Quantitative Determination of Cellulo Accessibility to Cellula Bad on Adsorption of a Nonhydrolytic Fusion Protein Containing
CBM and GFP with Its Applications
Jiong Hong,Xinhao Ye,and Y.-H.Percival Zhang*摆地摊的货源从哪里批发
Biological Systems Engineering Department,Virginia Tech,Blacksburg,Virginia24061
Recei V ed August20,2007.In Final Form:September18,2007
Heterogeneous cellulo accessibility is an important substrate characteristic,but all methods for determining cellulo accessibility to the large-size cellula molecule have some limitations.Characterization of cellulo accessibility to cellula(CAC)is vital for better understanding of the enzymatic cellulo hydrolysis mechanism(Zhang and Lynd, Biotechnol.Bioeng.2004,88,797-824;2006,94,888-898).Quantitative determination of cellulo accessibility to cellula(m2/g of cellulo)was established bad on the Langmuir adsorption of the fusion protein containing a cellulo-binding module(CBM)and a green fluorescent protein(GFP).One molecule of the recombinant fusion protein occupied21.2cellobio lattices on the
110face of bacterial cellulo nanofibers.The CAC values of veral cellulosic materials s regenerated amorphous cellulo(RAC),bacterial microcrystalline cellulo(BMCC),Whatman No.1filter paper,fibrous cellulo powder(CF1),and microcrystalline cellulo(Avicel)s were41.9,33.5,9.76,4.53, and2.38m2/g,respectively.The CAC value of amorphous cellulo made from Avicel was17.6-fold larger than that of crystalline cellulo s Avicel.Avicel enzymatic hydrolysis proceeded with a transition from substrate excess to substrate limited.The declining hydrolysis rates over conversion are mainly attributed to a combination of substrate consumption and a decrea in substrate reactivity.Declining heterogeneous cellulo reactivity is significantly attributed to a loss of CAC where the easily hydrolyzed cellulo fraction is digested first.
Introduction
Lignocellulo,a major component of the plant cell wall,is produced via photosynthesis.Photosynthesis fixes atmospheric CO2to produce living carbon compounds(mainly,lignocellulo); biodegradation of lignocellulo nds gaous CO2and methane back to the atmosphere.Cellulo,the primary component of lignocellulo,is the most abundant polymeric carbohydrate that human beings do not fully utilize.1,2Sustainable production of transportation ,cellulosic ethanol and hydrogen) from cellulosic materials would provide
benefits to the environ-ment,the economy,and national curity.1,3,4Efficient biological conversion of lignocellulosic biomass to biofuels and biobad chemicals involves three quential steps:lignocellulo pre-treatment,enzymatic cellulo hydrolysis,and fermentation, where enzymatic cellulo hydrolysis plays a central role in producing soluble fermentable sugars from the pretreated solid cellulosic feedstock.5
Enzymatic hydrolysis of crystalline cellulo,a complicated biological process,requires that three types of enzymes s endo-glucanas,exo-glucanas,and -glucosidas s work together.2,5 Cellulo(substrate)characteristics that influence enzymatic hydrolysis rates are believed to include substrate accessibility, crystallinity,degree of polymerization(DP),particle size,pore volume,etc.2,6
None of the methods for measuring cellulo accessibility s nitrogen adsorption-bad Brunauer-Emmett-Teller(BET),size exclusion chromatography,small-angle X-ray scattering(SAXS), microscopy s are perfectly applied in the enzymatic cellulo hydrolysis process becau(1)enzymatic cellulo hydrolysis occurs on the surface of hydrated solid matter in the aqueous ,dried cellulosic samples have completely different supramolecular structures from hydrated samples);2,7(2)cellulas are large-size molecules;and(3)cellula is preferentially adsorbed on the110face of cellulo fibers that cellula can hydrolyze.8-11Small-size molecule adsorption metho
ds,such as BET,water vapor sorption,alkali swelling,or the exchange of H to D atoms with D2H,could result in an overestimation of cellulo accessibility to cellula(CAC).Cellula-size exclusion chromatography can neither differentiate the effective cellulo surface for adsorption and hydrolysis nor account for the external surface.12
Quantitative determination of CAC is valuable for investigating complicated enzymatic cellulo hydrolysis mechanisms.Maxi-mum cellula adsorption capacity has been propod to reprent CAC2and the data in the literature have been summarized and ud before.2,13However,it is relatively difficult to obtain reliable and accurate data bad on adsorption of active cellulas on cellulo becau cellula could hydrolyze substrate during the adsorption measurement,especially for easily hydrolyzed cel-lulo,14resulting in rapid changes in substrate characteristics.15,16 Therefore,many adsorption studies have been conducted at a
*To whom correspondence should be addresd.E-mail:ypzhang@vt.edu. Tel.:540-231-7414.Fax:540-231-3199.
(1)Demain,A.L.;Newcomb,M.;Wu,J.H.D.Microbiol.Mol.Biol.Re V. 2005,69,124-154.
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12535
Langmuir2007,23,12535-12540
10.1021/la7025686CCC:$37.00©2007American Chemical Society
Published on Web11/08/2007
decread ,4°C)to minimize hydrolysis
effects.14,17-21But active cellula adsorption at low temperatures
could be significantly different from that at hydrolysis temper-
atures.17,19,22A new method has been developed for measuring
the scouring efficiencies of natural fibers bad on adsorption95夜色直播
of a fusion protein containing CBM and -glucuronida,but it
was not ud to provide quantitative data for the surface area of
cellulo.23
Cellulo reactivity hydrolyzed by cellula is an intrinsic
property of the solid substrate,associated with veral substrate
characteristics,such as CAC and DP,but it is independent of
enzyme denaturation and production inhibition.The mechanistic
mathematical model13with two substrate properties s DP and
CAC total s has been ud to elucidate a number of disparate phenomena in the literature,such as the effects of substrate
characteristics and experimental conditions on the degree of endo-/
exo-glucana synergy,but it is still unclear whether other
substrate characteristics could greatly influence substrate reactiv-
ity.
班会活动
In this study,we developed a new quantitative determination
for CAC bad on the adsorption of a nonhydrolytic fusion protein
containing thioredoxin,green fluorescent protein(GFP),and
cellulo-binding module(CBM)at a hydrolysis temperature
(50°C).With application to Avicel hydrolysis,we investigated the effects of CAC changes on hydrolysis rate and substrate reactivity.
Materials and Methods
Chemicals and Strains.All chemicals were reagent grade, purchad from Sigma(St.Louis,MO)and Fisher Sci.(Pittsburgh, PA),unless otherwi noted.Microcrystalline cellulo s Avicel PH105(20µm)s was obtained from FMC Corp.(Philadephia,PA). Cellula,Spezyme CP,was gifted from Genencor(P
alo Alto,CA). Escherichia coli TOP10(F-mcr A∆(mrr-hsd RMS-mcr BC)φ80Z∆M15,∆lac X74,rec A1,ara D139∆(ara-leu)7697gal U gal K rps L(Str R)end A1nup G)from Invitrogen(Carlsbad,CA)was ud as a host cell for all DNA manipulations.The li BL21Star(DE3)[F-ompT hsdS B(r B-m B-)gal dcm rne131(DE3)] was ud for recombinant protein expression.LB medium was ud for li growth with ampicillin added when necessary. Gluconacetobacter hannii(ATCC23769)was ud to produce bacterial cellulo(BC).Genomic DNA of Clostridium thermocellum was gifted by Dr.Mielenz at ORNL.The recombinant TGC protein was produced li BL21(pNT02).
Construction of Plasmids.The fusion protein expression plasmid (pNT02)containing thioredoxin-GFP-CBM(TGC)was constructed as shown in Figure1A.First,the green fluorescence protein(gfp) gene from pREST/EmGFP(Invitrogen)was amplified by PCR using a pair of primers(GFP-f,CACCATGGTGAGCAAGGGCGAG-GAGCTG,and GFP-r,GGATCCACGCGGAACCAGTGCCTTG-TACAGCTCGTCCATGCCGAG),and a DNA fragment was ligated into the Invitrogen pET102/D-TOPO vector.In the GFP-r primer, a protea(thrombin)cleavage site was added(Figure1B).This plasmid containing the gfp gene was designated as pNT01.Second, the Clostridium thermocellum cellulo-binding module DNA quence in the cipA gene was amplified by PCR using a pair of prim
ers(CBM3-fc,GATGACGAGCTCCCGGTATCAGGC-AATTTGAAGGTTG,and CBM3-rc,TCATATACCGGTTCAGC-CACCGGGTTCTTTACCCCATACAAG).After double digestion of Sac I and Age I,the CBM fragment was inrted into pNT01for constructing a new plasmid pNT02.The DNA quences of the plasmids were validated by the Virginia Bioinformatics Institute. Preparation of Recombinant Proteins.li BL21Star (pNT02)was grown in a1L flask containing200mL Luria-Bertani medium at37°C.The inducer isopropyl- -D-thiogalactopyranoside (IPTG)(0.2mM)was added until the OD reached0.5-0.6.After IPTG addition,the culture temperature was decread to room temperature and the recombinant TGC protein was expresd overnight.The cell pellets after centrifugation were suspended in 5mL of50mM Tris buffer(pH8.0)and then lyd by the Fisher Scientific Sonic Dismembrator(Model500)at a60%maximum strength for90s.The cell lysate was centrifuged at14000g for20 min.The supernatant containing TGC was precipitated by adding solid ammonia sulfate at50%saturated(NH4)2SO4.The precipitated TGC protein was dissolved in1×native buffer of the Invitrogen ProBond protein purification system.The TGC protein with a His tag was purified using the Ni-NTA column.To obtain the truncated proteins TG and CBM,the purified TGC was hydrolyzed by thrombin at a ratio of10units/mg of TGC at room temperature overnight.The hydrolyzed solution was loaded to a Ni-NTA column.The eluate contained the truncated CBM;the bound protein of TG on the resin was eluted by20mM imidazole.The TGC,TG,and CBM protein soluti
ons were dialyzed in a50mM sodium citric buffer(pH6.0). Preparation of Cellulo.Regenerated amorphous cellulo (RAC),high-reactivity nonsubstitution homologous cellulo,was prepared from Avicel PH105through cellulo slurry dissolution by concentrated phosphoric acid,as described previously.24Bacterial cellulo(BC)was isolated from G.hannii cultures growing on 0.5%peptone,0.5%yeast extract,and2%d-gluco.The bacterial microcrystalline cellulo(BMCC)was prepared from BC by quential treatment of NaOH and HCl.8
Adsorption and CAC Calculation.Protein adsorption on cellulo was conducted in200µL of2mg/mL cellulo suspension
(14)Steiner,W.;Sattler,W.;Esterbauer,H.
Biotechnol.Bioeng.1988,32,
853-865.
爱国诗歌现代诗
(15)Tanaka,M.;Nakamura,H.;Taniguchi,M.;Morita,T.;Matsuno,R.; Kamikubo,T.Appl.Microbiol.Biotechnol.1986,23,263-268.
(16)Fan,L.T.;Lee,Y.-H.;Beardmore,D.H.Biotechnol.Bioeng.1980,22, 177-199.
(17)Ooshima,H.;Sakata,M.;Harano,Y.Biotechnol.Bioeng.1983,25,3103-3114.
(18)Ryu,D.D.Y.;Kim,C.;Mandels,M.Biotechnol.Bioeng.1984,26, 488-496.
(19)Kyriacou,A.;Neufeld,R.J.;MacKenzie,C.R.Biotechnol.Bioeng.1989, 33,631-637.
(20)Reinikainen,T.;Teleman,O.;Teeri,T.T.Proteins:Struct.Funct.Genet. 1995,22,392-403.
(21)Medve,J.;Stahlberg,J.;Tjerneld,F.Appl.Biochem.Biotechnol.1997, 66,39-56.
(22)Kyriacou,A.;Neufeld,R.J.;MacKenzie,C.R.Enzyme Microb.Technol.
1988,10,675-681.
(23)Degani,O.;Gepstein,S.;Dosoretz,C.G.J.Biotechnol.2004,107,265-273.
(24)Zhang,Y.H.;Cui,J.;Lynd,L.R.;Kuang,L.R.Biomacromolecules 2006,7,644-648.
Figure1.Maps of(A)the plasmid pNT02producing a fusion protein TGC containing His-patch Thioredoxin,green fluorescence protein,and CBM;and(B)the fusion TGC protein,where the peptide-specific protea(thrombin)cleavage site was located in the linker between GFP and CBM.
12536Langmuir,Vol.23,No.25,2007Hong et al.
solution in a 50mM sodium citrate buffer (pH 6.0)at 50°C.After adsorption equilibrium (>30min)followed by centrifugation,the free protein molar concentrations of TGC and TG were measured bad on fluorescence readings by the BioTek multidetection microplate reader,and the free CBM molar concentrations were measured by the UV spectrophotometer with an extinction coefficient of 35410M -1cm -1at 280nm.The mass concentrations of TGC were calibrated using the Bradford method with bovine rum albumin as the protein standard.25For BMCC adsorption,2mg/mL BMCC was mixed with the protein concentrations up to 19µmol TGC/mL,13µmol TG/mL,and 24µmol CBM/mL.
Protein adsorption on the solid surface can be described by the Langmuir equation
in which E a is adsorbed cellula (µmol of cellula/L or mg/L),W max is the maximum cellula adsorption per liter (µmol or mg of cellula/L),and E f is free cellula (µmol or mg of cellula/L).The W max and K p values in eq 1can be calculated by a number of mathematical methods or software.26
Cellulo accessibility to cellula (CAC,m 2/g of cellulo)has been defined previously,2,13
where R is the number of cellobio lattices occupied by cellula,A max is the maximum cellula adsorption capacity (mol of cellula/g of cellulo),A max )W max /(106×S ),S is the cellulo concentration (g of cellulo/L),N A is Avogadro’s constant (6.023×1023molecules/mol),and A G2is the area of the cellobio lattice in the 110face (0.53×1.04nm )5.512×10-19m 2).2Total cellulo accessibility to cellula in terms of m 2/L is calculated as
On the other hand,the fraction of -glucosidic bonds accessible to cellula relative to the total number of glucosidic bonds (F a ,unitless)is defined elwhere,2,13
where MW anhydrogluco )162g/mol of anhydrogluco.
Enzymatic Hydrolysis of Avicel and Relevant Assays.Enzy-matic cellulo hydrolysis was conducted
in a 1L flask containing 400mL of 10g of Avicel/L in a 50mM citrate buffer (pH.4.8)with rotary shaking at 180rpm and 50°C.The enzyme loadings were 15FPU cellula/g of Avicel and 30IU cellobia/g of Avicel.The hydrolysate samples were withdrawn and stopped by mixing with 10M NaOH at a ratio of 20µL of alkali per mL of Avicel hydrolysate.27After centrifugation,the soluble sugars in the supernatant were measured by the phenol-sulfuric acid method.5The relationship between soluble sugars (g/L)vs hydrolysis time (h)was fitted by CurveExpert (Version 1.38).Hydrolysis rates at various times were calculated at the time t +10min minus sugar produced at the time t .After careful removal of the adsorbed cellula on the surface of cellulo (e the description below),characteristics of the remaining cellulosic pellets s substrate reactivity,DP,and CAC s were measured.The alkalinized solid cellulo pellets were suspended by 1.1%SDS solution and incubated at 80°C for 15min to further remove the adsorbed cellula on the surface of cellulo.After centrifugation,the solid samples were suspended and washed by 75%(v/v)ethanol three times and distilled water twice.The complete removal of adsorbed cellula from the surface of cellulo was confirmed by the Lowry Assay.28The residual cellulo was
suspended to produce 10g/L concentration by water or the adsorption buffer or the hydrolysis buffer.The concentration of reducing sugar ends in solid cellulo was determined by the modified B
CA method,as described previously.27The concentration of total gluco equivalent in solid cellulo was measured by the phenol-sulfuric acid method.27
Degree of Polymerization (DP)Determination .The number-average DP of cellulo was calculated by the ratio of glucosyl monomer concentration (determined by the phenol-sulfuric acid method)divided by the reducing-end concentration (determined by the modified BCA method).27
Substrate Reactivity Assay.Substrate reactivity was determined at the conditions (10g of cellulo/L in citrate buffer (pH 4.8),enzyme loadings of 15FPU cellula/g of cellulo and 30IU cellobia/g of cellulo)at a temperature of 50°C.Initial hydrolysis rates (within 10min)bad on the relead total soluble sugars (gluco equivalent)were ud to reprent substrate reactivity.Electrophoresis.SDS-PAGE was carried out in 12%Tris-HCl Gel.
Results
Protein Purification and Adsorption.Surface area is an important characteristic for heterogeneous materials.The existing surface area measurement means for cellulo are not suitable for quantitative determination of cellulo accessibility to cellula,a large-size molecule that preferentially adsorbs on the 110face of cellulo.Here we propod to produce a recombinant nonh
ydrolytic protein containing GFP and CBM.The recombinant protein expression plasmid (pNT02)was constructed bad on the T7promoter-bad vector for producing the fusion protein containing thioredoxin,GFP,and CBM,called TGC (in Figure 1B).The TGC protein has a molecular weight of 62kD,which is similar to that of Trichoderma reei endo -glucana I.2Large amounts of the recombinant protein were produced by li BL21Star strain after IPTG was added.The harvested cells were lyd by sonification,and then soluble TGC protein was purified through its His tag by Ni-NTA resin.
GFP is a convenient powerful tool for in vivo and in vitro applications.The GFP-containing TGC protein shows a strong green color and a very strong fluorescence under UV excitation (Figure 2A).No fluorescence was obrved from BMCC (Figure 2C).When TGC was mixed with BMCC,TGC was adsorbed on the surface of cellulo through CBM.After centrifugation,the BMCC with the adsorbed TGC shows a strong fluorescence (Figure 2B),suggesting that TGC can specifically bind on cellulo.The TGC protein with its strong fluorescence signal is convenient for high-nsitivity measurement.The CBM binding on the surface of cellulo was highly specific;no such binding was obrved on the control substrate s starch (data not shown).
(25)Bradford,M.M.
Anal.Biochem.1976,72,248-254.
(26)Bothwell,M.;Walker,L.P.Biores.Technol.1995,53,21-29.
(27)Zhang,Y.-H.P.;Lynd,L.R.Biomacromolecules 2005,6,1510-1515.(28)Lowry,O.;Robrough,N.J.;Farr,A.L.;Randall,R.J.J.Biol.Chem.1951,193,265-275.
E a )
W max K p E f 1+K p E f
(1)
CAC )R A max N A A G2
(2)
CAC Total )CAC ×S
(3)
F a )2R A max MW anhydrogluco
事半功倍造句(4)
Figure 2.Photos of the TGC solution (A),a mixture of TGC and BMCC after adsorption followed by precipitation (B),and the bacterial microcrystalline cellulo suspension solution (C)under UV excitation.
Determining Cellulo Accessibility to Cellula
Langmuir,Vol.23,No.25,200712537
Determination of Cellulo Accessibility to Cellula.The fusion TGC protein can be cleaved to two tr
uncated proteins s TG protein and CBM s by treatment with a peptide-specific protea s thrombin.The SDS-PAGE results show that the purified TGC and two truncated proteins have molecular weights of 62,45,and 17kD,respectively,which are consistent with their amino acid quences (data not shown).The adsorption curves of three proteins on BMCC are prented in Figure 3.TGC and CBM were adsorbed on cellulo,while TG did not adsorb on BMCC becau it had no cellulo-binding module.The adsorption data of TGC and CBM matched well with the Langmuir equations (Figure 3).The maximum protein adsorption capacities (A max )were 295(13mg of TGC/g of BMCC and 4.75(0.21µmol of TGC/g of BMCC,as well as 121(12mg of CBM/g of BMCC and 6.94(0.69µmol of CBM/g of BMCC,respectively (Table 1).
Cellulo-binding module or cellula,much larger than the cellobio lattice,occupies a number of cellobio lattices.The nanoribbons of BMCC have a cross ction with a dimension of 40×15nm.The edges of ribbons (15nm)are the 110face where cellulas adsorb and hydrolyze.2,8,9According to BMCC’s geometric shape and its specific density of crystalline cellulo (1.5g/cm 3),2the area of the 110face of BMCC is approximately 101µmol of cellobio lattice or 33.53m 2/g of BMCC,respectively.The R value of the TGC protein,the number of cellobio lattices occupied by the adsorbed TGC,was calculated to be 21.2by a ratio of the 101µmol of cellobio latti
ce/g of BMCC to 4.76µmol of TGC/g of BMCC.The small-size CBM protein had a slightly smaller R value of 14.6(Table 1),which was in agreement with the geometric structure of CBM (∼3×4nm).29
The CAC values of a number of cellulosic materials were determined for the first time according to eq 2.They are 2.38m 2/g of Avicel,4.53m 2/g of cellulo CF1,9.76m 2/g of Whatman No.1filter paper,33.53m 2/g of BMCC,and 41.91m 2/g of RAC (Table 2).Avicel (microcrystalline cellulo),which was made from wood pulp by removing the amorphous fraction through strong acid hydrolysis,has the lowest CAC.In contrast,RAC,made from regeneration of cellulo-solvent-dissolved Avicel,has the highest CAC.Some cellulosic materials (such as CF1and filter paper)have modest CAC values becau they may be regarded as a mixture of crystalline and amorphous cellulo.Enzymatic Hydrolysis of Avicel.Heterogeneous cellulo (Avicel)hydrolysis mediated by the fungal Trichoderma cellula was conducted with an enzyme loading of 15FPU +30IU -glucosida/g of Avicel at 50°C (Figure 4A).Cellulo conversion rapidly ro to 31%after the first 4h and then slowed.Total CAC decread drastically from 23.92to 13.13m 2/L at hour 4to 1.48m 2/L at hour 72,while reducing ends of solid cellulo decread from 231.4to 161.7µM at hour 4to 27.3µM at hour 72.Figure 4B prents a very drastic decline in hydrolysis rate with time,especially after the first veral hours,
(29)Bayer,E.A.;Chanzy,H.;Lamed,R.;Shoham,Y.
Curr.Opin.Struct.Biol.1998,8,548-557.
Figure 3.Adsorption of purified TGC,TG,and CBM proteins on 2mg/mL bacterial microcrystalline cellulo at 50°C.The dot curves were drawn by Langmuir equations bad on the parameter tting in Table 1.
Figure 4.Profile of Avicel (10g/L)enzymatic hydrolysis at an enzyme loading of 15FPU +30IU -glucosida per gram of cellulo in a 50mM sodium citrate buffer (pH 4.8)at 50°C.(A)Changes of cellulo conversion,reducing ends,and CAC of solid residual cellulo.(B)Changes in hydrolysis rate,enzyme dena-turation,and cellula/(A max S ),i.e.,E /S .
Table 1.Determination of the r Values for TGC,CBM,and TG Bad on Adsorption on the Standard Substrate s BMCC a
A max
protein mg/g µmol/g R TGC 295(13 4.76(0.2121.2(1.0CBM 121(12 6.94(0.6914.6(1.4
TG
a
Adsorption was conducted at 2mg of BMCC/mL in a 50mM citrate buffer with different protein concentrations at 50°C.
Figure 5.Profile of substrate reactivity,CAC,and DP as a function of substrate conversion.
12538Langmuir,Vol.23,No.25,2007Hong et al.
co-incident with a sharp ri in cellula/(A max S)from0.41to 1.15at hour12,a transition from substrate excess to substrate limited.Declining hydrolysis rates over conversion have been attributed
to a number of factors,such as enzyme denaturation, enzyme inhibition,changes in substrate characteristics,and so on.At the same time,enzyme denaturation was minor(<5%) for4days(Figure4B).All results suggest that rapid consumption in reducing ends for exo-glucanas and cellulo accessibility for endo-glucana could be major factors for the decreasing hydrolysis rates during the hydrolysis process.
To eliminate the effects of enzyme denaturation,product inhibition,and changes in E/S,we further investigated substrate characteristics and substrate reactivity at different conversion degrees.We developed a protocol that can efficiently remove residual cellula from the surface of cellulo by quentially using alkali,1%SDS at an elevated temperature(80°C),organic solvent(ethanol)washing,and water washing(e the details in Materials and Methods ction).The experimental data clearly indicated the complete removal of adsorbed cellula on the surface and no changes in cellulo reactivity(data not shown). We measured substrate reactivity bad on initial soluble sugar production rates by adding fresh cellula to a fixed concentration (10g of cellulo/L)of cellulosic materials that were withdrawn at different hydrolysis times,followed by careful removal of all adsorbed cellula from cellulosic samples.Avicel reactivity decread by∼80%with cellulo conversion,accompanied by a decrea in CAC from23.9to12.4m2/g and near
ly constant DPs over conversion(Figure5).Our results could for the first time provide clear quantitative evidence for the common belief that the easily hydrolyzed fraction of cellulo with larger CAC is hydrolyzed first.
Discussion
A new quantitative determination of CAC was established bad on the maximum adsorption capacity of the nonhydrolytic fluorescence fusion TGC protein,which is similar in size to the Trichoderma cellula.Recently,a fusion protein containing a fluorescent protein and a cellulo-binding module was developed to check plant cell wall substrate binding specificity,30but Ding et al.did not u it for quantitative determination of cellulo accessibility to cellula.Two other advantages of determining CAC bad on adsorption of the fusion protein containing GFP and CBM at hydrolysis temperatures were(1)low protein u becau the fluorescent protein assay has a higher signal than do protein mass concentration assays and(2)better Langmuir adsorption at low enzyme ,low interference between adsorbed protein and free protein).In our experimental condition (50°C),one molecule of the TGC protein occupied21.2cellobio lattices.This value was consistent with other results of∼16 cellobio lattices i cellobiohydrola I,2032.9per Cellulomonas fimi CenA,and27.9per C.fimi Cex.8Some variations in R value could be attributed to m
olecular size and
龙腾飞experimental conditions,such as temperature and ionic strength.20 The measured CAC value of Avicel(FMC PH105)was2.38
m2/g,which reasonably fell between0.3m2external area/g of
Avicel31and20m2gross area/g of Avicel measured by nitrogen
adsorption.32Our results suggest that87%accessible area of
Avicel to cellula is internal and that88%of gross area accessible
by nitrogen molecules is not accessible to large-size proteins.
CAC values in order were Avicel<CF1<filter paper<BMCC <RAC(Table2),in relatively good consistency with the results in the literature.2The substrate reactivity incread in the order
CF1<filter paper<Avicel<BMCC<RAC(data not shown).
Comparison of CAC and substrate reactivity suggest that CAC
is one of the key substrate characteristics,but not the sole primary
one,which impacts enzymatic cellulo hydrolysis rates. The enzymatic hydrolysis rate decread drastically as hydrolysis proceeded.Such decline has been attributed to the following(1)enzyme-associated factors,such as enzyme denaturation and enzyme product inhibition;(2)substrate reactivity,which is associated with substrate accessibility,degree of polymerization(DP),crystallinity,particle size,pore volume, etc.;(3)changes in enzyme/substrate ratio.A number of rearchers have reported minor effects of enzyme-associated factors on decread hydrolysis rates such as weak enzyme deactivation(within a range of veral days)and low product inhibitionatlowsugarconcentrationwithsufficient -glucosida.33-35 We have speculated that the availability of glucan and chain ends per gram of cellulo may decrea with conversion.2This is supported by our results(Figure4A).Although cellulo accessibility has long been believed to be important for the study of enzymatic cellulo hydrolysis mechanisms,surprisingly,to our knowledge,only two attempts have been made to measure cellulo accessibility changes over time by using the BET method16or by using active cellula adsorption.17Becau of the limitations of BET and active cellula adsorption methods, no clear quantitative conclusion has been made between hydrolysis rate and cellulo accessibility.A drastic
decrea in total CAC over conversion resulted in a transition from substrate excess[cellula/(A max S))0.41]to substrate limited[cellula/ (A max S))4.65],in good agreement with the commonly obrved fact that a large fraction of cellula is relead in the aqueous pha at the end of hydrolysis.35-37We may be the first to provide a quantitative value indicating such transition due to the low CAC of cellulo(low F a value in ,most -glucosida bonds of cellulo are not accessible to cellula).Different from soluble substrate bioconversion catalyzed by enzymes(substrate
(30)Ding,S.Y.;Xu,Q.;Ali,M.K.;Baker,J.O.;Bayer,E.A.;Barak,Y.; Lamed,R.;Sugiyama,J.;Rumbles,G.;Himmel,M.E.Biotechniques2006,41, 435-440.
(31)Weimer,P.J.;Lopez-Guisa,J.M.;French,A.D.Appl.En V iron.Microbiol. 1990,56,2421-2419.
(32)Marshall,K.;Sixsmith,D.Drug De V.Commun.1974,1,57-71.
(33)Zhang,S.;Wolfgang,D.E.;Wilson,D.B.Biotechnol.Bioeng.1999,66, 35-41.
(34)Desai,S.G.;Conver,A.O.Biotechnol.Bioeng.1997,56,650-655.
(35)Tu,M.;Chandra,R.P.;Saddler,J.N.Biotechnol.Prog.2007,23,398-406.
(36)Zhang,Y.-H.P.;Lynd,L.R.Anal.Chem.2003,75,219-227.
(37)Zhang,Y.-H.P.;Lynd,L.R.J.Bacteriol.2005,187,99-106.
Table2.Maximum Protein Adsorption Capacity,CAC,and Fraction of -Glucosidic Bond Accessible to Cellula(F a)for Various Cellulosic Materials Determined Bad on the Nonhydrolytic TGC Protein Adsorption
A max CAC F a淄博名吃
cellulo mg/gµmol/g(m2/g)(unitless)
Avicel21.0(0.850.338(0.014 2.38(0.0960.00232(0.00011 cellulo CF139.9(1.30.644(0.021 4.53(0.150.00442(0.00021 filter paper86.2(2.8 1.39(0.0459.76(0.320.00954(0.00045 BMCC295(13 4.76(0.2133.5(1.50.0327(0.0016
RAC369(19 5.97(0.3141.9(2.20.0410(0.0020 Determining Cellulo Accessibility to Cellula Langmuir,Vol.23,No.25,200712539
