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Njira zatsopano za immunological ndi misa spectrometric zowunikira zovuta za oligosaccharides mosalekeza mu stover ya chimanga pretreated ndi AFEX.Lignocellulosic biomass ndi njira yokhazikika yopangira mafuta oyaka kale ndipo imagwiritsidwa ntchito kwambiri popanga utekinoloje wachilengedwe popanga zinthu monga chakudya, chakudya, mafuta ndi mankhwala.Chinsinsi cha matekinolojewa ndikukula kwa njira zotsika mtengo zosinthira ma carbohydrate ovuta omwe amapezeka m'makoma a cell cell kukhala shuga wosavuta monga shuga, xylose ndi arabinose.Chifukwa lignocellulosic zotsalira zazomera ndi amakani kwambiri, ayenera pansi pa thermochemical mankhwala (mwachitsanzo, ammonia CHIKWANGWANI exfoliation (AFEX), kuchepetsa zidulo (DA), ionic zamadzimadzi (IL)) ndi mankhwala kwachilengedwenso (mwachitsanzo, enzymatic hydrolysis ndi tizilombo ting'onoting'ono nayonso mphamvu) osakaniza kupeza ankafuna mankhwala..Komabe, ma enzymes a fungal akagwiritsidwa ntchito mu hydrolysis, 75-85% yokha ya shuga wosungunuka wopangidwa ndi monosaccharides, ndipo otsala 15-25% amakhala osungunuka, oligosaccharides osasinthika, omwe sapezeka nthawi zonse kwa tizilombo tating'onoting'ono.M'mbuyomu, tapatula bwino ndikuyeretsa oligosaccharides osungunuka osungunuka pogwiritsa ntchito kuphatikizika kwa kaboni ndi diatomaceous kupatukana kwa dziko lapansi ndi kukula kwa chromatography, ndikufufuzanso zomwe zimalepheretsa ma enzyme.Tapeza kuti oligosaccharides okhala ndi digiri yapamwamba ya polymerization (DP) methylated uronic acid substitutes ndizovuta kwambiri popanga ndi malonda a enzyme blends kuposa DP otsika ndi oligosaccharides neutral.Apa tikuwonetsa kugwiritsa ntchito njira zingapo zowonjezera, kuphatikiza glycan profiling pogwiritsa ntchito ma monoclonal antibodies (mAbs) enieni obzala biomass glycans kuti awonetsere ma glycan bond mu makoma a cell cell ndi enzymatic hydrolysates, matrix-assisted laser desorption ionization, nthawi-ya-ndege mass- spectrometry..MALDI-TOF-MS) imagwiritsa ntchito nsonga zodziwikiratu zodziwikiratu zomwe zimapezedwa ndi spectroscopy pambuyo pakuwola kwachiwiri kwa ma ion negative, chromatography ya gas and mass spectrometry (GC-MS) kuzindikiritsa zomangira za oligosaccharide ndi popanda kutulutsa.Chifukwa cha kukula kwa oligosaccharides (DP 4-20), mamolekyuwa ndi ovuta kugwiritsira ntchito mAb kumanga ndi kufotokozera.Kuti tithane ndi vutoli, tidagwiritsa ntchito njira yatsopano ya biotin conjugation-based oligosaccharide immobilization yomwe idalemba bwino ma oligosaccharides ambiri osungunuka a DP pamtunda wa microplate, womwe udagwiritsidwa ntchito pamakina apamwamba a mAb pakuwunika mwachindunji.Njira yatsopanoyi ithandizira kupanga zoyezera zapamwamba kwambiri za glycome mtsogolomo zomwe zitha kugwiritsidwa ntchito kudzipatula ndikuzindikiritsa ma oligosaccharides omwe amapezeka muzolemba zamankhwala kuti adziwe matenda.
Lignocellulosic biomass, yopangidwa ndi ulimi, nkhalango, udzu ndi matabwa, ndi chakudya chomwe chingathe kupanga zinthu zochokera kumoyo, kuphatikizapo chakudya, chakudya, mafuta ndi mankhwala opangira mankhwala kuti apange zinthu zamtengo wapatali1.Zakudya zama carbohydrate (monga cellulose ndi hemicellulose) zomwe zimapezeka m'makoma a cellulose amasinthidwa kukhala ma monosaccharides popanga mankhwala ndi biotransformation (monga enzymatic hydrolysis ndi fermentation ya microbial).Mankhwala omwe amagwiritsidwa ntchito nthawi zambiri amaphatikizapo kuwonjezereka kwa ammonia fiber (AFEX), dilute acid (DA), ionic liquid (IL), ndi steam explosion (SE), yomwe imagwiritsa ntchito mankhwala osakanikirana ndi kutentha kuti achepetse kupanga lignocellulose potsegula makoma a chomera3,4.kuuma kwa nkhani, 5. Enzymatic hydrolysis ikuchitika pa katundu mkulu zolimba ntchito malonda yogwira zimam`patsa munali michere (CAZymes) ndi tizilombo ting'onoting'ono nayonso mphamvu ntchito transgenic yisiti kapena mabakiteriya kutulutsa zamera-based mafuta ndi mankhwala 6 .
CAZymes mu ma enzymes amalonda amapangidwa ndi kusakaniza kosakanikirana kwa michere yomwe imaphwanya molumikizana ma carbohydrate-shuga zomangira kupanga ma monosaccharides2,7.Monga tidanenera kale, maukonde ovuta a ma polima onunkhira a lignin okhala ndi chakudya amawapangitsa kukhala osasunthika, zomwe zimapangitsa kutembenuka kwa shuga kosakwanira, kudziunjikira 15-25% ya oligosaccharides ogonana omwe samapangidwa panthawi ya enzymatic hydrolysis ya biomass pretreated.Ili ndi vuto wamba ndi njira zosiyanasiyana zopangira ma biomass.Zifukwa zina zomwe zimalepheretsa izi ndi kuletsa kwa ma enzyme panthawi ya hydrolysis, kapena kusakhalapo kapena kuchepa kwa ma enzymes ofunikira omwe amafunikira kuti athyole zomangira za shuga muzomera.Kumvetsetsa kapangidwe kake ndi kapangidwe ka shuga, monga zomangira za shuga mu oligosaccharides, zitithandiza kusintha kutembenuka kwa shuga panthawi ya hydrolysis, kupanga njira za sayansi yachilengedwe kukhala zotsika mtengo ndi zinthu zochokera ku petroleum.
Kuzindikira kapangidwe ka chakudya chamafuta kumakhala kovuta ndipo kumafuna njira zingapo monga liquid chromatography (LC)11,12, nuclear magnetic resonance spectroscopy (NMR)13, capillary electrophoresis (CE)14,15,16 ndi mass spectrometry (MS)17.,khumi ndi zisanu ndi zitatu.Njira za MS monga nthawi ya kuthawa kwa misa spectrometry yokhala ndi laser desorption ndi ionization pogwiritsa ntchito matrix (MALDI-TOF-MS) ndi njira yosinthika yodziwira ma carbohydrate.Posachedwapa, Collision-Induced Dissociation (CID) tandem MS ya sodium ion adducts yakhala ikugwiritsidwa ntchito kwambiri pozindikira zala zala zomwe zimagwirizana ndi malo ophatikizira oligosaccharide, masinthidwe a anomeric, kutsatizana, ndi maudindo a nthambi 20, 21.
Kusanthula kwa Glycan ndi chida chabwino kwambiri chozindikiritsira mozama ma carbohydrate bond22.Njirayi imagwiritsa ntchito ma antibodies a monoclonal (mAbs) omwe amalunjikitsidwa kubzala ma cell khoma glycan ngati ma probes kuti amvetsetse kulumikizana kwamphamvu kwamafuta.Opitilira 250 mAbs akupezeka padziko lonse lapansi, opangidwa motsutsana ndi ma oligosaccharides osiyanasiyana amizere ndi nthambi pogwiritsa ntchito ma saccharides24 osiyanasiyana.Ma mAbs angapo akhala akugwiritsidwa ntchito kwambiri kuti awonetse mawonekedwe, mapangidwe, ndi kusintha kwa khoma la selo la zomera, popeza pali kusiyana kwakukulu malinga ndi mtundu wa maselo a zomera, chiwalo, zaka, siteji ya chitukuko, ndi kukula kwa chilengedwe25,26.Posachedwapa, njirayi yakhala ikugwiritsidwa ntchito kumvetsetsa kuchuluka kwa ma vesicle muzomera ndi zinyama komanso maudindo awo pamayendedwe a glycan monga momwe zimakhalira ndi zolembera zamagulu ang'onoang'ono, magawo otukuka, kapena zolimbikitsa zachilengedwe, komanso kudziwa zochita za enzymatic.Zina mwazinthu zosiyanasiyana za glycans ndi xylans zomwe zazindikirika ndi pectin (P), xylan (X), mannan (M), xyloglucans (XylG), ma glucans osakanikirana (MLG), arabinoxylan (ArbX), galactomannan (GalG) , glucuronic acid-arabinoxylan) arbinoxylan (9) Arbinoxylan (9) ndi arbinoxylan (9).
Komabe, ngakhale zoyesayesa zonsezi zofufuza, kafukufuku wochepa chabe adayang'ana pa chikhalidwe cha oligosaccharide kudzikundikira pa nthawi ya high solids load (HSL) hydrolysis, kuphatikizapo oligosaccharide kumasulidwa, oligomeric unyolo utali kusintha pa hydrolysis, zosiyanasiyana otsika DP ma polima, ndi ma curves awo.magawo 30,31,32.Pakalipano, ngakhale kuti kusanthula kwa glycan kwatsimikizira kuti ndi chida chothandiza pofufuza mwatsatanetsatane kapangidwe ka glycan, n'zovuta kuyesa DP oligosaccharides osungunuka m'madzi pogwiritsa ntchito njira za antibody.Ma oligosaccharides a DP ang'onoang'ono okhala ndi mamolekyu olemera osakwana 5-10 kDa samamangiriza ku mbale za ELISA 33, 34 ndipo amatsukidwa asanawonjezere antibody.
Pano, kwa nthawi yoyamba, tikuwonetsa kuyesa kwa ELISA pa mbale zophimbidwa ndi avidin pogwiritsa ntchito ma antibodies a monoclonal, kuphatikiza njira imodzi ya biotinylation ya oligosaccharides yosungunuka ndi kusanthula kwa glycome.Njira yathu pakuwunika kwa glycome idatsimikiziridwa ndi kusanthula kwa MALDI-TOF-MS ndi GC-MS pakulumikizana kowonjezera kwa oligosaccharide pogwiritsa ntchito trimethylsilyl (TMS) yotulutsa nyimbo za shuga wa hydrolyzed.Njira yatsopanoyi ikhoza kupangidwa ngati njira yopambana kwambiri m'tsogolomu ndikupeza ntchito yowonjezereka mu kafukufuku wamankhwala35.
Zosintha pambuyo pomasulira za michere ndi ma antibodies, monga glycosylation,36 zimakhudza ntchito yawo yachilengedwe.Mwachitsanzo, kusintha kwa glycosylation ya mapuloteni a seramu kumagwira ntchito yofunika kwambiri mu nyamakazi yotupa, ndipo kusintha kwa glycosylation kumagwiritsidwa ntchito ngati zizindikiro zozindikiritsa37.Ma glycans osiyanasiyana amalembedwa m'mabuku kuti awoneke mosavuta m'matenda osiyanasiyana, kuphatikizapo matenda otupa a m'mimba ndi chiwindi, matenda a tizilombo, ovarian, m'mawere, ndi khansa ya prostate38,39,40.Kumvetsetsa kapangidwe ka glycans pogwiritsa ntchito njira za glycan ELISA zokhala ndi antibody kudzapereka chidaliro chowonjezereka pakuzindikira matenda popanda kugwiritsa ntchito njira zovuta za MS.
Kafukufuku wathu wam'mbuyomu adawonetsa kuti oligosaccharides amakani adakhalabe opanda hydrolyzed pambuyo pa pretreatment ndi enzymatic hydrolysis (Chithunzi 1).M'ntchito yathu yomwe idasindikizidwa kale, tidapanga njira yopangira makala olimba kuti tisiyanitse oligosaccharides ku AFEX-pretreated corn stover hydrolyzate (ACSH)8.Pambuyo pochotsa koyamba ndi kupatukana, ma oligosaccharides adagawidwanso ndi kukula kwa chromatography (SEC) ndikusonkhanitsidwa motsatira kulemera kwa maselo.Ma monomers a shuga ndi oligomers omwe adatulutsidwa kuchokera kuzinthu zosiyanasiyana zochiritsira adawunikidwa ndi kusanthula kwamapangidwe a shuga.Poyerekeza zomwe zili ndi ma oligomers a shuga omwe amapezedwa ndi njira zosiyanasiyana zopangira mankhwala, kupezeka kwa oligosaccharides amakani ndi vuto wamba pa kutembenuka kwa zotsalira zazomera kukhala monosaccharides ndipo zingachititse kuchepetsa zokolola za shuga osachepera 10-15% ndipo ngakhale mpaka 18%.US.Njirayi imagwiritsidwa ntchito popanganso zigawo zazikulu za oligosaccharide.Zotsatira za ACH ndi tizigawo tating'ono tokhala ndi masikelo osiyanasiyana a mamolekyu adagwiritsidwa ntchito ngati zinthu zoyesera zowonetsera ma oligosaccharides mu ntchitoyi.
Pambuyo pa pretreatment ndi enzymatic hydrolysis, oligosaccharides kupitiriza anakhalabe unhydrolyzed.Pano (A) ndi njira yolekanitsa ya oligosaccharide yomwe oligosaccharides amasiyanitsidwa ndi AFEX-pretreated corn stover hydrolyzate (ACSH) pogwiritsa ntchito bedi lodzaza ndi carbon activated ndi diatomaceous earth;(B) Njira yolekanitsa oligosaccharides.Ma oligosaccharides adasiyanitsidwanso ndi kukula kwa chromatography (SEC);(C) Saccharide monomers ndi oligomers kumasulidwa ku pretreatments zosiyanasiyana (kuchepetsedwa asidi: DA, ionic madzi: IL ndi AFEX).Miyezo ya enzymatic hydrolysis: zolimba zodzaza ndi 25% (w/w) (pafupifupi 8% glucan loading), maola 96 hydrolysis, 20 mg/g commercial enzyme loading (Ctec2:Htec2:MP-2:1:1 ratio) ndi (D) Shuga monomers ndi oligomers a glucose, EXACS-core corn EXA-chimanga wotulutsidwa kuchokera ku xyraAFCSA-chimanga chotulutsidwa).
Kusanthula kwa Glycan kwatsimikizira kukhala chida chothandiza pakuwunikira mwatsatanetsatane ma glycans m'zigawo zosiyanitsidwa ndi zotsalira za biomass.Komabe, ma saccharides osungunuka m'madzi sayimiridwa mocheperapo pogwiritsa ntchito njira yachikhalidweyi41 chifukwa oligosaccharides otsika kwambiri a molekyulu ndizovuta kuti asasunthike pa mbale za ELISA ndipo amatsuka asanawonjezere antibody.Chifukwa chake, pomanga ma antibody ndi mawonekedwe, njira imodzi ya biotinylation idagwiritsidwa ntchito kuvala oligosaccharides osungunuka, osagwirizana ndi mbale za ELISA zokutira avidin.Njirayi idayesedwa pogwiritsa ntchito ACSH yathu yomwe idapangidwa kale komanso kagawo kakang'ono kutengera kulemera kwake kwa maselo (kapena digiri ya polymerization, DP).Gawo limodzi la biotinylation linagwiritsidwa ntchito kuti liwonjezere kuyanjana kwa oligosaccharide powonjezera biotin-LC-hydrazide mpaka kumapeto kwa carbohydrate (mkuyu 2).Poyankha, gulu la hemiacetal pamapeto ochepetsera limakumana ndi gulu la hydrazide la biotin-LC-hydrazide kupanga chomangira cha hydrazone.Pamaso pa wothandizira kuchepetsa NaCNBH3, chomangira cha hydrazone chimachepetsedwa kukhala chokhazikika chokhazikika cha biotinylated.Ndi kusinthidwa kwa mapeto ochepetsera shuga, kumangiriza kwa DP oligosaccharides otsika ku mbale za ELISA kunakhala kotheka, ndipo mu phunziro lathu izi zinachitidwa pa mbale zokhala ndi avidin pogwiritsa ntchito glycan-targeted mAbs.
Kuwunika kwa ma antibodies a monoclonal kutengera ELISA kwa oligosaccharides ya biotinylated.Apa (A) kuphatikiza biotinylation ya oligosaccharides ndi kuwunika kotsatira kwa ELISA ndi ma glycan-targeted mAbs pa NeutrAvidin coated plates ndipo (B) ikuwonetsa njira imodzi yokha ya biotinylation ya zinthu zomwe zimachitikira.
Ma mbale ophimbidwa ndi Avidin okhala ndi oligosaccharide-conjugated antibodies adawonjezeredwa ku ma antibodies oyambirira ndi achiwiri ndikutsuka mu sing'anga yowala ndi nthawi.Kumanga kwa antibody kukatha, onjezani gawo laling'ono la TMB kuti muyike mbale.Zomwezo zidayimitsidwa ndi sulfuric acid.Mambale olumikizidwawo adawunikidwa pogwiritsa ntchito wowerenga ELISA kuti adziwe mphamvu zomangirira za antibody iliyonse kuti azindikire kulumikizana kwamtundu wa antibody.Kuti mumve zambiri komanso magawo a kuyesako, onani gawo lofananira "Zida ndi Njira".
Tikuwonetsa kugwiritsa ntchito njira yomwe yangopangidwa kumeneyi kuti tigwiritse ntchito mwapadera pozindikiritsa ma oligosaccharides osungunuka omwe amapezeka mu ACSH komanso tizigawo ta oligosaccharide osakanizidwa ndi ma lignocellulosic hydrolysates.Monga momwe zasonyezedwera pa Chithunzi 3, ma xylans omwe amalowetsedwa m'malo mwa epitope odziwika mu ACSH pogwiritsa ntchito njira zoyezera glycome ya bioacylated nthawi zambiri amakhala uronic (U) kapena methyluronic (MeU) ndi pectic arabinogalactans.Ambiri aiwo adapezekanso mu kafukufuku wathu wam'mbuyomu pakuwunika kwa glycans of non-hydrolyzed solids (UHS)43.
Kuzindikira kwa recalcitrant oligosaccharide epitopes pogwiritsa ntchito monoclonal antibody yolunjika ku cell wall glycan.Gawo "losalowerera ndale" ndilo gawo la ACN ndipo gawo la "acidic" ndi FA.Zofiira zowoneka bwino pamapu otentha zimawonetsa kuchuluka kwa ma epitope, ndipo mabuluu owala amawonetsa maziko opanda kanthu.Makhalidwe amtundu pasikelo amatengera ma OD opangira ma formula N=2.Ma epitopes akuluakulu omwe amadziwika ndi ma antibodies akuwonetsedwa kumanja.
Mapangidwe osakhala a cellulosewa sakanatha kung'ambika ndi ma cellulases ndi ma hemicellulose omwe amayesedwa mu osakaniza oyesedwa a enzyme yamalonda, omwe amaphatikiza ma enzymes omwe amagwiritsidwa ntchito kwambiri.Chifukwa chake, ma enzymes atsopano amafunikira pa hydrolysis yawo.Popanda ma enzyme ofunikira omwe si a cellulose owonjezera, zomangira zopanda cellulosezi zimalepheretsa kutembenuka kwathunthu kukhala ma monosaccharides, ngakhale ma polima awo a shuga amapangidwa mochuluka ndi hydrolyzed kukhala tizidutswa tating'onoting'ono ndikusungunuka pogwiritsa ntchito ma enzyme osakanikirana.
Kufufuza kwina kwa kugawa chizindikiro ndi mphamvu zake zomangirira kunasonyeza kuti ma epitopes omangirira anali otsika kwambiri mu magawo a shuga a DP (A, B, C, DP mpaka 20 +) kusiyana ndi magawo otsika a DP (D, E, F, DP) mu dimers) (Mkuyu 1).Zidutswa za asidi zimakhala zofala kwambiri m'ma epitopes omwe si a cellulose kuposa zidutswa za ndale.Zochitika izi zimagwirizana ndi zomwe taziwona mu phunziro lathu lapitalo, pomwe ma DP apamwamba ndi magulu a asidi anali osagwirizana ndi enzymatic hydrolysis.Choncho, kukhalapo kwa ma epitopes a glycan omwe si a cellulose ndi ma U ndi MeU olowa m'malo angathandize kwambiri kukhazikika kwa oligosaccharides.Tiyenera kuzindikira kuti kumanga ndi kuzindikira bwino kungakhale kovuta kwa oligosaccharides otsika a DP, makamaka ngati epitope ndi dimeric kapena trimeric oligosaccharide.Izi zitha kuyesedwa pogwiritsa ntchito oligosaccharides zamalonda zautali wosiyanasiyana, iliyonse ili ndi epitope imodzi yokha yomwe imamangiriza ku mAb yeniyeni.
Chifukwa chake, kugwiritsa ntchito ma antibodies okhazikika kumawonetsa mitundu ina ya ma recalcitrant.Malingana ndi mtundu wa antibody womwe umagwiritsidwa ntchito, ndondomeko yoyenera yolumikizirana, ndi mphamvu ya chizindikiro chomwe chimapanga (chochuluka komanso chochepa kwambiri), ma enzyme atsopano amatha kudziwika ndi kuwonjezeredwa theka-quantitatively kusakaniza kwa enzyme kuti glycoconversion wathunthu.Kutengera kusanthula kwa oligosaccharides ACSH mwachitsanzo, titha kupanga nkhokwe ya ma glycan bond pamtundu uliwonse wa biomass.Tiyenera kuzindikira apa kuti kuyanjana kosiyana kwa ma antibodies kuyenera kuganiziridwa, ndipo ngati kugwirizana kwawo sikudziwika, izi zidzabweretsa zovuta zina poyerekezera zizindikiro za ma antibodies osiyanasiyana.Kuphatikiza apo, kufananiza zomangira za glycan zitha kugwira bwino ntchito pakati pa zitsanzo za antibody yomweyo.Zomangira zokakamirazi zitha kulumikizidwa ku database ya CAZyme, komwe titha kuzindikira ma enzymes, kusankha ma enzymes osankhidwa ndikuyesa ma enzymes osweka, kapena kupanga ma microbial system kuti afotokoze ma enzymes kuti agwiritsidwe ntchito mu biorefineries44.
Kuti tiwone momwe njira zotetezera chitetezo zimagwirizanirana ndi njira zina zowonetsera oligosaccharides otsika omwe amapezeka mu lignocellulosic hydrolysates, tinachita MALDI (mkuyu 4, S1-S8) ndi kusanthula ma saccharides opangidwa ndi TMS pogwiritsa ntchito GC-MS pagawo lomwelo (mkuyu 5) gawo la oligosaccharide.MALDI imagwiritsidwa ntchito kuyerekeza ngati kugawa kwakukulu kwa mamolekyu a oligosaccharide kumagwirizana ndi kapangidwe kake.Pa mkuyu.4 ikuwonetsa MC ya zigawo za ndale ACN-A ndi ACN-B.Kusanthula kwa ACN-A kunatsimikizira mitundu yambiri ya shuga ya pentose kuchokera ku DP 4-8 (mkuyu 4) mpaka DP 22 (mkuyu S1), omwe zolemera zake zimagwirizana ndi MeU-xylan oligosaccharides.Kusanthula kwa ACN-B kunatsimikizira mndandanda wa pentose ndi glucoxylan wokhala ndi DP 8-15.Muzinthu zowonjezera monga Chithunzi cha S3, mapu a FA-C acidic moiety mass distributions amasonyeza (Me) U m'malo mwa shuga wa pentose ndi DP ya 8-15 yomwe imagwirizana ndi ma xylans omwe amapezeka mu ELISA-based mAb screening.Ma epitopes ndi ofanana.
MALDI-MS sipekitiramu ya oligosaccharides yosungunuka yosavomerezeka yomwe ilipo mu ACS.Apa, (A) ACN-Magawo ochepa olemera omwe ali ndi methylated uronic acid (DP 4-8) m'malo mwa glucuroxylan oligosaccharides ndi (B) ACN-B xylan ndi methylated uronic acid oligosaccharides m'malo mwa glucuroxylan (DP 8-15).
Kusanthula kwa kapangidwe ka glycan zotsalira za oligosaccharides refractory.Apa (A) TMS saccharide yopangidwa ndi magawo osiyanasiyana a oligosaccharide omwe amapezeka pogwiritsa ntchito kusanthula kwa GC-MS.(B) Mapangidwe a shuga osiyanasiyana opangidwa ndi TMS omwe amapezeka mu oligosaccharides.ACN - gawo la acetonitrile lomwe lili ndi oligosaccharides osalowerera ndale ndi FA - gawo la ferulic acid lomwe lili ndi oligosaccharides acid.
Mfundo ina yochititsa chidwi inachokera ku LC-MS kusanthula kagawo kakang'ono ka oligosaccharide, monga momwe tawonetsera mu Chithunzi S9 (njira zikhoza kuwonetsedwa muzinthu zowonjezera zamagetsi).Zidutswa zamagulu a hexose ndi -OAc adawonedwa mobwerezabwereza panthawi yolumikizana ndi gawo la ACN-B.Kupeza kumeneku sikungotsimikizira kugawanika komwe kumawonedwa mu kusanthula kwa glycome ndi MALDI-TOF, komanso kumapereka chidziwitso chatsopano chokhudzana ndi zotumphukira zama carbohydrate mu pretreated lignocellulosic biomass.
Tidasanthulanso kuchuluka kwa shuga kwa gawo la oligosaccharide pogwiritsa ntchito kutulutsa shuga kwa TMS.Pogwiritsa ntchito GC-MS, tinatsimikiza kuti ma neural (non-derivative) amapangidwa ndi shuga (GluA ndi GalA) mu gawo la oligosaccharide (mkuyu 5).Glucuronic acid imapezeka mu zigawo za acidic C ndi D, pamene galacturonic acid imapezeka mu zigawo za acidic A ndi B, zonse zomwe zili ndi DP zambiri za shuga acidic.Zotsatirazi sizimangotsimikizira deta yathu ya ELISA ndi MALDI, komanso zimagwirizana ndi maphunziro athu akale a oligosaccharide accumulation.Choncho, timakhulupirira kuti njira zamakono zogwiritsira ntchito chitetezo cha mthupi pogwiritsa ntchito biotinylation ya oligosaccharides ndi kufufuza kwa ELISA kotsatira ndizokwanira kuti zizindikire oligosaccharides osungunuka a recalcitrant mu zitsanzo zosiyanasiyana zamoyo.
Popeza njira zowunikira za ELISA-mAb zatsimikiziridwa ndi njira zingapo zosiyanasiyana, tinkafuna kufufuzanso kuthekera kwa njira yatsopanoyi.Oligosaccharides awiri amalonda, xylohexasaccharide oligosaccharide (XHE) ndi 23-α-L-arabinofuranosyl-xylotriose (A2XX), anagulidwa ndi kuyesedwa pogwiritsa ntchito njira yatsopano ya mAb yolunjika pa khoma la cell glycan.Chithunzi 6 chikuwonetsa kulumikizana kwa mzere pakati pa chizindikiro chomangirira cha biotinylated ndi kuchuluka kwa chipika cha oligosaccharide, kutanthauza mtundu wa Langmuir adsorption.Pakati pa mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, ndi CCRC-M151 yogwirizana ndi XHE, ndi CCRC-M108, CCRC-M109, ndi LM11 yogwirizana ndi A2XX pamtundu wa 100 nmno.Chifukwa cha kupezeka kochepa kwa ma antibodies panthawi yoyesera, kuyesa kochepa kunachitidwa ndi gulu lililonse la oligosaccharide.Tiyenera kuzindikira apa kuti ma antibodies ena amachitira mosiyana kwambiri ndi oligosaccharide yemweyo monga gawo lapansi, mwinamwake chifukwa amamangiriza ma epitopes osiyana pang'ono ndipo akhoza kukhala ndi mgwirizano wosiyana kwambiri.Njira ndi zotsatira za chidziwitso cholondola cha epitope zidzakhala zovuta kwambiri pamene njira yatsopano ya mAb ikugwiritsidwa ntchito ku zitsanzo zenizeni.
Ma oligosaccharides awiri amalonda adagwiritsidwa ntchito kuti adziwe mitundu yosiyanasiyana ya ma glycan-targeting mAbs.Apa, maulalo amzere okhala ndi chipika cha oligosaccharide akuwonetsa mawonekedwe a Langmuir adsorption a (A) XHE ndi mAb ndi (B) A2XX ndi mAb.Ma epitopes ofananira akuwonetsa mapangidwe a oligosaccharides amalonda omwe amagwiritsidwa ntchito ngati magawo oyesa.
Kugwiritsa ntchito ma glycan-targeted monoclonal antibodies (glycocomic analysis kapena ELISA-based mAb screening) ndi chida champhamvu chofotokozera mozama ma glycans ambiri akuluakulu omwe amapanga zomera.Komabe, kusanthula kwakale kwa glycan kumangowonetsa ma glycans akuluakulu a cell, popeza oligosaccharides ambiri samakhazikika bwino pama mbale a ELISA.Mu kafukufukuyu, AFEX-pretreated chimanga stover anali enzymatic hydrolyzed pa zolimba kwambiri zili.Kusanthula shuga kunagwiritsidwa ntchito kuti adziwe momwe ma recalcitrant cell wall carbohydrate mu hydrolyzate amapangidwira.Komabe, kusanthula kwa mAb kwa oligosaccharides ang'onoang'ono m'ma hydrolysates sikumaganiziridwa, ndipo zida zowonjezera zimafunikira kuti zithetse bwino oligosaccharides pa mbale za ELISA.
Tikunena pano njira yatsopano komanso yothandiza ya oligosaccharide immobilization yowunikira mAb pophatikiza oligosaccharide biotinylation yotsatiridwa ndi kuwunika kwa ELISA pa mbale zokutira za NeutrAvidin™.The immobilized biotinylated oligosaccharides anasonyeza kuyanjana kokwanira kwa antibody kuti athe kuzindikira mwachangu komanso moyenera oligosaccharides recalcitrant.Kuwunika kwa mapangidwe a oligosaccharides okakamirawa ozikidwa pa misa spectrometry anatsimikizira zotsatira za njira yatsopanoyi ya immunoscreening.Choncho, maphunzirowa amasonyeza kuti kuphatikiza kwa oligosaccharide biotinylation ndi ELISA kuyang'ana ndi glycan-targeted monoclonal antibodies angagwiritsidwe ntchito kuti azindikire crosslinks mu oligosaccharides ndipo angagwiritsidwe ntchito kwambiri mu maphunziro ena a biochemical omwe amadziwika ndi mapangidwe a oligosaccharides.
Njira yofotokozera za glycan ya biotin iyi ndi lipoti loyamba lomwe lingathe kufufuza zomangira za recalcitrant carbohydrate za oligosaccharides zosungunuka muzomera za zomera.Izi zimathandiza kumvetsetsa chifukwa chomwe mbali zina za biomass zimakhala zolimba kwambiri pankhani yopanga mafuta a biofuel.Njirayi imadzaza kusiyana kofunikira mu njira zowunikira ma glycome ndikuwonjezera kugwiritsa ntchito kwake kumagulu ochulukirapo kuposa oligosaccharides.M'tsogolomu, tingagwiritse ntchito ma robotiki a biotinylation ndikugwiritsa ntchito njira yomwe tapanga kuti tifufuze zitsanzo zapamwamba pogwiritsa ntchito ELISA.
Udzu wa chimanga (CS) wobzalidwa kuchokera ku mbewu zosakanizidwa za Pioneer 33A14 adakololedwa mu 2010 kuchokera ku Kramer Farms ku Ray, Colorado.Ndi chilolezo cha eni malo, biomass iyi ingagwiritsidwe ntchito pofufuza. Zitsanzozo zidasungidwa zowuma <6% chinyezi m'matumba a zip-lock mu kutentha kwachipinda. Zitsanzozo zidasungidwa zowuma <6% chinyezi m'matumba a zip-lock mu kutentha kwachipinda. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Zitsanzo zinasungidwa zowuma pa <6% chinyezi m'matumba a zippered kutentha.樣品在室温下以干燥< 6% 的水分储存在自封袋中。样品在室温下以干燥< 6% Образцы хранят в пакетах с застежкой-молнией при комнатной температуре с влажностью < 6%. Zitsanzo zimasungidwa m'matumba a zipper kutentha kwa firiji ndi chinyezi <6%.Kafukufukuyu adatsatira malangizo a m'deralo ndi dziko lonse.Kusanthula kophatikiza kunachitika pogwiritsa ntchito protocol ya NREL.Zolembazo zinapezeka kuti zili ndi 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% mapuloteni ndi 13. 4% phulusa.
Cellic® CTec2 (138 mg protein / ml, lot VCNI 0001) ndi osakaniza osakaniza a cellulase, β-glucosidase ndi Cellic® HTec2 (157 mg protein / ml, lot VHN00001) kuchokera ku Novozymes (Franklinton, NC, USA)).Multifect Pectinase® (72 mg protein/mL), chosakaniza chovuta cha ma enzymes owononga pectin, adaperekedwa ndi DuPont Industrial Biosciences (Palo Alto, CA, USA).Kuchulukira kwa mapuloteni a enzyme kunatsimikiziridwa ndi kuyerekezera kuchuluka kwa mapuloteni (ndi kuchotsa chopereka cha nayitrogeni wopanda mapuloteni) pogwiritsa ntchito Kjeldahl nitrogen analysis (AOAC method 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA).Diatomaceous earth 545 idagulidwa kuchokera ku EMD Millipore (Billerica, MA).Activated carbon (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, ndi mankhwala ena onse anagulidwa ku Sigma-Aldrich (St. Louis, MO).
AFEX pretreatment idachitidwa ku GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA).Kukonzekera kusanachitike kunachitika pa 140 ° C. kwa mphindi 15.46 nthawi yokhala pa 1: 1 chiyerekezo cha anhydrous ammonia kupita ku biomass pa 60% (w/w) itakwezedwa muzitsulo zosapanga dzimbiri pa benchtop batch reactor (Parr Instruments Company).Zinatenga mphindi 30.The reactor inabweretsedwa ku 140 ° C ndipo ammonia inatulutsidwa mofulumira, kulola kuti biomass ibwerere mofulumira kutentha.Mapangidwe a AFEX pre-treated corn stover (ACS) anali ofanana ndi a corn stover (UT-CS).
Zolimba kwambiri ACSH 25% (w/w) (pafupifupi 8% dextran loading) inakonzedwa ngati chiyambi cha kupanga kwakukulu kwa oligosaccharides.Enzymatic hydrolysis ya ACS inkagwiritsidwa ntchito pogwiritsa ntchito mankhwala osakaniza a enzyme kuphatikizapo Cellic® Ctec2 10 mg mapuloteni / g glucan (mu pretreated biomass), Htec2 (Novozymes, Franklinton, NC), 5 mg mapuloteni / g glucan, ndi Multifect Pectinase (Genencor Inc, USA).).), 5 mg mapuloteni/g dextran.Enzymatic hydrolysis inkachitika mu 5-lita bioreactor ndi voliyumu yogwira ntchito ya malita 3, pH 4.8, 50 ° C ndi 250 rpm.Pambuyo hydrolysis kwa maola 96, ndi hydrolyzate anasonkhanitsidwa centrifugation pa 6000 rpm kwa mphindi 30 ndiyeno pa 14000 rpm kwa mphindi 30 kuchotsa zolimba unhydrolysed.Kenako, hydrolyzate idasefedwa kudzera mu beaker ya 0.22 mm.Hydrolyzate yosefedwa idasungidwa m'mabotolo osabala pa 4 ° C. kenako amagawidwa pa kaboni.
Kusanthula kwa kaphatikizidwe ka zitsanzo za biomass yochokera ku zoyambira molingana ndi njira zowunikira ma labotale a NREL: kukonzekera zitsanzo zowunikira (NREL/TP-510-42620) komanso kutsimikizika kwamafuta ampangidwe ndi lignin mu biomass (NREL/TP-510 - 42618)47.
Kusanthula kwa oligosaccharide kwa mtsinje wa hydrolyzate kunachitika pamlingo wa 2 ml pogwiritsa ntchito njira ya autoclave-based acid hydrolysis.Sakanizani chitsanzo cha hydrolyzate ndi 69.7 µl ya 72% sulfuric acid mu chubu cha 10 ml screw cap culture ndi kuumitsa 1 h pa benchtop pa 121 °C, kuziziritsa pa ayezi ndikusefa mu botolo lapamwamba kwambiri la liquid chromatography (HPLC).Kuchuluka kwa oligosaccharides kunatsimikiziridwa ndikuchotsa kuchuluka kwa ma monosaccharides mu zitsanzo zopanda hydrolyzed kuchokera ku kuchuluka kwa shuga mu zitsanzo za acid-hydrolyzed.
Glucose, xylose, ndi arabinose concentrations mu acid hydrolysed biomass adawunikidwa pogwiritsa ntchito Shimadzu HPLC system yokhala ndi autosampler, column heater, isocratic pump, ndi refractive index detector pagawo la Bio-Rad Aminex HPX-87H.Mzerewu unasungidwa pa 50 ° C ndipo umakhala ndi 0.6 ml / min 5 mM H2SO4 m'madzi.kuyenda.
Hydrolyzate supernatant idasungunuka ndikuwunikidwa kuti ikhale ndi monomer ndi oligosaccharide.Mashuga a monomeric omwe adapezedwa pambuyo pa enzymatic hydrolysis adawunikidwa ndi HPLC yokhala ndi gawo la Bio-Rad (Hercules, CA) Aminex HPX-87P ndi mzere woteteza phulusa.Kutentha kwa mzati kunasungidwa pa 80 ° C, madzi ankagwiritsidwa ntchito ngati gawo la mafoni ndi kuthamanga kwa 0.6 ml / min.Oligosaccharides adatsimikiziridwa ndi hydrolysis mu dilute acid pa 121 ° C molingana ndi njira zomwe zafotokozedwa mu refs.41, 48, 49.
Kusanthula kwa Saccharide kunkachitika pazaiwisi, AFEX yokonzedweratu ndi zotsalira zonse zopanda hydrolysed biomass (kuphatikizapo kupanga serial cell wall extracts and their mAb screening) pogwiritsa ntchito njira zomwe zafotokozedwa kale 27, 43, 50, 51 .Pakuwunika kwa glycome, zotsalira zosasungunuka za mowa zama cell cell khoma zimakonzedwa kuchokera ku zotsalira za biomass ndikuchotsedwa mosalekeza ndi zinthu zochulukirachulukira monga ammonium oxalate (50 mM), sodium carbonate (50 mM ndi 0.5% w/v), CON.(1M ndi 4M, onse ndi 1% w / v sodium borohydride) ndi asidi chlorite monga tafotokozera kale52,53.Zowonjezerazo zidaperekedwa kwa ELISA motsutsana ndi gulu lovuta la mAb50s lolunjika ku cell wall glycan, ndipo machitidwe omangirira a mAb adawonetsedwa ngati mapu otentha.mAbs yolunjika ku cell wall glycan idagulidwa kuchokera ku ma labotale (CCRC, JIM ndi MAC series).
Gawo limodzi la biotinylation ya oligosaccharides.Kuphatikizika kwa chakudya ndi biotin-LC-hydrazide kunachitika pogwiritsa ntchito njirayi.Biotin-LC-hydrazide (4.6 mg / 12 μmol) inasungunuka mu dimethyl sulfoxide (DMSO, 70 μl) ndi kusonkhezera mwamphamvu ndi kutentha pa 65 ° C. kwa 1 min.Glacial acetic acid (30 µl) anawonjezedwa ndipo osakaniza anatsanuliridwa pa sodium cyanoborohydride (6.4 mg/100 µmol) ndi kusungunuka kwathunthu pambuyo kutentha pa 65 ° C. kwa pafupifupi mphindi imodzi.Kenaka, kuchokera ku 5 mpaka 8 μl ya zomwe zimasakanikirana zinawonjezeredwa ku oligosaccharide zouma (1-100 nmol) kuti mupeze 10-fold or more molar over the label over reduction end.Zomwezo zidachitika pa 65 ° C kwa 2 h, pambuyo pake zitsanzozo zidayeretsedwa nthawi yomweyo.Palibe sodium cyanoborohydride yomwe idagwiritsidwa ntchito poyesa zolembera popanda kuchepetsa, ndipo zitsanzozo zinachitidwa pa 65 ° C. kwa maola 2.5.
Kupaka kwa ELISA ndikutsuka kwa zitsanzo za oligosaccharides biotinylated.25 μl ya zitsanzo za biotinylated (100 μl ya chitsanzo chilichonse chokhazikika chosungunuka mu 5 ml ya 0.1 M Tris buffer solution (TBS)) anawonjezedwa pa chitsime chilichonse cha mbale ya avidin.Zitsime zowongolera zidakutidwa ndi 50 μl ya biotin pamlingo wa 10 μg/ml mu 0.1 M TBS.Madzi a deionized ankagwiritsidwa ntchito ngati zokutira pamiyeso yopanda kanthu.Piritsiyo incubated kwa maola 2 firiji mu mdima.Sambani mbale katatu ndi 0.1% mkaka wosakanizidwa mu 0.1 M TBS pogwiritsa ntchito pulogalamu No.11 ya Grenier flat 3A.
Kuwonjezera ndi kutsuka kwa ma antibodies oyambirira.Onjezani 40 µl wa antibody yoyamba pachitsime chilichonse.Ikani microplate kwa ola limodzi pa kutentha kwapakati mumdima.Ma mbalewo adatsukidwa katatu ndi mkaka wa 0.1% mu 0.1M TBS pogwiritsa ntchito pulogalamu yosamba #11 ya Grenier Flat 3A.
Onjezani chitetezo chachiwiri ndikusamba.Onjezani 50 µl ya antibody yachiwiri ya mbewa/khoswe (yochepetsedwa 1:5000 mu mkaka 0.1% mu 0.1 M TBS) pachitsime chilichonse.Ikani microplate kwa ola limodzi pa kutentha kwapakati mumdima.Ma microplates adatsukidwa kasanu ndi mkaka wa 0.1% mu 0.1 M TBS pogwiritsa ntchito pulogalamu ya Grenier Flat 5A yotsuka mbale #12.
Kuwonjezera gawo lapansi.Onjezani 50 µl wa 3,3',5,5'-tetramethylbenzidine (TMB) kumunsi kwa gawo lapansi (powonjezera madontho awiri a bafa, madontho atatu a TMB, madontho awiri a hydrogen peroxide ku 15 ml ya madzi a deionized).Konzani gawo lapansi la TMB.ndi vortex musanagwiritse ntchito).Ikani microplate pa kutentha kwapakati kwa mphindi 30.Mumdima.
Malizitsani sitepe ndikuwerenga piritsi.Onjezani 50 µl ya 1 N sulfuric acid pachitsime chilichonse ndikulemba kuyamwa kuchokera pa 450 mpaka 655 nm pogwiritsa ntchito owerenga ELISA.
Konzani mayankho a 1 mg/ml a ma analyte awa m'madzi opangidwa ndi deionized: arabinose, rhamnose, fucose, xylose, galacturonic acid (GlA), glucuronic acid (GlcA), mannose, shuga, galactose, lactose, N-acetylmannosamine (manNAcgluc), N-coamine glucocorticosteroids.(glcNAc), N-acetylgalactosamine (galNAc), inositol (muyezo wamkati).Miyezo iwiri inakonzedwa powonjezerapo 1 mg / mL njira za shuga zomwe zikuwonetsedwa mu Table 1. Zitsanzo zimazizira ndi lyophilized pa -80 ° C. mpaka madzi onse achotsedwa (nthawi zambiri pafupifupi maola 12-18).
Onjezani 100–500 µg wa zitsanzo ku machubu opukutira pamlingo wowunika.Lembani ndalama zomwe zawonjezeredwa.Ndi bwino kusungunula chitsanzo mu ndende yeniyeni ya zosungunulira ndi kuwonjezera pa chubu monga madzi aliquot.Gwiritsani ntchito 20 µl ya 1 mg/ml inositol ngati muyezo wamkati wa chubu chilichonse.Kuchuluka kwa muyezo wamkati wowonjezeredwa ku chitsanzocho kuyenera kukhala kofanana ndi kuchuluka kwa mkati komwe kumawonjezeredwa ku chubu chokhazikika.
Onjezani 8 ml ya methanol ya anhydrous ku vial ya screw cap.Ndiye 4 ml ya 3 N. methanolic HCl njira, caped ndi kugwedezeka.Njirayi sigwiritsa ntchito madzi.
Onjezani 500 µl ya 1 M HCl methanol yankho ku zitsanzo za oligosaccharide ndi machubu wamba a TMS.Zitsanzo zinakulungidwa usiku wonse (maola 168) pa 80 ° C. mu chipika chotentha.Yanikani mankhwala a methanolysis kutentha kwa firiji pogwiritsa ntchito makina owumitsa.Onjezani 200 µl MeOH ndikuwumitsanso.Njirayi imabwerezedwa kawiri.Onjezani 200 µl wa methanol, 100 µl wa pyridine ndi 100 µl wa acetic anhydride ku chitsanzo ndikusakaniza bwino.Zitsanzo zidakulungidwa pa kutentha kwapakati kwa mphindi 30.ndi zouma.Onjezani 200 µl wa methanol ndikuwumitsanso.
Onjezani 200 µl wa Tri-Sil ndi chubu chophimbidwa ndi kutentha kwa mphindi 20.80 ° C, kenako utakhazikika kutentha kwa firiji.Gwiritsani ntchito chowumitsa chowirikiza kuti muumitsenso chitsanzocho mpaka kuchuluka kwa pafupifupi 50 µl.Ndikofunika kuzindikira kuti sitinalole kuti zitsanzo ziume kwathunthu.
Onjezerani 2 ml ya hexane ndikusakaniza bwino ndi vortexing.Lembani nsonga za Pasteur pipettes (5-8 mm) ndi chidutswa cha ubweya wagalasi poika ubweya wagalasi pamwamba pa pipette ya 5-3 / 4 inchi.Zitsanzo zinali centrifuged pa 3000 g kwa mphindi 2.Zotsalira zilizonse zosasungunuka zimawotchedwa.Yanikani chitsanzo mpaka 100-150 µl.Voliyumu pafupifupi 1 μl idayikidwa mu GC-MS pa kutentha koyambirira kwa 80 ° C komanso nthawi yoyamba ya mphindi 2.0 (Table 2).