Báo cáo khoa học: Surface density of cellobiohydrolase on crystalline celluloses A critical parameter to evaluate enzymatic kinetics at a solid–liquid interface ppt

Samejima, Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan Fax: +81 3 584152

A critical parameter to evaluate enzymatic kinetics at a solid–liquid interface

Kiyohiko Igarashi, Masahisa Wada, Ritsuko Hori and Masahiro Samejima

Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan

Cellulose degradation is one of the most important

processes in the carbon cycle, since cellulose is the

major component of the cell wall of plants and the

most abundant polymer in nature In addition to

the cell wall of terrestrial plants, cellulose is found in

marine algae, marine animals and bacteria, and it

gen-erally consists of a mixture of crystalline (cellulose I)

and disordered amorphous regions Cellulose I is

fur-ther classified into two polymorphs, triclinic cellulose

Ia and monoclinic cellulose Ib [1–3], whose detailed structures have been established recently through syn-chrotron X-ray and neutron fiber diffraction studies [4,5] Cellulose Iais metastable, and is irreversibly con-verted into cellulose Ib by hydrothermal treatment in alkaline solution [6]

To degrade cellulose, many organisms produce cellu-lases that hydrolyze b-1,4-glucosidic linkages of the polymer Almost all cellulases can act at amorphous

Keywords

cellobiohydrolase; cellobiose

dehydrogenase; crystalline cellulose;

glycoside hydrolase; solid–liquid interface

Correspondence

M Samejima, Department of Biomaterials

Sciences, Graduate School of Agricultural

and Life Sciences, The University of Tokyo,

1-1-1 Yayoi, Bunkyo-ku, Tokyo 113–8657,

Japan

Fax: +81 3 58415273

Tel: +81 3 58415255

E-mail: amsam@mail.ecc.u-tokyo.ac.jp

(Received 3 April 2006, revised 26 April

2006, accepted 2 May 2006)

doi:10.1111/j.1742-4658.2006.05299.x

The enzymatic kinetics of glycoside hydrolase family 7 cellobiohydrolase (Cel7A) towards highly crystalline celluloses at the solid–liquid interface was evaluated by applying the novel concept of surface density (q) of the enzyme, which is defined as the amount of adsorbed enzyme divided by the maximum amount of adsorbed enzyme When the adsorption levels of Trichoderma viride Cel7A on cellulose Iafrom Cladophora and cellulose Ib from Halocynthia were compared, the maximum adsorption of the enzyme

on cellulose Ib was
1.5 times higher than that on cellulose Ia, although the rate of cellobiose production from cellulose Ib was lower than that from cellulose Ia This indicates that the specific activity (k) of Cel7A adsorbed on cellulose Iais higher than that of Cel7A adsorbed on cellulose

Ib When k was plotted versus q, a dramatic decrease of the specific activity was observed with the increase of surface density (q-value), suggesting that overcrowding of enzyme molecules on a cellulose surface lowers their activ-ity An apparent difference of the specific activity was observed between crystalline polymorphs, i.e the specific activity for cellulose Ia was almost twice that for cellulose Ib When cellulose Iawas converted to cellulose Ib

by hydrothermal treatment, the specific activity of Cel7A decreased and became similar to that of native cellulose Ib at the same q-value These results indicate that the hydrolytic activity (rate) of bound Cel7A depends

on the nature of the crystalline cellulose polymorph, and an analysis that takes surface density into account is an effective means to evaluate cellulase kinetics at a solid–liquid interface

Abbreviations

BMCC, bacterial microcrystalline cellulose; CBD, cellulose-binding domain; CD, catalytic domain; CDH, cellobiose dehydrogenase; FT-IR, Fourier transform infrared spectrometer; GH, glycoside hydrolase; TEM, transmission electron microscope.

regions of cellulose, whereas only a limited number

can hydrolyze crystalline cellulose [7]

Cellobiohydro-lase, belonging to glycoside hydrolase (GH) family 7,

is the major secreted protein of many cellulolytic fungi

and is one of the best studied of the enzymes

hydrolyz-ing crystalline cellulose to cellobiose [7–11] These

enzymes have a two-domain structure: a
50 kDa

catalytic domain (CD) and a small (3 kDa)

cellulose-binding domain (CBD) connected by a highly

O-gly-cosylated linker region [12–15] Loss of the CBD

causes a significant decrease of crystalline cellulose

decomposition, but has less effect on the hydrolysis of

soluble or amorphous cellulose [16], suggesting that

the adsorption of the enzyme on the surface via the

CBD is important for the effective hydrolysis of

crys-talline cellulose [17–21] However, if an excess amount

of the enzyme is adsorbed, the CD is unable to bind

appropriately to the cellulose chain owing to steric

interference by other enzyme molecules This is called

nonproductive binding [15], and the hydrolysis of

crys-talline cellulose is inhibited, even though the amount

of bound enzyme is increased [22]

Although the kinetics of crystalline cellulose

hydro-lysis by cellulases has been investigated intensively, it

remains difficult to compare findings, because of the

variability of cellulose samples The main reason for

this variability is the difference of surface area between

celluloses from different sources and⁄ or different

prep-arations When the hydrolytic activity of cellulase for

one cellulose sample is higher than that for another, it

is difficult to determine whether this is because the

sample has a larger surface area available to the

cellu-lase, or whether the sample is indeed more susceptible

to degradation In the present study, we therefore

investigated a novel approach to evaluate cellulase

kin-etics on solid substrates by using the surface density of

the enzyme (defined as the adsorbed amount of the

enzyme divided by the maximum adsorption of the

enzyme) as a parameter, in order to avoid the influence

of heterogeneity of crystalline cellulose

Results

Analysis of highly crystalline celluloses

Highly crystalline celluloses, cellulose Iafrom

Cladopho-raand cellulose Ibfrom Halocynthia and from

hydro-thermally treated Cladophora, were characterized by

transmission electron microscope (TEM), synchrotron

diffraction, and Fourier transform infrared spectrometer

(FT-IR) Electron micrographs (Fig 1A–C) showed

that cellulose microcrystals prepared by hydrochloric

acid treatment appear as slender rods, more than 1 mm

in length and about 20 nm wide Although the micro-graphs are very similar, differences were observed in the synchrotron X-ray fiber diffraction diagrams (Fig 1D– F) The diagrams of crystalline celluloses from Halocyn-thia (Fig 1E) and hydrothermally treated Cladophora (Fig 1F) were typical of resolved Ib patterns, whereas that of Cladophora cellulose showed patterns of both cellulose Ia and Ib The FT-IR spectra of the samples were different (Fig 2) The characteristic peaks of cellu-lose Ia(3240 cm)1) and cellulose Ib (3270 cm)1) in the spectrum of Cladophora cellulose were consistent with a mixture of 70% cellulose Ia and 30% cellulose Ib

(Fig 2A), whereas only the peak at 3270 cm)1was seen

in the spectra of Halocynthia (Fig 2B) and hydrother-mally treated Cladophora (Fig 2C) celluloses This is because the hydrothermal treatment converted Clado-phoracellulose Iato cellulose Ib

Adsorption of Cel7A on crystalline celluloses The enzyme concentration dependence of adsorbed Cel7A was estimated at various time points of tion Figure 3A shows the results at 120 min of incuba-tion and Fig 3B is the Scatchard plot (A-A⁄ [F]) of the data in Fig 3A Cellulose Ibfrom Halocynthia showed the highest adsorption of Cel7A, which was approxi-mately 1.5 times higher than that of cellulose Iafrom Cladophoraat all Cel7A concentrations tested Since the Scatchard plots (Fig 3B) for the three cellulose samples were all nonlinear, the binding of Cel7A cannot be fitted

to a simple Langmuir equation; instead, a two-binding site model (Eqn 1) should be employed for simulation The adsorption parameters obtained by simulation using Eqn 1 are summarized in Table 1 Although the adsorption constants for high-affinity binding (Kad1) varied among substrates, those for low-affinity binding (Kad2) were all quite similar The hydrothermal treat-ment, which converts cellulose Ia to cellulose Ib, decreased Kad1and increased A1, but had no effect on

Kad2or A2 The maximum amount of adsorbed enzyme (Amax) for cellulose Ib from Halocynthia was 3.2 ± 0.4 nmolÆmg cellulose)1, which was 1.5 times higher than that for cellulose Ia from Cladophora (2.2 ± 0.2 nmolÆmg cellulose)1)

Hydrolysis of highly crystalline celluloses The time course of changes in cellobiose concentration was monitored for various concentrations of Cel7A using highly crystalline celluloses as substrates Figure 4 shows the degradation of cellulose Iafrom Cladophora

as a representative result The hydrolysis of the crystalline cellulose was well fitted by the double

expo-nential plot versus time (Eqn 7), which shows an initial

rapid increase followed by constant production of

cellobiose The cellobiose production increased with

increase of total Cel7A concentration up to 2.2 lm

(Abs280
0.2), but decreased at higher concentrations

The velocities of cellobiose production were estimated

by differentiation of cellobiose concentration in the

reaction mixture, as described in Experimental

proce-dures, then plotted versus Cel7A concentration

Figure 5 shows the results obtained at the incubation

time of 120 min As expected from the time course

of cellobiose concentration, cellobiose production by

Cel7A from cellulose Ia increased with increasing

enzyme concentration, reaching a maximum value

(0.56 lmolÆmin)1) at a free enzyme concentration, [F],

of 1.3 lm, and then decreasing with further increase of

enzyme concentration to 0.42 lmolÆmin)1 at [F]¼

6.9 lm Similar patterns were obtained using cellulose Ib

from Halocynthia and hydrothermally treated

Cladopho-ra as substrates, although the concentration

provid-ing maximum cellobiose production was lower

([F]
0.5 lm) than in the case of cellulose Ia from

Cladophora

Surface density plot of Cel7A

To analyze the difference between the hydrolytic properties towards cellulose Ia and cellulose Ib, the specific activity of adsorbed enzyme (k) was plotted versus surface density of Cel7A (q), as shown in Fig 6 The specific activity towards all crystalline celluloses was high at low surface density, but decreased with increase of the q-value, suggesting that the crowding of Cel7A on the surface of crys-talline celluloses causes a decrease of the activity The specific activity for cellulose Ia from Cladophora was approximately twice that for cellulose Ib from Halocynthia Interestingly, hydrothermal treatment caused a significant decrease of specific activity for Cladophora cellulose, and the q–k curve became quite similar to that for cellulose Ib from Halocynthia, although these celluloses had been prepared from different sources by different methods This suggests that the surface density plot compensates for the dif-ferent surface areas of crystalline celluloses, and reflects the specific activity of Cel7A for the crystal-line polymorphs

Fig 1 TEM pictures (top row) and synchrotron X-ray fiber diffraction diagrams (bottom row) of highly crystalline celluloses Bar indicates

500 nm (A) and (D) Cellulose I a from Cladophora; B and E, cellulose I b from Halocynthia; C and F, cellulose I b from hydrothermally treated Cladophora Circles in the bottom row indicate characteristic differences between cellulose Iaand cellulose Ib.

Cellobiose production and high- and low-affinity

absorption were plotted versus surface density, as

shown in Fig 7 The cellobiose production reached

maximum at q¼ 0.4 (cellulose Ia from Cladophora)

and q¼ 0.3 (cellulose Ib from Halocynthia and

hydro-thermally treated Cladophora), suggesting that

suffi-cient space for another 1.5 or 2.3 enzyme molecules

per adsorbed molecule must be left free in order to

achieve optimum hydrolysis of crystalline cellulose

The surface density dependence at high- and

low-affin-ity adsorption sites (solid and dashed lines,

respect-ively) showed that the high-affinity curve almost

reaches saturation at the q-value of 0.4 (cellulose Ia) or

0.3 (cellulose Ib), whereas the low-affinity curve rises

linearly with increase of q Moreover, the cellobiose

production increased at lower concentration, where the

high-affinity adsorption was observed, whereas it

declined with increase of low-affinity adsorption These

results may indicate that the high- and low-affinity

binding curves represent the amounts of productive

and nonproductive enzyme, respectively

Discussion

The hydrolysis of crystalline cellulose has generally been

evaluated using microcrystalline cellulose [(Avicel),

FMC Corp, Newark, DE] as a substrate, but heterogen-eity of the substrate often causes variable results in the case of cellobiohydrolase [7,20] To avoid this difficulty, bacterial microcrystalline cellulose (BMCC) has been used as a homogeneous crystalline cellulose substrate instead [22–24] However, as we have shown, the proper-ties of BMCC as a substrate of cellulase are strongly dependent on the preparation conditions [25] In the present study, we wished to compare the highly crystal-line celluloses from Cladophora and Halocynthia, and faced difficulties in evaluating their hydrolysis, presuma-bly because of the differences of surface area and⁄ or sur-face structure There are several techniques to estimate the surface area of solid cellulose from the amounts of

A

B

Fig 3 Enzyme concentration dependence of the amount of adsorbed Cel7A (A) and Scatchard plot (B). n, cellulose Ia from Cladophora; s, cellulose I b from Halocynthia; d, cellulose I b from hydrothermally treated Cladophora The adsorption of Cel7A was measured after incubation for 120 min with 1 mgÆmL)1of crystalline cellulose at 30
C as described in Experimental procedures The lines represent fitting the data to Eqn 1 in Experimental procedures.

A

B

C

Fig 2 FT-IR spectra of highly crystalline celluloses A, cellulose Ia

from Cladophora; B cellulose Ib from Halocynthia; C, cellulose Ib

from hydrothermally treated Cladophora Dotted line shows

charac-teristic peaks of cellulose Ia and cellulose Ibat 3240 cm)1 (right)

and 3270 cm)1(left), respectively.

bound small molecular compounds, such as nitrogen,

water or dye, but the results cannot be used to evaluate

the surface area available to cellulases, since CBDs are

adsorbed only on limited regions of crystalline cellulose,

mainly hydrophobic surfaces, as demonstrated

previ-ously [26–29] Therefore, we developed the novel

con-cept of using surface density as a parameter to express

the adsorption of cellobiohydrolase relative to the

maxi-mum amount of adsorption of the enzyme (Amax), in

order to obtain the specific activity of Cel7A for

crystal-line cellulose

This approach has several advantages: (1) Amax

pro-vides a measure of the surface area of crystalline

cellu-lose available as a substrate of cellulase It is reported

that cellulose Ibfrom Halocynthia has a greater

hydro-phobic surface than cellulose Ia from Cladophora [30]

Indeed, in the present study, Amax of Cel7A on

cellu-lose Ib from Halocynthia was 1.5 times higher than

that on cellulose Ia from Cladophora (2) Generally,

specific activity of cellulase is evaluated based on

the amount of added enzyme However, this is

Fig 5 Free enzyme concentration dependence of cellobiose pro-duction by Cel7A after incubation for 120 min.n, cellulose Iafrom Cladophora; s, cellulose Ibfrom Halocynthia; d, cellulose Ibfrom hydrothermally treated Cladophora The rate of cellobiose produc-tion was estimated by the fitting the time course of cellobiose con-centration to Eqn 7, and by calculation using Eqn 8.

Fig 6 Surface density dependence of specific activity for adsorbed enzyme after incubation for 120, 180, 240, and 320 min.n, cellu-lose I a from Cladophora; s, cellulose I b from Halocynthia; d, cellu-lose Ib from hydrothermally treated Cladophora q- and k-values were estimated by using Eqns 3 and 9, respectively.

Table 1 Adsorption parameters of highly crystalline celluloses for Cel7A The adsorption parameters were calculated by nonlinear fitting of the data after incubation for 120, 180, 240, 320 min to Eqn 1.

Hydrothermally

treated Cladophora

a

l M )1,b

nmolÆmg cellulose)1.

Fig 4 Time course of cellobiose production from Cladophora

cellu-lose by Cel7A The total concentration of Cel7A in the reaction

mix-ture was as follows:n, 0.40 l M ; d, 0.84 l M ; m, 1.3 l M ; h, 2.2 l M ;

s , 4.3 l M ; n, 8.6 l M The cellobiose concentration in the reaction

mixture was measured with a CDH–cytochrome c redox system as

described in Experimental procedures.

inappropriate for cellobiohydrolases, since only

adsorbed enzyme represents ‘working enzyme’ which

generates the product (cellobiose) Therefore, we

should evaluate the specific activity of adsorbed

enzyme (3) During the hydrolytic process, the shape

and surface area of the solid substrate should change

with the reaction time By using surface density as a

parameter, however, we can monitor the changes of

surface area and compensate for them, whether they

arise from the nature of the cellulose preparations, or

from changes during hydrolysis In the present study, indeed, the Amax values decreased slightly with increas-ing incubation time, perhaps because of a reduction of the surface area owing to enzymatic degradation (data not shown) However, cellobiose production also decreased correspondingly with increasing incubation time, suggesting that the surface density plot can allow for the real-time changes of the substrate caused by the enzymatic reaction

In nature, there are two crystalline polymorphs of cellulose, celluloses Iaand Ib[1–3], and cellulose Iahas been reported to be degraded much faster than cellu-lose Ib [31,32] To analyze the differences in degrada-bility in detail, we prepared three crystalline cellulose samples, Ia-rich crystalline cellulose from Cladophora, natural cellulose Ib from Halocynthia, and cellulose Ib generated by hydrothermal treatment of Cladophora cellulose, and we compared the hydrolysis of these samples by Cel7A The q–k plot of Cel7A (Fig 6) clearly indicates that the higher degradability of cellu-lose Iais mainly due to a higher specific activity of the enzyme for this substrate than for cellulose Ib, but is not due to a larger surface area As hydrothermal treatment does not cause any change of shape of cellu-lose microfibrils [33], differences of specific activity should reflect differences in the arrangements of cellu-lose chains in the two crystalline polymorphs Quite recently, the detailed structures of celluloses Ia and Ib

were solved by synchrotron X-ray and neutron fiber diffraction analyses [4,5] The top views of the hydro-phobic surfaces of celluloses Iaand Ibare compared in Fig 8 If cellulose chains of the first layer (colored cyan) are superimposed in the two crystalline poly-morphs, the cellobiose units in the second layer of cel-luloses Ia (colored yellow) are completely opposed to those of cellulose Ib(colored green) This suggests that Cel7A can distinguish this difference between the first and second layers of crystalline celluloses A possible reason for this is that the structural difference may cause a difference of steric hindrance at CBD or CD, and thus may affect the processivity of Cel7A on the crystalline celluloses [8,22,34]

The enzyme concentration dependence of absorp-tion ([F]–A plot; Fig 3A) fitted well to the two-bind-ing site equation reported by Sta˚hlberg et al [16] In addition, when the high- and low-affinity adsorption curves and cellobiose production were plotted versus surface density (Fig 7), it appeared that cellobiose production increased in the high-affinity phase of adsorption, whereas it was apparently inhibited with increase of low-affinity binding This may be because high-affinity adsorption involves both CD and CBD (productive binding), whereas low-affinity adsorption

A

B

C

Fig 7 Surface density dependence of high- (solid line) and

low-affinity (dashed line) adsorption of Cel7A with plot of cellobiose

pro-duction These lines are drawn using the parameters in Table 1,

and the plots were obtained from the results after incubation for

120, 180, 240, and 320 min A, cellulose Iafrom Cladophora; B,

cel-lulose Ibfrom Halocynthia; C, cellulose Ibfrom hydrothermally

trea-ted Cladophora.

may involve only CBD (nonproductive binding) In

Table 1, moreover, a higher Kad1-value was observed

for cellulose Ia than cellulose Ib, although Kad2 for

all samples were quite similar to each other This

phenomenon might be explained by the different

affinity of productive binding, i.e CD of Cel7A may

hold cellulose Ia more tightly than cellulose Ib,

resulting in higher cellobiose production from

cellu-lose Ia than cellulose Ib at same q-value Since low

affinity (nonproductive) binding contributes much

more to the total amount of adsorbed enzyme than

high-affinity (productive) binding, a drastic decrease

of specific activity is observed with increase of q, as

shown in Fig 7 To elucidate the relationship

between adsorption and hydrolysis, further

experi-ments with mutant enzymes and detailed kinetic

studies will be necessary

The simple analytical method used in the present

study, i.e measuring the adsorption of the enzyme and

the concentration of products in the same reaction

mixture, makes it possible to evaluate the enzyme

kin-etics at a solid–liquid interface This approach not only

provides novel insights into cellulose–cellulase

interac-tion, but also should be relevant to many other

enzymes acting on insoluble substrates having a limited

surface area

Experimental procedures

Cellulose and enzyme preparations

Cellulose samples from green alga Cladophora sp and

tunicate Halocynthia roretzi were used in this study They

were purified by repeated treatments with 5% KOH and 0.3% NaClO2 solutions [35], then broken into small frag-ments using a double-cylinder type homogenizer The Cladophora cellulose was further hydrothermally treated

in 0.1 m NaOH solution at 260
C [33] The cellulose samples thus obtained were hydrolyzed with 4 m HCl solution at 80
C for 6 h, and then suspensions of cellu-lose microcrystals dispersed in water were prepared as reported previously [36]

Cel7A from Trichoderma viride (formerly known as cello-biohydrolase I) was purified from a commercial cellulase mixture, Meicelase (Meiji Seika Kaisha Co., Ltd, Tokyo, Japan) as described previously [25,37] Recombinant cello-biose dehydrogenase (CDH) was produced by Pichia pastoris and purified from the culture filtrate as described previously [38] The purity of these enzymes was confirmed

by SDS⁄ PAGE No detectable contamination of b-glu-cosidase or hydroxyethylcellulose-degrading activity was observed in Cel7A or CDH

Analysis of highly crystalline celluloses

Dilute suspensions of crystalline celluloses were dropped on carbon-coated copper grids, allowed to dry, and observed with a JEOL 2000EX TEM (Jeol Ltd., Tokyo, Japan), operating at 200 kV under diffraction contrast in the bright-field mode [39]

For the X-ray fiber diffraction analysis, oriented films

of cellulose microcrystals were prepared as previously reported [40] The X-ray fiber patterns were obtained on

a flat imaging plate, R-AXIS IV++(Rigaku Corporation, Tokyo, Japan), at room temperature using synchrotron radiation with a wavelength of 0.1 nm in beam line BL40B2 at the SPring-8 facility in Japan

Fig 8 Views of the hydrophobic surfaces

of cellulose I a (left) and cellulose I b (right).

The cellulose chains in the first layer are

su-perimposed and colored cyan The chains in

the second layer are colored yellow

(cellu-lose I a ) and green (cellulose I b ) The

struc-tures are based on the results reported by

Nishiyama et al [4,5].

Dilute suspensions were cast on glass plates and the dried

films were analyzed with a JASCO FT-IR 615 spectrometer

(JASCO Corporation, Tokyo, Japan) in the region of

4000Ờ400 cm)1; 64 scans of 4 cm)1 resolution were

signal-averaged and stored

Adsorption of Cel7A on crystalline celluloses

Crystalline cellulose (0.1% w⁄ v) was incubated with various

concentrations of enzymes (total concentration,

Abs280
0.04Ờ1.6) in 1 mL of 50 mm sodium acetate buffer,

pH 5.0, at 30
C using an end-over-end mixer (12 r.p.m.)

The mixture was centrifuged (15 000 gở 30 s) to terminate

the reaction after incubation for 15, 30, 60, 120, 180, 240,

and 320 min, and the supernatant (900 lL) was collected

The absorbance at 280 nm of the supernatant was measured

after the termination of the enzymatic reaction, and the

con-centration of free enzyme [F] (lm) was determined based on

an absorption coefficient at 280 nm of 88 250 m)1ẳcm)1for

T viride Cel7A, estimated from the amino acid sequence

of the enzyme [41] The amount of adsorbed enzyme (A,

nmolẳmg cellulose)1) was calculated by subtraction of the

amount of free enzyme from the amount of added enzyme, as

described previously [16,22,23,42] The amount of adsorbed

enzyme was plotted versus free enzyme concentration, based

on a two-binding-site model for Cel7A analysis [16], using

the following equation:

AỬ A1=đ1=Kad1ợ ơFỡ ợ A2=đ1=Kad2ợ ơFỡ đ1ỡ

where A1and A2are the adsorption maxima of high- and

low-affinity binding (nmol⁄ mg-cellulose); Kad1 and Kad2are the

adsorption constants of the high- and low-affinity binding

sites (lm)1) The maximum amount of adsorbed enzyme

(Amax, nmolẳmg cellulose)1) and the surface density (q) of

Cel7A were defined according to the following equations:

Measurement of cellobiose formation

The concentration of cellobiose formed in the supernatant

was estimated from the amount of cytochrome c reduced by

CDH, as follows The supernatant (after incubation for 15,

30, 60, 120, 180, 240, and 320 min) was kept at 4
C for 18 h

to allow the anomeric configuration to reach equilibrium

The supernatant (100 lL) was then incubated for 3 min with

200 nm recombinant CDH and 50 lm cytochrome c (bovine

heart, Wako Pure Chemical Industries, Ltd, Osaka, Japan)

in 50 mm sodium acetate buffer, pH 4.0, at 30
C, and the

absorbance at 525.6 (Abs525.6: isosbestic point of oxidized

and reduced cytochrome c) and 550.0 nm (Abs550.0) were

measured The reduced cytochrome c concentrations were

calculated using the following equations

Abs550:0Ử eox

550:0ơCox ợ ered

550:0ơCred đ4ỡ Abs525:6Ử e525:6đơCox ợ ơCredỡ đ5ỡ

ơCred Ử đe525:6Abs550:0 eox

550:0ered 550:0Abs525:6ỡ=

e525:6đered 550:0 eox

where eox 550:0(Ử 7.80 mm)1ẳcm)1) and ered

550:0(Ử 25.8 mm)1ẳcm)1) are the absorption coefficients at 550.0 nm for oxidized and reduced cytochrome c, respectively; e525.6 (Ử 10.2

mm)1ẳcm)1) is the absorption coefficient of cytochrome c

at 525.6 nm; [Cox] and [Cred] are the concentrations of oxid-ized and reduced cytochrome c, respectively The proportion

of b-anomer in cellobiose was estimated to be 64.9 ổ 0.4%

at the temperature employed in the present study, and it was assumed that two moles of cytochrome c is reduced by one mole of b-anomeric cellobiose Examination of the cellobiose concentration after 18 h incubation at 4
C indicated that further hydrolysis was minimal (< 2 lm), and this was con-firmed by comparison of the cellobiose concentrations in reaction mixtures containing supernatant with and without ultrafiltration Since precipitation prevented the measure-ment of cellobiose concentration at the highest enzyme con-centration (Abs280
1.6), these data was eliminated from the results

Analysis of the rate of cellobiose production from crystalline celluloses

The rate of cellobiose production at various time points was estimated from fitting of cellobiose concentrations in the reaction mixtures to the following equation based on Vaẽljamaẽe et al [22]:

Pđtỡ Ử ađ1  ebtỡ ợ cđ1  edtỡ đ7ỡ where P(t) is the cellobiose concentration (lm); t is time (min); and a, b, c, and d are empirical constants The rate

of cellobiose production (v) was calculated by the differenti-ation of Eqn 7 as follows:

vỬ dPđtỡ=dt Ử a b ebtợ c d edt đ8ỡ Thus, the specific activity of adsorbed enzyme k (min)1) was defined as follows:

In order to evaluate the steady-state reaction of Cel7A, the rate of cellobiose production and the specific activity were calculated from the data points after incubation for 120,

180, 240, and 320 min It must be pointed out that we have used Eqns 7 and 8 only for estimating the rate of cellobiose production at each time point We do not include any phys-ical interpretation to the equations or the constants since they are empirical

The authors are grateful to Professor Gunnar

Johans-son (Department of Biochemistry, University of

Upp-sala) for valuable discussions about the kinetics of

cellobiohydrolases We thank Dr K Noguchi (Tokyo

University of Agriculture and Technology, Tokyo,

Japan) for his help during the synchrotron radiation

experiments, which were performed at BL40B2 in

SPring-8 with the approval of the Japan Synchrotron

Research Institute (JASRI) (Proposal no

2002A0435-NL2-np) This research was supported by a

Grant-in-Aid for Scientific Research to MS (no 17380102)

from the Japanese Ministry of Education, Culture,

Sports and Technology, and a Research Fellowship to

RH from the Japan Society for the Promotion of

Science

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