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Http://www.abbs.info e-mail:[email protected] ISSN 0582-9879
ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(1):
13-18
CN 31-1300/Q |
Non-hydrolytic
Disruption of Crystalline Structure of Cellulose by Cellulose
( State key Laboratory of Microbial
Technology, Shandong University, Jinan 250100, China )
The
CBHI enzyme, like many cellulolytic enzymes, consists of two distinct domains
connected by a linker region: a
relatively small non-catalytic cellulose-binding domain (CBD), and a catalytic
domain (CD). The ultrastructure of CBD from CBHI has been studied at high
resolution[6,7]. Based on the structure and subsequent mutagenesis
studies, Linder et al[7] proposed that the CBDs relied on
several aromatic amino acids for high binding capacity to the crystalline
cellulose surface and might help to enhance the activity of cellulolytic
enzymes. However the exact mode of interaction between CBD and cellulose is not
yet understood[7,8]. In addition, the exact role of linker is also
unclear.
Removal
of the linker sequence from CBHI of T. reesei did not alter either the
hydrolytic rate of enzymes or their ability of binding cellulose[9].
We now present evidence for the first time that the isolated functional region
of the cellulose binding domain with its linker, called “CBDCBHI”
can lead to non-hydrolytic disruption of the crystalline structure of cellulose
and therefore help to enhance the activity of endoglucanase towards crystalline
cellulose.
1.1 Limited
proteolysis of LacZ-CBDCBHI fusion protein and purification of CBDCBHI
The
detailed procedure of subcloning and expression of the coding region of
LacZ-CBDCBHI fusion in E. coli and its production and
purification has been reported elsewhere[10]. 0.5 ml of papain
solution (500 mg/L, Sigma) in phosphate buffer (pH 7.0, 40 mmol/L) containing 5
mmol/L L-cysteine and 2 mmol/L EDTA was added to 4 ml of Lac Z-CBDCBHI
fusion protein solution (500 mg/L) in an acetate buffer (pH 5.0, 50 mmol/L) and
incubated at 37 ºC. The peptide of CBDCBHI released from LacZ-CBDCBHI
fusion protein was first isolated on a Bio-gel P-4 column (0.8 cm´80
cm) eluted with acetate buffer (pH 5.0, 50 mmol/L) at a flow rate of 6 ml/h, 2
ml per tube. The activity of CBDCBHI was determined by the increase
of turbidity caused by short fiber formation during cellulose degradation[11].
The fraction containing CBDCBHI was further purified by gel
filtration on Sephadex G-25 column (0.8 cm´60
cm) with water as elution buffer at the same flow rate as above. The
homogeneity of CBDCBHI obtained was determined with a LC-18-DB HPLC
column (Pharmacia, Sweden) in a HPLC system (Waters, USA), eluted by
methanol/water (7:3).
1.2 Scanning
electron microscopy (SEM) investigation of morphological changes of cotton
fibers after binding CBDCBHI
Cellulose
fibers after binding CBDCBHI was prepared for the SEM experiments as
follows: the reaction mixture
containing 2 g/L of de-waxed cotton fiber powders in sodium acetate buffer (pH
6.0, 0.05 mmol/L) and 0.5 ml CBDCBHI solution (100 mg/L) in a total
volume of 1.0 ml (with buffer alone as control) was incubated at 45 ºC with
gentle agitation. At different time points, the cellulose fibers were collected
on a glass filter and washed with distilled water, and dried in an oven at 80 ºC,
then coated with 4-6
nm gold for scanning electron microscopy (S-520, Hitachi, Japan).
1.3
IR spectroscopy and X-ray diffraction studies on the changes in
structural parameters of cotton fibers after binding CBDCBHI
The
IR spectra were obtained using a FT-IR 710 infrared spectrometer (Nicolet).
Spectra were recorded in the transmission mode with a resolution of 4 cm-1
in the range of 4 000-400
cm-1.
Because the infrared spectra of cellulose are relatively difficult to interpret
in the region of 800-600
cm-1,
the absorbance values at 1 160 cm-1
were measured, which correspond to the C-O-C spectra. Samples were prepared in
the same way as described for the SEM experiments.
The
X-ray diffraction of each sample was recorded using a RIGAKU RADI System
Diffractometer (D/Max-rB, Japan). The wavelength of the Cu Ka radiation source
was 0.154 nm, and the spectra were obtained at 30 mA with a voltage of 40 kV.
For each sample, the integral intensity, integral width and face width half
mold (FWHM) were estimated from the analysis of the diffraction profiles by a
least square peak fitting program.
1.4
Determination of thermal activity of crystal-line cellulose powder
during the binding process of CBDCBHI
The
thermograms were determined under the conditions of constant temperature,
volume, reaction system and dissolved oxygen using a heat-flow microcalorimeter
(2277 thermal activity monitor, Thermo Metric AB, Sweden). The reaction
suspension contained 1.0 mg of crystalline cellulose powder (Sigmacell, Type
50, Sigma), and 50 mg of CBDCBHI was made up to a total of l.0 ml in
potassium phosphate buffer (50 mmol/L, pH 6.0). The samples were incubated at 45
ºC. The procedures for the complete cleaning and sterilization of the flow
tubing were carried out as described by Zhang et al[12].
To
compare the effect of CBHI with that of EGI, the same experiments were carried
out using CBHI and EGI, except that acetate buffer (pH 4.8) was used for EGI
instead of phosphate buffer. The purified components of CBHI and EGI were
obtained from T. pseudokoningii[13].
1.5
Synergism between CBDCBHI and EGI during hydrolysis of
different cellulose substrates
Synergism
between CBDCBHI and EGI from T. pseudokoningii S38 was
investigated by analyzing the production of soluble reducing sugars on
different cellulose substrates. The reaction solution contained 20 mg of CBDCBHI
alone or combined with 30 mg of EGI and 50 mg of cotton fiber powders in 1.0 ml
of phosphate buffer (pH 6.0, 20 mmol/L), which was incubated at 45 ºC. Soluble
reducing sugars were determined by the DNS method[11].
2.1
Limited proteolysis of LacZ-CBDCBHI fusion protein and
purification of CBDCBHI
As
mentioned previously, since only the exposed regions in a protein are usually
attacked, limited proteolytic digestion often yields intact structural domains.
The primary sites of proteolysis in cellulase by papain are close to the linker
and adjacent to the oligopeptides composed of the three types residues Gly, Ser
and Thr. In addition, Gly-Gly-y oligopeptides were also found to be a
proteolytic site, where y was a hydrophobic residue[14]. For the
CBHI from P. janthinellum[10, 15] the residues GGT, adjacent
to the N-terminus of the linker were the proteolytic site:
After
the LacZ-CBDCBHI fusion protein was proteolyzed by papain, the
proteolysate was isolated with gel filtration on Bio-gel P-4 column. SDS-PAGE
analysis indicated that a 60-min digestion yielded a complete proteolyic
cleavage of the fusion protein. The chromatographs and the activity analysis
showed that prolonging the incubation time did not cause the CBDCBHI
peptide to be further digested. This result was similar to that of CBD from T.
pseudokoningii[14]. When the fraction containing CBDCBHI
was further purified on Sephadex G-25 column, a symmetrical elution peak of CBDCBHI
was obtained. HPLC analysis revealed that the purified CBDCBHI
peptide was homogeneous (data not shown).
2.2
Disruption of cotton fiber structure after incubation with CBDCBHI
The
use of SEM permitted the visualization of morphological changes of the cotton
fibers incubated with purified CBDCBHI. It was observed that after
binding/adsorbing the cellulose fibers, CBDCBHI caused a significant
roughening and swelling of the substrate, and eventually led to the release of
short fibers. These structural changes of cotton fibers could not be monitored
by biochemical studies. This phenomenon of morphological changes of cellulose
fibers was called "deaggregation", "defibrillation", or "dispersion"
in the literature[2,3,16]. Fig.1(A),(B) showed the SEM images of the
native cotton fiber (fibrils). The lateral diameter of the fibrils ranged from
10 to 15 mm, and had a relatively smooth and uniform surface. After 6 h
incubation with CBDCBHI, several disruptions occurred on the outer
surface of the fibers [Fig.1(C)], and numerous splits appeared along their long
axes [Fig.1(D)]. As a result of a long time treatment with CBDCBHI,
the release of small fragments from the fibers was also observed. This could be
quantified by the turbidity analysis (data not shown). This result was similar
to that reported for the bacterial CBD of CenA by Din et al[17].
Fig.1 Scanning electron micrographs
(SEM) of cotton fibers before and after being treated by CBDCBHI
(A)
Native cotton fibrils (6000´); (B) Native cotton fibrils (2000´); (C) Cotton fibrils after 6 h incubation
with CBDCBHI (6000´); (D) Cotton fibrils after 10 h
incubation with CBDCBHI (2000´).
As
shown in Fig.2, the highly crystalline, native cellulose has a characteristic
IR spectrum[18], which differs from that binding CBDCBHI,
and the major difference was shown to be around the broad bands of 3 600-3
200 cm–1, corresponding to the strong -OH
stretching and flexural vibration frequencies of the intra- and inter-
molecular hydrogen bonds of cellulose[18]. The relative intensities
of these bands were reduced by 11.3% after binding CBDCBHI
(calculated by the baseline method). The intensities of bands near 1 206 cm-1
and 663 cm-1,
which reflected the -OH
group flexural vibration were also changed. These results may indicate that
some hydrogen bonds between -OH groups in cellulose were disrupted after
binding CBDCBHI.
Fig.2 IR spectra of cotton fibers
before (A) and after (B) incubation with CBDCBHI
Fig.3
shows the comparison of X-ray diffraction of cotton fibers before and after
binding CBDCBHI. The diffraction pattern in Fig.3(A) indicated a
typical crystalline structure of native cotton cellulose, and there were no
discernible differences between the two samples[18]. Binding CBDCBHI
only gave a slight decrease of diffraction intensity as well as peak width of
002 peak (peak 4, at 2q=22.5 º) [Fig.3(B)]. This suggested that the crystalline
order of the treated sample was decreased.
Fig.3 X-ray diffraction spectra of cotton
fibers before (A) and after (B) incubation with CBDCBHI
As
shown in Fig.4(A), the thermometric curve of crystalline cellulose after binding
CBDCBHI revealed a heat-absorption effect (thermonegative reaction)
during the whole process. In the system of CBHI enzyme, it absorbed energy at
the beginning and then turned to an exothermic effect in the subsequent stage
[Fig.4(B)]. That reflected the different thermodynamic properties of two
domains of CBHI i.e. a heat-absorption effect for CBD and an exothermic effect
for catalytic domain (CD), and the free energy released from hydrolysis of
b-1,4-glycosidic bond was more than absorption for CBDCBHI binding
cellulose. While the EGI always appeared as an exothermic effect [Fig.4(C)].
These findings were in good agreement with the theoretical prediction that the
hydrolysis of b-1,4 glucan link was an exothermic reaction[19] and
energy was necessary during the adsorption process of CBD.
Fig.4 Thermometric changes during
incubation processes of crystalline cellulose with CBDCBHI or
different cellulases
(A)
CBDCBHI; (B) CBHI; (C) EGI.
The
experiments showed that CBDCBHI alone did not release soluble
reducing sugars from cotton fibers, and EGI itself could only produce few soluble
reducing sugars. However, an obvious synergistic effect was observed if the
cellulose substrates were treated by the two components simultaneously. That
is, more soluble reducing sugars were produced than those produced by EGI
itself. The synergism of CBDCBHI with EGI was only detectable when
crystalline cellulose was used as the substrate, such as Avicel (Sigmacell,
Type 50) and cotton fibers, but not for amorphous cellulose, such as phosphoric
acid-swollen crystalline cellulose (data not shown).
In
comparison with soluble substrates, cellulose degradation has many special
requirements for enzyme systems because it is in a crystalline state. For a
long time it has been suggested that the synergism is one of the most
remarkable features of cellulose systems and the synergistic action between
CBHI and EGI is necessary for efficiently solubilizing crystalline cellulose[4,
5]. However the molecular mechanisms are still poorly understood[1].
From the thermodynamic viewpoint, during the hydrolysis of cellulose by
cellulases, energy is required to disrupt the cellulose crystallite prior to
the hydrolysis of the glucan chain[16, 19, 20]. Recently, Sinnott[19]
proposed a hypothesis that the CBHs are preferential disrupters of crystalline
cellulose: they use the free
energy released from hydrolysis of the b-1,4-glycosidic bonds between sugars to
disrupt the crystallites. But it is not clear where the energy comes from
during the cellulolytic processes. Now from the thermodynamic studies in the present
work (See Fig.4), we can assume that endoglucanase may attack cellulose in a
some manner at the beginning of the cellulolytic process and from which free
energy is released without the production of soluble reducing sugars. Then CBHs
can utilize this energy to bind cellulose.
Another
question is what is the reason for the phenomenon of defibrillation (short
fiber formation) in cellulose degradation? As early as 1980s, Chanzy et al[21]
suggested that the first step during the enzymatic degradation of cellulose is
defibrillation or dispersion of crystalline cellulose, due to the action of
CBHI. According to White and Brown[22], defibrillation of cellulose
was caused by endoglucanase. We have recently confirmed that the short fiber
formation is generally an initial step in cellulose biodegradation[12, 22].
As reported earlier, family II CBDs, such as CBD from bacterial CenA which
consisted of more then 200 amino acids could disrupt the surface of cellulose
fibers and release fine particles from cotton or Avicel[7, 17], but
the family I CBDs, such as CBD from T. reesei and P. janthinellum
which only contained about 36 amino acids can not disrupt the cellulose
structure[7,22,23]. As mentioned above, the present work revealed
that the binding of CBDCBHI from P. janthinellum would lead
to the formation of short fibers, which might make it easier for cellulases to
penetrate into the crystalline regions and further produce new fiber surfaces
and ends of cellulose chains, thereby facilitate the rapid degradation of
crystalline cellulose.
Acknowledgments We are grateful to
Prof. Shao Z-F (Dept. of Molecular Physiology & Biological Physics,
University of Virginia, USA) and Dr. Chen H-Z (Department of Biochemistry and Molecular
Biology and Center for Biological Resource Recovery, The University of Georgia,
USA) for the critical reading of the manuscript. We are also indebted to Wang L
S for his help in the preparation of the manuscript.
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Received: July 3, 2000 Accepted: August 31, 2000
This work was supported by a grant from
National Natural Science Foundation of China, No.39430020
*Corresponding author: Tel, 86-531-8564429; Fax, 86-531-8565234; e-mail, [email protected]