Lignocellulose Biodegradation - American Chemical Society

Chapter 9

Productive Cellulase Adsorption on Cellulose Hanshu Ding and Feng Xu*

Downloaded by COLUMBIA UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: July 29, 2004 | doi: 10.1021/bk-2004-0889.ch009

Novozymes Biotech, 1445 Drew Avenue, Davis, C A 95616

Hydrolysis of insoluble polymeric cellulose by cellulases requires enzyme adsorption onto the substrate. Being heterogeneous on its surface, cellulose in general has different sites for different cellulases to adsorb. Only a portion of the adsorbed cellulase is productive, able to lead to subsequent glycosidic bond cleavage. We applied the dependence of initial hydrolysis rate on the concentration of enzyme and substrate to probe the "productive" adsorption of four different cellulases on two types of cellulose. We found that different celluloses could have significantly different cellulase accessibility; that different cellulases, even from the same family, could have significantly different "productive" adsorption on cellulose; and that functionally analogous cellulases could have overlapping adsorption sites, while functionally dissimilar cellulases could have separate adsorption sites. The usefulness of the method for mechanistic study and industrial application of cellulases was discussed.

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© 2004 American Chemical Society

In Lignocellulose Biodegradation; Saha, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.


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To efficiently hydrolyze cellulose, cellulases are often required to tightly bind onto their insoluble substrate (1-6). Many cellulases are modularly structured, possessing a catalytic core and one or more carbohydrate-binding modules (CBM) (2, 3, 7-9). The function of CBM, which has high affinity for crystalline cellulose, is believed to include anchoring the catalytic core onto the surface of a cellulose microfibril, disrupting hydrogen bonds in cellulose, and facilitating the binding of the cellulose chain at core's active site. For cellobiohydrolases (CBH), the typical catalytic cycle involves binding at the end of a cellulose chain, cleavage of a β-1,4 glycosidic bond to yield cellobiose, and finally translocation along the chain for the next cycle ("processivity") or desorption into solution when the chain end is reached. Trichoderma reesei and Humicola insolens CBH-I (Cel7A) act from the reducing end of a cellulose chain, while their CBH-II (Cel6A) prefer the non-reducing end (10, 11). For endo-P-l,4-glucanases (EG), the typical catalytic cycle involves random binding along a cellulose chain, cleavage of a β-1,4 bond to fragment the chain or release soluble cellooligomers, and desorption from the chain before the next cycle. It has been proposed that it is the core, rather than CBM, that specifies where on the cellulose the cellulase adsorbs (12). In the past two decades, tremendous progress has been made in determining the structure of cellulases (2, 13-16). Many cellulase families have been discovered based on sequence homology (17). Available crystallographic data indicate that cellulases belonging to the same family share highly homologous 3D structures. Substrate specificity, reactivity and mechanism of cellulases have been probed by various techniques including X-ray crystallography and sitedirected mutagenesis. For CBH, their processivity has been related to the tunnel morphology of their catalytic site where long loops can open/close to facilitate CBH to adsorb to, slide along, or desorb from a cellulose chain (18, 19). Principles of cellulase action on cellulose have been established (7, 2, 5, 20). Several mathematic models have been developed to analyze the multiphasic kinetics of the heterogeneous cellulose hydrolysis by cellulase (6,10,21-28). Despite the significant progress achieved so far, several mechanistic details governing CBM-cellulose interaction and its effect on catalysis remain unclear (3, 12, 16, 29-31). All fungal CBM belong to CBM family 1 because of their sequence homology (16). Yet different fungal cellulases can have significantly different cellulose adsorption in terms of adsorption rate, affinity constant (K ) and reversibility. For instance, T. reesei CBH-I binds cellulose reversibly while T. reesei CBH-II binds irreversibly (32, 33). It is not known whether CBH-I and CBH-II, or even CBH-I analogs from different fungi, adsorb onto the same or different regions of a cellulose microfibril. A general correlation between cellulose affinity and catalytic reactivity for cellulases has yet to be established. Conventionally, cellulase adsorption onto (or accessibility for) cellulose, such as that of Γ. reesei cellulases onto amorphous/crystalline/lingo-cellulose, has been investigated by Langmuir-type isotherms (20, 34). They measure overall cellulase protein adsorption, assuming a uniform cellulose surface, a



156

adsorption enthalpy independent on surface coverage, and negligible interaction among adsorbed molecules. The main shortcoming of the methods is their inability of differentiating "productive" adsorption, which leads to subsequent β1,4 glycosidic bond cleavage, from "nonproductive" adsorption, which leads to either desorption or enzyme-inactivating immobilization (70, 35). We developed a new method capable of measuring the "productive" cellulase adsorption, based on the dependence of initial hydrolysis rate on the concentration of cellulase and cellulose. In contrast to the "static" cellulose accessibility revealed by Langmuir isotherm, the accessibility detected by the new method is "kinetic" since it relates to the surface region hydrolysable by adsorbed cellulase. Four representative cellulases (CBH-I from T. reesei and Κ insolens, CBH-II from H. insolens and EG-I (Cel7B) from 7! reesei) were studied for their "kinetic" accessibility (related to their "productive" adsorption) towards two representative celluloses (amorphous phosphoric acid-swollen cellulose (PASC) and microcrystalline Avicel), in comparison with their "static" Langmuir accessibility. In addition to the cellulose accessibility, we also probed whether these cellulases bind onto the same or different regions of the celluloses.

Mathematic modeling for cellulase.cellulose interaction When CBH encounters cellulose, it first adsorbs/binds onto cellulose, complexes a cellooligomeric chain at its active site, hydrolyzes a β-1,4 bond to release a cellobiose product, then slides along the chain processively for the next hydrolysis (7, 2, 75-75, 36). At the initial stage of hydrolysis, when product inhibition is negligible, the reaction can be represented by the simplified mechanism shown in Figure 1, with the following symbols: E, free enzyme; S, enzyme-accessible/hydrolysable cellobiosyl unit in cellulose; β, number of the cellobiosyl units covered by a bound CBH molecule; E ^ S , adsorbed enzyme; "Ε·β8", pseudo-Michaelis intermediate ("pseudo" because of the insoluble nature of substrate); P, released hydrolysis product (cellobiose); E -(P-l)S, enzymexellulose complex right after cellobiose release. Four major steps are depicted in Figure 1 : Enzyme adsorption onto cellulose (Κ = k\lk.{) substrate activation (pseudo-Michaelis constant K = (k.2^)/k ), hydrolysis (rate constant £ ), and processive translocation (procession constant K = Α4/Λ.4). a

a

Λ

m

3

y

2

p

Figure 1. Simplified mechanism of cellulose-catalyzed cellulose hydrolysis.


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Under steady-state conditions, the hydrolysis step becomes rate-limiting, while other steps reach equilibrium, yielding the following kinetic equations: d[EpS]/dt=* [E pS]-(*. +* )[EpS]=0, or [E^S]=ik.2+k )mS]/k^K [E^S] (1) 2

a

2

3

3

m

[E][pS]/[E pS]=P[E][S]/[E„pS]=p/^, or [E]=[E pS]/(/: [S])=/: [EpS]/(/: [S]) (2) a

a

a

nl

a

[E pS]/[E (P-l)S] = K , or [E (p- 1)S] = [E pS]/u: = K [EpS]/Kp a


a

p

a

a

p

(3)

m

From eqs 1 to 3 and the mass balance [E] = [E]+[E pS]+[EpS]+[E (P-l)S], in which [E] is the total enzyme concentration, eq 4 is obtained: 0

a

a

0

[EpS] = [E] [S]/{(l + /:*)(a + [S]}

(4)

0

in which K* = K (\ + 1/K ), a = KJ{K (l + K*)}. The mass balance [S] = [S] + P[E pS] + p[EpS] + (p-l)[E (p-l)S] + [P], in which [S] is the total enzyme-accessible/hydrolysable cellobiosyl unit concentration, applies to the substrate. Because β ~ 39 (22, 37, 38), 1/p « 1. When hydrolysis extent is low, [P] is negligible, thus [S]o=[S]+p{[E pS]+[EpS] +[E (p-l)S]}=tS]+p[EpS]{^ +l+A: //:p}. Considering eq 4, this leads to: m

P

a

0

a

a

0

a

a

ra

n

2

[S]o=[S](a+p[E] +[S])/(a+[S]), or [S] + (a + p[E]„ - [S] )[S] - a[S] = 0 (5) 0

0

0

Because the hydrolysis rate ν = £ [EpS], eqs 4 and 5 can lead to eq 6: 3

v = ^E] [S]o/(a + p[E] + [S]) 0

(6)

0

in which Κ = k /(l + l/K*). Since cellulase adsorption on cellulose is generally very strong (1/Κ -> 0), α becomes negligible. Assuming [S]=[S], in which [S] is the total cellobiosyl unit concentration of substrate, then the initial rate can be expressed as: 3

Λ

0

t

t

v = ^[E] [S] /(P[E] + [S]) t

0

0

(7)

in which [S] is a function of [S] according to [S] = [S]o/§ and eq 5. For non-processive EG, the i , k. reactions in Figure 1 can be omitted, and the E -(|3-l)S can be replaced by Ε + P. Eqs 6 and 7 can be derived with α = KJ{K*(\ +K )} andK = A /(l + 1 / ^ . t

t

4

4

e

m

3


158

Dependence of initial rate on concentration When initial substrate concentration [S] is given, the ν dependence on initial enzyme concentration [E] can be characterized by an initial linear phase and a final saturation phase: When [E] « [S] , [S] « [S] and eq 7 becomes ν ~ K[E] ; when [E] » [S] , eq 7 becomes ν ~ A^>[S]/p. The two lines intersect at [E] , leading to: t

0

0

0

0

t

t

t

t

S

0

s

[E] /[S] = /p 0

(8)

t

When initial enzyme concentration [E] is given, the ν dependence on initial substrate concentration [S] can also be characterized by an initial linear phase and a final saturation phase: When [S] « [E] , eq 7 becomes ν ~ ^[S] /P; when [S] » [E]o, [S] ~ §[S] and eq 7 becomes ν ~ K[E] . The two lines intersect at [S] , leading to


0

t

t

t

t

0

t

0

s

t

IFWiStf-W

(8')

Based on crystallographic data, the number of cellobiose unit covered by cellulose-absorbed CBH, β, can be estimated as -39 (22, 37, 38). Thus both rate-concentration dependences can reveal φ, a parameter reflecting the initial "kinetic" accessibility of cellulose for cellulase.

Difference between φ and Langmuir-type binding capability Langmuir-type isotherms, governed by an adsorption constant and a capability N , are widely used to measure the adsorption of cellulase onto cellulose. The parameter N reflects the portion of surface cellobiosyl/lattice units accessible for cellulase adsorption, regardless whether or not the adsorption is followed by hydrolysis. In contrast to the "static" N , φ is "kinetic" and measures those "productive" cellulase adsorptions that can lead to subsequent β1,4 bond cleavage. It is generally believed that EG cleave β-1,4 bonds randomly on the surface of cellulose, and CBH cleave β-1,4 bonds in a processive mannerfromeither the reducing or non-reducing end of a cellulose chain (2,11,13,15). It is not clear, however, whether different cellulases, belonging to either the same or different functional group/family, adsorb initially at the same cellulose surface region. Measuring φ for individual and mixed cellulases would address this question. 0

0

0


159


Accessibility measurement H. insolens CBH-I and CBH-II were purified as reported (39). T. reesei CBH-I and EG-I were purified to apparent electrophoretic homogeneity by chromatographical methods similar to those published (40). Briefly, a commercial T. reesei cellulase mix, Celluclast (activity: ~60 Filter-PaperUnit/mL, protein content: ~100 mg/mL, Major component: Cel7A, 6A, 7B and 5A, Novozymes), was first buffer-exchanged by ultrafiltration (Pall Filtron ultrafiltration apparatus and polysulfone membrane of 10K M W cutoff), then subjected to Q-Sepharose (pH 7) and Mono-S (pH 4) chromatography (on a Pharmacia FPLC). Activefractionswere monitored by carboxymethyl cellulose (CMC) and PASC-hydrolyzing activity at pH 5 and 30 °C. Purity was measured by SDS-PAGE and IEF, and identity was verified by mass spectrometry-based protein sequencing. Purified T. reesei CBH-I, T. reesei EG-I, H. insolens CBH-I and H. insolens CBH-II showed respectively a molecular mass of 65000, 60000, 72000 and 65000, isoelectric point of 4.1, 4.8, 4.5 and 4.9, initial PASChydrolyzing activity of 0.86, 36, 2.0 and 5.5 IU/mg, Avicel-hydrolyzing activity of 0.12, 0.3, 0.12 and 0.2 IU/mg, and CMC-hydrolyzing activity of 0.6, 84, 0.4 and 0.1 IU/mg. Table I summarizes the extended CBH-I activity on PASC and Avicel in the presence of Aspergillus oryzae β-glucosidase (to prevent cellobiose inhibition).

Table I. Extent og long-term cellulose hydrolysis by CBH-I CBH-I T. reesei H. insolens

Avicel

PASC 2h 49% 54%

24 h 99% 83%

2h 3% 1%

24 h 19% 5%

Buffer: 50 mM Na-acetate, pH 5. Temperature: 40 °C. Concentration: 1.8 g/L PASC, 10 g/L Avicel, 50 nM A. oryzae β-glucosidase, 3 μΜ CBH-I.

Chemicals used as substrate, chromogenic agent, or buffer were commercial products of at least reagent grade. PASC was prepared from Avicel (PHI01 from FMC) (39). Acid pretreated corn stover (PCS) was obtainedfromNational Renewable Energy Laboratory, with a composition of ~57% cellulose, 28% lignin, 5% hemicellulose and 4% protein. Cellulose-cellulase incubation was done in 50 mM Na-acetate of pH 5 with 0.5 g/L bovine serum albumin (BSA; to reduce nonspecific binding of cellulase on inner surface of reaction vessel) at 40 °C. To study ν dependence on [cellulase], 0.3-15 μΜ cellulase was reacted with 1.8 g/L cellulose. To study ν


160

dependence on cellulose dose, denoted as "[cellulose]" (amount divided by suspension volume), 0.2-10 g/L cellulose was reacted with 5 μΜ cellulase. Reducing sugar production was monitored with p-hydroxybenzoic acid hydrazide (PHBAH) (41). First, 100 \kh sample was mixed with 50 μι 1.5 % PHBAH in 0.5 M NaOH in a 96-well plate. After 10 min at 95 °C, 50 μΐ. H 0 was added and 100 μL solution was read at 410 nm in another 96-well plate. DGlucose served as quantification standard. Initial ν was derived from five samplings taken during the first hour of hydrolysis. Because of its endocellulase nature, T. reesei EG-I could hydrolyze a cellulose chain without releasing soluble sugar. To detect such endo- reactivity, insoluble cellulose, after being incubated with EG-I, decanted and washed, was subjected to PHBAH reaction. Reduced PHBAH in solution was measured colorimetrically. Under our conditions, the majority (~90%) products of EG-Fs initial reaction was found in solution, likely as glucose (36). As detected by HPLC, the main initial product from < 1 and > 4 μΜ EG-I was cellobiose and glucose, respectively; with > 15 μΜ EG-I, glucose accounted for more than 85% of the initial products. When initial rate ν was plotted against initial enzyme concentration [EG-I] , two ν "saturations" were observed: The first one took place at ~2 μΜ [EG-I] and the second at ~11 μΜ [EG-I] . Likely thefirstand second saturation corresponded to cellobiose and glucose production by EG-I, respectively. Only the first [EG-I] was used to derive φ for EG-I.


2

0

0

0

S

0

Langmuir adsorption measurement Cellulose-cellulase incubation was made at 25 °C with 1 mL of 50 mM Naacetate of pH 5, 0.8 - 62 μΜ cellulase, 1.8 g/L cellulose and 8 μΜ BSA in 1.2mL Pierce ImmunoWare tubes. After 30 min, the supernatant was decanted, filtered (by 0.45 μ Millipore Multi Screen-HV), and assayed by PASC hydrolysis activity for free cellulase. Langmuir adsorption equation, l/[E] j bed = 1/([E] - [ Ε ] ) = 1/(NOA:'[E]) + β/Νο (38), was then applied to calculate apparent capacity N (in mole/mole) and adsorption constant K\ For PASC hydrolysis assay, 10 μL sample was mixed with 190 μL solution containing 2.1 g/L PASC and 8 μΜ BSA in 50 mM Na-acetate of pH 5 in a 96well plate. After 30 min at 50 °C, 50 μΐ 0.5 M NaOH was added to stop hydrolysis. After 5 min centrifugation at 2000 rpm, 100 μL supernatant was subjected to PHBAH reducing sugar assay. a< sor

0

0


161

Accessibility of individual cellulase Under our conditions, mixing cellulase with cellulose led to immediate release of reducing sugar. Figure 2A shows the dependence of initial rate ν on initial enzyme concentration [CBH-I] for the PASC hydrolysis by T. reesei CBH-I. At low [CBH-I] , ν was proportional to [CBH-I] . At high [CBH-I] , however, a "saturating" phase was achieved where ν became independent on [CBH-I]o. Crossing the ν oc [CBH-I] and ν = constant lines, a [CBH-I] was obtained and φ was deduced according to eq 8. A similar situation was observed when [PASC] varied (Figure 2B). The φ obtained from [PASC] and eq 8' was close to that obtained from [CBH-I] and eq 8. Table II summarizes φ measured for the four cellulases and two celluloses. 0

0

0

0

S

0

0

s

t

t

S


0

300,

§

/ο

I I

c

Ι

901

•

Ο

._o

!° Β

1

100

'2 _J ι ι ι 5

r-

1

/-—σ™*-—-Φ ο

200]

H

I

120

/ - /

6 1

1

10

15

...J

1

1

L_

5

20

[CBH-I]o,mM

10

15

[PASC] , m M t

Figure 2. PASC hydrolysis by T. reesei CBH-L Initial rate ν dependence on (A) [CBH-I]ο or (B) [PASC] . (A): [PASC] : 5 mM cellobiose equivalent (1.8 g/L). Lines ν = 30[CBH-I] and ν = 255 intercept at [CBH-I]£ = 8.5 μΜ (0.55 g/L). (B); [CBH-I] : 4.6 μΜ (0.3 g/L). Lines ν = 35.5[PASC] and ν = 103 intercept at [PASCJf = 2.8 mM cellobiose equivalent (1 g/L). For clarity purpose, "saturating" ν data beyond the depicted concentration range are not included. t

t

0

0

t

For a given cellulase, Avicel's φ was less than PASC's φ, suggesting less "productive" cellulase adsorption on more crystalline cellulose surface. With crystalline cellulose substrate, it could be more difficult for the cellulase to thread a cellulose chain into its active site cleft/tunnel, since more inter-chain Η bonds would need to be broken in a highly crystalline region than in an amorphous/chain end region.


162 Table II. Cellulase accessibilitytowardscellulose


Cellulase T. reesei CBH-I T. reesei EG-I Κ insolens CBH-I H. insolens CBH-II T. reesei CBH-I T. reesei EG-I H. insolens CBH-I H. insolens CBH-II T. reesei CBH-I

Cellulose PASC PASC PASC PASC Avicel Avicel Avicel Avicel PCS

s

[E]

Lignocellulose Biodegradation - American Chemical Society

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