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1.
Paul Zhu Jose M. Moran-Mirabal Jeremy S. Luterbacher Larry P. Walker Harold G. Craighead 《Cellulose (London, England)》2011,18(3):749-758
We report on studies of Thermobifida fusca cellulases Cel5A, Cel6B and Cel9A binding to pretreated wood particles using Confocal Laser Scanning Microscopy (CLSM). Hydro-thermal
pretreated wood particles were immobilized on borosilicate substrates before fluorescently-labeled cellulase solutions at
various concentrations were added. Time-lapse CLSM revealed that cellulases Cel5A, Cel6B and Cel9A quickly bound to certain
areas of wood particles, slowly diffused into and adsorbed to less accessible areas, but showed little affinity for other
areas of the wood. Cellulase-to-substrate association constants were estimated using a transient enzyme binding kinetics model,
and were found to be in agreement with published values. In order to accurately account for the fluorescence signal of labeled
enzyme mixed with wood autofluorescence, we also developed a spectral deconvolution method to separate signals from multiple
fluorochromes. 相似文献
2.
It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In
this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce
(SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products,
hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly
with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically,
and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition
was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with
Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests
that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis
rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that
extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive
action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains
can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved
model is supported by adsorption studies during hydrolysis. 相似文献
3.
Baker John O. Adney William S. Nleves Rafael A. Thomas Steven R. Wilson David B. Himmel Michael E. 《Applied biochemistry and biotechnology》1994,(1):245-256
A new thermostable endoglucanase,Acidothermus cellulolyticus E1, and another bacterial endoglucanase, E5 fromThermomonospora fusca, each exhibit striking synergism with a fungal cellobiohydrolase (Trichoderma reesei CBH I) in the saccharification of microcrystalline cellulose. In neither case did the ratio of endoglucanase to exoglucanase
that demonstrated maximum synergism coincide exactly with the ratio that actually released the maximum quantity of soluble
sugar for a given total cellulase loading. The difference between the two ratios, after significant hydrolysis of the substrate,
was considerably larger in the case ofA. cellulolyticus E1. For both endoglucanase pairings with CBH I, the offset between the ratio for maximum synergism and the ratio for maximal
soluble sugar production was found to be a function of digestion time. 相似文献
4.
Alina I. Chiriac Francisco I. Javier Pastor Valentin I. Popa Magdalena Aflori Diana Ciolacu 《Cellulose (London, England)》2014,21(1):203-219
A newly identified cellulase with a high polysaccharide degrading potential and a processive mode of action, has been evaluated on cellulose fibers. Cellulase Cel9B from Paenibacillus barcinonensis is a modular endoglucanase with the domain structure GH9-CBM3c-Fn3-CBM3b, consisting of a family nine catalytic module GH9, an auxiliary module CBM3c, a fibronectin-like module Fn3, and a functional cellulose binding module CBM3b. The whole cellulase Cel9B (E1) and two truncated forms of the enzyme that consist of the catalytic module linked to the auxiliary module, GH9-CBM3c (E2), and of the cellulose binding module of the enzyme, CBM3b (CBD), were applied to softwood dissolving pulp. The changes in the supramolecular structure and morphology of the fibres after the enzymatic treatment were evaluated by viscosimetry, X-ray diffraction (XRD), thermogravimetric analysis, differential scanning calorimetry and scanning electron microscopy (SEM). XRD studies provided the crystallite size, interplanar distances and crystallinity index of the samples before and after the enzymatic treatment. The treatment with cellulases E1 and E2 decreased the degree of polymerization and increased the crystallinity index of the pulp. Both E1 and E2 had a pronounced capacity for removing fuzz and improved the smoothness and surface appearance of the fibers, as shown by SEM. On the other hand, CBD proved to be less effective under the tested conditions. Moreover, the solubility of dissolving pulp in alkaline solutions has been evaluated as an indirect measure of cellulose accessibility. A notable enhancement in alkaline solubility of the samples treated with the cellulases was observed. 相似文献
5.
José M. Escovar-Kousen David Wilson Diana Irwin 《Applied biochemistry and biotechnology》2004,113(1-3):287-297
Cellulases are a complex group of enzymes that are fundamental for the degradation of amorphous and crystalline cellulose
in lignocellulosic material. Unfortunately, cellulases have a low catalytic efficiency on their substrates when compared to
similar enzymes such as amylases, which has led to a strong interest in improving their activities. Thermobifida fusca secretes six cellulose degrading enzymes: two exo- and three endocellulases and an endo/exocellulase Cel9A (formerly called
E4). Cel9A shows unique properties because of its endo- and exocellulase characteristics, strong activity on crystalline cellulose,
and good synergistic properties. Therefore, it is an excellent target for mutagenesis techniques to improve crystalline cellulose
degradation. In this article, we describe research conducted to improve Cel9A catalytic efficiency using a rational design
and computer modeling. A computer model of Cel9A was created using the program CHARMM plus its PDB structure and a cellohexose
molecule attached to the catalytic site as a starting model. Initially molecular graphics and energy minimization were used
to extend the cellulose chain to 18 glucose residues spanning the catalytic domain and cellulose-binding domain (CBD). The
interaction between this cellulose chain and conserved CBD residues was determined in the model, and mutations likely to improve
the binding properties of the CBD were selected. Site-directed mutations were carried out using the pET vector pET26b, Escherichia coli DH5-α, and the QuickChange mutagenesis method. E. coli BL21-DE3 was used for protein production and expression. The purified proteins were assayed for enzymatic activity on filter
paper, swollen cellulose, bacterial microcrystalline cellulose, and carboxymethylcellulose (CMC). Mutation of the conserved
residue F476 to Y476 gave a 40% improved activity in assays with soluble and amorphous cellulose such as CMC and swollen cellulose. 相似文献
6.
Yao Chen Arthur J. Stipanovic William T. Winter David B. Wilson Young-Jun Kim 《Cellulose (London, England)》2007,14(4):283-293
In this study we employed Size Exclusion Chromatography (SEC) and X-ray diffraction to monitor the molecular weight and crystallinity
of bacterial cellulose I and II (BC-I, BC-II) and microcrystalline cellulose (MCC) digested with three “pure” Thermobifida fusca cellulases (Cel6A, Cel6B, and Cel9A ). For each enzyme, cellulose crystallinity was found to increase modestly with treatment
time. The digestion rate of BC-II was higher than that of BC-I for Cel6A and Cel9A, both endocellulases. SEC results show
that the endocellulases create a very rapid decrease in cellulose molecular weight while a slower molecular weight loss was
observed with Cel6B, an exocellulase. This work suggests that conversion of native cellulose I to cellulose II by mercerization
may beneficially impact the rate of sugar release by cellulases from biomass. In general, lower conversion rates are observed
for MCC compared to BC, possibly due to a higher initial crystallinity for MCC. Surface area effects may also be important. 相似文献
7.
McCarter Suzanne L. Adney William S. Vinzant Todd B. Jennings Edward Eddy Fannie Posey Decker Stephen R. Baker John O. Sakon Joshua Himmel Michael E. 《Applied biochemistry and biotechnology》2002,98(1-9):273-287
Understanding the interactions between cellulases and cellulosic substrates is critical to the development of an efficient
artificial cellulase system for conversion of biomass to sugars. We directed specific mutations to the interactive surface
of the Acidothermus cellulolyticus EI endoglucanase catalytic domain. The cellulose-binding domain is not translated in these mutants. Amino acid mutations
were designed either to change the surface charge of the protein or to modify the potential for hydrogen bonding with cellulose.
The relationship between cellulase-to-cellulose (Avicel PH101) binding and hydrolysis activity was determined for various
groupings of mutations. While a significant increase in hydrolysis activity was not observed, certain clusters of residues
did significantly alter substrate binding and some interesting correlations emerged. In the future, these observations may
be used to aid the design of endoglucanases with improved performance on pretreated biomass. 相似文献
8.
Hyungil Jung Ho Geun Yoon Woojun Park Cheol Choi David B. Wilson Dong Hoon Shin Young Jun Kim 《Cellulose (London, England)》2008,15(3):465-471
The synergistic action between Thermobifida fusca exocellulase Cel6B and endocellulase Cel5A on sodium hydroxide pretreated bacterial cellulose (BC) was determined. The activities
of Cel6B and Cel5A were tested singly and both activities were dramatically increased on pretreated BC, especially in the
early stage of hydrolysis. Cel5A, which attacks the cellulose chain randomly, showed a larger increase on NaOH treated BC
than Cel6B. Mixtures of the two enzymes were also able to degrade NaOH treated BC faster than BC and the kinetics of the mixture
differed from that of the individual enzymes. The degree of synergistic effect (DSE) on BC decreased dramatically with time
of hydrolysis. However, the DSE on NaOH treated BC was almost constant throughout the incubation, with a smaller effect at
higher NaOH concentrations. The change caused by NaOH did not increase the DSE, although each individual cellulase activity
increased. This showed that synergistic activity was more effective on recalcitrant cellulose, which requires effective cooperation
between the cellulase components for hydrolysis. 相似文献
9.
The ethanol effect on the Trichoderma reesei cellulases was studied to quantify and clarify this inhibition type. To determine inhibition parameters of crude cellulase
and purified exoglucanase Cel7A, integrated Michaelis-Menten equations were used assuming the presence of two inhibitors:
cellobiose as the reaction product and ethanol as a possible bioproduct of cellulose fermentation.
It was found that hydrolysis of cellulose by crude enzyme follows a model that considers noncompetitive inhibition by ethanol,
whereas Cel7A is very slightly competitively inhibited. Crude cellulase is much more inhibited (K
iul=K
icl=151.9 mM) than exoglucanase Cel7A (K
icl=1.6 × 1015 mM). Also, calculated inhibition constants showed that cellobiose inhibition is more potent than ethanol inhibition both for
the crude enzyme as well as exoglucanase Cel7A. 相似文献
10.
David B. Wilson 《Cellulose (London, England)》2009,16(4):723-727
There are two well studied mechanisms that are used by cellulolytic microorganisms to degrade the cellulose present in plant
cell walls and a third less well studied oxidative mechanism used by brown rot fungi. The well studied mechanisms use cellulases
to hydrolyze the β-1,4 linkages present in cellulose, however the way in which cellulases are presented to the environment
are quite different for each mechanism. Most aerobic microorganisms secrete a set of cellulases outside the cell (free cellulase
mechanism) while most anaerobic microorganisms produce large multi enzyme complexes on their outer surface (cellulosomal mechanism).
Their genomic sequences suggest that the aerobic bacterium, Cytophaga hutchinsonii and the anaerobic bacterium, Fibrobacter succinogenes, do not use either of these mechanisms for degrading cellulose, as these organisms only code for normal endocellulases not
for processive cellulases like exocellulases and processive endocellulases which are used in both of the well studied mechanisms. 相似文献
11.
The cost of cellulase is still a problem for bioethanol production. As the cellulase of Trichoderma reesei is applicable for producing ethanol from cellulosic materials, the cellulase productivity of this fungus should be increased.
Therefore, we attempted to develop a system to isolate the strain with higher degrading ability of a filter paper and superior
proliferation characteristics among the conidia treated with the mitotic arrester, colchicine. When green mature conidia of
T.
reesei RUT C-30 were swollen, autopolyploidized, and incubated in the double-layer selection medium containing Avicel, colonies
appeared on the surface earlier than the original strain. When such colonies and the original colony were incubated on the
Avicel plates, strain B5, one of the colonies derived from the colchicine-treated conidia, showed superior proliferation characteristics.
Moreover, when strain B5 and the original strain were compared in the filter paper degrading ability and the cellulose hydrolyzing
activity, strain B5 was also superior to the original strain. It was suspected that superior proliferation characteristics
of strain B5 reflects higher filter paper degrading ability. Thus, we concluded that the Trichoderma strain with higher degrading ability of a filter paper and superior proliferation characteristics can be isolated using Avicel
plates and the double-layer selection medium. 相似文献
12.
Nieves Rafael A. Chou Yat-Chen Himmel Michael E. Thomas Steven R. 《Applied biochemistry and biotechnology》1995,(1):211-223
Two distinct quantitative indirect ELISAs were developed to determine the concentration of recombinant cellulase enzymes in
culture filtrates. A monoclonal antibody (E1P7) was used as the primary antibody in developing an ELISA specific forAcidothermus cellulolyticus E1 endoglucanase. Likewise, a polyclonal rabbit serum (Ab684) was used to develop an ELISA specific forThermomonospora fusca E3 exoglucanase. Dose-response curves indicated a dynamic range for both assays between 0.01 and 0.08 μg/mL (1–8 ng/assay) when
purified enzymes were used as standards. These assays have been used to estimate concentrations of secreted recombinant E1
and/or E3 in culture supernatants ofStreptomyces lividans strain TK24 in which the corresponding genes have been cloned and expressed. 相似文献
13.
Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this
study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase
activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects
for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning
method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform
with the capacity of secretive expression of multiple cellulases. 相似文献
14.
Louime C Abazinge M Johnson E Latinwo L Ikediobi C Clark AM 《Applied biochemistry and biotechnology》2007,141(1):127-138
Cytophaga hutchinsonii was originally isolated from sugarcane piles. This microorganism therefore probably produces an array of enzymes allowing
it to digest cellulosic substrates. C. hutchinsonii thus represents a rich source of potentially effective cellulase enzymes that can be harnessed for conversion of biomass
to simple sugars. These sugars can then be used as feedstock for ethanol production or other chemical syntheses. In this study,
we report the PCR cloning of an endoglucanase gene (Cel9A) from C. hutchinsonii using degenerated primers directed at the catalytic domain. Alignment of the amino acids sequence revealed that Cel9A has
a gene structure totally different from the other known cellulose degraders. The most striking feature of this cloned protein
is the absence of a cellulose-binding domain (CBD), which to date was believed to be imperative in cellulose hydrolysis. Consequently,
the Cel9A gene, encoding β-1,4 endoglucanase from C. hutchinsonii was over-expressed in Escherichia coli with a His-Tag based expression vector. The resulting polypeptide, with a molecular mass of 105 KDa, was purified from cell
extracts by affinity chromatography on cellulose. Mature Cel9A was optimally active at pH 5.0 and 45°C. The enzyme efficiently
hydrolyzes carboxymethyl-cellulose (CMC). Analysis of CMC and filter paper hydrolysis suggests that Cel9A is a nonprocessive
enzyme with endo-cellulase activities. 相似文献
15.
Cheng G Datta S Liu Z Wang C Murton JK Brown PA Jablin MS Dubey M Majewski J Halbert CE Browning JF Esker AR Watson BJ Zhang H Hutcheson SW Huber DL Sale KL Simmons BA Kent MS 《Langmuir : the ACS journal of surfaces and colloids》2012,28(22):8348-8358
A study of the interaction of four endoglucanases with amorphous cellulose films by neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) is reported. The endoglucanases include a mesophilic fungal endoglucanase (Cel45A from H. insolens), a processive endoglucanase from a marine bacterium (Cel5H from S. degradans ), and two from thermophilic bacteria (Cel9A from A. acidocaldarius and Cel5A from T. maritima ). The use of amorphous cellulose is motivated by the promise of ionic liquid pretreatment as a second generation technology that disrupts the native crystalline structure of cellulose. The endoglucanases displayed highly diverse behavior. Cel45A and Cel5H, which possess carbohydrate-binding modules (CBMs), penetrated and digested within the bulk of the films to a far greater extent than Cel9A and Cel5A, which lack CBMs. While both Cel45A and Cel5H were active within the bulk of the films, striking differences were observed. With Cel45A, substantial film expansion and interfacial broadening were observed, whereas for Cel5H the film thickness decreased with little interfacial broadening. These results are consistent with Cel45A digesting within the interior of cellulose chains as a classic endoglucanase, and Cel5H digesting predominantly at chain ends consistent with its designation as a processive endoglucanase. 相似文献
16.
Molar excess volume V
E
and enthalpy H
E
data have been measured at 25°C for pyridine A saturated with anhydrous cupric chloride (S) [A(S)]+ B [where B is aniline or o-toluidine (OT) or formamide (FD) or N, N-dimethylformamide (NND)] mixtures on the assumption that while the standard state of B is that of pure components B, the standard state of A(S) is that of A saturated with the salt S. The excess volume or enthalpy data for an equimolar mixture at a given temperature have been utilized to evaluate the interactional parameter X12 of the Sanchez and Lacombe theory of fluid mixtures at that temperature, and the same has been combined with V
E
(x
A
) data for a good prediction not only of the coresponding H
E
(x
A
) data for the mixture but also the extent of unlike interactions between the A(S) and B components of these A(S)+B mixtures. 相似文献
17.
Min Keun Kim Tae Ho Kang Jungho Kim Hoon Kim Han Dae Yun 《Applied biochemistry and biotechnology》2012,168(7):1834-1848
This study was conducted to assess the gene duplication and diversification of tandem cellulase genes in thermophilic bacteria. The tandem cellulase genes cel5C and cel5D were cloned from Thermotoga maritima MSB8, and a survey of the thermophilic bacterial genome for tandem cel genes from the databases was carried out. A clone having 2.3?kb fragment from T. maritima MSB8 showed cellulase activity, which had two open reading frames in tandem (cel5C and cel5D). The cel5C gene has 954?bp, which encodes a protein of 317 amino acid residues with a signal peptide of 23 amino acids, and the other gene cel5D consisting of 990?bp encoding a protein of 329 amino acid residues. These two proteins have similarity with the enzymes of glycosyl hydrolase family 5. From the enzyme assay, it was observed that Cel5C was extracellular and Cel5D was intracellular cellulase. Phylogenetic and homology matrix analyses of DNA and protein sequences revealed that family 12 cellulase enzymes Cel12A and Cel12B displayed higher homology (>50?%), but Cel5C and Cel5D enzymes belong to family 5 displayed lower homology (<30?%). In addition, repeated and mirror sequences in tandem genes are supposed to show the existence of gene duplication and recombination. 相似文献
18.
Divanery Rodriguez-Gomez Linda Lehmann Nadja Schultz-Jensen Anne Belinda Bjerre Timothy John Hobley 《Applied biochemistry and biotechnology》2012,166(8):2051-2063
Plasma-assisted pretreated wheat straw was investigated for cellulase and xylanase production by Trichoderma reesei fermentation. Fermentations were conducted with media containing washed and unwashed plasma-assisted pretreated wheat straw
as carbon source which was sterilized by autoclavation. To account for any effects of autoclavation, a comparison was made
with unsterilized media containing antibiotics. It was found that unsterilized washed plasma-assisted pretreated wheat straw
(which contained antibiotics) was best suited for the production of xylanases (110 IU ml−1) and cellulases (0.5 filter paper units (FPU) ml−1). Addition of Avicel boosted enzyme titers with the highest cellulase titers (1.5 FPU ml−1) found with addition of 50 % w/w Avicel and with the highest xylanase production (350 IU ml−1) reached in the presence of 10 % w/w Avicel. Comparison with enzyme titers from other nonrefined feedstocks suggests that plasma pretreated wheat straw is a promising
and suitable substrate for cellulase and hemicellulase production. 相似文献
19.
A computer docking study was carried out on the (110) crystal surface of the cellulose Iα crystal model for the carbohydrate
binding module (CBM) of cellobiohydrolase Cel6A, which is produced by the filamentous fungus Trichoderma reesei. Three-dimensional structures of the CBM were constructed by the homology modeling method using the Cel7A CBM, which is another
cellobiohydrolase from T. reesei, as a template, and refined by molecular dynamics calculations in the solution state. Among the three models tested, those
with three disulfide bonds were selected for a docking analysis. The binding free energy maps represented changes in non-covalent
interactions and solvation free energies with respect to the CBM position. These indicated two minimum positions within the
unit cell for both the parallel and antiparallel orientation modes of the CBM with respect to the cellulose fiber axis. Molecular
dynamics calculations under an explicit solvent system were performed for the four complex models derived from the minimum
positions of the binding free energy maps. The complex models with CBM in the parallel orientation had the lowest binding
energies. 相似文献
20.
Heterogeneous cellulose accessibility is an important substrate characteristic, but all methods for determining cellulose accessibility to the large-size cellulase molecule have some limitations. Characterization of cellulose accessibility to cellulase (CAC) is vital for better understanding of the enzymatic cellulose hydrolysis mechanism (Zhang and Lynd, Biotechnol. Bioeng. 2004, 88, 797-824; 2006, 94, 888-898). Quantitative determination of cellulose accessibility to cellulase (m2/g of cellulose) was established based on the Langmuir adsorption of the fusion protein containing a cellulose-binding module (CBM) and a green fluorescent protein (GFP). One molecule of the recombinant fusion protein occupied 21.2 cellobiose lattices on the 110 face of bacterial cellulose nanofibers. The CAC values of several cellulosic materials -- regenerated amorphous cellulose (RAC), bacterial microcrystalline cellulose (BMCC), Whatman No. 1 filter paper, fibrous cellulose powder (CF1), and microcrystalline cellulose (Avicel) -- were 41.9, 33.5, 9.76, 4.53, and 2.38 m2/g, respectively. The CAC value of amorphous cellulose made from Avicel was 17.6-fold larger than that of crystalline cellulose - 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 decrease in substrate reactivity. Declining heterogeneous cellulose reactivity is significantly attributed to a loss of CAC where the easily hydrolyzed cellulose fraction is digested first. 相似文献