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Njira zatsopano zowunikira chitetezo chamthupi komanso kuchuluka kwa ma spectrometric pofufuza zovuta za oligosaccharides zomwe zimapitilira mu chimanga chophimbidwa kale ndi AFEX. Lignocellulosic biomass ndi njira ina yokhazikika m'malo mwa mafuta osungiramo zinthu zakale ndipo imagwiritsidwa ntchito kwambiri popanga ma biotechnology popanga zinthu monga chakudya, chakudya, mafuta ndi mankhwala. Chinsinsi cha ukadaulo uwu ndi kupanga njira zopikisana mtengo zosinthira chakudya chovuta chomwe chili m'makoma a maselo a zomera kukhala shuga wosavuta monga shuga, xylose ndi arabinose. Chifukwa lignocellulosic biomass ndi yovuta kwambiri, iyenera kuthandizidwa ndi thermochemical (monga, ammonia fiber exfoliation (AFEX), dilute acids (DA), ionic liquids (IL)) ndi mankhwala achilengedwe (monga enzymatic hydrolysis ndi microbial fermentation) pamodzi kuti mupeze chinthu chomwe mukufuna. Komabe, pamene ma enzyme a bowa amalonda akugwiritsidwa ntchito mu njira ya hydrolysis, 75-85% yokha ya shuga wosungunuka wopangidwa ndi monosaccharides, ndipo 15-25% yotsalayo ndi ma oligosaccharides osungunuka, osasinthika, omwe nthawi zonse sapezeka kwa tizilombo toyambitsa matenda. Kale, tinatha kusiyanitsa ndi kuyeretsa ma oligosaccharides osungunuka pogwiritsa ntchito kuphatikiza kwa carbon ndi diatomaceous earth separation ndi size exclusion chromatography, komanso kufufuza za mphamvu zawo zoletsa ma enzyme. Tapeza kuti ma oligosaccharides okhala ndi digiri yapamwamba ya polymerization (DP) methylated uronic acid substitutions ndi ovuta kwambiri kuwagwiritsa ntchito ndi ma enzyme blends amalonda kuposa ma DP otsika ndi ma oligosaccharides osalowerera. Pano tikupereka lipoti la kugwiritsa ntchito njira zingapo zowonjezera, kuphatikizapo glycan profiling pogwiritsa ntchito ma monoclonal antibodies (mAbs) apadera kwa plant biomass glycans kuti azindikire ma glycan bonds m'makoma a maselo a zomera ndi ma enzymatic hydrolysates, matrix-assisted laser desorption ionization, time-of-flight mass-spectrometry. MALDI-TOF-MS) amagwiritsa ntchito njira yodziwira kapangidwe kake ka zinthu zomwe zimapezeka pogwiritsa ntchito spectroscopy pambuyo pa kuwonongeka kwachiwiri kwa ma ion oipa, gas chromatography ndi mass spectrometry (GC-MS) kuti azindikire ma bond a oligosaccharide okhala ndi komanso opanda derivatization. Chifukwa cha kukula kochepa kwa oligosaccharides (DP 4-20), mamolekyu awa ndi ovuta kugwiritsa ntchito pomanga ndi kusiyanitsa ma mAb. Kuti tithetse vutoli, tinagwiritsa ntchito njira yatsopano yolumikizira oligosaccharide yochokera ku biotin yomwe inalemba bwino ma oligosaccharides ambiri otsika a DP pamwamba pa microplate, omwe kenako adagwiritsidwa ntchito mu high throughput mAb system kuti ayese kuyika ma ligation. Njira yatsopanoyi ithandiza pakupanga ma high throughput glycome assays mtsogolo omwe angagwiritsidwe ntchito kudzipatula ndikuwonetsa ma oligosaccharides omwe ali mu biomarkers kuti azindikire.
Lignocellulosic biomass, yopangidwa ndi ulimi, nkhalango, udzu ndi zinthu zamatabwa, ndi chakudya chomwe chingagwiritsidwe ntchito popanga zinthu zopangidwa ndi zamoyo, kuphatikizapo chakudya, chakudya, mafuta ndi mankhwala opangira zinthu kuti apange zinthu zamtengo wapatali1. Chakudya (monga cellulose ndi hemicellulose) chomwe chili m'makoma a maselo a zomera chimachotsedwa mu ma monosaccharides mwa kukonza mankhwala ndi kusintha kwa biotransformation (monga enzymatic hydrolysis ndi microbial fermentation). Mankhwala ofala kwambiri a pre-treatment ndi monga ammonia fiber expansion (AFEX), dilute acid (DA), ionic liquid (IL), ndi steam explosion (SE), zomwe zimagwiritsa ntchito kuphatikiza kwa mankhwala ndi kutentha kuti zichepetse kupanga lignocellulose potsegula makoma a maselo a zomera3,4. kuuma kwa zinthu, 5. Enzymatic hydrolysis imachitika pamlingo waukulu wa solids pogwiritsa ntchito ma enzymes okhala ndi carbohydrate (CAZymes) ndi microbial fermentation pogwiritsa ntchito yisiti ya transgenic kapena mabakiteriya kuti apange mafuta ndi mankhwala opangidwa ndi zamoyo6.
Ma CAZyme mu ma enzyme amalonda amapangidwa ndi kusakaniza kovuta kwa ma enzyme omwe amagawaniza ma carbohydrate-shuga bonds kuti apange ma monosaccharides2,7. Monga tidanenera kale, maukonde ovuta a ma polima a aromatic a lignin ndi ma carbohydrate amawapangitsa kukhala osasinthika kwambiri, zomwe zimapangitsa kuti shuga isasinthe mokwanira, zomwe zimapangitsa kuti 15-25% ya oligosaccharides achiwerewere omwe sapangidwa panthawi ya hydrolysis ya enzymatic biomass yokonzedwa kale. Ili ndi vuto lofala ndi njira zosiyanasiyana zochizira biomass. Zifukwa zina za vutoli ndi monga kuletsa ma enzyme panthawi ya hydrolysis, kapena kusowa kapena kuchepa kwa ma enzyme ofunikira omwe amafunika kuswa ma shuga bonds mu zomera. Kumvetsetsa kapangidwe ndi mawonekedwe a shuga, monga ma shuga bonds mu oligosaccharides, kudzatithandiza kusintha kusintha kwa shuga panthawi ya hydrolysis, zomwe zimapangitsa kuti njira za biotechnology zikhale zokwera mtengo ndi zinthu zochokera ku mafuta.
Kudziwa kapangidwe ka chakudya cham'thupi n'kovuta ndipo kumafuna njira zosiyanasiyana monga madzi 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 yowuluka mass spectrometry yokhala ndi laser desorption ndi ionization pogwiritsa ntchito matrix (MALDI-TOF-MS) ndi njira yodziwika bwino yodziwira kapangidwe ka chakudya cham'thupi. Posachedwapa, Collision-Induced Dissociation (CID) tandem MS ya sodium ion adducts yagwiritsidwa ntchito kwambiri kuzindikira zala zogwirizana ndi malo olumikizirana a oligosaccharide, mawonekedwe a anomeric, ma sequences, ndi malo olumikizirana 20, 21.
Kusanthula kwa Glycan ndi chida chabwino kwambiri chodziwira mozama ma carbohydrate bond22. Njirayi imagwiritsa ntchito ma antibodies a monoclonal (mAbs) omwe amalunjika ku glycan ya khoma la maselo a chomera ngati ma probe kuti amvetsetse kulumikizana kovuta kwa carbohydrate. Ma mAb opitilira 250 alipo padziko lonse lapansi, opangidwa motsutsana ndi oligosaccharides osiyanasiyana olunjika ndi nthambi pogwiritsa ntchito ma saccharides osiyanasiyana24. Ma mAb angapo agwiritsidwa ntchito kwambiri pofotokoza kapangidwe, kapangidwe, ndi kusintha kwa khoma la maselo a chomera, chifukwa pali kusiyana kwakukulu kutengera mtundu wa maselo a chomera, chiwalo, zaka, gawo la chitukuko, ndi malo okulira25,26. Posachedwapa, njira iyi yagwiritsidwa ntchito kumvetsetsa kuchuluka kwa ma vesicle m'machitidwe a zomera ndi nyama komanso ntchito zawo pakunyamula ma glycan monga momwe zimakhalira ndi zizindikiro za subcellular, magawo a chitukuko, kapena zoyambitsa zachilengedwe, komanso kudziwa ntchito ya enzymatic. Zina mwa kapangidwe kosiyana ka glycans ndi xylan zomwe zapezeka ndi monga pectin (P), xylan (X), mannan (M), xyloglucans (XylG), mixed bond glucans (MLG), arabinoxylan (ArbX), galactomannan (GalG), glucuronic acid-arabinoxylan (GArbX) ndi arabino-galactan (ArbG)29.
Komabe, ngakhale kuti kafukufukuyu wachitika motere, kafukufuku wochepa chabe ndi amene wayang'ana kwambiri za kuchuluka kwa oligosaccharide panthawi ya hydrolysis yamphamvu kwambiri (HSL), kuphatikizapo kutulutsidwa kwa oligosaccharide, kusintha kwa unyolo wa oligomeric panthawi ya hydrolysis, ma polima osiyanasiyana otsika a DP, ndi ma curve awo. 30,31,32. Pakadali pano, ngakhale kuti kusanthula kwa glycan kwakhala chida chothandiza pakusanthula kwathunthu kapangidwe ka glycan, n'kovuta kuwunika ma oligosaccharide otsika a DP osungunuka m'madzi pogwiritsa ntchito njira za antibody. Ma oligosaccharide ang'onoang'ono a DP okhala ndi kulemera kwa molekyulu kosakwana 5-10 kDa samalumikizana ndi mbale za ELISA 33, 34 ndipo amatsukidwa asanawonjezere ma antibody.
Pano, koyamba, tikuwonetsa mayeso a ELISA pa mbale zophimbidwa ndi avidin pogwiritsa ntchito ma antibodies a monoclonal, kuphatikiza njira imodzi yopangira biotinylation ya ma oligosaccharide osungunuka ndi kusanthula kwa glycome. Njira yathu yowunikira glycome idatsimikiziridwa ndi kusanthula kochokera ku MALDI-TOF-MS ndi GC-MS kwa maulumikizidwe owonjezera a oligosaccharide pogwiritsa ntchito trimethylsilyl (TMS) derivatization ya hydrolyzed sugar compositions. Njira yatsopanoyi ikhoza kupangidwa ngati njira yapamwamba kwambiri mtsogolo ndikupeza kugwiritsidwa ntchito kwakukulu mu kafukufuku wa zamankhwala35.
Kusintha kwa ma enzyme ndi ma antibodies pambuyo pomasulira, monga glycosylation,36 kumakhudza ntchito yawo yachilengedwe. Mwachitsanzo, kusintha kwa glycosylation ya mapuloteni a seramu kumachita gawo lofunika kwambiri pa kutupa kwa nyamakazi, ndipo kusintha kwa glycosylation kumagwiritsidwa ntchito ngati zizindikiro zodziwira matenda37. Ma glycans osiyanasiyana anenedwa m'mabuku kuti amapezeka mosavuta m'matenda osiyanasiyana, kuphatikizapo matenda osatha otupa am'mimba ndi chiwindi, matenda opatsirana ndi mavairasi, khansa ya m'chiberekero, ya m'mawere, ndi ya prostate38,39,40. Kumvetsetsa kapangidwe ka ma glycans pogwiritsa ntchito njira za glycan ELISA zochokera ku ma antibodies kumapereka chidaliro chowonjezera pakuzindikira matenda popanda kugwiritsa ntchito njira zovuta za MS.
Kafukufuku wathu wakale adawonetsa kuti ma oligosaccharide osasunthika adakhalabe osasungunuka pambuyo pochiza ndi enzyme hydrolysis (Chithunzi 1). Mu ntchito yathu yomwe idasindikizidwa kale, tidapanga njira yotulutsira ma charcoal solid-phase yogwira ntchito kuti tichotse ma oligosaccharide kuchokera ku AFEX-pretreated corn stover hydrolyzate (ACSH)8. Pambuyo pochotsa ndi kulekanitsa koyamba, ma oligosaccharide adagawidwanso m'magulu pogwiritsa ntchito kukula kwa exclusion chromatography (SEC) ndikusonkhanitsidwa motsatira kulemera kwa mamolekyulu. Ma monomers a shuga ndi ma oligomers omwe adatulutsidwa kuchokera ku pretreatments osiyanasiyana adawunikidwa pogwiritsa ntchito kusanthula kwa kapangidwe ka shuga. Poyerekeza zomwe zili mu ma oligomers a shuga zomwe zimapezeka pogwiritsa ntchito njira zosiyanasiyana pretreatment, kupezeka kwa ma oligosaccharide osasunthika ndi vuto lofala pakusintha kwa biomass kukhala ma monosaccharide ndipo kungayambitse kuchepa kwa zokolola za shuga ndi osachepera 10-15% komanso mpaka 18%. US. Njirayi imagwiritsidwa ntchito popanga ma oligosaccharide fractions ambiri. Mu ntchito iyi, ACH ndi magawo ake otsatira omwe ali ndi kulemera kosiyanasiyana kwa mamolekyulu adagwiritsidwa ntchito ngati zinthu zoyesera pofotokozera ma oligosaccharides.
Pambuyo pochiza ndi hydrolysis ya enzymatic, ma oligosaccharide okhazikika anakhalabe osathiridwa madzi. Apa (A) pali njira yolekanitsira oligosaccharide momwe ma oligosaccharide amapatulidwa kuchokera ku AFEX-pretreated corn stover hydrolysate (ACSH) pogwiritsa ntchito bedi lodzaza la activated carbon ndi diatomaceous earth; (B) Njira yolekanitsira ma oligosaccharide. Ma oligosaccharide adapatulidwanso ndi kukula kwa chromatography (SEC); (C) Ma monomers a Saccharide ndi ma oligomer omwe adatulutsidwa kuchokera ku mitundu yosiyanasiyana ya pretreatments (diluted acid: DA, ionic liquid: IL ndi AFEX). Mikhalidwe ya hydrolysis ya enzymatic: kuchuluka kwa zinthu zolimba zomwe zimayikidwa mu 25% (w/w) (pafupifupi 8% ya glucan yomwe imayikidwa), hydrolysis ya maola 96, 20 mg/g ya enzyme yogulitsa (Ctec2:Htec2:MP-2:1:1 ratio) ndi (D) Ma monomers a shuga ndi oligomers a glucose, xylose ndi arabinose omwe amatulutsidwa kuchokera ku AFEX pre-treated corn stover (ACS).
Kusanthula kwa Glycan kwakhala chida chothandiza pakusanthula kwathunthu kapangidwe ka glycans mu zotulutsa zomwe zachotsedwa ku zotsalira zolimba za biomass. Komabe, ma saccharides osungunuka m'madzi sakufotokozedwa mokwanira pogwiritsa ntchito njira yachikhalidwe iyi41 chifukwa ma oligosaccharides otsika a molekyulu ndi ovuta kuletsa kuyenda pa mbale za ELISA ndipo amatsukidwa asanawonjezere ma antibody. Chifukwa chake, pakumanga ndi kuwonetsa ma antibody, njira imodzi ya biotinylation idagwiritsidwa ntchito kuphimba ma oligosaccharides osungunuka, osagwirizana ndi ma mbale a ELISA okhala ndi avidin. Njirayi idayesedwa pogwiritsa ntchito ACSH yomwe idapangidwa kale komanso gawo lochokera pa kulemera kwake kwa molekyulu (kapena digiri ya polymerization, DP). Biotinylation imodzi idagwiritsidwa ntchito kukulitsa oligosaccharide binding affinity powonjezera biotin-LC-hydrazide kumapeto kwa carbohydrate (Chithunzi 2). Poyankha, gulu la hemiacetal kumapeto kochepetsera limayankha ndi gulu la hydrazide la biotin-LC-hydrazide kuti lipange hydrazone bond. Pamaso pa NaCNBH3 yochepetsera, chomangira cha hydrazone chimachepetsedwa kukhala chinthu chokhazikika cha biotinylated. Ndi kusintha kwa chomangira chochepetsera shuga, kumangirira kwa oligosaccharides otsika a DP ku mbale za ELISA kunakhala kotheka, ndipo mu kafukufuku wathu izi zidachitika pa mbale zophimbidwa ndi avidin pogwiritsa ntchito ma Abs olunjika a glycan.
Kuwunika ma antibodies a monoclonal kutengera ELISA kuti mupeze ma oligosaccharide opangidwa ndi biotinylated. Apa (A) kuphatikiza biotinylation ya ma oligosaccharide ndi kuwunika kwa ELISA pambuyo pake ndi ma mAbs olunjika a glycan pa mbale zophimbidwa ndi NeutrAvidin ndipo (B) kukuwonetsa njira imodzi yokha yopangira biotinylation ya zinthu zomwe zimayambitsa matendawa.
Ma plates ophimbidwa ndi Avidin okhala ndi ma antibodies ogwirizana ndi oligosaccharide adawonjezedwa ku ma antibodies oyambira ndi achiwiri ndikutsukidwa mu chipangizo chopepuka komanso chosavuta kugwiritsa ntchito nthawi. Pambuyo pomaliza kumanga ma antibodies, onjezani TMB substrate kuti muyike mbaleyo. Pomaliza pake idayimitsidwa ndi sulfuric acid. Ma plates ophimbidwa adawunikidwa pogwiritsa ntchito ELISA reader kuti adziwe mphamvu yomangirira ya antibody iliyonse kuti azindikire kulumikizana kwa antibody. Kuti mudziwe zambiri ndi magawo a kuyesera, onani gawo lofananalo "Zida ndi Njira".
Tikuwonetsa kufunika kwa njira yatsopanoyi yopangidwira ntchito zinazake mwa kufotokoza ma oligosaccharides osungunuka omwe ali mu ACSH komanso m'magawo a oligosaccharide osaphikidwa omwe amachokera ku lignocellulosic hydrolysates. Monga momwe zasonyezedwera mu Chithunzi 3, ma xylans odziwika kwambiri omwe amapezeka mu ACSH pogwiritsa ntchito njira za bioacylated glycome assay nthawi zambiri amakhala uronic (U) kapena methyluronic (MeU) ndi pectic arabinogalactans. Ambiri mwa iwo adapezekanso mu kafukufuku wathu wakale wokhudza kusanthula kwa glycans ya non-hydrolyzed solids (UHS)43.
Kuzindikira ma epitopes a recalcitrant oligosaccharide pogwiritsa ntchito monoclonal antibody yolunjika ku cell wall glycan. Gawo la "neutral" ndi gawo la ACN ndipo gawo la "acidic" ndi gawo la FA. Ma red owala kwambiri pa heatmap amasonyeza kuchuluka kwa epitope, ndipo buluu owala kwambiri amasonyeza maziko opanda kanthu. Ma color values ​​​​pa sikelo amachokera ku ma OD values ​​​​atsopano a formulations N=2. Ma epitopes akuluakulu omwe amadziwika ndi ma antibodies akuwonetsedwa kumanja.
Mapangidwe osakhala a cellulose awa sakanatha kugawikana ndi ma cellulase ndi ma hemicellulase odziwika bwino omwe ali mu enzyme yogulitsa yoyesedwa, yomwe imaphatikizapo ma enzymes ogulitsa omwe amagwiritsidwa ntchito kwambiri. Chifukwa chake, ma enzymes atsopano othandizira amafunikira kuti hydrolysis yawo ichitike. Popanda ma enzymes ofunikira osakhala a cellulose, ma bond awa osakhala a cellulose amalepheretsa kusintha kwathunthu kukhala ma monosaccharides, ngakhale ma polima a shuga omwe amawayambitsa amasinthidwa kwambiri kukhala zidutswa zazifupi ndikusungunuka pogwiritsa ntchito zosakaniza za ma enzyme ogulitsa.
Kafukufuku wowonjezereka wa kufalikira kwa zizindikiro ndi mphamvu yake yomangirira wasonyeza kuti ma epitopes omangira anali otsika m'magawo a shuga a DP ambiri (A, B, C, DP mpaka 20+) kuposa m'magawo a DP ochepa (D, E, F, DP) mu ma dimers) (Chithunzi 1). Zidutswa za asidi zimapezeka kwambiri m'ma epitopes osakhala a cellulose kuposa zidutswa zopanda mbali. Zochitikazi zikugwirizana ndi momwe zidawonedwera mu kafukufuku wathu wakale, komwe ma DP ambiri ndi ma acid moieties anali otsutsana kwambiri ndi enzyme hydrolysis. Chifukwa chake, kupezeka kwa ma epitopes osakhala a cellulose glycan ndi ma substitutions a U ndi MeU kungathandize kwambiri kukhazikika kwa ma oligosaccharides. Tiyenera kudziwa kuti kugwira ntchito bwino kwa ma epitopes ndi kuzindikira kungakhale kovuta kwa ma oligosaccharides otsika a DP, makamaka ngati epitope ndi dimeric kapena trimeric oligosaccharide. Izi zitha kuyesedwa pogwiritsa ntchito ma oligosaccharides ogulitsa akutalika kosiyana, iliyonse ili ndi epitope imodzi yokha yomwe imalumikizana ndi mAb inayake.
Motero, kugwiritsa ntchito ma antibodies enieni a kapangidwe kake kunavumbula mitundu ina ya ma recalcitrant bonds. Kutengera mtundu wa antibody yomwe imagwiritsidwa ntchito, mawonekedwe oyenera a ligation, ndi mphamvu ya chizindikiro chomwe chimapanga (chambiri komanso chocheperako), ma enzyme atsopano amatha kuzindikirika ndikuwonjezedwa pang'ono ku chisakanizo cha enzyme kuti glycoconversion ikhale yokwanira. Potengera kusanthula kwa ACSH oligosaccharides mwachitsanzo, titha kupanga database ya ma glycan bonds pa chinthu chilichonse cha biomass. Tiyenera kudziwa apa kuti kuyanjana kosiyana kwa ma antibodies kuyenera kuganiziridwa, ndipo ngati kuyanjana kwawo sikudziwika, izi zipanga zovuta zina poyerekeza zizindikiro za ma antibodies osiyanasiyana. Kuphatikiza apo, kufananiza ma glycan bonds kungagwire ntchito bwino pakati pa zitsanzo za antibody yomweyo. Ma bond osasunthika awa amatha kulumikizidwa ku database ya CAZyme, komwe tingathe kuzindikira ma enzyme, kusankha ma enzyme oyenera ndikuyesa ma enzyme osweka, kapena kupanga ma microbial systems kuti afotokoze ma enzyme awa kuti agwiritsidwe ntchito mu biorefineries44.
Kuti tiwone momwe njira zodzitetezera thupi zimathandizira njira zina zodziwira oligosaccharides otsika a molekyulu omwe ali mu lignocellulosic hydrolysates, tinachita MALDI (Chithunzi 4, S1-S8) ndi kusanthula kwa ma saccharides ochokera ku TMS kutengera GC-MS pagawo lomwelo (Chithunzi 5) la oligosaccharide. MALDI imagwiritsidwa ntchito kuyerekeza ngati kufalikira kwa mamolekyulu a oligosaccharide kukugwirizana ndi kapangidwe kake. Pa chithunzi 4 chikuwonetsa MC ya zigawo zosalowerera za ACN-A ndi ACN-B. Kusanthula kwa ACN-A kunatsimikizira mitundu yosiyanasiyana ya shuga wa pentose kuyambira DP 4–8 (Chithunzi 4) mpaka DP 22 (Chithunzi S1), yomwe kulemera kwake kumafanana ndi MeU-xylan oligosaccharides. Kusanthula kwa ACN-B kunatsimikizira mndandanda wa pentose ndi glucoxylan ndi DP 8-15. Mu zinthu zowonjezera monga Chithunzi S3, mamapu ogawa a FA-C acidic moiety mass akuwonetsa mitundu yosiyanasiyana ya shuga wa pentose wosinthidwa (Me)U wokhala ndi DP ya 8-15 womwe umagwirizana ndi ma xylan osinthidwa omwe amapezeka mu ELISA-based mAb screening. Ma epitopes ndi ofanana.
MALDI-MS spectrum ya oligosaccharides yosasinthika yomwe ili mu ACS. Apa, (A) ACN-A low weight range fractions yokhala ndi methylated uronic acid (DP 4-8) inalowa m'malo mwa glucuroxylan oligosaccharides ndi (B) ACN-B xylan ndi methylated uronic acid oligosaccharides inalowa m'malo mwa glucuroxylan (DP 8-15).
Kusanthula kapangidwe ka glycan residue ya refractory oligosaccharides. Apa (A) Kapangidwe ka TMS saccharide ya magawo osiyanasiyana a oligosaccharide omwe apezeka pogwiritsa ntchito kusanthula kwa GC-MS. (B) Kapangidwe ka shuga wosiyanasiyana wochokera ku TMS womwe umapezeka mu oligosaccharides. ACN - gawo la acetonitrile lomwe lili ndi oligosaccharides osalowererapo ndi gawo la FA - ferulic acid lomwe lili ndi acid oligosaccharides.
Chotsatira china chosangalatsa chinachokera ku kusanthula kwa LC-MS kwa gawo la oligosaccharide, monga momwe zasonyezedwera pa Chithunzi S9 (njira zitha kuwoneka muzinthu zowonjezera zamagetsi). Zidutswa za magulu a hexose ndi -OAc zidawonedwa mobwerezabwereza panthawi yolumikizana kwa gawo la ACN-B. Kupeza kumeneku sikungotsimikizira kugawikana komwe kumawoneka mu kusanthula kwa glycome ndi MALDI-TOF, komanso kumapereka chidziwitso chatsopano chokhudza zomwe zingayambitse matenda a carbohydrate mu lignocellulosic biomass yothandizidwa kale.
Tinasanthulanso kapangidwe ka shuga ka gawo la oligosaccharide pogwiritsa ntchito TMS shuga derivatization. Pogwiritsa ntchito GC-MS, tinapeza kapangidwe ka shuga wa neural (osachokera ku chinthu china) ndi acidic (GluA ndi GalA) mu gawo la oligosaccharide (Chithunzi 5). Glucuronic acid imapezeka mu zigawo za acidic C ndi D, pomwe galacturonic acid imapezeka mu zigawo za acidic A ndi B, zonse zomwe ndi zigawo za DP zambiri za shuga wa acidic. Zotsatirazi sizikungotsimikizira deta yathu ya ELISA ndi MALDI, komanso zikugwirizana ndi maphunziro athu am'mbuyomu okhudza kuchuluka kwa oligosaccharide. Chifukwa chake, tikukhulupirira kuti njira zamakono zodzitetezera pogwiritsa ntchito biotinylation ya oligosaccharides ndi ELISA yotsatira ndizokwanira kuzindikira oligosaccharides wosungunuka m'zitsanzo zosiyanasiyana zamoyo.
Popeza njira zowunikira za mAb zochokera ku ELISA zatsimikiziridwa ndi njira zingapo zosiyanasiyana, tinkafuna kufufuza kwambiri kuthekera kwa njira yatsopanoyi yowerengera. Ma oligosaccharides awiri ogulitsa, xylohexasaccharide oligosaccharide (XHE) ndi 23-α-L-arabinofuranosyl-xylotriose (A2XX), adagulidwa ndikuyesedwa pogwiritsa ntchito njira yatsopano ya mAb yolunjika ku cell wall glycan. Chithunzi 6 chikuwonetsa kulumikizana kwa mzere pakati pa chizindikiro chomangira cha biotinylated ndi kuchuluka kwa oligosaccharide, zomwe zikusonyeza kuti pali njira yoti Langmuir adsorption ichitike. Pakati pa mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, ndi CCRC-M151 yogwirizana ndi XHE, ndipo CCRC-M108, CCRC-M109, ndi LM11 yogwirizana ndi A2XX pamtundu wa 1 nm mpaka 100 nano. Chifukwa cha kupezeka kochepa kwa ma antibodies panthawi yoyesera, mayeso ochepa adachitika ndi kuchuluka kwa oligosaccharide iliyonse. Tiyenera kudziwa apa kuti ma antibodies ena amachita mosiyana kwambiri ndi oligosaccharide yomweyo ngati substrate, mwina chifukwa amalumikizana ndi ma epitopes osiyana pang'ono ndipo amatha kukhala ndi ma affinities osiyana kwambiri. Njira ndi zotsatira za kuzindikira epitope molondola zidzakhala zovuta kwambiri pamene njira yatsopano ya mAb ikugwiritsidwa ntchito pa zitsanzo zenizeni.
Ma oligosaccharide awiri amalonda adagwiritsidwa ntchito kudziwa kuchuluka kwa ma mAbs osiyanasiyana omwe amalimbana ndi glycan. Pano, kulumikizana kolunjika ndi kuchuluka kwa oligosaccharide komwe kumawonetsa mawonekedwe a Langmuir adsorption a (A) XHE ndi mAb ndi (B) A2XX ndi mAb. Ma epitopes ofanana akuwonetsa kapangidwe ka oligosaccharide amalonda omwe amagwiritsidwa ntchito ngati substrates mu assay.
Kugwiritsa ntchito ma antibodies a monoclonal omwe amalimbana ndi glycan (kusanthula kwa glycocomic kapena ELISA-based mAb screening) ndi chida champhamvu chofotokozera mozama ma glycan ambiri akuluakulu a makoma a maselo omwe amapanga biomass ya zomera. Komabe, kusanthula kwa glycan kwachikhalidwe kumangowonetsa ma glycan akuluakulu a makoma a maselo okha, chifukwa ma oligosaccharides ambiri sakhazikika bwino pama mbale a ELISA. Mu kafukufukuyu, AFEX-pretreated corn stover idasinthidwa ndi enzymatically hydrolyzed pamlingo wambiri wa solids. Kusanthula kwa shuga kunagwiritsidwa ntchito kudziwa kapangidwe ka ma carbohydrate a makoma a maselo osinthika mu hydrolyzate. Komabe, kusanthula kwa mAb kwa ma oligosaccharides ang'onoang'ono mu ma hydrolysates sikunaganiziridwe bwino, ndipo zida zina zimafunika kuti ma oligosaccharides azitha kuyenda bwino pama mbale a ELISA.
Pano tikulongosola njira yatsopano komanso yothandiza yochepetsera kusuntha kwa oligosaccharide poyesa mAb mwa kuphatikiza biotinylation ya oligosaccharide kutsatiridwa ndi kuyesa ELISA pa mbale zophimbidwa ndi NeutrAvidin™. Ma oligosaccharide osasuntha omwe ali ndi biotinylated adawonetsa kuyanjana kokwanira kwa antibody kuti athe kuzindikira mwachangu komanso moyenera ma oligosaccharides obwezeretsanso. Kusanthula kwa kapangidwe ka ma oligosaccharides osasunthika awa kutengera ma mass spectrometry kwatsimikizira zotsatira za njira yatsopanoyi yowunikira chitetezo chamthupi. Chifukwa chake, maphunzirowa akuwonetsa kuti kuphatikiza kwa oligosaccharide biotinylation ndi kuyesa ELISA ndi ma antibodies a monoclonal omwe ali ndi glycan kungagwiritsidwe ntchito kuzindikira maulalo olumikizana mu oligosaccharides ndipo kungagwiritsidwe ntchito kwambiri mu maphunziro ena a biochemical omwe amafotokozera kapangidwe ka oligosaccharides.
Njira iyi yopezera ma glycan yochokera ku biotin ndiyo lipoti loyamba lomwe lingathe kufufuza za ma carbohydrate bond osungunuka a oligosaccharides mu biomass ya zomera. Izi zimathandiza kumvetsetsa chifukwa chake magawo ena a biomass ndi olimba kwambiri pankhani yopanga biofuel. Njira iyi imadzaza mpata wofunikira mu njira zowunikira glycome ndipo imakulitsa kugwiritsidwa ntchito kwake kuzinthu zambiri kupatula oligosaccharides ya zomera. M'tsogolomu, tingagwiritse ntchito robotics pa biotinylation ndikugwiritsa ntchito njira yomwe tapanga pofufuza zitsanzo pogwiritsa ntchito ELISA.
Udzu wa chimanga (CS) womwe umalimidwa kuchokera ku mbewu zosakanikirana za Pioneer 33A14 unasonkhanitsidwa mu 2010 kuchokera ku Kramer Farms ku Ray, Colorado. Ndi chilolezo cha mwini malo, zomerazi zitha kugwiritsidwa ntchito pofufuza. Zitsanzozo zinasungidwa zouma <6% chinyezi m'matumba otsekedwa ndi zipu kutentha kwa chipinda. Zitsanzozo zinasungidwa zouma <6% chinyezi m'matumba otsekedwa ndi zipu kutentha kwa chipinda. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Zitsanzo zinasungidwa zouma pa chinyezi cha <6% m'matumba okhala ndi zipi kutentha kwa chipinda.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 chipinda ndi chinyezi <6%.Kafukufukuyu adatsatira malangizo am'deralo ndi a dziko lonse. Kusanthula kapangidwe kake kunachitika pogwiritsa ntchito njira ya NREL. Kapangidwe kake kanapezeka kuti kali ndi 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% protein ndi 13.4% phulusa.
Cellic® CTec2 (138 mg protein/ml, lot VCNI 0001) ndi chisakanizo chovuta cha cellulase, β-glucosidase ndi Cellic® HTec2 (157 mg protein/ml, lot VHN00001) kuchokera ku Novozymes (Franklinton, NC, USA)). Multifect Pectinase® (72 mg protein/mL), chisakanizo chovuta cha ma enzymes owononga pectin, chinaperekedwa ndi DuPont Industrial Biosciences (Palo Alto, CA, USA). Kuchuluka kwa mapuloteni a enzyme kunatsimikiziridwa poyesa kuchuluka kwa mapuloteni (ndi kuchotsa zomwe nayitrogeni wosakhala wa mapuloteni amapereka) pogwiritsa ntchito Kjeldahl nitrogen analysis (AOAC method 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA). Diatomaceous earth 545 inagulidwa ku EMD Millipore (Billerica, MA). Kaboni wopangidwa (DARCO, ma mesh granules 100), Avicel (PH-101), beech xylan, ndi mankhwala ena onse adagulidwa ku Sigma-Aldrich (St. Louis, MO).
Chithandizo cha AFEX chisanachitike chinachitikira ku GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). Chithandizo chisanachitike chinachitikira pa 140°C. kwa mphindi 15. Nthawi yokhazikika ya 46 pa chiŵerengero cha 1:1 cha ammonia ya anhydrous ndi biomass pa 60% (w/w) yodzaza mu reactor yachitsulo chosapanga dzimbiri (Parr Instruments Company). Zinatenga mphindi 30. Reactor inafika pa 140°C ndipo ammonia inatulutsidwa mwachangu, zomwe zinalola kuti biomass ibwerere mwachangu kutentha kwa chipinda. Kapangidwe ka AFEX pre-treated corn stover (ACS) kanali kofanana ndi ka corn stover yosakonzedwa (UT-CS).
Zolimba kwambiri za ACSH 25% (w/w) (pafupifupi 8% dextran loading) zinakonzedwa ngati zinthu zoyambira kupanga oligosaccharides pamlingo waukulu. Enzymatic hydrolysis ya ACS idachitika pogwiritsa ntchito kusakaniza kwa enzyme yogulitsa kuphatikizapo Cellic® Ctec2 10 mg protein/g glucan (mu biomass yokonzedwa kale), Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan, ndi Multifect Pectinase (Genencor Inc, USA). ), 5 mg protein/g dextran. Enzymatic hydrolysis idachitika mu bioreactor ya malita 5 yokhala ndi voliyumu yogwira ntchito ya malita 3, pH 4.8, 50°C ndi 250 rpm. Pambuyo pa hydrolysis kwa maola 96, hydrolyzate idasonkhanitsidwa pogwiritsa ntchito centrifugation pa 6000 rpm kwa mphindi 30 kenako pa 14000 rpm kwa mphindi 30 kuti ichotse zolimba zosasungunuka. Kenako hydrolyzate inasefedwa pogwiritsa ntchito chidebe choyezera cha 0.22 mm. Hydrolyzate yosefedwayo inasungidwa m'mabotolo osawonongeka pa 4°C. kenako inadulidwa pa carbon.
Kusanthula kapangidwe ka zitsanzo za biomass zochokera ku zotulutsa malinga ndi njira zowunikira za labotale ya NREL: kukonzekera zitsanzo kuti ziwunike kapangidwe kake (NREL/TP-510-42620) ndi kudziwa chakudya cham'thupi ndi lignin mu biomass (NREL/TP-510 - 42618)47.
Kusanthula kwa oligosaccharide pa mtsinje wa hydrolyzate kunachitika pa sikelo ya 2 ml pogwiritsa ntchito njira ya autoclave-based acid hydrolysis. Sakanizani chitsanzo cha hydrolyzate ndi 69.7 µl ya 72% sulfuric acid mu chubu chokulitsa cha screw cap cha 10 ml ndikuchiyika kwa ola limodzi pa benchtop pa 121 °C, kuzizizira pa ayezi ndikusefa mu vial ya high performance liquid chromatography (HPLC). Kuchuluka kwa oligosaccharides kunadziwika pochotsa kuchuluka kwa monosaccharides mu chitsanzo chosapangidwa kuchokera ku kuchuluka konse kwa shuga mu chitsanzo chopangidwa ndi acid-hydrolyzed.
Kuchuluka kwa shuga, xylose, ndi arabinose mu acid hydrolyzed biomass kunawunikidwa pogwiritsa ntchito njira ya Shimadzu HPLC yokhala ndi autosampler, column heater, isocratic pump, ndi refractive index detector pa Bio-Rad Aminex HPX-87H column. Column inasungidwa pa 50°C ndipo inachotsedwa ndi 0.6 ml/min 5 mM H2SO4 m'madzi. flow.
Supanatant ya hydrolyzate inachepetsedwa ndi kufufuzidwa kuti ione kuchuluka kwa monomer ndi oligosaccharide. Shuga wa monomeric wopezeka pambuyo pa hydrolysis ya enzymatic unafufuzidwa ndi HPLC yokhala ndi Bio-Rad (Hercules, CA) Aminex HPX-87P column ndi ash guard column. Kutentha kwa column kunasungidwa pa 80°C, madzi anagwiritsidwa ntchito ngati gawo loyenda ndi flow rate ya 0.6 ml/min. Oligosaccharides anadziwika ndi hydrolysis mu dilute acid pa 121°C malinga ndi njira zomwe zafotokozedwa mu refs. 41, 48, 49.
Kusanthula kwa Saccharide kunachitika pa zotsalira za biomass zosaphika, zokonzedwa kale za AFEX komanso zonse zomwe sizinagwiritsidwe ntchito (kuphatikiza kupanga zotulutsa za khoma la maselo otsatizana ndi kuyesa kwawo kwa mAb) pogwiritsa ntchito njira zomwe zafotokozedwa kale 27, 43, 50, 51. Pa kusanthula kwa glycome, zotsalira zosasungunuka za khoma la maselo a zomera zimakonzedwa kuchokera ku zotsalira za biomass ndipo zimachotsedwa serial ndi ma reagents amphamvu kwambiri monga ammonium oxalate (50 mM), sodium carbonate (50 mM ndi 0.5% w/v), CON. (1M ndi 4M, zonse ndi 1% w/v sodium borohydride) ndi acid chlorite monga momwe tafotokozera kale52,53. Zotsitsazo kenako zidayikidwa ku ELISA motsutsana ndi gulu lovuta la mAb50s lolunjika ku cell wall glycan, ndipo ma mAb binding reactions adaperekedwa ngati mapu otentha. mAbs yolunjika ku khoma la maselo a zomera idagulidwa kuchokera ku stocks za labotale (CCRC, JIM ndi MAC series).
Kusakaniza kwa oligosaccharides mu njira imodzi. Kuphatikizika kwa chakudya ndi biotin-LC-hydrazide kunachitika pogwiritsa ntchito njira yotsatirayi. Biotin-LC-hydrazide (4.6 mg/12 μmol) inasungunuka mu dimethyl sulfoxide (DMSO, 70 μl) mwa kusakaniza mwamphamvu ndikutenthetsa pa 65° C. kwa mphindi imodzi. Glacial acetic acid (30 µl) inawonjezedwa ndipo chisakanizocho chinathiridwa pa sodium cyanoborohydride (6.4 mg/100 µmol) ndikusungunuka kwathunthu mutatenthetsa pa 65° C. kwa mphindi imodzi. Kenako, kuyambira 5 mpaka 8 μl ya chisakanizocho chinawonjezedwa ku oligosaccharide youma (1-100 nmol) kuti pakhale kuchuluka kwa molar kwa nthawi 10 kapena kuposerapo kwa chizindikirocho kumapeto kwa kuchepetsa. Kuyankhako kunachitika pa 65°C kwa maola awiri, pambuyo pake zitsanzozo zinayeretsedwa nthawi yomweyo. Palibe sodium cyanoborohydride yomwe idagwiritsidwa ntchito poyesa kulemba zilembo popanda kuchepetsa, ndipo zitsanzozo zidayankhidwa pa 65°C. kwa maola 2.5.
Kupaka ndi kutsuka zitsanzo za oligosaccharides zopangidwa ndi biotinylated ku ELISA. 25 μl ya zitsanzo zopangidwa ndi biotinylated (100 μl ya chitsanzo chilichonse chosungunuka mu 5 ml ya 0.1 M Tris buffer solution (TBS)) zinawonjezedwa ku chitsime chilichonse cha mbale yophimbidwa ndi avidin. Zitsime zowongolera zinapakidwa ndi 50 μl ya biotin pamlingo wa 10 μg/ml mu 0.1 M TBS. Madzi oyeretsedwa anagwiritsidwa ntchito ngati chophimba cha miyeso yopanda kanthu. Piritsilo linayikidwa mu incubator kwa maola awiri kutentha kwa chipinda mumdima. Tsukani mbale katatu ndi mkaka wopanda mafuta wa 0.1% mu 0.1 M TBS pogwiritsa ntchito pulogalamu nambala 11 ya Grenier flat 3A.
Kuwonjezera ndi kutsuka ma antibodies oyambira. Onjezani 40 µl ya ma antibodies oyambira pachitsime chilichonse. Ikani microplate kwa ola limodzi kutentha kwa chipinda mumdima. Kenako mbalezo zinatsukidwa katatu ndi mkaka wa 0.1% mu 0.1M TBS pogwiritsa ntchito pulogalamu yotsuka #11 ya Grenier Flat 3A.
Onjezani antibody yachiwiri ndikutsuka. Onjezani 50 µl ya antibody yachiwiri ya mbewa/makoswe (yochepetsedwa 1:5000 mu mkaka wa 0.1% mu 0.1 M TBS) ku chitsime chilichonse. Ikani microplate kwa ola limodzi kutentha kwa chipinda mumdima. Kenako microplates anatsukidwa kasanu ndi kamodzi ndi mkaka wa 0.1% mu 0.1 M TBS pogwiritsa ntchito pulogalamu ya Grenier Flat 5A plate wash #12.
Kuwonjezera gawo lapansi. Onjezani 50 µl ya 3,3′, 5,5′-tetramethylbenzidine (TMB) ku gawo lapansi loyambira (powonjezera madontho awiri a buffer, madontho atatu a TMB, madontho awiri a hydrogen peroxide ku 15 ml ya madzi oyeretsedwa). Konzani gawo lapansi la TMB. ndi vortex musanagwiritse ntchito). Ikani microplate pa kutentha kwa chipinda kwa mphindi 30. Mumdima.
Malizitsani sitepeyo ndikuwerenga piritsi. Onjezani 50 µl ya 1 N sulfuric acid pachitsime chilichonse ndikulemba momwe imayamwira kuyambira 450 mpaka 655 nm pogwiritsa ntchito chowerengera cha ELISA.
Konzani mayankho a 1 mg/ml a ma analytes awa m'madzi osaphatikizidwa: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine. (glcNAc), N-acetylgalactosamine (galNAc), inositol (muyezo wamkati). Miyezo iwiri inakonzedwa powonjezera mayankho a shuga a 1 mg/mL omwe akuwonetsedwa mu Table 1. Zitsanzo zimasungidwa mufiriji ndikusungidwa pa -80° C. mpaka madzi onse atachotsedwa (nthawi zambiri pafupifupi maola 12-18).
Onjezani 100–500 µg ya chitsanzo ku machubu ophimba pamlingo wowunikira. Lembani kuchuluka komwe kwawonjezeredwa. Ndi bwino kusungunula chitsanzocho mu kuchuluka kwina kwa solvent ndikuchiyika mu chubu ngati aliquot yamadzimadzi. Gwiritsani ntchito 20 µl ya 1 mg/ml inositol ngati muyezo wamkati pa chubu chilichonse cha chitsanzo. Kuchuluka kwa muyezo wamkati wowonjezeredwa ku chitsanzocho kuyenera kukhala kofanana ndi kuchuluka kwa muyezo wamkati wowonjezeredwa ku chubu chokhazikika.
Onjezani 8 ml ya methanol yosaphwanyika mu botolo lophimba ndi screw cap. Kenako 4 ml ya 3 N. methanolic HCl solution, muyiike pamwamba ndikuyigwedeza. Njirayi sigwiritsa ntchito madzi.
Onjezani 500 µl ya yankho la methanol la 1 M HCl ku zitsanzo za oligosaccharide ndi machubu okhazikika a TMS. Zitsanzo zinayikidwa usiku wonse (maola 168) pa 80° C. mu thermal block. Umitsani mankhwala a methanolysis kutentha kwa chipinda pogwiritsa ntchito manifold owumitsa. Onjezani 200 µl MeOH ndikuumitsanso. Njirayi imabwerezedwa kawiri. Onjezani 200 µl ya methanol, 100 µl ya pyridine ndi 100 µl ya acetic anhydride ku chitsanzocho ndikusakaniza bwino. Zitsanzo zinayikidwa kutentha kwa chipinda kwa mphindi 30 ndikuumitsa. Onjezani 200 µl ya methanol ndikuumitsanso.
Onjezani 200 µl ya Tri-Sil ndikutenthetsa chubu chotsekedwa kwa mphindi 20. 80°C, kenako muziziziritse kutentha kwa chipinda. Gwiritsani ntchito chida chowumitsira kuti muumitsenso chitsanzocho mpaka kufika pa voliyumu ya pafupifupi 50 µl. Ndikofunikira kudziwa kuti sitinalole zitsanzozo kuti ziume kwathunthu.
Onjezani 2 ml ya hexane ndikusakaniza bwino potulutsa vortex. Dzazani nsonga za Pasteur pipettes (5-8 mm) ndi chidutswa cha ubweya wagalasi poyika ubweya wagalasi pamwamba pa pipette ya mainchesi 5-3/4. Zitsanzozo zinayikidwa mu centrifuge pa 3000 g kwa mphindi ziwiri. Zotsalira zilizonse zosasungunuka zimaphwanyidwa. Umitsani chitsanzocho mpaka 100-150 µl. Voliyumu ya pafupifupi 1 μl inalowetsedwa mu GC-MS pa kutentha koyambirira kwa 80 °C ndi nthawi yoyambirira ya mphindi 2.0 (Table 2).