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Sabbin hanyoyin rigakafin rigakafi da taro don hadaddun bincike na oligosaccharides na ci gaba a cikin murhun masara da aka riga aka yi da AFEX.Lignocellulosic biomass madadin mai dorewa ne ga burbushin mai kuma ana amfani dashi sosai don haɓaka fasahar kere kere don samar da kayayyaki kamar abinci, abinci, mai da sinadarai.Makullin waɗannan fasahohin shine haɓaka hanyoyin haɓaka farashi don canza hadaddun carbohydrates da ke cikin ganuwar tantanin halitta zuwa sikari mai sauƙi kamar glucose, xylose da arabinose.Saboda lignocellulosic biomass yana da taurin kai, dole ne a yi shi da magungunan thermochemical (misali, ammonia fiber exfoliation (AFEX), dilute acid (DA), ruwa mai ion (IL)) da kuma jiyya na nazarin halittu (misali, enzymatic hydrolysis da fermentation microbial) a hade don samun samfurin da ake so..Koyaya, lokacin da ake amfani da enzymes na fungal na kasuwanci a cikin tsarin hydrolysis, kawai 75-85% na sukari masu narkewa da aka kafa sune monosaccharides, sauran 15-25% kuma suna iya narkewa, oligosaccharides, waɗanda ba koyaushe suke samuwa ga microorganisms ba.A baya can, mun sami nasarar keɓewa da tsabtace masu taurin oligosaccharides masu narkewa ta hanyar amfani da haɗin carbon da diatomaceous rarrabuwar ƙasa da girman ware chromatography, kuma mun bincika abubuwan hana enzyme su.Mun gano cewa oligosaccharides dauke da matsayi mafi girma na polymerization (DP) methylated uronic acid maye gurbin sun fi wuya a aiwatar da haɗin gwiwar enzyme na kasuwanci fiye da ƙananan DP da tsaka-tsakin oligosaccharides.Anan mun ba da rahoton amfani da ƙarin ƙarin hanyoyin, gami da bayanin martabar glycan ta amfani da ƙwayoyin rigakafi na monoclonal (mAbs) musamman don shuka glycans biomass don siffanta haɗin glycan a cikin ganuwar tantanin halitta da enzymatic hydrolysates, ionization Laser desorption na matrix-taimaka, lokaci-na-jirgin taro-spectrometry..MALDI-TOF-MS) yana amfani da kololuwar bincike mai ba da labari da aka samu ta hanyar spectroscopy bayan lalata na biyu na korau ions, chromatography gas da mass spectrometry (GC-MS) don siffanta haɗin oligosaccharides tare da kuma ba tare da rarrabuwa ba.Saboda ƙananan ƙananan oligosaccharides (DP 4-20), waɗannan kwayoyin suna da wuya a yi amfani da su don haɗakar mAb da halayyar.Don shawo kan wannan matsala, mun yi amfani da sabuwar hanyar biotin conjugation-based oligosaccharides immobilization wanda ya sami nasarar lakafta mafi yawan ƙananan DP soluble oligosaccharides a kan microplate surface, wanda aka yi amfani da shi a cikin babban tsarin mAb na kayan aiki don takamaiman bincike na ligation.Wannan sabuwar hanyar za ta sauƙaƙe haɓaka haɓakar haɓakar haɓakar haɓakar glycome mafi girma a nan gaba waɗanda za a iya amfani da su don keɓewa da kuma siffata oligosaccharides da ke cikin biomarkers don dalilai na bincike.
Lignocellulosic biomass, wanda ya ƙunshi aikin gona, gandun daji, ciyawa da kayan itace, shine yuwuwar abinci don samar da samfuran tushen halittu, gami da abinci, abinci, man fetur da abubuwan sinadarai don samar da samfuran ƙima mafi girma1.Carbohydrates (kamar cellulose da hemicellulose) da ke cikin ganuwar tantanin halitta an lalata su zuwa monosaccharides ta hanyar sarrafa sinadarai da biotransformation (irin su enzymatic hydrolysis da fermentation microbial).Magani na yau da kullum sun hada da fadada fiber ammonia (AFEX), dilute acid (DA), ruwa na ionic (IL), da fashewar tururi (SE), wanda ke amfani da haɗin sunadarai da zafi don rage yawan samar da lignocellulose ta hanyar bude ganuwar tantanin halitta 3,4.taurin kwayoyin halitta, 5. Enzymatic hydrolysis ne da za'ayi a wani babban daskararru lodi ta yin amfani da kasuwanci aiki carbohydrate-dauke da enzymes (CAZymes) da kuma microbial fermentation ta yin amfani da transgenic yeasts ko kwayoyin don samar da bio-tushen habaka da sunadarai 6.
CAZymes a cikin enzymes na kasuwanci sun ƙunshi hadadden cakuda enzymes waɗanda ke haɗa hadaddun hadaddun carbohydrate-sukari don samar da monosaccharides2,7.Kamar yadda muka ruwaito a baya, hadaddun cibiyar sadarwa na polymers na lignin tare da carbohydrates suna sa su zama mai saurin kamuwa da su, wanda ke haifar da canjin sukari da bai cika ba, yana tara 15-25% na jima'i oligosaccharides waɗanda ba a samar da su a lokacin enzymatic hydrolysis na pretreated biomass.Wannan matsala ce ta gama gari tare da hanyoyi daban-daban na pretreatment bioomass.Wasu dalilai na wannan kwalban sun haɗa da hana enzyme yayin hydrolysis, ko rashi ko ƙananan matakan mahimman enzymes masu mahimmanci waɗanda ake buƙata don karya haɗin sukari a cikin kwayoyin halitta.Fahimtar abun da ke ciki da sifofin sifofi na masu ciwon sukari, irin su haɗin sukari a cikin oligosaccharides, zai taimaka mana haɓaka canjin sukari a lokacin hydrolysis, yin ayyukan fasahar kere-kere masu tsada tare da samfuran da aka samu na mai.
Ƙayyade tsarin tsarin carbohydrates yana da ƙalubale kuma yana buƙatar haɗuwa da hanyoyi kamar ruwa chromatography (LC) 11,12, nukiliya magnetic resonance spectroscopy (NMR) 13, capillary electrophoresis (CE) 14,15,16 da mass spectrometry (MS)17., sha takwas.Hanyoyin MS irin su lokacin-lokacin-jirgin taro mai yawa tare da desorption laser da ionization ta amfani da matrix (MALDI-TOF-MS) hanya ce mai dacewa don gano tsarin carbohydrate.Kwanan nan, rashin daidaituwa-jawo rarrabuwa (CID) Tandem Ms na Mawallafin Sidar Sodium, da jerin abubuwan hannu, jerin abubuwa, da kuma manyan matsayi 20 20, 21.
Binciken Glycan kyakkyawan kayan aiki ne don gano zurfin gano abubuwan haɗin carbohydrate22.Wannan hanyar tana amfani da ƙwayoyin rigakafin monoclonal (mAbs) waɗanda aka ba da umarnin shuka bangon glycan a matsayin bincike don fahimtar hadaddun haɗin gwiwar carbohydrate.Fiye da 250 mAbs ana samun su a duk duniya, an ƙirƙira su da layin layi daban-daban da reshe na oligosaccharides ta amfani da saccharides24 daban-daban.An yi amfani da mAbs da yawa don kwatanta tsari, abun da ke ciki, da gyare-gyare na bangon tantanin halitta, saboda akwai bambance-bambance masu mahimmanci dangane da nau'in kwayar halitta, sashin jiki, shekaru, matakin ci gaba, da yanayin girma25,26.Kwanan nan, an yi amfani da wannan hanyar don fahimtar yawan vesicle a cikin tsarin tsire-tsire da dabbobi da kuma matsayinsu a cikin sufuri na glycan kamar yadda aka ƙaddara ta hanyar alamar ƙananan ƙwayoyin cuta, matakan ci gaba, ko abubuwan da suka shafi muhalli, da kuma ƙayyade aikin enzymatic.Wasu daga cikin sifofi daban-daban na glycans da xylans waɗanda aka gano sun haɗa da pectin (P), xylan (X), mannan (M), xyloglucans (XylG), glucans na haɗin gwiwa (MLG), arabinoxylan (ArbX), galactomannan (GalG) , glucuronic acid-arabinoxylan (GarbX) da lactan9.
Duk da haka, duk da waɗannan yunƙurin bincike, ƙananan binciken ne kawai suka mayar da hankali kan yanayin tarin oligosaccharides a lokacin babban nauyin nauyin nauyi (HSL), ciki har da sakin oligosaccharides, canjin sarkar oligomeric a lokacin hydrolysis, daban-daban ƙananan DP polymers, da masu lankwasa.rabawa 30,31,32.A halin yanzu, kodayake bincike na glycan ya tabbatar da zama kayan aiki mai amfani don cikakken bincike na tsarin glycan, yana da wuya a kimanta ƙarancin DP oligosaccharides mai narkewar ruwa ta amfani da hanyoyin antibody.Ƙananan DP oligosaccharides tare da nauyin kwayoyin halitta na kasa da 5-10 kDa ba sa ɗaure zuwa faranti na ELISA 33, 34 kuma ana wanke su kafin ƙarin antibody.
Anan, a karon farko, muna nuna gwajin ELISA akan faranti mai rufi avidin ta amfani da ƙwayoyin rigakafi na monoclonal, tare da haɗa tsarin biotinylation na mataki ɗaya don oligosaccharides mai narkewa tare da nazarin glycome.Hanyarmu ta nazarin glycome ta sami ingantacciyar hanyar MALDI-TOF-MS da GC-MS tushen bincike na haɗin gwiwar oligosaccharides ta amfani da trimethylsilyl (TMS) ƙaddamar da abubuwan sukari na hydrolyzed.Za a iya haɓaka wannan sabuwar hanyar a matsayin babbar hanyar da za a iya aiwatarwa a nan gaba kuma a sami ƙarin aikace-aikace a cikin binciken nazarin halittu35.
Canje-canjen bayan-fassara na enzymes da ƙwayoyin rigakafi, irin su glycosylation,36 suna shafar ayyukansu na rayuwa.Misali, canje-canje a cikin glycosylation na sunadaran jini suna taka muhimmiyar rawa a cikin cututtukan cututtukan fata, kuma ana amfani da canje-canje a cikin glycosylation azaman alamun bincike37.An ba da rahoton glycans daban-daban a cikin wallafe-wallafen don bayyana a hankali a cikin cututtuka daban-daban, ciki har da cututtuka masu kumburi na gastrointestinal tract da hanta, cututtuka na kwayar cuta, ovarian, nono, da prostate cancers38,39,40.Fahimtar tsarin glycans ta amfani da hanyoyin ELISA na tushen glycan zai ba da ƙarin tabbaci game da gano cutar ba tare da amfani da hanyoyin MS masu rikitarwa ba.
Bincikenmu na baya ya nuna cewa oligosaccharides masu taurin kai sun kasance marasa ruwa bayan pretreatment da enzymatic hydrolysis (Figure 1).A cikin aikin da aka buga a baya, mun ƙirƙiri hanyar hakar gawayi mai ƙarfi don ware oligosaccharides daga AFEX-pretreated masara stover hydrolyzate (ACSH)8.Bayan hakar farko da rabuwa, oligosaccharides sun kara raguwa ta hanyar chromatography girman keɓancewa (SEC) kuma an tattara su don nauyin kwayoyin halitta.Sugar monomers da oligomers da aka saki daga magunguna daban-daban an bincika su ta hanyar nazarin abubuwan sukari.Lokacin kwatanta abun ciki na oligomers masu ciwon sukari da aka samu ta hanyoyi daban-daban na pretreatment, kasancewar oligosaccharides masu taurin kai shine matsala ta gama gari a cikin jujjuyawar biomass zuwa monosaccharides kuma yana iya haifar da raguwar yawan sukari aƙalla 10-15% har ma har zuwa 18%.AmurkaAna amfani da wannan hanyar don ƙara yawan samar da ɓangarorin oligosaccharides.Sakamakon ACH da sassan da suka biyo baya tare da ma'auni daban-daban an yi amfani da su azaman kayan gwaji don halayyar oligosaccharides a cikin wannan aikin.
Bayan pretreatment da enzymatic hydrolysis, m oligosaccharides kasance unhydrolysed.Anan (A) shine hanyar rabuwa na oligosaccharides wanda oligosaccharides ke ware daga AFEX-pretreated masara stover hydrolyzate (ACSH) ta amfani da gado mai cike da carbon da aka kunna da diatomaceous ƙasa;(B) Hanyar don rabuwa da oligosaccharides.An kara raba oligosaccharides ta hanyar chromatography girma (SEC);(C) Saccharide monomers da oligomers da aka saki daga wasu pretreatments (diluted acid: DA, ionic liquid: IL da AFEX).Enzymatic hydrolysis yanayi: high daskararru loading na 25% (w / w) (kimanin 8% glucan loading), 96 hours hydrolysis, 20 mg / g kasuwanci enzyme loading (Ctec2: Htec2: MP-2: 1: 1 rabo) da kuma (D) Sugar monomers da oligomers na glucose, xylose da aka saki daga CS EXcorn.
Binciken Glycan ya tabbatar da zama kayan aiki mai amfani don ingantaccen tsarin bincike na glycans a cikin abubuwan da aka keɓe daga ƙaƙƙarfan ragowar biomass.Koyaya, saccharides masu narkewar ruwa ba su da wakilci ta amfani da wannan hanyar gargajiya41 saboda ƙananan nauyin oligosaccharides suna da wahalar haɓaka akan faranti na ELISA kuma ana wanke su kafin ƙari na antibody.Sabili da haka, don ɗaure antibody da sifa, an yi amfani da hanyar biotinylation mataki ɗaya don shafa mai narkewa, oligosaccharides marasa dacewa akan faranti na ELISA mai avidin.An gwada wannan hanyar ta amfani da ACSH da aka samar da shi a baya da kuma juzu'i dangane da nauyin kwayoyin halitta (ko digiri na polymerization, DP).An yi amfani da biotinylation na mataki daya don ƙara haɓaka haɗin gwiwar oligosaccharides ta ƙara biotin-LC-hydrazide zuwa rage ƙarshen carbohydrate (Fig. 2).A cikin bayani, ƙungiyar hemiacetal a ƙarshen raguwa yana amsawa tare da ƙungiyar hydrazide na biotin-LC-hydrazide don samar da haɗin hydrazone.A gaban wakili mai rage NaCNBH3, haɗin hydrazone yana raguwa zuwa ingantaccen samfurin biotinylated.Tare da gyare-gyare na ƙarshen rage ciwon sukari, ɗaurin ƙananan DP oligosaccharides zuwa faranti na ELISA ya zama mai yiwuwa, kuma a cikin bincikenmu an yi wannan a kan faranti mai rufi avidin ta amfani da mAbs glycan.
Nuna ƙwayoyin rigakafin monoclonal bisa ELISA don oligosaccharides biotinylated.Anan (A) haɗe biotinylation na oligosaccharides da gwajin ELISA na gaba tare da glycan-niyya mAbs akan faranti mai rufi na NeutrAvidin da (B) yana nuna hanyar mataki ɗaya don biotinylation na samfuran amsawa.
An saka faranti mai rufaffiyar Avidin tare da ƙwayoyin rigakafin oligosaccharides-conjugated a cikin ƙwayoyin rigakafi na farko da na sakandare kuma an wanke su a cikin matsakaici mai haske da lokaci.Bayan an gama daurin antibody, ƙara TMB substrate don shigar da farantin.A ƙarshe an dakatar da amsa tare da sulfuric acid.An bincika faranti da aka haɗa ta amfani da mai karanta ELISA don tantance ƙarfin ɗaure kowane antibody don gano takamaiman haɗin kai na antibody.Don cikakkun bayanai da sigogi na gwajin, duba sashin da ya dace "Kayan aiki da Hanyoyi".
Muna nuna amfanin wannan sabuwar hanyar da aka haɓaka don takamaiman aikace-aikace ta hanyar siffanta oligosaccharides masu narkewa da ke cikin ACSH da kuma a cikin ɗanyen da aka tsarkake da ɓarke ​​​​oligosaccharides keɓe daga lignocellulosic hydrolysates.Kamar yadda aka nuna a cikin Hoto na 3, mafi yawan xylans masu maye gurbin epitope da aka gano a cikin ACSH ta amfani da hanyoyin assay na bioacylated glycome yawanci uronic (U) ko methyluronic (MeU) da pectic arabinogalactans.Yawancin su kuma an samo su a cikin bincikenmu na baya game da nazarin glycans na abubuwan da ba na ruwa ba (UHS)43.
Gano na recalcitrant oligosaccharides epitopes ta amfani da monoclonal antibody kai tsaye zuwa ga cell bango glycan.Matsakaicin "tsaka-tsaki" shine juzu'in ACN kuma sashin "acid" shine juzu'in FA.Jajayen haske masu haske akan taswirar zafi suna nuna mafi girman abun ciki na epitope, kuma shuɗi masu haske suna nuna babu komai.Ƙimar launi a kan sikelin sun dogara ne akan ƙimar OD don ƙirar N = 2.Ana nuna manyan epitopes da ƙwayoyin rigakafi suka gane a hannun dama.
Waɗannan sifofin da ba na cellulose ba ba za a iya raba su ta hanyar mafi yawan cellulases da hemicellulases a cikin cakuda enzyme da aka gwada, wanda ya haɗa da enzymes na kasuwanci da aka fi amfani da su.Sabili da haka, ana buƙatar sababbin enzymes masu taimako don hydrolysis.Ba tare da dole ba-cellulose m enzymes, wadannan wadanda ba cellulose shaidu hana cikakken tuba zuwa monosaccharides, ko da iyayensu sugar polymers suna da yawa hydrolyzed cikin guntu guntu da kuma narkar da ta amfani da kasuwanci enzyme cakuda.
Ƙarin nazarin rarraba sigina da ƙarfin ɗaurinsa ya nuna cewa ɗaurin epitopes sun kasance ƙasa a cikin manyan ɓangarorin DP sugar (A, B, C, DP har zuwa 20 +) fiye da ƙananan ƙananan DP (D, E, F, DP) a cikin dimers) (Fig. 1).Gutsutsun acid sun fi kowa a cikin epitopes waɗanda ba cellulose ba fiye da gutsure tsaka.Wadannan abubuwan mamaki sun yi daidai da tsarin da aka lura a cikin bincikenmu na baya, inda manyan DP da kwayoyin acid sun fi tsayayya ga enzymatic hydrolysis.Saboda haka, kasancewar wadanda ba cellulose glycan epitopes da U da MeU maye gurbinsu na iya taimakawa sosai ga kwanciyar hankali na oligosaccharides.Ya kamata a lura cewa haɓakawa da haɓakawa na iya zama matsala ga ƙananan DP oligosaccharides, musamman ma idan epitope ya kasance dimeric ko trimeric oligosaccharides.Ana iya gwada wannan ta amfani da oligosaccharides na kasuwanci na tsayi daban-daban, kowanne yana ɗauke da epitope ɗaya kawai wanda ke ɗaure ga takamaiman mAb.
Don haka, yin amfani da ƙayyadaddun ƙwayoyin rigakafin ƙwayoyin cuta sun bayyana wasu nau'ikan haɗin kai.Dangane da nau'in antibody da aka yi amfani da shi, tsarin ligation da ya dace, da ƙarfin siginar da yake samarwa (mafi yawa kuma mafi ƙarancin yawa), ana iya gano sabbin enzymes kuma an ƙara su da ƙima zuwa ga cakuda enzyme don ƙarin cikakkiyar glycoconversion.Ɗaukar nazarin ACSH oligosaccharides a matsayin misali, za mu iya ƙirƙirar bayanan bayanan glycan bond don kowane abu na halitta.Ya kamata a lura a nan cewa ya kamata a yi la'akari da bambancin kusanci na ƙwayoyin cuta, kuma idan ba a san dangantakar su ba, wannan zai haifar da wasu matsaloli yayin kwatanta alamun cututtuka daban-daban.Bugu da ƙari, kwatanta haɗin gwiwar glycan na iya yin aiki mafi kyau tsakanin samfurori don maganin rigakafi iri ɗaya.Ana iya haɗa waɗannan taurin kai zuwa bayanan CAZyme, daga inda za mu iya gano enzymes, zaɓi enzymes ɗan takara da gwada enzymes masu karya haɗin gwiwa, ko haɓaka tsarin ƙwayoyin cuta don bayyana waɗannan enzymes don amfani da su a biorefineries44.
Don kimanta yadda hanyoyin rigakafi suka dace da madadin hanyoyin da za a iya kwatanta ƙananan nauyin kwayoyin oligosaccharides da ke cikin lignocellulosic hydrolysates, mun yi MALDI (Fig. 4, S1-S8) da kuma nazarin saccharides da aka samo TMS bisa GC-MS a kan wannan panel (Fig. 5) oligosaccharides part.Ana amfani da MALDI don kwatanta ko yawan rarraba kwayoyin oligosaccharides ya dace da tsarin da aka yi niyya.A kan fig.4 yana nuna MC na abubuwan tsaka tsaki ACN-A da ACN-B.Binciken ACN-A ya tabbatar da nau'in ciwon sukari na pentose daga DP 4-8 (Fig. 4) zuwa DP 22 (Fig. S1), wanda nauyinsa ya dace da MeU-xylan oligosaccharides.Binciken ACN-B ya tabbatar da jerin pentose da glucoxylan tare da DP 8-15.A cikin ƙarin kayan kamar Hoto S3, taswirar rarraba kayan acidic na FA-C suna nuna kewayon (Me) U maye gurbin sukarin pentose tare da DP na 8-15 waɗanda suka yi daidai da maye gurbin xylans da aka samu a cikin gwajin mAb na tushen ELISA.Alamun sun yi daidai.
MALDI-MS bakan na oligosaccharides masu narkewa marasa dacewa da ke cikin ACS.Anan, (A) ACN-A ƙananan nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in) nauyin nauyi) ya maye gurbin glucuroxylan oligosaccharides da (B) ACN-B xylan da methylated uronic acid oligosaccharides wanda aka maye gurbinsu da glucuroxylan (DP 8-15).
Binciken abun da ke ciki na ragowar glycan na refractory oligosaccharides.Anan (A) TMS saccharide abun da ke ciki na nau'ikan ɓangarorin oligosaccharides daban-daban da aka samu ta amfani da bincike na GC-MS.(B) Tsarin nau'ikan nau'ikan sukari da aka samu TMS da ke cikin oligosaccharides.ACN - kashi na acetonitrile mai dauke da tsaka tsaki oligosaccharides da FA - ferulic acid juzu'i mai dauke da oligosaccharides acid.
Wani ƙarshe mai ban sha'awa an zana shi daga nazarin LC-MS na ɓangaren oligosaccharides, kamar yadda aka nuna a cikin Hoto S9 (hanyoyi za a iya gani a cikin ƙarin kayan lantarki).An lura da gutsuttsura na ƙungiyoyin hexose da -OAc akai-akai yayin haɗar juzu'in ACN-B.Wannan binciken ba wai kawai ya tabbatar da rarrabuwar kawuna da aka gani a cikin glycome da bincike na MALDI-TOF ba, har ma yana ba da sabbin bayanai game da yuwuwar abubuwan da ake samu na carbohydrate a cikin ƙwayoyin halittar lignocellulosic da aka riga aka rigaya.
Mun kuma bincika abun da ke ciki na sukari na juzu'in oligosaccharides ta amfani da cirewar sukari na TMS.Yin amfani da GC-MS, mun ƙaddara abubuwan da ke tattare da kwayoyin halitta (marasa asali) da acidic sugars (GluA da GalA) a cikin ɓangaren oligosaccharides (Fig. 5).Ana samun Glucuronic acid a cikin abubuwan acidic C da D, yayin da ana samun galacturonic acid a cikin abubuwan acidic A da B, duka biyun manyan abubuwan DP ne na sukari na acidic.Waɗannan sakamakon ba kawai tabbatar da bayanan ELISA da MALDI ɗinmu ba, amma kuma sun yi daidai da karatunmu na baya na tarin oligosaccharides.Sabili da haka, mun yi imanin cewa hanyoyin rigakafi na zamani ta amfani da biotinylation na oligosaccharides da gwajin ELISA na gaba sun isa don gano oligosaccharides mai narkewa mai narkewa a cikin samfuran halitta daban-daban.
Tun da tushen ELISA hanyoyin tantance mAb an inganta su ta hanyoyi daban-daban, muna son ƙara bincika yuwuwar wannan sabuwar hanyar ƙididdigewa.Kasuwancin oligosaccharides guda biyu, xylohexasaccharide oligosaccharides (XHE) da 23-α-L-arabinofuranosyl-xylotriose (A2XX), an saya da gwada su ta amfani da sabon tsarin mAb wanda ke niyya ga glycan tantanin halitta.Hoto na 6 yana nuna ma'auni na layi tsakanin siginar dauri na biotinylated da tarin log na maida hankali na oligosaccharides, yana nuna yiwuwar tallan Langmuir.Daga cikin mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, da CCRC-M151 sun haɗa da XHE, da CCRC-M108, CCRC-M109, da LM11 sun haɗa da A2XX a kan kewayon 1 na nm.Saboda ƙayyadaddun kasancewar ƙwayoyin rigakafi yayin gwajin, an yi iyakacin gwaje-gwaje tare da kowane ƙwayar oligosaccharides.Ya kamata a lura a nan cewa wasu ƙwayoyin rigakafi suna amsawa daban-daban ga oligosaccharides iri ɗaya a matsayin substrate, mai yiwuwa saboda suna ɗaure zuwa epitopes daban-daban kuma suna iya samun alaƙa daban-daban.Hanyoyi da abubuwan da ke haifar da ingantacciyar ganewar epitope za su kasance da rikitarwa sosai lokacin da aka yi amfani da sabuwar hanyar mAb akan samfuran gaske.
An yi amfani da oligosaccharides na kasuwanci guda biyu don tantance kewayon gano nau'ikan mAbs masu niyya glycan.Anan, daidaitawar layi tare da tattara tarin oligosaccharides yana nuna alamun tallan Langmuir don (A) XHE tare da mAb da (B) A2XX tare da mAb.Abubuwan da suka dace suna nuna sifofin oligosaccharides na kasuwanci da aka yi amfani da su azaman madogara a cikin kima.
Amfani da glycan-niyya monoclonal antibodies (glycocomic analysis ko ELISA-based mAb screening) kayan aiki ne mai ƙarfi don ƙididdigewa mai zurfi na mafi yawan manyan glycans na bangon tantanin halitta waɗanda ke samar da kwayoyin halitta.Duk da haka, nazarin glycan na gargajiya kawai yana kwatanta glycans mafi girma na cell cell, kamar yadda yawancin oligosaccharides ba su da kyau a kan faranti na ELISA.A cikin wannan binciken, AFEX-pretreated masara stover an enzymatically hydrolyzed a wani babban daskararru abun ciki.An yi amfani da bincike na sukari don ƙayyade abubuwan da ke tattare da ƙwayoyin carbohydrates na bango na recalcitrant a cikin hydrolyzate.Duk da haka, nazarin mAb na ƙananan oligosaccharides a cikin hydrolysates ba a yi la'akari da shi ba, kuma ana buƙatar ƙarin kayan aiki don hana oligosaccharides daidai a kan faranti na ELISA.
Muna ba da rahoto anan wani labari kuma ingantaccen hanyar hana oligosaccharides don gwajin mAb ta hanyar haɗa oligosaccharides biotinylation wanda ke biye da gwajin ELISA akan faranti mai rufi na NeutrAvidin™.The immobilized biotinylated oligosaccharides ya nuna isashen kusanci ga antibody don ba da damar gano sauri da ingantaccen gano oligosaccharides na recalcitrant.Binciken abubuwan da ke tattare da waɗannan oligosaccharides masu taurin kai dangane da yawan spectrometry sun tabbatar da sakamakon wannan sabuwar hanyar rigakafin rigakafi.Don haka, waɗannan nazarin sun nuna cewa haɗin oligosaccharides biotinylation da ELISA nunawa tare da glycan-niyya monoclonal antibodies za a iya amfani da su gano crosslinks a oligosaccharides kuma za a iya amfani da ko'ina a cikin sauran biochemical nazarin halittu characterizes tsarin oligosaccharides.
Wannan hanyar bayanan glycan ta tushen biotin ita ce rahoton farko da ke da ikon bincikar haɗin gwiwar carbohydrate mai narkewa na oligosaccharides mai narkewa a cikin kwayoyin halitta.Wannan yana taimakawa wajen fahimtar dalilin da yasa wasu sassa na biomass ke da taurin kai idan ana maganar samar da biofuel.Wannan hanya ta cika muhimmiyar rata a cikin hanyoyin bincike na glycome kuma ta ƙaddamar da aikace-aikacensa zuwa nau'in nau'i mai yawa fiye da oligosaccharides na shuka.A nan gaba, za mu iya amfani da mutum-mutumi don biotinylation da kuma amfani da hanyar da muka ɓullo da domin high-samar bincike na samfurori ta amfani da ELISA.
Bambaron masara (CS) da aka girma daga nau'in nau'in nau'in Pioneer 33A14 an girbe shi a cikin 2010 daga gonakin Kramer a Ray, Colorado.Tare da izinin mai gida, ana iya amfani da wannan biomass don bincike. An adana samfuran bushe <6% danshi a cikin jakunkuna-kulle a cikin zafin jiki. An adana samfuran bushe <6% danshi a cikin jakunkuna-kulle a cikin zafin jiki. Образцы хранились сухими при влажности <6% в пакетах с застежкой-молнией при комнатной температуре. An adana samfurori a bushe a <6% zafi a cikin jakunkuna masu zira a zafin jiki.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%. Ana adana samfurori a cikin jakunkuna na zik a dakin da zafin jiki tare da zafi <6%.Binciken ya bi ka'idodin gida da na ƙasa.An yi nazarin abubuwan haɗin kai ta amfani da ka'idar NREL.An gano abun da ya ƙunshi 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% protein da 13. 4% ash.
Cellic® CTec2 (138 mg protein/ml, lot VCNI 0001) wani hadadden cakuda cellulase, β-glucosidase da Cellic® HTec2 (157 mg protein / ml, kuri'a VHN00001) daga Novozymes (Franklinton, NC, Amurka)).Multifect Pectinase® (72 MG protein/mL), hadadden cakuda pectin enzymes masu lalata, an ba da gudummawa ta DuPont Industrial Biosciences (Palo Alto, CA, USA).An ƙaddara yawan adadin furotin na enzyme ta hanyar ƙididdige abubuwan da ke cikin furotin (da kuma rage gudunmawar nitrogen maras gina jiki) ta amfani da nazarin Kjeldahl nitrogen (Hanyar AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA).Diatomaceous earth 545 an saya daga EMD Millpore (Billerica, MA).Carbon da aka kunna (DARCO, granules mesh 100), Avicel (PH-101), beech xylan, da duk sauran sinadarai an siya daga Sigma-Aldrich (St. Louis, MO).
AFEX pretreatment an yi a GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, Amurka).An riga an gudanar da magani a 140 ° C. na mintina 15.Lokacin zama na 46 a 1: 1 rabo na ammonia anhydrous zuwa biomass a 60% (w / w) lodi a cikin ma'aunin ƙarfe na benchtop batch reactor (Kamfanin Instruments Parr).Ya ɗauki mintuna 30.An kawo reactor zuwa 140 ° C kuma an saki ammonia da sauri, wanda ya ba da damar kwayar halitta ta dawo da sauri zuwa zafin jiki.Abubuwan da aka haɗa na AFEX pre-treated masara stover (ACS) yayi kama da na masarar masara mara magani (UT-CS).
Babban daskararru ACSH 25% (w / w) (kimanin 8% dextran loading) an shirya shi azaman kayan farawa don manyan sikelin samar da oligosaccharides.Enzymatic hydrolysis na ACS da aka yi ta amfani da wani kasuwanci enzyme cakuda ciki har da Cellic® Ctec2 10 MG protein / g glucan (a pretreated biomass), Htec2 (Novozymes, Franklinton, NC), 5 MG protein / g glucan, da kuma Multifect Pectinase (Genencor Inc, USA).).5 MG protein/g dextran.Enzymatic hydrolysis da aka za'ayi a cikin 5-lita bioreactor tare da wani aiki girma na 3 lita, pH 4.8, 50 ° C da 250 rpm.Bayan hydrolysis na 96 hours, da hydrolyzate aka tattara ta centrifugation a 6000 rpm na minti 30 sa'an nan a 14000 rpm na 30 minutes don cire unhydrolysed daskararru.Bayan haka, an sanya hydrolyzate ɗin zuwa tacewa mara kyau ta hanyar ƙwanƙolin tacewa na 0.22 mm.An adana hydrolyzate da aka tace a cikin kwalabe maras kyau a 4 ° C. sa'an nan kuma an rarraba shi akan carbon.
Analysis na abun da ke ciki na tsantsa tushen biomass samfurori bisa ga NREL dakin gwaje-gwaje hanyoyin bincike hanyoyin: shirye-shiryen da samfurori don abun da ke ciki analysis (NREL / TP-510-42620) da kayyade tsarin carbohydrates da lignin a biomass (NREL / TP-510 - 42618) 47.
An gudanar da bincike na Oligosaccharides na rafin hydrolyzate akan sikelin 2 ml ta hanyar amfani da hanyar hydrolysis na tushen autoclave.Mix da samfurin hydrolyzate tare da 69.7 µl na 72% sulfuric acid a cikin 10 ml dunƙule hula al'ada tube da kuma incubate for 1 h a kan benci a 121 ° C, sanyi a kan kankara da kuma tace a cikin wani babban yi ruwa chromatography (HPLC) vial.An ƙaddamar da ƙaddamar da oligosaccharides ta hanyar rage yawan ƙwayar monosaccharides a cikin samfurin da ba na ruwa ba daga jimlar sukari a cikin samfurin acid-hydrolyzed.
Glucose, xylose, da arabinose maida hankali a cikin acid hydrolysed biomass aka bincika ta amfani da Shimadzu HPLC tsarin sanye take da autosampler, shafi hita, isocratic famfo, da refractive index ganowa a kan wani Bio-Rad Aminex HPX-87H shafi.An kiyaye ginshiƙi a 50 ° C kuma an cire shi tare da 0.6 ml / min 5 mM H2SO4 a cikin ruwa.kwarara.
An diluted supernatant hydrolyzate kuma an bincika don monomer da oligosaccharides abun ciki.Monomeric sugars samu bayan enzymatic hydrolysis aka bincikar ta HPLC sanye take da Bio-Rad (Hercules, CA) Aminex HPX-87P shafi da kuma ash ginshiƙi.An kiyaye zafin jiki na ginshiƙi a 80 ° C, an yi amfani da ruwa azaman lokaci na wayar hannu tare da ƙimar 0.6 ml / min.Oligosaccharides an ƙaddara ta hanyar hydrolysis a cikin dilute acid a 121 ° C bisa ga hanyoyin da aka bayyana a cikin refs.41, 48, 49.
An gudanar da bincike na saccharide a kan raw, AFEX da aka riga aka yi da shi da duk sauran abubuwan da ba su da ruwa (ciki har da samar da sassan bango na serial cell da kuma su mAb) ta amfani da hanyoyin da aka bayyana a baya 27, 43, 50, 51.Don nazarin glycome, barasa-insoluble sharanan shuka cell bango abu an shirya daga biomass sharan gona da kuma hõre serial hakar tare da ƙara m reagents kamar ammonium oxalate (50 mM), sodium carbonate (50 mM da 0.5% w / v), CON.(1M da 4M, duka tare da 1% w/v sodium borohydride) da acid chlorite kamar yadda aka bayyana a baya52,53.An gabatar da abubuwan da aka cirewa zuwa ELISA a kan wani hadadden panel na mAb50s wanda aka kai ga glycan bangon tantanin halitta, kuma an gabatar da halayen ɗaurin mAb azaman taswirar zafi.An sayi mAbs da ke niyya ga glycan cell cell daga hannun jari (CCRC, JIM da MAC jerin).
Biotinylation mataki daya na oligosaccharides.An gudanar da haɗin gwiwar carbohydrates tare da biotin-LC-hydrazide ta amfani da hanya mai zuwa.An narkar da Biotin-LC-hydrazide (4.6 mg / 12 μmol) a cikin dimethyl sulfoxide (DMSO, 70 μl) ta hanyar motsawa mai karfi da dumama a 65 ° C. don 1 min.An ƙara Glacial acetic acid (30 µl) kuma an zuba cakuda a kan sodium cyanoborohydride (6.4 mg/100 µmol) kuma an narkar da shi gaba daya bayan dumama a 65 ° C. na kimanin minti 1.Sa'an nan kuma, daga 5 zuwa 8 μl na cakuda dauki an ƙara zuwa busassun oligosaccharides (1-100 nmol) don samun nauyin 10-ninka ko fiye na lakabin akan ƙarshen ragewa.An gudanar da aikin a 65 ° C na 2 h, bayan haka an wanke samfurori nan da nan.Ba a yi amfani da sodium cyanoborohydride a cikin gwaje-gwajen lakabi ba tare da raguwa ba, kuma an yi amfani da samfurori a 65 ° C. na 2.5 hours.
ELISA shafi da wanke samfurori na oligosaccharides biotinylated.25 μl na samfurori na biotinylated (100 μl na kowane samfurin da aka tattara da aka diluted a cikin 5 ml na 0.1 M Tris buffer bayani (TBS)) an ƙara zuwa kowace rijiyar avidin mai rufi.An rufe rijiyoyin sarrafawa tare da 50 μl na biotin a matakin 10 μg / ml a cikin 0.1 M TBS.An yi amfani da ruwan da aka lalatar da shi azaman sutura don ma'auni mara kyau.An shigar da kwamfutar hannu na tsawon sa'o'i 2 a dakin da zafin jiki a cikin duhu.A wanke farantin sau 3 tare da madara mai 0.1% a cikin 0.1 M TBS ta amfani da shirin No.11 don Grenier flat 3A.
Ƙarawa da wanke magungunan rigakafi na farko.Ƙara 40 µl na maganin rigakafi na farko zuwa kowace rijiya.Sanya microplate na awa 1 a cikin zafin jiki a cikin duhu.Sannan an wanke faranti sau 3 tare da madara 0.1% a cikin 0.1M TBS ta amfani da shirin wankewa #11 don Grenier Flat 3A.
Ƙara maganin rigakafi na sakandare a wanke.Ƙara 50 µl na linzamin kwamfuta / bera na biyu antibody (diluted 1:5000 a cikin 0.1% madara a cikin 0.1 M TBS) zuwa kowace rijiya.Sanya microplate na awa 1 a cikin zafin jiki a cikin duhu.Sannan an wanke microplates ɗin sau 5 tare da madara 0.1% a cikin 0.1 M TBS ta amfani da shirin wanke faranti na Grenier Flat 5A #12.
Ƙara substrate.Ƙara 50 µl na 3,3′,5,5′-tetramethylbenzidine (TMB) zuwa tushen tushe (ta ƙara 2 saukad da buffer, 3 saukad da TMB, 2 saukad da hydrogen peroxide zuwa 15 ml na deionized ruwa).Shirya TMB substrate.da vortex kafin amfani).Sanya microplate a zafin jiki na tsawon mintuna 30.A cikin duhu.
Kammala mataki kuma karanta kwamfutar hannu.Ƙara 50 µl na 1 N sulfuric acid zuwa kowace rijiya kuma yin rikodin abin sha daga 450 zuwa 655 nm ta amfani da mai karanta ELISA.
Shirya maganin 1 MG / ml na waɗannan nazarin a cikin ruwa mai narkewa: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine.(glcNAc), N-acetylgalactosamine (galNAc), inositol (misali na ciki).An shirya ma'auni guda biyu ta hanyar ƙara 1 mg / mL masu ciwon sukari da aka nuna a cikin Table 1. Samfurori suna daskarewa da lyophilized a -80 ° C. har sai an cire duk ruwa (yawanci game da 12-18 hours).
Ƙara 100-500 μg na samfur don dunƙule bututun hula akan ma'aunin nazari.Yi rikodin adadin da aka ƙara.Zai fi kyau a narkar da samfurin a cikin ƙayyadaddun ƙayyadaddun ƙaura kuma ƙara shi a cikin bututu azaman aliquot ruwa.Yi amfani da 20 µl na 1 mg/ml inositol azaman ma'auni na ciki don kowane samfurin bututu.Adadin ma'auni na ciki da aka ƙara zuwa samfurin dole ne ya zama daidai da adadin ma'auni na ciki da aka ƙara zuwa bututu.
Add 8 ml na methanol anhydrous a cikin dunƙule hula vial.Sa'an nan 4 ml na 3 N. methanol HCl bayani, capped da girgiza.Wannan tsari baya amfani da ruwa.
Ƙara 500 µl na 1 M HCl maganin methanol zuwa samfuran oligosaccharides da daidaitattun bututun TMS.An yi amfani da samfurori a cikin dare (168 hours) a 80 ° C. a cikin shinge na thermal.Bushe samfurin methanolysis a zazzabi na ɗaki ta amfani da nau'in bushewa.Ƙara 200 µl MeOH kuma a sake bushewa.Ana maimaita wannan tsari sau biyu.Ƙara 200 µl na methanol, 100 μl na pyridine da 100 μl na acetic anhydride a cikin samfurin kuma gauraya sosai.An yi amfani da samfurori a cikin zafin jiki na minti 30.da bushewa.Ƙara 200 µl na methanol a sake bushewa.
Ƙara 200 µl na Tri-Sil da zafi da aka rufe na minti 20.80 ° C, sannan a sanyaya zuwa zafin jiki.Yi amfani da nau'in bushewa don ƙara bushe samfurin zuwa ƙarar kusan 50 µl.Yana da mahimmanci a lura cewa ba mu ƙyale samfuran su bushe gaba ɗaya ba.
Ƙara 2 ml na hexane kuma haɗuwa da kyau ta hanyar vortexing.Cika tukwici na Pasteur pipettes (5-8 mm) tare da guntun ulun gilashi ta hanyar saka ulun gilashin a saman wani diamita na 5-3/4 inch pipette.An yi amfani da samfurori a cikin 3000 g na minti 2.Duk sauran abubuwan da ba a iya narkewa suna haɗe.Busa samfurin zuwa 100-150 µl.An yi allurar girma na kusan 1 μl a cikin GC-MS a farkon zafin jiki na 80 ° C da farkon lokacin mintuna 2.0 (Table 2).