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Sabbin hanyoyin rigakafi da yawa don nazarin hadaddun oligosaccharides masu dorewa a cikin tukunyar masara da aka riga aka yi wa magani da AFEX. Lignocellulosic biomass madadin man fetur ne mai dorewa kuma ana amfani da shi sosai don haɓaka fasahar halittu don samar da kayayyaki kamar abinci, abinci, mai da sinadarai. Mabuɗin waɗannan fasahohin shine haɓaka hanyoyin da ke da tsada don canza hadaddun carbohydrates da ke cikin bangon ƙwayoyin shuka zuwa sukari mai sauƙi kamar glucose, xylose da arabinose. Saboda biomass na lignocellulosic yana da taurin kai sosai, dole ne a yi masa maganin thermochemical (misali, ammonia fiber exfoliation (AFEX), diluted acids (DA), ionic liquids (IL)) da kuma maganin halittu (misali, enzymatic hydrolysis da microbial fermentation) a hade don samun samfurin da ake so. . Duk da haka, lokacin da aka yi amfani da enzymes na fungal na kasuwanci a cikin tsarin hydrolysis, kashi 75-85% kawai na sukari mai narkewa da aka samar sune monosaccharides, kuma sauran kashi 15-25% sune oligosaccharides masu narkewa, masu sauƙin narkewa, waɗanda ba koyaushe ake samu ga ƙananan halittu ba. A baya, mun sami nasarar ware kuma an tsarkake oligosaccharides masu taurin kai ta amfani da haɗin rabuwar carbon da diatomaceous earth da kuma chromatography na cire girman, sannan kuma mun binciki halayen hana enzyme ɗinsu. Mun gano cewa oligosaccharides waɗanda ke ɗauke da mafi girman matakin polymerization (DP) methylated uronic acid suna da wahalar sarrafawa tare da gaurayen enzyme na kasuwanci fiye da ƙananan oligosaccharides na DP da tsaka tsaki. A nan mun bayar da rahoton amfani da ƙarin hanyoyi da dama, gami da bayanin glycan ta amfani da monoclonal antibodies (mAbs) musamman ga shuke-shuken biomass glycans don siffanta haɗin glycan a cikin bangon ƙwayoyin shuka da enzymatic hydrolysates, matrix-assisted laser desorption ionization, time-of-flight mass-spectrometry. . MALDI-TOF-MS) yana amfani da kololuwar ganewar tsari-bayani da aka samu ta hanyar spectroscopy bayan lalacewa ta biyu na ions marasa kyau, gas chromatography da mass spectrometry (GC-MS) don siffanta haɗin oligosaccharides tare da kuma ba tare da derivatization ba. Saboda ƙaramin girman oligosaccharides (DP 4–20), waɗannan ƙwayoyin suna da wahalar amfani da su don ɗaurewa da kuma siffanta mAb. Don shawo kan wannan matsalar, mun yi amfani da sabuwar hanyar hana oligosaccharides bisa biotin conjugation wadda ta yi nasarar yiwa yawancin oligosaccharides masu narkewa a cikin ƙananan DP a saman microplate alama, wanda aka yi amfani da shi a cikin tsarin mAb mai yawan fitarwa don takamaiman nazarin ligation. Wannan sabuwar hanyar za ta sauƙaƙe haɓaka gwaje-gwajen glycome masu yawan fitarwa a nan gaba waɗanda za a iya amfani da su don ware da kuma siffanta oligosaccharides da ke cikin alamun biomarkers don dalilai na ganewar asali.
Lignocellulosic biomass, wanda ya ƙunshi kayan aikin gona, dazuzzuka, ciyawa da kayan itace, yana da yuwuwar samar da samfuran da suka dogara da halittu, gami da abinci, abinci, mai da sinadarai masu mahimmanci don samar da samfuran da suka fi daraja1. Carbohydrates (kamar cellulose da hemicellulose) da ke cikin bangon ƙwayoyin shuka ana canza su zuwa monosaccharides ta hanyar sarrafa sinadarai da kuma canza halittu (kamar su enzyme hydrolysis da fermentation microbial). Magungunan da aka saba amfani da su kafin a fara amfani da su sun haɗa da faɗaɗa zare ammonia (AFEX), diluted acid (DA), ionic liquid (IL), da tururi fashewa (SE), waɗanda ke amfani da haɗin sinadarai da zafi don rage samar da lignocellulose ta hanyar buɗe bangon ƙwayoyin shuka3,4. taurin kai na abu, 5. Ana gudanar da enzyme hydrolysis a cikin babban nauyin daskararru ta amfani da enzymes masu ɗauke da carbohydrate masu aiki na kasuwanci (CAZymes) da fermentation na ƙwayoyin cuta ta amfani da yisti ko ƙwayoyin cuta masu canzawa don samar da man fetur da sinadarai masu tushen halittu 6.
CAZymes a cikin enzymes na kasuwanci sun ƙunshi cakuda enzymes masu rikitarwa waɗanda ke raba haɗin carbohydrates-sukari masu rikitarwa don samar da monosaccharides2,7. Kamar yadda muka ruwaito a baya, hanyar sadarwa mai rikitarwa ta polymers masu ƙanshi na lignin tare da carbohydrates yana sa su zama masu wahala sosai, wanda ke haifar da rashin cikar juyar sukari, yana tara kashi 15-25% na oligosaccharides na jima'i waɗanda ba a samar da su yayin hydrolysis na enzymatic na biomass da aka riga aka yi wa magani. Wannan matsala ce da aka saba da ita tare da hanyoyi daban-daban na premassing biomass. Wasu dalilan wannan matsala sun haɗa da hana enzyme yayin hydrolysis, ko rashin ko ƙarancin matakan enzymes masu mahimmanci waɗanda ake buƙata don karya haɗin sukari a cikin biomass na shuka. Fahimtar abubuwan da ke ciki da halayen tsarin sukari, kamar haɗin sukari a cikin oligosaccharides, zai taimaka mana inganta juyar sukari yayin hydrolysis, yana sa hanyoyin fasahar halittu su zama masu tsada tare da samfuran da aka samo daga man fetur.
Tantance tsarin carbohydrates abu ne mai wahala kuma yana buƙatar haɗakar hanyoyi kamar liquid chromatography (LC)11,12, nuclear magnetic resonance spectroscopy (NMR)13, capillary electrophoresis (CE)14,15,16 da mass spectrometry (MS)17. ,18. Hanyoyin MS kamar time-of-flight mass spectrometry tare da laser desorption da ionization ta amfani da matrix (MALDI-TOF-MS) hanya ce mai amfani don gano tsarin carbohydrate. Kwanan nan, an fi amfani da Collision-Induced Dissociation (CID) tandem MS na sodium ion adducts don gano yatsu masu dacewa da matsayin haɗe-haɗe na oligosaccharide, tsarin anomeric, jerin abubuwa, da matsayin reshe 20, 21.
Binciken Glycan kayan aiki ne mai kyau don gano zurfin haɗin carbohydrate22. Wannan hanyar tana amfani da ƙwayoyin rigakafi na monoclonal (mAbs) waɗanda aka yi niyya don glycan na bangon ƙwayoyin shuka a matsayin bincike don fahimtar haɗin carbohydrate mai rikitarwa. Fiye da mAbs 250 suna samuwa a duk duniya, waɗanda aka tsara akan oligosaccharides masu layi da rassan daban-daban ta amfani da saccharides24 daban-daban. An yi amfani da mAbs da yawa sosai don siffanta tsari, abun da ke ciki, da gyare-gyare na bangon ƙwayoyin shuka, saboda akwai bambance-bambance masu mahimmanci dangane da nau'in ƙwayoyin shuka, gabobi, shekaru, matakin ci gaba, da yanayin girma25,26. Kwanan nan, an yi amfani da wannan hanyar don fahimtar yawan vesicle a cikin tsarin tsirrai da dabbobi da kuma rawar da suka taka a cikin jigilar glycan kamar yadda alamun ƙananan ƙwayoyin halitta suka ƙaddara, matakan ci gaba, ko abubuwan da ke motsa muhalli, da kuma tantance ayyukan enzymatic. Wasu daga cikin nau'ikan glycans da xylans daban-daban da aka gano sun haɗa da pectin (P), xylan (X), mannan (M), xyloglucans (XylG), mixed bond glucans (MLG), arabinoxylan (ArbX), galactomannan (GalG), glucuronic acid-arabinoxylan (GArbX) da arabino-galactan (ArbG)29.
Duk da haka, duk da duk waɗannan ƙoƙarin bincike, ƙananan bincike ne kawai suka mayar da hankali kan yanayin tarin oligosaccharide a lokacin hydrolysis mai yawa (HSL), gami da sakin oligosaccharide, canje-canjen tsawon sarkar oligomeric yayin hydrolysis, nau'ikan polymers masu ƙarancin DP, da lanƙwasa. rarrabawa 30,31,32. A halin yanzu, kodayake nazarin glycan ya tabbatar da cewa kayan aiki ne mai amfani don cikakken nazarin tsarin glycan, yana da wuya a kimanta ƙananan oligosaccharides na DP masu narkewa cikin ruwa ta amfani da hanyoyin rigakafi. Ƙananan oligosaccharides na DP waɗanda ke da nauyin kwayoyin halitta ƙasa da 5-10 kDa ba sa ɗaurewa da faranti na ELISA 33, 34 kuma ana wanke su kafin a ƙara ƙwayoyin cuta.
A nan, a karon farko, mun nuna gwajin ELISA akan faranti masu rufi da avidin ta amfani da ƙwayoyin rigakafi na monoclonal, tare da haɗa hanyar biotinylation mataki ɗaya don oligosaccharides masu narkewa tare da nazarin glycome. Hanyarmu ta nazarin glycome an tabbatar da ita ta hanyar nazarin MALDI-TOF-MS da GC-MS bisa ga haɗin gwiwa na oligosaccharide mai dacewa ta amfani da trimethylsilyl (TMS) na abubuwan da suka haɗa da sukari da aka samar da hydrolyzed. Wannan sabuwar hanyar za a iya haɓaka ta azaman hanyar aiki mai girma a nan gaba kuma a sami fa'ida a cikin binciken biomedical35.
Canje-canjen bayan fassara na enzymes da ƙwayoyin rigakafi, kamar glycosylation,36 suna shafar ayyukansu na halitta. Misali, canje-canje a cikin glycosylation na sunadaran jini suna taka muhimmiyar rawa a cikin kumburin amosanin gabbai, kuma ana amfani da canje-canje a cikin glycosylation azaman alamun gano cuta37. An ruwaito glycans daban-daban a cikin wallafe-wallafen don bayyana cikin sauƙi a cikin cututtuka daban-daban, gami da cututtukan kumburi na yau da kullun na hanyoyin ciki da hanta, cututtukan ƙwayoyin cuta, cututtukan ovarian, nono, da prostate38,39,40. Fahimtar tsarin glycans ta amfani da hanyoyin ELISA na glycan mai tushen rigakafi zai samar da ƙarin kwarin gwiwa a cikin ganewar cututtuka ba tare da amfani da hanyoyin MS masu rikitarwa ba.
Binciken da muka yi a baya ya nuna cewa oligosaccharides masu taurin kai ba su da isasshen ruwa bayan an yi musu magani kafin a yi musu magani da kuma an yi musu aikin hydrolysis na enzymatic (Hoto na 1). A cikin aikinmu da aka buga a baya, mun ƙirƙiro wata hanyar cire gawayi mai ƙarfi don ware oligosaccharides daga AFEX-pretreated corn stover hydrolyzate (ACSH)8. Bayan cirewa da rabuwa da farko, an ƙara raba oligosaccharides ta hanyar girman cirewa (SEC) kuma an tattara su bisa ga nauyin kwayoyin halitta. An yi nazarin monomers na sukari da oligomers da aka fitar daga magunguna daban-daban ta hanyar nazarin abubuwan da suka shafi sukari. Lokacin da aka kwatanta abubuwan da ke cikin oligomers na sukari da aka samu ta hanyoyi daban-daban na magani kafin a yi musu magani, kasancewar oligosaccharides masu taurin kai matsala ce ta gama gari a cikin sauya biomass zuwa monosaccharides kuma yana iya haifar da raguwar yawan sukari na akalla 10-15% har ma har zuwa 18%. Ana amfani da wannan hanyar don ƙarin samar da ƙananan sassan oligosaccharides. An yi amfani da ACH da ɓangarorinsa masu zuwa tare da nauyin kwayoyin halitta daban-daban azaman kayan gwaji don siffanta oligosaccharides a cikin wannan aikin.
Bayan an riga an yi magani kafin a yi amfani da shi da kuma an yi amfani da sinadarin enzyme hydrolysis, sai aka daina amfani da sinadarin oligosaccharides. A nan (A) akwai wata hanyar rabuwa da oligosaccharides wadda ake ware oligosaccharides daga AFEX-pretreated corn stover hydrolysate (ACSH) ta amfani da wani akwati mai cike da carbon da diatomaceous earth; (B) Hanyar rabuwa da oligosaccharides. An ƙara raba oligosaccharides ta hanyar amfani da chromatography na cire girman (SEC); (C) Monomers na Saccharide da oligomers da aka fitar daga magunguna daban-daban (diluted acid: DA, ionic liquid: IL da AFEX). Yanayin hydrolysis na enzymatic: yawan nauyin daskararru na 25% (w/w) (kusan kashi 8% na glucan loading), hydrolysis na awanni 96, 20 mg/g enzyme loading na kasuwanci (Ctec2:Htec2:MP-2:1:1 rabo) da (D) Monomers na sukari da oligomers na glucose, xylose da arabinose da aka fitar daga AFEX corn stover da aka riga aka yi amfani da shi (ACS).
Binciken Glycan ya tabbatar da cewa kayan aiki ne mai amfani don cikakken nazarin tsarin glycans a cikin abubuwan da aka cire daga ragowar biomass masu ƙarfi. Duk da haka, ba a nuna su sosai ta amfani da wannan hanyar gargajiya ba41 saboda ƙananan oligosaccharides masu nauyin kwayoyin halitta suna da wahalar hana motsi a kan faranti ELISA kuma ana wanke su kafin a ƙara ƙwayoyin rigakafi. Saboda haka, don ɗaure ƙwayoyin rigakafi da halayyarsu, an yi amfani da hanyar biotinylation mataki ɗaya don shafa oligosaccharides masu narkewa, waɗanda ba su dace da juna ba akan faranti ELISA da aka shafa da avidin. An gwada wannan hanyar ta amfani da ACSH ɗinmu da aka samar a baya da kuma wani ɓangare bisa ga nauyin ƙwayoyin halittarsa ​​(ko matakin polymerization, DP). An yi amfani da biotinylation mataki ɗaya don ƙara alaƙar ɗaure oligosaccharide ta hanyar ƙara biotin-LC-hydrazide zuwa ƙarshen rage carbohydrate (Hoto na 2). A cikin maganin, ƙungiyar hemiacetal a ƙarshen ragewa tana amsawa da ƙungiyar hydrazide na biotin-LC-hydrazide don samar da haɗin hydrazone. A gaban sinadarin rage sinadarin NaCNBH3, haɗin hydrazone zai ragu zuwa wani samfurin ƙarshe mai ƙarfi wanda aka yi amfani da shi wajen samar da biotinylated. Tare da gyaran ƙarshen rage sukari, ɗaure ƙananan oligosaccharides na DP zuwa faranti ELISA ya zama mai yiwuwa, kuma a cikin bincikenmu an yi hakan akan faranti masu rufi da avidin ta amfani da mAbs masu niyya ga glycan.
Binciken ƙwayoyin rigakafi na monoclonal bisa ga ELISA don ƙwayoyin oligosaccharides masu biotinylated. A nan (A) haɗakar biotinylation na oligosaccharides da kuma gwajin ELISA na gaba tare da mAbs da aka yi niyya ga glycan akan faranti masu rufi na NeutrAvidin da (B) yana nuna hanya ɗaya ta biotinylation na samfuran amsawa.
Daga nan aka ƙara faranti masu rufi da Avidin tare da ƙwayoyin rigakafi masu haɗin gwiwa da oligosaccharide zuwa ga ƙwayoyin rigakafi na farko da na sakandare kuma aka wanke su a cikin wani wuri mai haske da kuma mai sauƙin ɗauka. Bayan an gama ɗaure ƙwayoyin rigakafi, sai a ƙara sinadarin TMB don ya yi ƙulli a faranti. A ƙarshe an dakatar da amsawar da sinadarin sulfuric. An yi nazarin faranti masu ƙulli ta amfani da mai karanta ELISA don tantance ƙarfin ɗaurewar kowane ƙwayoyin rigakafi don gano haɗin gwiwa na musamman tsakanin ƙwayoyin rigakafi. Don cikakkun bayanai da sigogi na gwajin, duba sashin da ya dace "Kayayyaki da Hanyoyi".
Mun nuna amfanin wannan sabuwar hanyar da aka ƙirƙiro don takamaiman aikace-aikace ta hanyar siffanta oligosaccharides masu narkewa da ke cikin ACSH da kuma a cikin sassan oligosaccharides da aka tsarkake da aka ware daga lignocellulosic hydrolysates. Kamar yadda aka nuna a Hoto na 3, mafi yawan xylans da aka maye gurbin epitope da aka gano a cikin ACSH ta amfani da hanyoyin gwajin bioacylated glycome yawanci sune uronic (U) ko methyluronic (MeU) da pectic arabinogalactans. Yawancinsu an kuma same su a cikin bincikenmu na baya kan nazarin glycans na daskararru marasa hydrolyzed (UHS)43.
Gano epitopes masu recalcitrant oligosaccharide ta amfani da wani monoclonal antibody da aka kai wa glycan bangon tantanin halitta. Kashi na "tsaka tsaki" shine kashin ACN kuma kashin "mai acidic" shine kashin FA. Ja masu haske akan taswirar zafi suna nuna yawan abun ciki na epitope, kuma shuɗi mai haske yana nuna bango mara komai. Ƙimar launi akan sikelin ta dogara ne akan ƙimar OD da ba a sarrafa ba don tsari N=2. Manyan epitopes da aka gane ta hanyar ƙwayoyin rigakafi an nuna su a dama.
Waɗannan tsarin da ba na cellulose ba ba za a iya raba su ta hanyar ƙwayoyin halitta da hemicellulases da aka fi sani a cikin cakuda enzyme na kasuwanci da aka gwada ba, wanda ya haɗa da enzymes na kasuwanci da aka fi amfani da su. Saboda haka, ana buƙatar sabbin enzymes na taimako don hydrolysis ɗinsu. Ba tare da enzymes na haɗi waɗanda ba na cellulose ba, waɗannan haɗin da ba na cellulose ba suna hana canzawa gaba ɗaya zuwa monosaccharides, koda kuwa iyayensu na sukari polymers an haɗa su sosai zuwa gajerun gutsuttsura kuma an narkar da su ta amfani da gaurayen enzyme na kasuwanci.
Ƙarin bincike kan rarraba sigina da ƙarfin ɗaurewar sa ya nuna cewa epitopes masu ɗaure sun yi ƙasa a cikin manyan sassan sukari na DP (A, B, C, DP har zuwa 20+) fiye da ƙananan sassan DP (D, E, F, DP) a cikin dimers) (Hoto na 1). Guraben acid sun fi yawa a cikin epitopes marasa cellulose fiye da guraben tsaka tsaki. Waɗannan abubuwan sun yi daidai da tsarin da aka gani a cikin bincikenmu na baya, inda manyan sassan DP da acid sun fi juriya ga hydrolysis na enzymatic. Saboda haka, kasancewar epitopes marasa cellulose glycan da maye gurbin U da MeU na iya ba da gudummawa sosai ga kwanciyar hankali na oligosaccharides. Ya kamata a lura cewa ingancin ɗaurewa da ganowa na iya zama matsala ga ƙananan oligosaccharides na DP, musamman idan epitope dimeric ko trimeric oligosaccharides ne. Ana iya gwada wannan ta amfani da oligosaccharides na kasuwanci masu tsayi daban-daban, kowannensu yana ɗauke da epitope ɗaya kawai wanda ke ɗaure da takamaiman mAb.
Saboda haka, amfani da ƙwayoyin rigakafi na musamman na tsari ya bayyana wasu nau'ikan haɗin gwiwa masu sassauci. Dangane da nau'in ƙwayoyin rigakafi da aka yi amfani da su, tsarin ɗaurewa da ya dace, da ƙarfin siginar da yake samarwa (mafi yawa kuma mafi ƙarancin yawa), ana iya gano sabbin enzymes kuma a ƙara su a cikin rabin adadi zuwa ga cakuda enzyme don ƙarin cikakken canjin glycoconversion. Idan muka ɗauki nazarin ACSH oligosaccharides a matsayin misali, za mu iya ƙirƙirar bayanai na haɗin glycan don kowane kayan biomass. Ya kamata a lura a nan cewa ya kamata a yi la'akari da bambancin alaƙar ƙwayoyin rigakafi, kuma idan ba a san alaƙar su ba, wannan zai haifar da wasu matsaloli yayin kwatanta siginar ƙwayoyin rigakafi daban-daban. Bugu da ƙari, kwatanta haɗin glycan na iya aiki mafi kyau tsakanin samfuran ƙwayoyin rigakafi iri ɗaya. Waɗannan haɗin gwiwa masu taurin kai za a iya haɗa su da bayanan CAZyme, daga inda za mu iya gano enzymes, zaɓar enzymes masu takara da gwada enzymes masu karya haɗin gwiwa, ko haɓaka tsarin ƙwayoyin cuta don bayyana waɗannan enzymes don amfani a cikin biorefineries44.
Domin kimanta yadda hanyoyin rigakafi ke ƙara wa wasu hanyoyin da za a iya gano ƙananan ƙwayoyin cuta masu nauyin ƙwayoyin cuta da ke cikin lignocellulosic hydrolysates, mun yi MALDI (Hoto na 4, S1-S8) da kuma nazarin saccharides da aka samo daga TMS bisa ga GC-MS a kan wannan ɓangaren oligosaccharide (Hoto na 5). Ana amfani da MALDI don kwatanta ko rarrabawar ƙwayoyin oligosaccharide ya dace da tsarin da aka nufa. A kan Hoto na 4 yana nuna MS na sassan tsaka tsaki ACN-A da ACN-B. Binciken ACN-A ya tabbatar da nau'ikan sukari na pentose daga DP 4-8 (Hoto na 4) zuwa DP 22 (Hoto na S1), waɗanda nauyinsu ya yi daidai da oligosaccharides na MeU-xylan. Binciken ACN-B ya tabbatar da jerin pentose da glucoxylan tare da DP 8-15. A cikin ƙarin kayan aiki kamar Hoto na S3, taswirar rarrabawar yawan acidic moiety na FA-C tana nuna nau'ikan sukari pentose da aka maye gurbinsu da (Me)U tare da DP na 8-15 waɗanda suka yi daidai da waɗanda aka maye gurbinsu da aka samu a cikin gwajin mAb na ELISA. Epitopes ɗin suna da daidaito.
Bambance-bambancen MALDI-MS na oligosaccharides masu narkewa waɗanda ba sa bin ƙa'idodin ACS. A nan, (A) ACN-A ƙananan juzu'i na kewayon nauyi wanda ke ɗauke da methylated uronic acid (DP 4-8) an maye gurbin glucuroxylan oligosaccharides da (B) ACN-B xylan da methylated uronic acid oligosaccharides da aka maye gurbinsu da glucuroxylan (DP 8-15).
Binciken abubuwan da ke cikin ragowar glycan na oligosaccharides masu rarrafe. A nan (A) Tsarin TMS saccharide na ɓangarori daban-daban na oligosaccharide da aka samu ta amfani da nazarin GC-MS. (B) Tsarin nau'ikan sukari daban-daban da aka samo daga TMS da ke cikin oligosaccharides. ACN - ɓangaren acetonitrile wanda ke ɗauke da oligosaccharides masu tsaka-tsaki da kuma ɓangaren FA - ferulic acid wanda ke ɗauke da oligosaccharides na acid.
An samo wani ƙarshe mai ban sha'awa daga nazarin LC-MS na ɓangaren oligosaccharide, kamar yadda aka nuna a Hoto na S9 (ana iya ganin hanyoyin a cikin kayan ƙarin kayan lantarki). An lura da guntu-guntun ƙungiyoyin hexose da -OAC akai-akai yayin haɗa ɓangaren ACN-B. Wannan binciken ba wai kawai yana tabbatar da rarrabuwar da aka lura a cikin nazarin glycome da MALDI-TOF ba, har ma yana ba da sabon bayani game da yiwuwar abubuwan da suka samo asali daga carbohydrates a cikin biomass na lignocellulosic da aka riga aka yi wa magani.
Mun kuma yi nazarin abubuwan da ke cikin glycan na sassan oligosaccharide ta amfani da TMS glycan derivatization. Ta amfani da GC-MS, mun tantance abubuwan da ke cikin jijiyoyi (marasa tushe) da sukari mai acidic (GluA da GalA) a cikin sassan oligosaccharide (Hoto na 5). Ana samun Glucuronic acid a cikin sassan acidic C da D, yayin da ake samun galacturonic acid a cikin sassan acidic A da B, waɗanda duka manyan abubuwan DP ne na sukari mai acidic. Waɗannan sakamakon ba wai kawai suna tabbatar da bayanan ELISA da MALDI ba ne, har ma sun yi daidai da bincikenmu na baya game da tarin oligosaccharide. Saboda haka, mun yi imanin cewa hanyoyin rigakafi na zamani ta amfani da biotinylation na oligosaccharides da kuma gwajin ELISA na gaba sun isa don gano oligosaccharides masu narkewa a cikin samfuran halittu daban-daban.
Tunda an tabbatar da hanyoyin tantance mAb da aka dogara da ELISA ta hanyoyi daban-daban, mun so mu ƙara bincika yuwuwar wannan sabuwar hanyar adadi. An sayi oligosaccharides guda biyu na kasuwanci, xylohexasaccharide oligosaccharide (XHE) da 23-α-L-arabinofuranosyl-xylotriose (A2XX), kuma an gwada su ta amfani da sabuwar hanyar mAb da ke nufin glycan na bangon tantanin halitta. Hoto na 6 yana nuna alaƙar layi tsakanin siginar ɗaure biotinylated da yawan log na yawan oligosaccharide, yana nuna yiwuwar samfurin shaƙar Langmuir. Daga cikin mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, da CCRC-M151 sun yi alaƙa da XHE, da CCRC-M108, CCRC-M109, da LM11 sun yi alaƙa da A2XX a cikin kewayon nano 1 nm zuwa 100. Saboda ƙarancin wadatar ƙwayoyin rigakafi yayin gwajin, an yi gwaje-gwaje masu iyaka tare da kowane yawan oligosaccharide. Ya kamata a lura a nan cewa wasu ƙwayoyin rigakafi suna amsawa daban-daban ga oligosaccharide iri ɗaya a matsayin substrate, wataƙila saboda suna ɗaurewa da ɗan bambance-bambancen epitopes kuma suna iya samun alaƙa daban-daban. Hanyoyi da tasirin gano ainihin epitope zai fi rikitarwa lokacin da aka yi amfani da sabuwar hanyar mAb ga samfuran gaske.
An yi amfani da oligosaccharides guda biyu na kasuwanci don tantance kewayon gano mAbs daban-daban da ke niyya ga glycan. A nan, alaƙar layi tare da yawan log na yawan oligosaccharides yana nuna tsarin shaye-shaye na Langmuir don (A) XHE tare da mAb da (B) A2XX tare da mAb. Epitopes masu dacewa suna nuna tsarin oligosaccharides na kasuwanci da aka yi amfani da su azaman substrates a cikin gwajin.
Amfani da ƙwayoyin rigakafi na monoclonal da aka yi niyya ga glycan (binciken glycocomic ko gwajin mAb na ELISA) kayan aiki ne mai ƙarfi don zurfafa ganewar yawancin manyan glycans na bangon tantanin halitta waɗanda ke samar da biomass na tsire-tsire. Duk da haka, nazarin glycan na gargajiya yana nuna manyan glycans na bangon tantanin halitta ne kawai, saboda yawancin oligosaccharides ba su da ƙarfi sosai akan faranti na ELISA. A cikin wannan binciken, an sanya murhun masara da aka riga aka yi wa magani na AFEX hydrolyzed a cikin enzyme a cikin babban abun ciki na tauri. An yi amfani da nazarin sukari don tantance abun da ke cikin carbohydrates na bangon tantanin halitta masu sake canzawa a cikin hydrolyzate. Duk da haka, ba a rage girman nazarin mAb na ƙananan oligosaccharides a cikin hydrolysates ba, kuma ana buƙatar ƙarin kayan aiki don rage motsi na oligosaccharides akan faranti na ELISA yadda ya kamata.
Mun bayar da rahoton wata sabuwar hanyar hana motsi ta oligosaccharide don tantance mAb ta hanyar haɗa biotinylation na oligosaccharide sannan kuma a tantance ELISA akan faranti masu rufi na NeutrAvidin™. Kwayoyin halittar da aka hana motsi sun nuna isasshen ƙarfi ga ƙwayoyin halittar don ba da damar gano oligosaccharides masu sassauci cikin sauri da inganci. Binciken abubuwan da ke cikin waɗannan ƙwayoyin halittar da suka taurare bisa ga mass spectrometry ya tabbatar da sakamakon wannan sabuwar hanyar tantance garkuwar jiki. Don haka, waɗannan nazarin sun nuna cewa haɗin biotinylation na oligosaccharide da gwajin ELISA tare da ƙwayoyin halittar monoclonal masu niyya ga glycan za a iya amfani da su don gano hanyoyin haɗin gwiwa a cikin oligosaccharides kuma ana iya amfani da su sosai a cikin wasu nazarin halittu da ke nuna tsarin oligosaccharides.
Wannan hanyar tantance glycan mai tushen biotin ita ce rahoto na farko da ke da ikon bincika haɗin carbohydrate mai narkewa na oligosaccharides mai narkewa a cikin biomass na shuka. Wannan yana taimakawa wajen fahimtar dalilin da yasa wasu sassan biomass suke da taurin kai idan ana maganar samar da man fetur. Wannan hanyar tana cike gibi mai mahimmanci a cikin hanyoyin nazarin glycome kuma tana faɗaɗa aikace-aikacenta zuwa ga nau'ikan substrates daban-daban fiye da oligosaccharides na shuka. A nan gaba, za mu iya amfani da robotics don biotinylation kuma mu yi amfani da hanyar da muka ƙirƙiro don nazarin samfura masu yawa ta amfani da ELISA.
An girbe bambaro na masara (CS) da aka noma daga iri na Pioneer 33A14 a shekarar 2010 daga Kramer Farms da ke Ray, Colorado. Da izinin mai gona, ana iya amfani da wannan biomass don bincike. An adana samfuran a busasshe ƙasa da danshi na ƙasa da kashi 6% a cikin jakunkunan kulle zip a zafin ɗaki. An adana samfuran a busasshe ƙasa da danshi na ƙasa da kashi 6% a cikin jakunkunan kulle zip a zafin ɗaki. Образцы хранились сухими при влажности <6% в пакетах с застежкой-молнией при комнатной температуре. An adana samfuran a busasshe a yanayin zafi sama da kashi 6% a cikin jakunkuna masu zipper a zafin ɗaki.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 samfuran a cikin jakunkunan zip a zafin ɗaki tare da ɗan zafi ƙasa da 6%.Binciken ya bi ƙa'idodin gida da na ƙasa. An gudanar da nazarin abubuwan da aka haɗa ta amfani da ƙa'idar NREL. An gano cewa abubuwan da aka haɗa sun ƙunshi 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% furotin da 13.4% toka.
Cellic® CTec2 (138 mg furotin/ml, VCNI 0001) cakuda ce mai rikitarwa ta cellulase, β-glucosidase da Cellic® HTec2 (157 mg furotin/ml, lot VHN00001) daga Novozymes (Franklinton, NC, Amurka)). DuPont Industrial Biosciences (Palo Alto, CA, Amurka) ne ya bayar da gudummawar Multifect Pectinase® (72 mg furotin/mL), wani hadadden hadadden enzymes masu lalata pectin. An tantance yawan furotin ta hanyar kimanta adadin furotin (da kuma rage gudummawar nitrogen mara furotin) ta amfani da nazarin nitrogen na Kjeldahl (hanyar AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, Amurka). An sayi ƙasa mai suna Diatomaceous 545 daga EMD Millipore (Billerica, MA). An sayi sinadarin carbon da aka kunna (DARCO, granules 100 na raga), Avicel (PH-101), beech xylan, da duk wasu sinadarai daga Sigma-Aldrich (St. Louis, MO).
An yi maganin AFEX kafin a yi amfani da shi a GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, Amurka). An yi maganin kafin a yi amfani da shi a zafin jiki na 140°C na tsawon mintuna 15. Lokacin zama 46 a rabon ammonia mai hana ruwa zuwa biomass 1:1 a cikin nauyin 60% (w/w) a cikin wani injin ƙarfe mai amfani da ƙarfe (Parr Instruments Company). Ya ɗauki mintuna 30. An kawo na'urar zuwa 140°C kuma an saki ammonia cikin sauri, wanda ya ba da damar biomass ya koma zafin ɗaki da sauri. Abun da ke cikin na'urar murhu ta masara da aka riga aka yi amfani da ita (ACS) yayi kama da na na'urar murhu ta masara da ba a yi amfani da ita ba (UT-CS).
An shirya babban sinadarin ACSH 25% (w/w) (kusan kashi 8% na nauyin dextran) a matsayin kayan farawa don samar da oligosaccharides mai yawa. An yi amfani da cakuda enzyme na enzymatic hydrolysis na ACS ta amfani da cakuda enzyme na kasuwanci wanda ya haɗa da Cellic® Ctec2 10 mg protein/g glucan (a cikin biomass da aka riga aka yi wa magani), Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan, da Multifect Pectinase (Genencor Inc, Amurka). ), 5 mg protein/g dextran. An gudanar da hydrolysis na enzymatic a cikin bioreactor mai lita 5 tare da girman aiki na lita 3, pH 4.8, 50°C da 250 rpm. Bayan hydrolysis na tsawon awanni 96, an tattara hydrolyzate ta hanyar centrifugation a 6000 rpm na tsawon mintuna 30 sannan a 14000 rpm na tsawon mintuna 30 don cire daskararrun da ba a hydrolyzed ba. Daga nan aka yi wa hydrolyzate tacewa ta hanyar amfani da beaker mai girman 0.22 mm. An adana hydrolyzate ɗin da aka tace a cikin kwalaben da ba a tace ba a zafin digiri 4 na Celsius sannan aka raba shi da carbon.
Binciken abubuwan da ke cikin samfuran biomass da aka samo daga tushen cirewa bisa ga hanyoyin nazarin dakin gwaje-gwaje na NREL: shirya samfuran don nazarin abubuwan da ke ciki (NREL/TP-510-42620) da kuma tantance carbohydrates masu tsari da lignin a cikin biomass (NREL/TP-510 - 42618)47.
An gudanar da binciken oligosaccharide na kwararar hydrolyzate akan sikelin 2 ml ta amfani da hanyar hydrolysis ta acid mai tushen autoclave. A haɗa samfurin hydrolyzate da 69.7 µl na 72% sulfuric acid a cikin bututun al'ada na murfin sukurori na 10 ml sannan a saka a cikin kwano na tsawon awa 1 a kan benci a 121 °C, a sanyaya a kan kankara sannan a tace a cikin kwalbar chromatography mai aiki (HPLC). An ƙayyade yawan oligosaccharides ta hanyar cire yawan monosaccharides a cikin samfurin da ba a hydrolyzed ba daga jimlar yawan sukari a cikin samfurin da aka hydrolyzed acid.
An yi nazarin yawan glucose, xylose, da arabinose a cikin biomass ɗin da aka yi wa acid hydrolyzed ta amfani da tsarin Shimadzu HPLC wanda aka sanye shi da autosampler, hita na shafi, famfon isocratic, da na'urar gano firikwensin haske a kan ginshiƙin Bio-Rad Aminex HPX-87H. An kiyaye ginshiƙin a 50°C kuma an cire shi da ruwa mai gudana 0.6 ml/min 5 mM H2SO4.
An narkar da sinadarin hydrolyzate supernatant kuma an yi nazari kan abubuwan da ke cikin monomer da oligosaccharide. An yi nazarin sukari mai kama da monomeric da aka samu bayan an samar da sinadarin enzymatic hydrolysis ta hanyar HPLC wanda aka sanye da Bio-Rad (Hercules, CA) Aminex HPX-87P ginshiƙi da ginshiƙi mai kariya daga toka. An kiyaye zafin ginshiƙi a 80°C, an yi amfani da ruwa a matsayin matakin motsi tare da saurin kwarara na 0.6 ml/min. An tantance oligosaccharides ta hanyar hydrolysis a cikin acid mai narkewa a 121°C bisa ga hanyoyin da aka bayyana a cikin refs. 41, 48, 49.
An gudanar da binciken Saccharide akan ragowar biomass na ɗanyen AFEX, waɗanda aka riga aka yi musu magani da kuma duk ragowar biomass ɗin da ba a yi musu hydrolyze ba (gami da samar da cirewar bangon tantanin halitta da kuma tantance mAb ɗinsu) ta amfani da hanyoyin da aka bayyana a baya 27, 43, 50, 51. Don nazarin glycome, ana shirya ragowar bangon tantanin halitta marasa narkewar barasa daga ragowar biomass kuma ana yin su ne da cirewa ta hanyar amfani da sinadarai masu ƙarfi 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. Daga nan aka yi wa cirewar ELISA a kan wani tsari mai rikitarwa na mAb50s da aka kai wa glycan bangon tantanin halitta, kuma an gabatar da halayen ɗaure mAb a matsayin taswirar zafi. An sayi mAbs da ke niyya ga glycan bangon tantanin halitta daga hannun jari na dakin gwaje-gwaje (CCRC, JIM da MAC jerin).
Biotinylation na oligosaccharides mataki ɗaya. An yi haɗa carbohydrates tare da biotin-LC-hydrazide ta amfani da wannan hanya. An narkar da Biotin-LC-hydrazide (4.6 mg/12 μmol) a cikin dimethyl sulfoxide (DMSO, 70 μl) ta hanyar juyawa da ƙarfi da dumama a 65°C. na tsawon minti 1. 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 ɗaya bayan an dumama a 65°C. na kimanin minti 1. Sannan, daga 5 zuwa 8 μl na cakuda amsawar an ƙara shi a cikin busasshen oligosaccharide (1-100 nmol) don samun ƙarin molar na lakabin ninki 10 ko fiye a ƙarshen da aka rage. An gudanar da amsawar a 65°C na tsawon awanni 2, bayan haka an tsarkake samfuran nan da nan. Ba a yi amfani da sinadarin sodium cyanoborohydride a gwaje-gwajen lakabi ba tare da rage yawan sinadarin ba, kuma an yi amfani da samfuran a zafin jiki na 65°C na tsawon awanni 2.5.
Rufin ELISA da wanke samfuran oligosaccharides masu biotinylated. An ƙara μl 25 na samfuran biotinylated (100 μl na kowane samfurin da aka tattara a cikin 5 ml na maganin Tris buffer 0.1 M (TBS)) a kowace rijiya ta farantin da aka rufe da avidin. An shafa rijiyoyin sarrafawa da 50 μl na biotin a cikin yawan 10 μg/ml a cikin 0.1 M TBS. An yi amfani da ruwan da aka cire ionized a matsayin shafi don aunawa mara komai. An saka kwamfutar a cikin daki na tsawon awanni 2 a zafin ɗaki a cikin duhu. A wanke farantin sau 3 da madarar da aka cire 0.1% a cikin 0.1 M TBS ta amfani da shirin lamba 11 don Grenier flat 3A.
Ƙara da wanke ƙwayoyin rigakafi na farko. Ƙara 40 µl na ƙwayoyin rigakafi na farko a kowace rijiya. A saka ƙaramin farantin na tsawon awa 1 a zafin ɗaki a cikin duhu. Sannan an wanke faranti sau 3 da madara 0.1% a cikin 0.1M TBS ta amfani da shirin wankewa #11 don Grenier Flat 3A.
Sai a zuba maganin rigakafi na biyu sannan a wanke. A zuba maganin rigakafi na 50 µl na maganin rigakafi na beraye/bera (wanda aka narkar da shi 1:5000 a cikin madara 0.1% a cikin 0.1 M TBS) a kowace rijiya. A zuba maganin rigakafi na beraye na tsawon awa 1 a zafin ɗaki a cikin duhu. Sannan a wanke maganin rigakafi sau 5 da madara 0.1% a cikin 0.1 M TBS ta amfani da shirin wanke farantin Grenier Flat 5A #12.
Ƙara wani abu mai kama da ƙasa. Ƙara 50 µl na 3,3′, 5,5′-tetramethylbenzidine (TMB) a cikin tushen ƙasa (ta hanyar ƙara ɗigo 2 na buffer, ɗigo 3 na TMB, ɗigo 2 na hydrogen peroxide a cikin 15 ml na ruwan da aka cire daga ion). Shirya TMB substrate. kuma girgiza kafin amfani). A saka ƙaramin farantin a zafin ɗaki na minti 30. A cikin duhu.
Kammala matakin kuma ka karanta kwamfutar. Ƙara 50 µl na 1 N sulfuric acid a kowace rijiya sannan ka rubuta yawan shan ruwa daga 450 zuwa 655 nm ta amfani da na'urar karanta ELISA.
Shirya maganin 1 mg/ml na waɗannan masu nazarin a cikin ruwan da aka cire daga ion: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine. (glcNAc), N-acetylgalactosamine (galNAc), inositol (ma'aunin ciki). An shirya ma'auni biyu ta hanyar ƙara maganin sukari 1 mg/mL da aka nuna a cikin Jadawali na 1. Ana daskare samfuran kuma ana shafa su a cikin -80°C. har sai an cire dukkan ruwa (yawanci kimanin awanni 12-18).
A ƙara 100–500 µg na samfurin don a murƙushe bututun murfin a kan ma'aunin nazari. A rubuta adadin da aka ƙara. Zai fi kyau a narkar da samfurin a cikin takamaiman adadin ruwan da ke narkewa sannan a ƙara shi a cikin bututun a matsayin wani abu na ruwa. Yi amfani da 20 µl na 1 mg/ml inositol a matsayin ma'aunin ciki ga kowane bututun samfurin. Adadin ma'aunin ciki da aka ƙara wa samfurin dole ne ya zama iri ɗaya da adadin ma'aunin ciki da aka ƙara wa bututun da aka saba.
Sai a zuba 8 ml na methanol mai ruwa a cikin kwalbar murfi. Sai a zuba 4 ml na maganin HCl na N. methanol 3, a rufe a girgiza. Wannan tsari ba ya amfani da ruwa.
Ƙara 500 µl na maganin methanol na HCl 1 M a cikin samfuran oligosaccharide da bututun TMS na yau da kullun. An saka samfuran a cikin firiji na dare ɗaya (awanni 168) a zafin jiki na 80°C. a cikin toshe mai zafi. Busar da samfurin methanolysis a zafin ɗaki ta amfani da man shafawa mai bushewa. Ƙara 200 µl MeOH kuma 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 a gauraya sosai. A saka samfuran a zafin ɗaki na minti 30 sannan a busar. Ƙara 200 µl na methanol sannan a sake busar da su.
Sai a zuba 200 µl na Tri-Sil sannan a dafa bututun da aka rufe na tsawon minti 20. 80°C, sannan a sanyaya zuwa zafin ɗaki. Yi amfani da injin busarwa don ƙara busar da samfurin zuwa girman kusan µl 50. Yana da mahimmanci a lura cewa ba mu bar samfuran su bushe gaba ɗaya ba.
Sai a zuba 2 ml na hexane a gauraya sosai ta hanyar yin vortexing. A cika ƙarshen Pasteur pipettes (5-8 mm) da wani yanki na ulu na gilashi ta hanyar saka ulu na gilashi a saman pipette mai diamita inci 5-3/4. An saka samfuran a centrifuge a 3000 g na tsawon minti 2. Duk wani ragowar da ba ya narkewa za a haƙa shi. A busar da samfurin zuwa 100-150 µl. An saka kimanin μl 1 a cikin GC-MS a zafin farko na 80 °C da kuma lokacin farko na minti 2.0 (Tebur 2).