiwe kuri spectrometry y’ubunini ryemeje ibyavuye muri ubu buryo bushya bwo gusuzuma ubudahangarwa bw’umubiri. Bityo, ubu bushakashatsi bwerekana ko guhuza biotinylation ya oligosaccharide na ELISA hamwe na antibodies za monoclonal zigamije glycan bishobora gukoreshwa mu kumenya imikoranire muri oligosaccharide kandi bishobora gukoreshwa cyane mu bundi bushakashatsi bwa biochemical bugaragaza imiterere ya oligosaccharide.
Ubu buryo bwo gusesengura glycan bushingiye kuri biotin ni bwo raporo ya mbere ishobora gukora iperereza ku migozi ya karubohidrati idashobora guhindurwa ya oligosaccharide ishongeshwa mu bimera. Ibi bifasha gusobanukirwa impamvu bimwe mu bice bya biomass bigora cyane iyo bigeze ku ikorwa rya biofuel. Ubu buryo bwuzuza icyuho gikomeye mu buryo bwo gusesengura glycome kandi bukanagura ikoreshwa ryabwo ku bintu bitandukanye birenze oligosaccharide z'ibimera. Mu gihe kizaza, dushobora gukoresha robotics mu gusesengura biotinylation no gukoresha uburyo twakoze mu gusesengura ingero hakoreshejwe ELISA.
Ibishishwa by'ibigori (CS) byahinzwe mu mbuto z'ubwoko bwa Pioneer 33A14 byasaruwe mu 2010 muri Kramer Farms i Ray, muri Colorado. Binyuze ku ruhushya rwa nyir'ubutaka, iyi biomass ishobora gukoreshwa mu bushakashatsi. Ingero zabitswe zumye <6% by'ubushuhe mu mifuka ifunze neza mu bushyuhe bw'icyumba. Ingero zabitswe zumye <6% by'ubushuhe mu mifuka ifunze neza mu bushyuhe bw'icyumba. Об କାନцы хранились сух мин при влажности <6% в пакакх с застежкой-молнией при комнатной мае. Ingero zabitswe zumye ku bushyuhe buri munsi ya 6% mu mifuka ifite zipu ku bushyuhe bw'icyumba.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% Об କାନцы хранят в пак сех застежкой-молнией при комнатной мае с влажностью <6%. Ingero zibikwa mu mifuka ya zipu ku bushyuhe bw'icyumba burimo ubushuhe buri munsi ya 6%.Ubushakashatsi bwakurikije amabwiriza y’ibanze n’ay’igihugu. Isesengura ry’ibikubiye mu bi ...
Cellic® CTec2 (138 mg poroteyine / ml, lot VCNI 0001) ni uruvange rw'urusobe rwa cellulase, β-glucosidase na Cellic® HTec2 (157 mg poroteyine / ml, lot VHN00001) bya Novozymes (Franklinton, NC, USA)). Multifect Pectinase® (72 mg poroteyine / mL), uruvange rw'urusobe rwa enzymes za pectin, yatanzwe na DuPont Industrial Biosciences (Palo Alto, CA, USA). Ingano ya poroteyine ya enzyme yagenwe hapimwa ingano ya poroteyine (hakurwamo uruhare rwa azote itari poroteyine) hakoreshejwe isesengura rya azote rya Kjeldahl (uburyo bwa AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA). Diatomaceous earth 545 yaguzwe muri EMD Millipore (Billerica, MA). Karuboni ikora (DARCO, utubumbe 100 tw’umuyoboro), Avicel (PH-101), beech xylan, n’indi miti yose yaguzwe muri Sigma-Aldrich (St. Louis, MO).
Ubuvuzi bwa AFEX bwakorewe muri GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). Ubuvuzi bwakozwe ku bushyuhe bwa 140°C. mu gihe cy'iminota 15. Igihe cyo gutura cya 46 ku gipimo cya 1:1 cya ammonia ya anhydrous na biomass kuri 60% (w/w) mu cyuma gishyushya icyuma kidafunze (Parr Instruments Company). Byatwaye iminota 30. Reactor yagejejwe kuri 140°C maze ammonia irekurwa vuba, bituma biomass isubira vuba ku bushyuhe bw'icyumba. Imiterere ya AFEX pre-treated corn stover (ACS) yari isa n'iya AFEX pre-treated corn stover (UT-CS).
Ibinyabutabire byinshi bya ACSH 25% (w/w) (hafi 8% by'umutwaro wa dextran) byateguwe nk'ibikoresho byo gutangira gukora oligosaccharides nyinshi. Hydrolysis ya enzimatike ya ACS yakozwe hakoreshejwe uruvange rwa enzyme irimo Cellic® Ctec2 10 mg protein/g glucan (muri biomass yavuwe mbere), Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan, na Multifect Pectinase (Genencor Inc, USA). ). ), 5 mg protein/g dextran. Hydrolysis ya enzimatike yakozwe muri bioreactor ya litiro 5 ifite ingano y'akazi ya litiro 3, pH 4.8, 50°C na 250 rpm. Nyuma yo guhidrolysis mu masaha 96, hydrolyzate yakusanyijwe hakoreshejwe centrifugation kuri 6000 rpm mu minota 30 hanyuma kuri 14000 rpm mu minota 30 kugira ngo ikuremo ibinyabutabire bitarahishijwe. Hanyuma hydrolyzate yashyizwe mu icupa rya 0.22 mm rya filter. Hydrolyzate yayunguruwe yabikwaga mu macupa ya vinyl ku bushyuhe bwa 4°C. hanyuma ishyirwa mu bice bya carbon.
Isesengura ry'imiterere y'ibikomoka kuri biomass bishingiye ku bimera hakurikijwe uburyo bwo gusesengura muri laboratwari ya NREL: gutegura ingero zo gusesengura imiterere y'ibimera (NREL/TP-510-42620) no kumenya imiterere ya karubohidrati na lignin muri biomass (NREL/TP-510 - 42618)47.
Isesengura rya Oligosaccharide ku mugezi wa hydrolyzate ryakozwe ku gipimo cya mililitiro 2 hakoreshejwe uburyo bwa autoclave-based acid hydrolysis. Vanga icyitegererezo cya hydrolyzate na 69.7 µl ya aside sulfurike 72% mu muyoboro wa screw cap wa mililitiro 10 hanyuma ubishyire mu cyuma mu gihe cy'isaha imwe ku ntebe kuri 121 °C, ubikonjeshe ku rubura hanyuma ubishyire mu gacupa ka HPLC gafite ubushobozi bwo hejuru. Igipimo cya oligosaccharide cyagenwe hakurwamo igipimo cya monosaccharide mu gipimo kitari hydrolyzed mu gipimo cy'isukari yose mu gipimo cya aside hydrolyzed.
Igipimo cya glucose, xylose, na arabinose mu bimera bya aside hidrolised byasesenguwe hakoreshejwe sisitemu ya Shimadzu HPLC ifite autosampler, column heater, isocratic pump, na refractive index detector ku nkingi ya Bio-Rad Aminex HPX-87H. Inkingi yagumye kuri 50°C kandi yakuweho 0.6 ml/min 5 mM H2SO4 mu mazi.
Isukari ya hydrolyzate yagabanijwe ikanasesengurwa kugira ngo harebwe ingano ya monomer na oligosaccharide. Isukari ya monomeric yabonetse nyuma ya hydrolysis ya enzymatic yasesenguwe na HPLC ifite inkingi ya Bio-Rad (Hercules, CA) Aminex HPX-87P n'inkingi yo kurinda ivu. Ubushyuhe bw'inkingi bwagumye kuri 80°C, amazi yakoreshejwe nk'ikigero cyo kugenda hamwe n'umuvuduko wa 0.6 ml/min. Oligosaccharides zagenwe hakoreshejwe hydrolysis muri aside dilute kuri 121°C hakurikijwe uburyo bwasobanuwe mu mareferensi 41, 48, 49.
Isesengura rya Saccharide ryakozwe ku bisigazwa bya biomass bibisi, bya AFEX byavuwe mbere ndetse n’ibidakoreshejwe amazi (harimo no gukora ibisigazwa by’urukuta rw’uturemangingo hamwe no gusuzuma mAb) hakoreshejwe uburyo bwavuzwe mbere 27, 43, 50, 51. Mu isesengura rya glycome, ibisigazwa bidashonga by’uturemangingo tw’uturemangingo tw’ibimera bitegurwa bivuye ku bisigazwa by’uturemangingo tw’ibimera kandi bigakurwamo ibintu byinshi birimo imbaraga nyinshi nka ammonium oxalate (50 mM), sodium carbonate (50 mM na 0.5% w/v), CON. (1M na 4M, byombi hamwe na 1% w/v sodium borohydride) na aside chlorite nk'uko byasobanuwe mbere52,53. Ibisigazwa byashyizwe muri ELISA ku gice cy’ingenzi cya mAb50s cyoherejwe kuri glycan y’urukuta rw’uturemangingo, maze uburyo mAb ifatana nabyo bwagaragajwe nk'ikarita y'ubushyuhe. MAbs igamije glycan y’urukuta rw’uturemangingo tw’ibimera byaguzwe mu bubiko bwa laboratwari (CCRC, JIM na MAC series).
Uburyo bumwe bwo guhuza oligosaccharide. Guhuza karubohidrati na biotin-LC-hydrazide byakozwe hakoreshejwe uburyo bukurikira. Biotin-LC-hydrazide (4.6 mg/12 μmol) yashongeshejwe muri dimethyl sulfoxide (DMSO, 70 μl) binyuze mu gukangura no gushyushya cyane kuri 65° C. mu gihe cy'umunota 1. Hongewemo aside acetic (30 µl) hanyuma uruvange rusukwa kuri sodium cyanoborohydride (6.4 mg/100 µmol) hanyuma rushonga burundu nyuma yo gushyushya kuri 65° C. mu gihe cy'umunota 1. Hanyuma, kuva kuri 5 kugeza kuri 8 μl by'uruvange rw'ibisubizo byongewe kuri oligosaccharide yumye (1-100 nmol) kugira ngo haboneke molar excess ikubye inshuro 10 cyangwa zirenzeho ku gice cyo kugabanya. Ibisubizo byakozwe kuri 65° C mu gihe cy'amasaha 2, hanyuma ingero zihita zisukurwa. Nta cyanoborohydride ya sodium yakoreshejwe mu igerageza ryo gushyira ibimenyetso ku bipimo nta kugabanya, kandi ingero zasubiwemo kuri dogere selisiyusi 65 mu gihe cy'amasaha 2.5.
Gusiga no koza ingero za oligosaccharide za biotinyle. 25 μl by'ingero za biotinyle (100 μl bya buri ngero yashyizwemo muri 5 ml y'umuti wa Tris buffer wa 0.1 M (TBS)) byongewe kuri buri kiroba cy'isahani yashyizwemo avidin. Imbobo zo kugenzura zasizwe 50 μl bya biotin ku gipimo cya 10 μg/ml muri 0.1 M TBS. Amazi yakuwemo iion yakoreshejwe nk'igipimo cyo gupima ubusa. Ikinini cyashyizwe mu icupa ry'amazi amasaha 2 ku bushyuhe bw'icyumba mu mwijima. Oza isahani inshuro 3 ukoresheje amata ya 0.1% yakuwemo icupa muri 0.1 M TBS ukoresheje gahunda nimero 11 ya Grenier flat 3A.
Kongeramo no koza antibodies z’ibanze. Ongeraho 40 µl ya antibodies z’ibanze kuri buri mwobo. Shyira microplate mu gihe cy’isaha 1 ku bushyuhe bw’icyumba mu mwijima. Hanyuma amasahani yogejwe inshuro 3 hamwe n’amata ya 0.1% muri 0.1M TBS hakoreshejwe gahunda yo koza #11 ya Grenier Flat 3A.
Shyiramo antibody ya kabiri hanyuma woge. Shyiramo 50 µl ya antibody ya kabiri y'imbeba/imbeba (yavanzwemo 1:5000 mu mata ya 0.1% mu mata ya 0.1 M TBS) kuri buri mwobo. Shyira microplate mu gihe cy'isaha 1 ku bushyuhe bw'icyumba mu mwijima. Hanyuma microplates zogejwe inshuro 5 hamwe n'amata ya 0.1% mu mata ya 0.1 M TBS hakoreshejwe gahunda ya Grenier Flat 5A yoza plate #12.
Kongeramo substrate. Ongeraho 50 µl ya 3,3′, 5,5′-tetramethylbenzidine (TMB) ku substrate y'ibanze (wongeramo ibitonyanga 2 bya buffer, ibitonyanga 3 bya TMB, ibitonyanga 2 bya hydrogen peroxide kuri ml 15 z'amazi ya deionized). Tegura substrate ya TMB. na vortex mbere yo kuyikoresha). Shyira microplate ku bushyuhe bw'icyumba mu gihe cy'iminota 30. Mu mwijima.
Uzuza intambwe hanyuma usome ikinini. Ongeraho 50 µl ya aside sulfurike 1 N kuri buri ruzi hanyuma wandike uko amazi ashoka kuva kuri 450 kugeza kuri 655 nm ukoresheje icyuma gisoma ELISA.
Tegura 1 mg/ml y'ibi bisesengura mu mazi yakuwemo ioni: arabinose, rhamnose, fucose, xylose, aside galacturonic (GalA), aside glucuronic (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine. (glcNAc), N-acetylgalactosamine (galNAc), inositol (igipimo cy'imbere). Ibipimo bibiri byateguwe hongewemo 1 mg/mL isukari igaragara ku mbonerahamwe ya 1. Ingero zirakonjeshwa kandi zigashyirwa kuri -80° C. kugeza amazi yose akuweho (ubusanzwe amasaha 12-18).
Ongeraho µg 100–500 z'icyitegererezo mu miyoboro y'ifatizo ku buryo bwo gusesengura. Andika ingano y'icyitegererezo. Ni byiza gushonga icyitegererezo mu gipimo runaka cya solvent hanyuma ukagishyira mu muyoboro nk'amazi. Koresha µl 20 za 1 mg/ml inositol nk'icyitegererezo cy'imbere kuri buri muyoboro w'icyitegererezo. Ingano y'icyitegererezo cy'imbere cyongewe ku cyitegererezo igomba kuba imwe n'ingano y'icyitegererezo cy'imbere cyongewe ku muyoboro usanzwe.
Shyiramo mililitiro 8 za methanoli idafite amazi mu gacupa k'umupfundikizo. Hanyuma shyiramo mililitiro 4 z'umuti wa HCl wa 3 N. methanolique, upfundikishe hanyuma unyeganyeze. Ubu buryo ntibukoresha amazi.
Shyiramo 500 µl ya 1 M HCl methanol mu bipimo bya oligosaccharide n'imiyoboro isanzwe ya TMS. Ibipimo byashyizwe mu icuba ijoro ryose (amasaha 168) kuri 80°C. mu gice cy'ubushyuhe. Kumisha methanolysis product ku bushyuhe bw'icyumba ukoresheje icyuma cyo kumisha. Shyiramo 200 µl MeOH hanyuma wongere wumishe. Iki gikorwa gisubirwamo kabiri. Shyiramo 200 µl ya methanol, 100 µl ya pyridine na 100 µl ya acetic anhydride mu gipimo hanyuma uvange neza. Shyiramo ibipimo ku bushyuhe bw'icyumba mu minota 30. hanyuma wumishe. Shyiramo 200 µl ya methanol hanyuma wongere wumishe.
Ongeraho 200 µl ya Tri-Sil hanyuma ushyushye umuyoboro upfundikiye mu gihe cy'iminota 20. 80°C, hanyuma ukonjeshe kugeza ku bushyuhe bw'icyumba. Koresha agakoresho ko kumisha kugira ngo wongere wumishe icyitegererezo kugeza ku gipimo cya 50 µl. Ni ngombwa kumenya ko tutaretse icyitegererezo cyuma burundu.
Ongeramo mililitiro 2 za hexane hanyuma uvange neza ukoresheje vortex. Uzuza igice cy'ubwoya bw'ikirahure ku mitwe ya Pasteur pipettes (milimetero 5-8) ushyiramo ubwoya bw'ikirahure hejuru y'umuyoboro wa santimetero 5-3/4 z'umurambararo. Ingero zashyizwe muri centrifuge kuri 3000 g mu gihe cy'iminota 2. Ibisigazwa byose bidashonga birashongeshwa. Kumisha icyitegererezo kugeza kuri 100-150 µl. Ingano ya 1 μl yashyizwe muri GC-MS ku bushyuhe bwa mbere bwa 80 °C n'igihe cya mbere cya iminota 2.0 (Imbonerahamwe ya 2).