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Tshiab immunological thiab loj spectrometric txoj kev rau txoj kev tsom xam ntawm oligosaccharides tsis tu ncua nyob rau hauv pob kws stover pretreated nrog AFEX.Lignocellulosic biomass yog ib txoj hauv kev ruaj khov rau cov fossil fuels thiab tau siv dav los tsim biotechnologies rau kev tsim cov khoom xws li zaub mov, pub, roj thiab tshuaj.Tus yuam sij rau cov thev naus laus zis no yog kev txhim kho cov txheej txheem sib tw nqi rau kev hloov cov carbohydrates nyuaj uas muaj nyob rau hauv cov phab ntsa ntawm cov nroj tsuag rau hauv cov suab thaj yooj yim xws li qabzib, xylose thiab arabinose.Vim tias lignocellulosic biomass tawv tawv heev, nws yuav tsum raug rau cov kev kho mob thermochemical (xws li, ammonia fiber exfoliation (AFEX), dilute acids (DA), ionic kua (IL)) thiab kev kho mob lom (xws li, enzymatic hydrolysis thiab microbial fermentation) ua ke kom tau cov khoom xav tau..Txawm li cas los xij, thaum kev lag luam fungal enzymes tau siv rau hauv cov txheej txheem hydrolysis, tsuas yog 75-85% ntawm cov suab thaj soluble tsim yog monosaccharides, thiab cov seem 15-25% yog soluble, intractable oligosaccharides, uas tsis yog ib txwm muaj rau cov kab mob.Yav dhau los, peb tau ua tiav kev sib cais thiab ua kom huv si cov tawv tawv oligosaccharides siv kev sib xyaw ntawm cov pa roj carbon thiab diatomaceous lub ntiaj teb sib cais thiab qhov loj me ntawm chromatography, thiab tseem tshawb xyuas lawv cov enzyme inhibitory zog.Peb tau pom tias oligosaccharides uas muaj qib siab dua ntawm polymerization (DP) methylated uronic acid hloov yog qhov nyuaj rau kev ua lag luam nrog cov enzyme sib xyaw dua li cov DP qis thiab nruab nrab oligosaccharides.Ntawm no peb qhia txog kev siv ntau txoj hauv kev ntxiv, suav nrog glycan profileing siv cov tshuaj tiv thaiv kab mob monoclonal (mAbs) tshwj xeeb rau cov nroj tsuag biomass glycans los ua tus cwj pwm glycan bonds hauv cov phab ntsa ntawm tes thiab enzymatic hydrolysates, matrix-pab laser desorption ionization, lub sij hawm-ntawm-flight mass-spectrometry..MALDI-TOF-MS) siv cov qauv-cov ntaub ntawv txheeb xyuas qhov siab tshaj plaws tau txais los ntawm spectroscopy tom qab qhov kev puas tsuaj ntawm cov ions tsis zoo, roj chromatography thiab huab hwm coj spectrometry (GC-MS) los ua tus cwj pwm oligosaccharide bonds nrog thiab tsis muaj derivatization.Vim yog qhov me me ntawm oligosaccharides (DP 4-20), cov molecules no nyuaj rau siv rau mAb khi thiab tus cwj pwm.Txhawm rau kov yeej qhov teeb meem no, peb siv biotin conjugation-raws li oligosaccharide immobilization txoj kev uas tau sau npe rau feem ntau ntawm cov tsis tshua muaj DP soluble oligosaccharides ntawm microplate nto, uas tau siv nyob rau hauv high throughput mAb system rau kev ligation tsom xam.Txoj kev tshiab no yuav pab txhawb txoj kev loj hlob ntawm kev nce qib siab dhau los ntawm kev ntsuas glycome yav tom ntej uas tuaj yeem siv los cais tawm thiab ua tus yam ntxwv ntawm oligosaccharides tam sim no hauv biomarkers rau kev kuaj mob.
Lignocellulosic biomass, muaj li ntawm kev ua liaj ua teb, hav zoov, nyom thiab ntoo, yog ib lub peev xwm feedstock rau zus tau tej cov bio-based khoom, nrog rau cov zaub mov, pub, roj thiab tshuaj precursors los tsim cov khoom muaj nqis dua 1.Carbohydrates (xws li cellulose thiab hemicellulose) tam sim no nyob rau hauv cov nroj tsuag cell phab ntsa yog depolymerized rau hauv monosaccharides los ntawm tshuaj ua thiab biotransformation (xws li enzymatic hydrolysis thiab microbial fermentation).Cov kev kho mob ua ntej muaj xws li ammonia fiber expansion (AFEX), dilute acid (DA), ionic kua (IL), thiab chav tawg (SE), uas siv cov tshuaj sib xyaw ua ke thiab cua sov kom txo qis lignocellulose ntau lawm los ntawm kev qhib lub cell phab ntsa3,4.tawv tawv ntawm cov teeb meem, 5. Enzymatic hydrolysis yog nqa tawm ntawm cov khib nyiab siab uas siv cov khoom lag luam nquag carbohydrate-muaj enzymes (CAZymes) thiab microbial fermentation siv transgenic poov xab los yog kab mob los tsim bio-based fuels thiab tshuaj 6.
CAZymes nyob rau hauv kev lag luam enzymes yog tsim los ntawm ib tug complex sib tov ntawm enzymes synergistically cleave complex carbohydrate-qab zib daim ntawv cog lus rau tsim monosaccharides2,7.Raws li peb tau tshaj tawm ua ntej, lub network complex ntawm aromatic polymers ntawm lignin nrog carbohydrates ua rau lawv heev intractable, uas ua rau kom tsis tiav qab zib conversion, accumulating 15-25% ntawm kev sib deev oligosaccharides uas tsis tau tsim thaum lub sij hawm enzymatic hydrolysis ntawm pretreated biomass.Qhov no yog ib qho teeb meem tshwm sim nrog ntau yam biomass pretreatment txoj kev.Qee qhov laj thawj rau lub cev tsis muaj zog no suav nrog enzyme inhibition thaum lub sij hawm hydrolysis, los yog qhov tsis muaj lossis qis ntawm cov enzymes tseem ceeb uas yuav tsum tau ua kom tawg cov piam thaj hauv cov nroj tsuag biomass.Kev nkag siab txog qhov muaj pes tsawg leeg thiab cov yam ntxwv ntawm cov suab thaj, xws li cov piam thaj hauv cov oligosaccharides, yuav pab peb txhim kho cov piam thaj hloov thaum lub sij hawm hydrolysis, ua cov txheej txheem biotechnological nqi sib tw nrog cov khoom siv roj av.
Kev txiav txim siab cov qauv ntawm carbohydrates yog qhov nyuaj thiab yuav tsum muaj kev sib xyaw ua ke xws li kua chromatography (LC)11,12, nuclear magnetic resonance spectroscopy (NMR)13, capillary electrophoresis (CE) 14,15,16 thiab huab hwm coj spectrometry (MS)17., kaum yim.MS txoj kev xws li lub sij hawm ntawm-flight huab hwm coj spectrometry nrog laser desorption thiab ionization siv ib tug matrix (MALDI-TOF-MS) yog ib tug ntau yam txoj kev los txheeb xyuas cov carbohydrate qauv.Tsis ntev los no, kev sib tsoo-Induced Dissociation (CID) tandem MS ntawm sodium ion adducts tau siv dav tshaj plaws los txheeb xyuas cov ntiv tes sib xws rau oligosaccharide txuas txoj haujlwm, anomeric configurations, sequences, thiab branching txoj hauj lwm 20, 21 .
Glycan tsom xam yog ib qho cuab yeej zoo heev rau kev txheeb xyuas qhov tob ntawm carbohydrate bonds22.Txoj kev no siv cov tshuaj tiv thaiv kab mob monoclonal (mAbs) qhia rau cog cell phab ntsa glycan raws li kev sojntsuam kom nkag siab txog kev sib txuas ntawm cov carbohydrate.Ntau tshaj 250 mAbs muaj nyob thoob plaws ntiaj teb, tsim los tiv thaiv ntau yam kab thiab ceg oligosaccharides siv ntau yam saccharides24.Ob peb mAbs tau siv dav los ua tus yam ntxwv ntawm cov qauv, muaj pes tsawg leeg, thiab kev hloov kho ntawm cov nroj tsuag cell phab ntsa, vim muaj qhov sib txawv tseem ceeb nyob ntawm cov nroj tsuag ntawm tes, lub cev, hnub nyoog, theem kev loj hlob, thiab kev loj hlob ib puag ncig25,26.Tsis ntev los no, txoj kev no tau siv los nkag siab txog cov vesicle pej xeem hauv cov nroj tsuag thiab tsiaj txhu thiab lawv lub luag haujlwm hauv kev thauj glycan raws li kev txiav txim siab los ntawm cov cim subcellular, theem kev loj hlob, lossis ib puag ncig stimuli, thiab txiav txim siab ua haujlwm enzymatic.Qee cov qauv sib txawv ntawm glycans thiab xylans uas tau txheeb xyuas muaj xws li pectin (P), xylan (X), mannan (M), xyloglucans (XylG), sib xyaw cov nyiaj glucans (MLG), arabinoxylan (ArbX), galactomannan (GalG), glucuronic acid-arabinoxylan (Grabinogalan (GArab.9)) thiab a.
Txawm li cas los xij, txawm tias tag nrho cov kev tshawb fawb no, tsuas yog qee qhov kev tshawb fawb tau tsom mus rau qhov xwm txheej ntawm oligosaccharide tsub zuj zuj thaum lub sij hawm siab load (HSL) hydrolysis, suav nrog oligosaccharide tso tawm, oligomeric saw ntev hloov thaum lub sij hawm hydrolysis, ntau yam tsawg DP polymers, thiab lawv cov nkhaus.30, 31, 32.Lub caij no, txawm hais tias kev tshuaj xyuas glycan tau ua pov thawj tias yog ib qho cuab yeej tseem ceeb rau kev tsom xam ntawm glycan qauv, nws nyuaj rau kev ntsuas cov dej-soluble qis DP oligosaccharides siv cov tshuaj tiv thaiv kab mob.Cov DP oligosaccharides me me uas muaj qhov hnyav dua li 5-10 kDa tsis khi rau ELISA daim hlau 33, 34 thiab raug ntxuav tawm ua ntej cov tshuaj tiv thaiv ntxiv.
Ntawm no, thawj zaug, peb ua qauv qhia ELISA qhov kev ntsuam xyuas ntawm avidin-coated daim hlau siv cov tshuaj tiv thaiv monoclonal, sib txuas cov txheej txheem biotinylation ib-kauj ruam rau soluble refractory oligosaccharides nrog glycome tsom xam.Peb txoj hauv kev rau kev soj ntsuam glycome tau raug lees paub los ntawm MALDI-TOF-MS thiab GC-MS raws li kev tshuaj xyuas ntawm kev sib txuas ntawm oligosaccharide uas siv trimethylsilyl (TMS) derivatization ntawm hydrolyzed qab zib compositions.Txoj hauv kev tshiab no tuaj yeem tsim los ua ib txoj hauv kev zoo tshaj plaws rau yav tom ntej thiab nrhiav kev siv dav hauv kev tshawb fawb biomedical35.
Kev hloov pauv tom qab kev hloov pauv ntawm cov enzymes thiab cov tshuaj tiv thaiv kab mob, xws li glycosylation, 36 cuam tshuam rau lawv txoj haujlwm lom neeg.Piv txwv li, kev hloov pauv hauv glycosylation ntawm cov ntshav cov protein ua lub luag haujlwm tseem ceeb hauv kev mob caj dab, thiab cov kev hloov hauv glycosylation yog siv los ua cov cim ntsuas 37.Ntau yam glycans tau raug tshaj tawm nyob rau hauv cov ntaub ntawv kom nkag mus rau hauv ntau yam kab mob, nrog rau cov kab mob ntev ntawm cov kab mob hauv plab thiab lub siab, kab mob kis, zes qe menyuam, mis, thiab qog nqaij hlav prostate38,39,40.Kev nkag siab txog cov qauv ntawm glycans siv cov tshuaj tiv thaiv kab mob raws li glycan ELISA yuav muab kev ntseeg siab ntxiv rau kev kuaj mob yam tsis tas siv cov txheej txheem MS nyuaj.
Peb txoj kev tshawb fawb yav dhau los tau pom tias tawv ncauj oligosaccharides tseem tsis muaj dej tom qab kev kho mob thiab enzymatic hydrolysis (Daim duab 1).Hauv peb qhov kev tshaj tawm yav dhau los, peb tau tsim ib txoj kev siv hluav taws xob hluav taws xob-theem rho tawm kom cais cov oligosaccharides los ntawm AFEX-pretreated pob kws stover hydrolyzate (ACSH)8.Tom qab thawj zaug rho tawm thiab sib cais, cov oligosaccharides tau muab faib ntxiv los ntawm qhov loj me me chromatography (SEC) thiab khaws cia hauv qhov kev txiav txim ntawm qhov hnyav molecular.Qab Zib monomers thiab oligomers tso tawm los ntawm ntau yam pretreatments tau soj ntsuam los ntawm kev txheeb xyuas cov piam thaj.Thaum muab piv cov ntsiab lus ntawm cov piam thaj oligomers tau los ntawm ntau txoj kev kho ua ntej, qhov muaj cov tawv nqaij oligosaccharides yog ib qho teeb meem tshwm sim hauv kev hloov pauv ntawm biomass rau monosaccharides thiab tuaj yeem ua rau txo qis cov piam thaj tsawg kawg 10-15% thiab txawm tias mus txog 18%.Teb Chaws Asmeskas.Txoj kev no yog siv rau kev loj hlob ntxiv ntawm oligosaccharide feem.Qhov tshwm sim ACH thiab nws cov seem tom qab nrog qhov sib txawv ntawm qhov hnyav molecular tau siv los ua cov khoom siv sim rau cov yam ntxwv ntawm oligosaccharides hauv txoj haujlwm no.
Tom qab pretreatment thiab enzymatic hydrolysis, tsis tu ncua oligosaccharides tseem unhydrolysed.Ntawm no (A) yog txoj kev sib cais oligosaccharide uas oligosaccharides raug cais tawm ntawm AFEX-pretreated pob kws stover hydrolyzate (ACSH) siv lub txaj ntim ntawm cov pa roj carbon activated thiab diatomaceous lub ntiaj teb;(B) Txoj kev sib cais ntawm oligosaccharides.Cov oligosaccharides tau sib cais ntxiv los ntawm qhov loj me me tsis suav nrog chromatography (SEC);(C) Saccharide monomers thiab oligomers tso tawm los ntawm ntau yam pretreatments (diluted acid: DA, ionic kua: IL thiab AFEX).Enzymatic hydrolysis tej yam kev mob: siab loading ntawm 25% (w / w) (kwv yees li 8% glucan loading), 96 teev hydrolysis, 20 mg / g coj mus muag enzyme loading (Ctec2: Htec2: MP-2: 1: 1 ratio) thiab (D) Qab Zib monomers thiab oligomers ntawm qabzib AF, xylostre, AC lub qhov cub tso tawm (AC)
Kev tsom xam Glycan tau ua pov thawj tias yog ib qho cuab yeej muaj txiaj ntsig rau kev txheeb xyuas cov qauv ntawm glycans hauv cov khoom rho tawm los ntawm cov khoom biomass residues.Txawm li cas los xij, cov dej-soluble saccharides tsis tshua muaj npe siv cov qauv no 41 vim tias qhov hnyav molecular oligosaccharides tsis yooj yim rau immobilize ntawm ELISA daim hlau thiab raug ntxuav tawm ua ntej cov tshuaj tiv thaiv ntxiv.Yog li ntawd, rau cov tshuaj tiv thaiv kev khi thiab kev ua tus cwj pwm, ib txoj hauv kev biotinylation ib-kauj ruam tau siv los coated soluble, tsis ua raws li oligosaccharides ntawm avidin-coated ELISA daim hlau.Txoj kev no tau sim siv peb cov ACSH yav dhau los ua thiab ib feem raws li nws qhov hnyav molecular (los yog degree ntawm polymerization, DP).Ib-kauj ruam biotinylation tau siv los ua kom oligosaccharide binding affinity los ntawm kev ntxiv biotin-LC-hydrazide rau txo qhov kawg ntawm cov carbohydrate (Fig. 2).Hauv kev daws teeb meem, pawg hemiacetal ntawm qhov txo qhov kawg reacts nrog hydrazide pawg ntawm biotin-LC-hydrazide los tsim ib daim ntawv cog lus hydrazone.Nyob rau hauv lub xub ntiag ntawm tus txo tus neeg sawv cev NaCNBH3, hydrazone daim ntawv cog lus yog txo mus rau ib tug ruaj khov biotinylated kawg khoom.Nrog rau kev hloov kho cov piam thaj txo qhov kawg, kev khi ntawm qis DP oligosaccharides rau ELISA daim hlau tau ua tau, thiab hauv peb txoj kev tshawb fawb no tau ua tiav ntawm avidin-coated daim hlau siv glycan-targeted mAbs.
Kev tshuaj xyuas cov tshuaj tiv thaiv monoclonal raws li ELISA rau biotinylated oligosaccharides.Ntawm no (A) ua ke biotinylation ntawm oligosaccharides thiab tom qab ELISA kev tshuaj ntsuam nrog glycan-targeted mAbs ntawm NeutrAvidin coated daim hlau thiab (B) qhia ib kauj ruam rau biotinylation ntawm cov tshuaj tiv thaiv cov khoom.
Avidin-coated daim hlau nrog oligosaccharide-conjugated cov tshuaj tiv thaiv tom qab tau ntxiv rau cov tshuaj tiv thaiv thawj thiab theem nrab thiab ntxuav hauv lub teeb- thiab lub sijhawm-rhiab.Tom qab cov tshuaj tiv thaiv kab mob ua tiav, ntxiv TMB substrate los tsim cov phaj.Cov tshuaj tiv thaiv thaum kawg nres nrog sulfuric acid.Cov phaj incubated tau txheeb xyuas siv tus nyeem ntawv ELISA los txiav txim siab lub zog khi ntawm txhua cov tshuaj tiv thaiv kab mob txhawm rau txheeb xyuas cov tshuaj tiv thaiv kab mob sib kis.Rau cov ntsiab lus thiab qhov ntsuas ntawm qhov kev sim, saib cov ntu "Cov ntaub ntawv thiab cov txheej txheem".
Peb ua qauv qhia txog kev siv hluav taws xob ntawm txoj kev tsim tshiab no rau cov ntawv thov tshwj xeeb los ntawm kev qhia txog cov soluble oligosaccharides tam sim no nyob rau hauv ACSH nrog rau cov khoom nyoos thiab purified oligosaccharide feem cais los ntawm lignocellulosic hydrolysates.Raws li pom nyob rau hauv daim duab 3, feem ntau cov epitope-hloov xylans pom nyob rau hauv ACSH siv bioacylated glycome assay txoj kev yog feem ntau uronic (U) los yog methyluronic (MeU) thiab pectic arabinogalactans.Lawv feem ntau kuj tau pom nyob rau hauv peb txoj kev tshawb fawb dhau los ntawm kev tsom xam glycans ntawm cov khoom tsis-hydrolyzed (UHS) 43.
Kev kuaj pom ntawm recalcitrant oligosaccharide epitopes siv cov tshuaj tiv thaiv monoclonal coj mus rau phab ntsa ntawm tes glycan.Qhov "tsis nruab nrab" feem yog ACN feem thiab "acidic" feem yog FA feem.Cov xim liab ci dua ntawm daim duab tshav kub qhia tias cov ntsiab lus epitope siab dua, thiab lub ntsej muag ci dua qhia tias muaj keeb kwm yav dhau los.Xim qhov tseem ceeb ntawm qhov ntsuas yog raws li raw OD qhov tseem ceeb rau cov qauv N = 2.Lub ntsiab epitopes lees paub los ntawm cov tshuaj tiv thaiv yog qhia ntawm sab xis.
Cov qauv tsis-cellulose no tsis tuaj yeem raug tshem tawm los ntawm cov cellulases thiab hemicellulases feem ntau hauv cov khoom lag luam enzyme sib xyaw, uas suav nrog cov khoom lag luam feem ntau siv.Yog li ntawd, cov enzymes tshiab yog xav tau rau lawv cov hydrolysis.Yog tsis muaj qhov tsim nyog tsis yog-cellulose accessory enzymes, cov ntawv cog lus tsis yog cellulose tiv thaiv kev hloov pauv mus rau monosaccharides, txawm tias lawv niam lawv txiv cov piam thaj polymers tau siv ntau hydrolyzed rau hauv luv luv thiab yaj siv cov khoom lag luam enzyme.
Kev tshawb fawb ntxiv ntawm cov teeb liab kev faib tawm thiab nws txoj kev khi lub zog tau pom tias kev khi epitopes qis dua hauv cov qib siab DP qab zib feem ntau (A, B, C, DP mus txog 20+) dua li cov DP feem tsawg (D, E, F, DP) hauv dimers) (Fig. 1).Acid fragments muaj ntau dua nyob rau hauv uas tsis yog-cellulose epitopes dua li tsis muaj feem.Cov xwm txheej no tau ua raws li cov qauv uas tau pom hauv peb txoj kev tshawb fawb yav dhau los, qhov twg DP siab thiab cov kua qaub moieties tau tiv taus ntau dua rau enzymatic hydrolysis.Yog li, muaj cov tsis-cellulose glycan epitopes thiab U thiab MeU hloov pauv tuaj yeem ua rau muaj kev ruaj ntseg ntawm oligosaccharides.Nws yuav tsum raug sau tseg tias kev sib khi thiab kev tshawb pom kev ua haujlwm tuaj yeem muaj teeb meem rau DP oligosaccharides qis, tshwj xeeb tshaj yog tias cov epitope yog dimeric lossis trimeric oligosaccharide.Qhov no tuaj yeem kuaj tau siv cov khoom lag luam oligosaccharides ntawm qhov ntev sib txawv, txhua qhov muaj tsuas yog ib qho epitope uas khi rau ib qho tshwj xeeb mAb.
Yog li, kev siv cov qauv tshwj xeeb cov tshuaj tiv thaiv tau qhia qee yam ntawm cov ntawv cog lus recalcitrant.Nyob ntawm seb hom tshuaj tiv thaiv kab mob siv li cas, tus qauv ligation tsim nyog, thiab lub zog ntawm lub teeb liab nws tsim (feem ntau thiab tsawg tshaj plaws), cov enzymes tshiab tuaj yeem txheeb xyuas thiab ntxiv ib nrab rau cov enzyme sib xyaw kom tiav glycoconversion.Ua piv txwv ntawm ACSH oligosaccharides, peb tuaj yeem tsim cov ntaub ntawv ntawm glycan bonds rau txhua yam khoom siv biomass.Nws yuav tsum tau muab sau tseg ntawm no hais tias qhov sib txawv affinity ntawm cov tshuaj tiv thaiv yuav tsum tau coj mus rau hauv tus account, thiab yog hais tias lawv affinity yog tsis paub, qhov no yuav tsim ib co teeb meem thaum muab piv cov cim ntawm txawv antibodies.Tsis tas li ntawd, kev sib piv ntawm glycan bonds tuaj yeem ua haujlwm zoo tshaj plaws ntawm cov qauv rau tib cov tshuaj tiv thaiv.Cov ntawv cog lus tawv tawv no tuaj yeem txuas mus rau CAZyme database, los ntawm qhov peb tuaj yeem txheeb xyuas cov enzymes, xaiv cov neeg sib tw enzymes thiab kuaj rau cov enzymes sib tsoo, lossis tsim cov kab mob microbial los qhia cov enzymes siv hauv biorefineries44.
Txhawm rau tshuaj xyuas seb cov txheej txheem tiv thaiv kab mob li cas ntxiv rau lwm txoj hauv kev rau tus yam ntxwv qis molecular yuag oligosaccharides tam sim no hauv lignocellulosic hydrolysates, peb tau ua MALDI (Fig. 4, S1-S8) thiab kev tshuaj xyuas ntawm TMS-derived saccharides raws li GC-MS ntawm tib lub vaj huam sib luag (Fig. 5) oligos.MALDI yog siv los sib piv seb qhov loj ntawm oligosaccharide molecules sib piv cov qauv tsim.Ntawm daim duab.4 qhia MS ntawm cov khoom nruab nrab ACN-A thiab ACN-B.ACN-A tsom xam tau lees paub ntau yam ntawm cov suab thaj pentose xws li DP 4–8 (Daim duab 4) txog DP 22 (Daim duab S1), uas nws qhov hnyav sib haum rau MeU-xylan oligosaccharides.Kev tshuaj xyuas ACN-B tau lees paub cov pentose thiab glucoxylan series nrog DP 8-15.Hauv cov khoom siv ntxiv xws li daim duab S3, FA-C acidic moiety mass distribution maps qhia ntau yam (Me) U hloov pentose qab zib nrog DP ntawm 8-15 uas zoo ib yam nrog cov xylans hloov tau pom hauv ELISA-based mAb kev tshuaj ntsuam.Cov epitopes zoo ib yam.
MALDI-MS spectrum ntawm soluble non-compliant oligosaccharides tam sim no nyob rau hauv ACS.Ntawm no, (A) ACN-A cov khoom hnyav qis uas muaj cov methylated uronic acid (DP 4-8) hloov glucuroxylan oligosaccharides thiab (B) ACN-B xylan thiab methylated uronic acid oligosaccharides hloov nrog glucuroxylan (DP 8-15).
Kev soj ntsuam ntawm cov muaj pes tsawg leeg ntawm glycan residue ntawm refractory oligosaccharides.Ntawm no (A) TMS saccharide muaj pes tsawg leeg ntawm ntau yam oligosaccharide feem uas tau siv GC-MS tsom xam.(B) Cov qauv ntawm ntau yam TMS-derived qab zib muaj nyob rau hauv oligosaccharides.ACN - acetonitrile feem uas muaj nruab nrab oligosaccharides thiab FA - ferulic acid feem uas muaj cov kua qaub oligosaccharides.
Lwm qhov kev txiav txim siab nthuav tawm tau kos los ntawm LC-MS tsom xam ntawm oligosaccharide feem, raws li qhia hauv daim duab S9 (txoj kev tuaj yeem pom hauv cov khoom siv hluav taws xob ntxiv).Fragments ntawm hexose thiab -OAc pawg tau rov pom dua thaum lub sij hawm ligation ntawm ACN-B feem.Qhov kev tshawb pom no tsis tsuas yog lees paub qhov kev tawg ntawm kev soj ntsuam hauv glycome thiab MALDI-TOF, tab sis kuj tseem muab cov ntaub ntawv tshiab txog cov khoom noj muaj peev xwm carbohydrate derivatives hauv pretreated lignocellulosic biomass.
Peb kuj tau ua glycan muaj pes tsawg leeg tsom xam ntawm oligosaccharide feem siv TMS glycan derivatization.Siv GC-MS, peb txiav txim siab muaj pes tsawg leeg ntawm neural (non-derivative) thiab acidic qab zib (GluA thiab GalA) nyob rau hauv oligosaccharide feem (Fig. 5).Glucuronic acid muaj nyob rau hauv acidic Cheebtsam C thiab D, thaum galacturonic acid yog pom nyob rau hauv acidic Cheebtsam A thiab B, ob qho tib si uas yog siab DP Cheebtsam ntawm acidic qab zib.Cov txiaj ntsig no tsis tsuas yog lees paub peb cov ntaub ntawv ELISA thiab MALDI, tab sis kuj tseem ua raws li peb cov kev tshawb fawb yav dhau los ntawm oligosaccharide tsub zuj zuj.Yog li ntawd, peb ntseeg tias niaj hnub immunological txoj kev siv biotinylation ntawm oligosaccharides thiab tom ntej ELISA kev tshuaj ntsuam yog txaus los soj ntsuam cov soluble recalcitrant oligosaccharides nyob rau hauv ntau yam kev kuaj lom.
Txij li thaum ELISA-raws li mAb kev tshuaj ntsuam txoj kev tau raug lees paub los ntawm ntau txoj kev sib txawv, peb xav tshawb ntxiv txog qhov muaj peev xwm ntawm cov qauv tshiab no.Ob lub lag luam oligosaccharides, xylohexasaccharide oligosaccharide (XHE) thiab 23-α-L-arabinofuranosyl-xylotriose (A2XX), tau yuav thiab sim siv mAb tshiab rau kev tsom mus rau lub cell phab ntsa glycan.Daim duab 6 qhia txog kev sib raug zoo ntawm biotinylated binding teeb liab thiab lub cav concentration ntawm oligosaccharide concentration, qhia txog qhov ua tau Langmuir adsorption qauv.Ntawm cov mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, thiab CCRC-M151 correlated nrog XHE, thiab CCRC-M108, CCRC-M109, thiab LM11 correlated nrog A2XX tshaj li ntawm ntau yam.Vim tias muaj cov tshuaj tiv thaiv tsawg tsawg thaum lub sijhawm sim, kev sim tsawg tau ua nrog txhua qhov oligosaccharide concentration.Nws yuav tsum raug sau tseg ntawm no tias qee cov tshuaj tiv thaiv kab mob sib txawv heev rau tib lub oligosaccharide raws li lub substrate, txawm tias vim lawv khi rau cov epitopes sib txawv me ntsis thiab tuaj yeem muaj qhov sib txawv sib txawv heev.Cov txheej txheem thiab qhov cuam tshuam ntawm kev txheeb xyuas qhov tseeb epitope yuav nyuaj dua thaum txoj hauv kev tshiab mAb siv rau cov qauv tiag.
Ob lub lag luam oligosaccharides tau siv los txiav txim siab qhov kev tshawb pom ntawm ntau yam glycan-targeting mAbs.Ntawm no, cov kev sib raug zoo nrog cov log concentration ntawm oligosaccharide concentration qhia Langmuir adsorption qauv rau (A) XHE nrog mAb thiab (B) A2XX nrog mAb.Cov epitopes sib thooj qhia cov qauv ntawm cov lag luam oligosaccharides siv los ua cov substrates hauv qhov kev ntsuam xyuas.
Kev siv glycan-targeted monoclonal antibodies (glycocomic tsom xam lossis ELISA-raws li mAb tshuaj ntsuam) yog ib qho cuab yeej muaj zog rau kev ua kom pom tseeb ntawm feem ntau ntawm cov phab ntsa loj ntawm tes glycans uas ua rau cov nroj tsuag biomass.Txawm li cas los xij, classical glycan tsom xam tsuas yog tus cwj pwm ntawm cov phab ntsa ntawm cov phab ntsa loj dua, vim tias feem ntau cov oligosaccharides tsis zoo immobilized ntawm ELISA daim hlau.Hauv txoj kev tshawb no, AFEX-pretreated pob kws stover tau enzymatically hydrolyzed ntawm cov ntsiab lus siab.Kev tsom xam qab zib tau siv los txiav txim qhov muaj pes tsawg leeg ntawm cov cell phab ntsa carbohydrate recalcitrant hauv hydrolyzate.Txawm li cas los xij, kev tsom xam mAb ntawm cov oligosaccharides me me hauv hydrolysates yog qhov tsis txaus ntseeg, thiab cov cuab yeej ntxiv yuav tsum tau ua kom zoo immobilize oligosaccharides ntawm ELISA daim hlau.
Peb qhia ntawm no ib qho tshiab thiab muaj txiaj ntsig oligosaccharide immobilization txoj kev rau mAb kev tshuaj ntsuam los ntawm kev sib txuas oligosaccharide biotinylation ua raws li ELISA kev tshuaj ntsuam ntawm NeutrAvidin™ coated daim hlau.Cov immobilized biotinylated oligosaccharides tau pom tias muaj kev sib raug zoo txaus rau cov tshuaj tiv thaiv kab mob kom ua kom sai thiab ua tau zoo ntawm cov recalcitrant oligosaccharides.Kev soj ntsuam ntawm cov muaj pes tsawg leeg ntawm cov tawv ncauj oligosaccharides raws li huab hwm coj spectrometry tau lees paub cov txiaj ntsig ntawm txoj hauv kev tshiab no rau immunoscreening.Yog li, cov kev tshawb fawb no qhia tau hais tias kev sib xyaw ntawm oligosaccharide biotinylation thiab ELISA kev tshuaj ntsuam nrog glycan-targeted monoclonal antibodies tuaj yeem siv los txheeb xyuas cov kab sib txuas hauv oligosaccharides thiab tuaj yeem siv dav hauv lwm cov kev tshawb fawb biochemical characterizing tus qauv ntawm oligosaccharides.
Qhov biotin-based glycan profileing txoj kev no yog thawj daim ntawv tshaj tawm muaj peev xwm tshawb xyuas cov ntaub ntawv rov qab ntawm cov carbohydrate ntawm soluble oligosaccharides hauv cov nroj tsuag biomass.Qhov no yuav pab kom nkag siab tias vim li cas qee qhov ntawm biomass tawv ncauj thaum nws los txog rau biofuel ntau lawm.Txoj kev no ua rau muaj qhov sib txawv tseem ceeb hauv kev tshuaj ntsuam glycome thiab txuas ntxiv nws daim ntawv thov mus rau ntau hom substrates tshaj li cov nroj tsuag oligosaccharides.Nyob rau hauv lub neej yav tom ntej, peb yuav siv neeg hlau rau biotinylation thiab siv txoj kev peb tau tsim rau high-throughput tsom xam ntawm cov qauv siv ELISA.
Pob kws straw (CS) zus los ntawm Pioneer 33A14 hybrid noob tau sau xyoo 2010 los ntawm Kramer Farms hauv Ray, Colorado.Nrog kev tso cai ntawm tus tswv av, qhov biomass no tuaj yeem siv rau kev tshawb fawb. Cov qauv tau khaws cia qhuav <6% noo noo nyob rau hauv zip-lock hnab hauv chav tsev kub. Cov qauv tau khaws cia qhuav <6% noo noo nyob rau hauv zip-lock hnab hauv chav tsev kub. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Cov qauv tau khaws cia qhuav ntawm <6% av noo hauv cov hnab zippered ntawm chav tsev kub.样品在室温下以干燥< 6% 的水分储存在自封袋中。Cov nyiaj tau los <6% Образцы хранят в пакетах с застежкой-молнией при комнатной температуре с влажностью < 6%. Cov qauv muab khaws cia rau hauv lub hnab zipper ntawm chav tsev kub nrog cov av noo <6%.Txoj kev tshawb no tau ua raws li cov lus qhia hauv zos thiab hauv tebchaws.Kev txheeb xyuas cov ntsiab lus tau ua tiav siv NREL raws tu qauv.Cov muaj pes tsawg leeg tau pom tias muaj 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% protein thiab 13. 4% tshauv.
Cellic® CTec2 (138 mg protein/ml, lot VCNI 0001) yog ib qho kev sib xyaw ntawm cellulase, β-glucosidase thiab Cellic® HTec2 (157 mg protein/ml, lot VHN00001) los ntawm Novozymes (Franklinton, NC, USA).Multifect Pectinase® (72 mg protein / mL), kev sib xyaw ua ke ntawm pectin degrading enzymes, tau pub los ntawm DuPont Industrial Biosciences (Palo Alto, CA, USA).Enzyme protein concentrations tau txiav txim siab los ntawm kev kwv yees cov ntsiab lus ntawm cov protein (thiab rho tawm qhov kev koom tes ntawm cov tsis muaj protein ntau nitrogen) siv Kjeldahl nitrogen tsom xam (AOAC txoj kev 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA).Diatomaceous ntiaj teb 545 tau yuav los ntawm EMD Millipore (Billerica, MA).Cov pa roj carbon activated (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, thiab tag nrho lwm yam tshuaj tau yuav los ntawm Sigma-Aldrich (St. Louis, MO).
AFEX pretreatment tau ua tiav ntawm GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA).Kev kho ua ntej tau ua tiav ntawm 140 ° C. rau 15 feeb.46 lub sijhawm nyob ntawm 1: 1 piv ntawm anhydrous ammonia rau biomass ntawm 60% (w / w) thauj khoom hauv stainless hlau benchtop batch reactor (Parr Instruments Company).Nws siv 30 feeb.Lub reactor tau coj mus rau 140 ° C thiab ammonia tau tso tawm sai sai, tso cai rau biomass rov qab mus rau chav sov sai.Cov muaj pes tsawg leeg ntawm AFEX pre-kho pob kws stover (ACS) zoo ib yam li cov pob kws tsis kho (UT-CS).
Cov khib nyiab siab ACSH 25% (w / w) (kwv yees li 8% dextran loading) tau npaj los ua cov khoom pib rau kev loj hlob ntawm oligosaccharides.Enzymatic hydrolysis ntawm ACS tau ua tiav siv cov khoom lag luam enzyme sib xyaw suav nrog Cellic® Ctec2 10 mg protein / g glucan (hauv pretreated biomass), Htec2 (Novozymes, Franklinton, NC), 5 mg protein / g glucan, thiab Multifect Pectinase (Genencor Inc, USA).).), 5 mg protein / g dextran.Enzymatic hydrolysis tau ua nyob rau hauv 5-liter bioreactor nrog lub ntim ua haujlwm ntawm 3 litres, pH 4.8, 50 ° C thiab 250 rpm.Tom qab hydrolysis rau 96 teev, cov hydrolyzate tau sau los ntawm centrifugation ntawm 6000 rpm rau 30 feeb thiab tom qab ntawd ntawm 14000 rpm rau 30 feeb kom tshem tawm cov khoom tsis muaj dej.Tom qab ntawd cov hydrolyzate tau raug ua kom tsis muaj menyuam pom los ntawm 0.22 mm lim beaker.Cov lim dej hydrolyzate tau muab cia rau hauv lub raj mis tsis muaj menyuam ntawm 4 ° C. thiab tom qab ntawd muab faib rau cov pa roj carbon.
Kev soj ntsuam ntawm cov qauv ntawm cov khoom siv biomass raws li NREL cov txheej txheem kuaj xyuas: kev npaj cov qauv rau kev tshuaj ntsuam xyuas muaj pes tsawg leeg (NREL / TP-510-42620) thiab kev txiav txim siab ntawm cov qauv carbohydrates thiab lignin hauv biomass (NREL / TP-510 - 42618) 47.
Oligosaccharide tsom xam ntawm cov kwj dej hydrolyzate tau ua ntawm 2 ml nplai siv autoclave-based acid hydrolysis txoj kev.Sib tov cov qauv hydrolyzate nrog 69.7 µl ntawm 72% sulfuric acid nyob rau hauv 10 ml ntsia hlau cap kab lis kev cai raj thiab incubate rau 1 h ntawm ib tug benchtop ntawm 121 ° C, txias rau dej khov thiab lim mus rau hauv ib tug high efficiency kua chromatography (HPLC) vial.Qhov concentration ntawm oligosaccharides tau txiav txim siab los ntawm kev rho tawm cov concentration ntawm monosaccharides hauv cov qauv uas tsis yog hydrolyzed los ntawm tag nrho cov piam thaj concentration hauv cov qauv acid-hydrolyzed.
Glucose, xylose, thiab arabinose concentrations nyob rau hauv cov kua qaub hydrolysed biomass tau soj ntsuam siv lub Shimadzu HPLC system nruab nrog ib tug autosampler, kem rhaub, isocratic twj tso kua mis, thiab refractive index detector ntawm ib tug Bio-Rad Aminex HPX-87H kem.Kem tau khaws cia ntawm 50 ° C thiab eluted nrog 0.6 ml / min 5 mM H2SO4 hauv dej.ntws.
Lub hydrolyzate supernatant tau diluted thiab tshuaj xyuas rau cov ntsiab lus monomer thiab oligosaccharide.Monomeric qab zib tau txais tom qab enzymatic hydrolysis tau soj ntsuam los ntawm HPLC nruab nrog Bio-Rad (Hercules, CA) Aminex HPX-87P kem thiab ib qho tshauv guard kem.Qhov ntsuas kub tau khaws cia ntawm 80 ° C, dej tau siv los ua lub sijhawm txawb nrog tus nqi ntws ntawm 0.6 ml / min.Oligosaccharides tau txiav txim siab los ntawm hydrolysis hauv dilute acid ntawm 121 ° C raws li txoj kev tau piav qhia hauv refs.41, 48, 49 ib.
Saccharide tsom xam tau ua tiav ntawm raw, AFEX pre-kho thiab tag nrho cov uas tsis yog-hydrolysed biomass residues (nrog rau kev tsim cov serial cell phab ntsa rho tawm thiab lawv cov mAb tshuaj ntsuam xyuas) siv cov txheej txheem yav dhau los piav qhia 27, 43, 50, 51 .Rau kev tsom xam glycome, cawv-insoluble residues ntawm cov nroj tsuag cell phab ntsa khoom yog npaj los ntawm biomass residues thiab raug serial extraction nrog nce aggressive reagents xws li ammonium oxalate (50 mM), sodium carbonate (50 mM thiab 0.5% w / v), CON.(1M thiab 4M, ob qho tib si nrog 1% w / v sodium borohydride) thiab acid chlorite raws li tau piav qhia dhau los 52,53.Cov kev rho tawm tau raug xa mus rau ELISA tiv thaiv lub vaj huam sib luag nyuaj ntawm mAb50s qhia rau ntawm phab ntsa ntawm tes glycan, thiab mAb kev sib txuas lus tau nthuav tawm raws li daim ntawv qhia tshav kub.mAbs lub hom phiaj cog cell phab ntsa glycan tau yuav los ntawm cov chaw kuaj mob (CCRC, JIM thiab MAC series).
Ib-kauj ruam biotinylation ntawm oligosaccharides.Kev sib txuas ntawm carbohydrates nrog biotin-LC-hydrazide tau ua los ntawm cov txheej txheem hauv qab no.Biotin-LC-hydrazide (4.6 mg / 12 μmol) tau yaj hauv dimethyl sulfoxide (DMSO, 70 μl) los ntawm kev nplawm thiab cua sov ntawm 65 ° C rau 1 min.Glacial acetic acid (30 µl) tau ntxiv thiab qhov sib tov yog nchuav rau hauv sodium cyanoborohydride (6.4 mg / 100 µmol) thiab yaj tag nrho tom qab cua sov ntawm 65 ° C rau li 1 feeb.Tom qab ntawd, los ntawm 5 mus rau 8 μl ntawm cov tshuaj tiv thaiv sib tov tau ntxiv rau cov qhuav oligosaccharide (1-100 nmol) kom tau 10-fold los yog ntau dua molar tshaj ntawm daim ntawv lo rau qhov txo qhov kawg.Cov tshuaj tiv thaiv tau ua tiav ntawm 65 ° C rau 2 h, tom qab ntawd cov qauv raug ntxuav tam sim ntawd.Tsis muaj sodium cyanoborohydride tau siv rau hauv cov ntawv xeem uas tsis muaj kev txo qis, thiab cov qauv tau tshwm sim ntawm 65 ° C. rau 2.5 teev.
ELISA txheej thiab ntxuav cov qauv ntawm biotinylated oligosaccharides.25 μl ntawm cov qauv biotinylated (100 μl ntawm txhua tus qauv concentrated diluted hauv 5 ml ntawm 0.1 M Tris buffer solution (TBS)) tau ntxiv rau txhua qhov dej ntawm avidin-coated phaj.Cov dej tswj tau coated nrog 50 μl ntawm biotin ntawm qhov concentration ntawm 10 μg / ml hauv 0.1 M TBS.Cov dej deionized tau siv los ua cov txheej txheem rau kev ntsuas qhov khoob.Cov ntsiav tshuaj tau incubated rau 2 teev ntawm chav tsev kub hauv qhov tsaus ntuj.Ntxuav lub phaj 3 zaug nrog 0.1% skimmed mis nyuj hauv 0.1 M TBS siv qhov program no.11 rau Grenier tiaj tus 3A.
Ntxiv thiab ntxuav cov tshuaj tiv thaiv thawj zaug.Ntxiv 40 µl ntawm thawj cov tshuaj tiv thaiv rau txhua qhov dej.Incubate lub microplate rau 1 teev ntawm chav tsev kub hauv qhov tsaus ntuj.Cov phiajcim tau raug ntxuav 3 zaug nrog 0.1% mis nyuj hauv 0.1M TBS siv cov kev pabcuam ntxuav #11 rau Grenier Flat 3A.
Ntxiv cov tshuaj tiv thaiv kab mob thib ob thiab ntxuav.Ntxiv 50 µl ntawm nas/rat Secondary antibody (diluted 1:5000 hauv 0.1% mis nyuj hauv 0.1 M TBS) rau txhua qhov dej.Incubate lub microplate rau 1 teev ntawm chav tsev kub hauv qhov tsaus ntuj.Cov microplates raug ntxuav 5 zaug nrog 0.1% mis nyuj hauv 0.1 M TBS siv Grenier Flat 5A phaj ntxuav qhov kev pab cuam #12.
Ntxiv ib lub substrate.Ntxiv 50 µl ntawm 3,3′,5,5′-tetramethylbenzidine (TMB) rau lub hauv paus substrate (los ntawm kev ntxiv 2 tee ntawm tsis, 3 tee ntawm TMB, 2 tee ntawm hydrogen peroxide rau 15 ml ntawm dej deionized).Npaj TMB substrate.thiab vortex ua ntej siv).Incuate lub microplate ntawm chav tsev kub rau 30 feeb.Hauv qhov tsaus ntuj.
Ua kom tiav cov kauj ruam thiab nyeem cov ntsiav tshuaj.Ntxiv 50 µl ntawm 1 N sulfuric acid rau txhua qhov dej thiab sau cov absorbance ntawm 450 mus rau 655 nm siv ELISA nyeem ntawv.
Npaj 1 mg / ml ntawm cov tshuaj analytes hauv dej deionized: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, qabzib, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylg.(glcNAc), N-acetylgalactosamine (galNAc), inositol (tus qauv sab hauv).Ob qho qauv tau npaj los ntawm kev ntxiv 1 mg / mL qab zib cov ntsiab lus qhia nyob rau hauv Table 1. Cov qauv yog khov thiab lyophilized ntawm -80 ° C. kom txog thaum tag nrho cov dej tshem tawm (feem ntau yog li 12-18 teev).
Ntxiv 100-500 µg ntawm cov qauv rau cov ntsia hlau cap raj ntawm qhov ntsuas ntsuas.Sau tus nqi ntxiv.Nws yog qhov zoo tshaj plaws kom yaj cov qauv hauv ib qho tshwj xeeb ntawm cov kuab tshuaj thiab ntxiv rau hauv lub raj ua kua aliquot.Siv 20 µl ntawm 1 mg / ml inositol ua tus qauv sab hauv rau txhua tus qauv raj.Tus nqi ntawm cov txheej txheem sab hauv ntxiv rau cov qauv yuav tsum yog tib yam li cov qauv sab hauv ntxiv rau cov qauv raj.
Ntxiv 8 ml ntawm anhydrous methanol rau hauv lub hau hau vial.Tom qab ntawd 4 ml ntawm 3 N. methanolic HCl tov, capped thiab shaken.Cov txheej txheem no tsis siv dej.
Ntxiv 500 µl ntawm 1 M HCl methanol tov rau cov qauv oligosaccharide thiab tus qauv TMS raj.Cov qauv raug muab tso rau ib hmos (168 teev) ntawm 80 ° C. nyob rau hauv ib lub thermal thaiv.Qhuav cov khoom methanolysis ntawm chav tsev kub siv lub tshuab ziab khaub ncaws.Ntxiv 200 µl MeOH thiab qhuav dua.Cov txheej txheem no rov ua dua ob zaug.Ntxiv 200 μl ntawm methanol, 100 μl ntawm pyridine thiab 100 μl ntawm acetic anhydride rau cov qauv thiab sib tov zoo.Incubate cov qauv ntawm chav tsev kub rau 30 feeb.thiab qhuav.Ntxiv 200 µl ntawm methanol thiab qhuav dua.
Ntxiv 200 µl ntawm Tri-Sil thiab cua sov capped raj rau 20 feeb.80 ° C, ces txias rau chav tsev kub.Siv lub tshuab ziab khaub ncaws kom qhuav cov qauv mus rau qhov ntim ntawm kwv yees li 50 µl.Nws yog ib qho tseem ceeb kom nco ntsoov tias peb tsis tau tso cai rau cov qauv kom qhuav tag.
Ntxiv 2 ml ntawm hexane thiab sib tov zoo los ntawm vortexing.Sau cov lus qhia ntawm Pasteur pipettes (5-8 mm) nrog ib daim iav ntaub plaub los ntawm kev ntxig cov iav ntaub plaub rau saum lub 5-3/4 nti inch pipette.Cov qauv raug centrifuged ntawm 3000 g rau 2 feeb.Txhua yam insoluble residues yog precipitated.Qhuav tus qauv rau 100-150 µl.Ib qhov ntim ntawm kwv yees li 1 μl tau txhaj rau hauv GC-MS ntawm qhov pib kub ntawm 80 ° C thiab lub sijhawm pib ntawm 2.0 feeb (Table 2).