Cellulase activity of a haloalkaliphilic anaerobic bacterium, strain Z-7026

Summary The cellulolytic activity of an alkaliphilic obligate anaerobic bacterium, Z-7026, which was isolated from the microbial community of soda-lake sediments and belongs to the cluster III of Clostridia with low G+C content, was studied. The bacterium was capable of growing in media with cellulose or cellobiose as the sole energy sources. Its maximal growth rate on cellobiose (0.042–0.046 h^–1) was observed at an initial pH value of 8.5–9.0, whereas the maximal rate of cellulase synthesis, assayed by using a novel fluorimetric approach, was found to be 0.1 h^–1 at pH 8–8.5. Secreted proteins revealed high affinity for cellulose and were represented by two major forms of molecular masses of 75 and 84 kDa, whereas the general protein composition of the precipitated and cellulose-bound preparations was similar to cellulosome subunits of Clostridium thermocellum. The optimum pH of the partially purified enzyme preparation towards both amorphous and crystalline cellulose was in the range 6–9, with more than 70% and less than 50% of maximal activity being retained at pH 9.2 and 5.0, respectively.     

Spot synthesis: observations and optimizations.

Positionally addressable syntheses of peptides on continuous cellulose membranes (spot synthesis) have often been reported in detail, but important questions dealing with synthesis quality, reproducibility and subsequent binding assays have largely been under-emphasized. In this report we have investigated some of these problems. The most important results were: (i) the signal intensity of ligate binding to cellulose-bound peptides and the affinity of the corresponding soluble peptides show good correlation, illustrated by three different ligate binding assays; (ii) reducing peptide density on the cellulose avoids the 'ring spot' effect, i.e. where less binding is observed in the spot-center compared to the rim. We recommend a peptide density of 10 nmol/cm2 as a reasonable starting point for further optimization; (iii) statistical analysis of binding assay reproducibility with more than 15000 peptides resulted in a mean standard signal deviation of 0.18; and (iv) optimization of side-chain deprotection revealed that a 30-min pretreatment of the cellulose with 90% trifluoroacetic acid followed by the standard deprotection protocol resulted in higher purity of the synthesized products.     

Parameter and process significance in mechanistic modeling of cellulose hydrolysis

A process-based model relevant to landfill and anaerobic digesters was developed, which included a novel approach to biomass transfer between a cellulose-bound biofilm and biomass in the bulk liquid. Model results highlighted the significance of the bacterial colonization of cellulose particles by attachment through contact in solution. Simulations revealed that both enhanced colonization and cellulose degradation are associated with reduced cellulose particle size, increased biomass populations in solution and increased cellulose-binding ability of the biomass. This suggests that transportation of biomass into the system from elsewhere and/or bacterial inoculation of such systems could enhance degradation significantly. A sensitivity analysis of the system parameters revealed the biological rate and yield properties of the hydrolyzing bacteria are most significant with regard to cellulose degradation in the system.     

Widely different off rates of two closely related cellulose-binding domains from Trichoderma reesei.

The filamentous fungus Trichoderma reesei produces two cellobiohydrolases (CBHI and CBHII). These, like most other cellulose-degrading enzymes, have a modular structure consisting of a catalytic domain linked to a cellulose-binding domain (CBD). The isolated catalytic domains bind poorly to cellulose and have a much lower activity towards cellulose than the intact enzymes. For the CBDs, no function other than binding to cellulose has been found. We have previously described the reversibility and exchange rate for the binding of the CBD of CBHI to cellulose. In this work, we studied the binding of the CBD of CBHII and showed that it differs markedly from the behaviour of that of CBHI. The apparent binding affinities were similar, but the CBD of CBHII could not be dissociated from cellulose by buffer dilution and did not show a measurable exchange rate. However, desorption could be triggered by shifting the temperature. The CBD of CBHII bound reversibly to chitin. Two variants of the CBHII CBD were made, in which point mutations increased its similarity to the CBD of CBHI. Both variants were found to bind reversibly to cellulose.     

Oligomerization,membrane anchoring,and cellulose-binding characteristics of AbpS,a receptor-like Streptomyces protein

Streptomyces reticuli produces a 34.6-kDa surface-anchored protein (AbpS) whose surface-exposed N terminus binds strongly to Avicel, a dominantly crystalline type of cellulose. The generation of a large set of mutated abpS-genes and the subsequent analysis of the corresponding proteins in vitro as well as in vivo in a Streptomyces host allow the assignment of the following characteristics for AbpS. (i) Amino acid residues participating directly in the cellulose-interaction are located at the N terminus. (ii) As ascertained by cross-linking experiments, AbpS forms homotetramers in its soluble as well as cellulose-bound form. (iii) The intermolecular assembly of four AbpS molecules is governed by two domains (including amino acids 60-110 and 161-212). Both domains possess large portions of alpha-helical regions in which hydrophobic amino acids are located on one side as known from coiled-coil proteins. (iv) The C-terminal part of AbpS comprising 35 amino acids contains a transmembrane domain. Due to the surface-exposed N terminus of AbpS and the presence of transmembrane helix the C terminus has to be situated in the cytoplasm of the S. reticuli hyphae. Thus AbpS connects the interior of the mycelia with the extracellular space and binds cellulose using a unique cellulose-binding module.     

Enzymatic production of ethanol from cellulose using soluble cellulose acetate as an intermediate

A two-stage process for the enzymatic conversion of cellulose to ethanol is proposed as an alternative to currently incomplete and relatively slow enzymatic conversion processes employing natural insoluble cellulose. This alternative approach is designed to promote faster and more complete conversion of cellulose to fermentable sugars through the use of a homogeneous enzymatic hydrolysis reaction. Cellulose is chemically dissolved in the first stage to form water-soluble cellulose acetate (WSCA). The WSCA is then converted to ethanol in a simultaneous saccharification-fermentation with Pestal-otiopsis westerdijkii enzymes (containing cellulolytic and acetyl esterase components) and yeast.Water-soluble cellulose acetate was successfully prepared from purified wood cellulose (Solka Floe) and chemical reagents. Enzyme pretreatment of WSCAto form metabolizable sugars was a necessary step in achieving practical conversion of WSCA to ethanol using yeast. The results showed that WSCA has a low enzyme requirement and a high convertibility to reducing sugars with enzymes from P. westerdijkii fungus. Pestalotiopsis westerdijkii enzymes were found to be superior to enzymes from Trichoderma viride in producing metabolizable glucose from WSCA. The yeast utilized 55-70% of the hydrolyzate sugars that were produced by P. westerrlijkii enzymes on WSCA and produced ethanol. The acetate that was liberated into solution by the action of acetyl esterase enzymes on WSCA was found to have a stimulatory effect on ethanol production in yeast. This is an important feature that can be used to advantage in manipulating the conversion to maximize the production of ethanol. Hence, the simultaneous saccharification-fermentation of WSCA to ethanol using P. westerdijkii enzymes and yeast has features that are highly desirable for developing an economical cellulose conversion process.     

Preparation, analysis and antibody binding studies of simultaneously synthesized soluble and cellulose-bound HIV-1 p24 peptide epitope libraries

Summary Epitope libraries of the HIV-1 p24 epitope GATPQDLNTM, recognized by the murine monoclonal antibody CB 4-1, were prepared by simultaneous synthesis on single resin supports (solution phase library) and on a continuous cellulose membrane support (solid phase-bound library) Each position of the epitope was replaced by 19 l-amino acids (cysteine omitted) in the soluble library or by 20 l-amino acids in the cellulose-bound library. The soluble library was synthesized by simultaneously incorporating equimolar amino acid mixtures at each position of the epitope or by synthesizing single epitope analogues. The peptide mixtures were subsequently analyzed by HPLC, CZE and MALDI-TOF mass spectrometry. Double coupling of equimolar amino acid mixtures of either 0.8 equiv (coupling at epitope positions 6–10) or 1.5 equiv (coupling at epitope positions 1–5) resulted in approximately equimolar incorporation of all single components of the mixture. The mixtures were then separated by preparative HPLC, and the peptides or peptide mixtures of single fractions were isolated and analyzed for binding CB 4-1. The results were compared with those obtained from antibody binding studies using the cellulose-bound epitope library. The affinity constants of the soluble peptide variants qualitatively correlated with the binding of CB 4-1 to single cellulose-bound analogues. Both approaches allowed the rapid identification of key residues in antibody binding, thus giving insight into the molecular nature of this antibody-peptide interaction.     

Ultrastructure of the cell surface cellulosome of Clostridium thermocellum and its interaction with cellulose.

The ultrastructural distribution of the cellulosome (a cellulose-binding, multicellulase-containing protein complex) on the cell surface of Clostridium thermocellum YS was examined by cytochemical techniques and immunoelectron microscopy. When cells of the bacterium were grown on cellobiose, cellulosome complexes were compacted into quiescent exocellular protuberant structures. However, when the same cells were grown on cellulose, these polycellulosomal organelles underwent extensive structural transformation; after attachment to the insoluble substrate, the protuberances protracted rapidly to form fibrous "contact corridors." The contact zones mediated physically between the cellulosome (which was intimately attached to the cellulose matrix) and the bacterial cell surface (which was otherwise detached from its substrate). In addition, cell-free cellulosome clusters coated the surface of the cellulose substrate. The cellulose-bound cellulosome clusters appear to be the site of active cellulolysis, the products of which are conveyed subsequently to the cell surface via the exocellular contact zones.     

Soluble and immobilized clostridial aminopeptidase and aminopeptidase P as metal-requiring enzymes

The dependence of enzymatic activity on Co2+ concentration was found to be bell-shaped for the soluble and immobilized clostridial aminopeptidase (alpha-aminoacyl-peptide hydrolase, EC 3.4.11.13) and aminopeptidase P (aminoacylpropyl-peptide hydrolase, EC 3.4.11.9), with maxima in the 3-18 microM range of Co2+ concentration. The Co2+-enzyme association constants derived from the activation of soluble, glass- and cellulose-bound clostridial aminopeptidase by Co2+ were KE-Co = 5.2 X 10(5), 4.5 X 10(6) and 2.0 X 10(5) M-1, respectively; for soluble and glass-bound aminopeptidase P, the KE-Co were 1.5 X 10(5) and 8.2 X 10(5) M-1, respectively. Kinetic measurements indicate the involvement of Co2+ in the enzyme-substrate binding. Cobalt-citrate (Co-cit) acted as a useful metallobuffer and protected both enzymes against inhibition by high concentrations of CoSO4. For association of citrate with Co2+ under the assay conditions, KCo-cit was determined as (5.3 +/- 1.4) X 10(3) M-1 by anodic stripping polarography. In contrast to the rapid association of Co2+ with soluble and glass-bound clostridial aminopeptidase (less than 1 min at 4 degrees C), the dissociation process was very slow (hours to days), being slower for the glass-bound than for the soluble and cellulose-bound enzyme. For aminopeptidase P, both processes were rapid. All the interactions were shown to be reversible.     

Purification and Characterization of a Cellulose-Binding (beta)-Glucosidase from Cellulose-Degrading Cultures of Phanerochaete chrysosporium

Extracellular (beta)-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium was purified by DEAE-Sephadex chromatography, by Sephacryl S-200 chromatography, and by fast protein liquid chromatography (FPLC) using a Mono Q anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of FPLC-purified (beta)-glucosidase indicated the presence of three enzyme forms with molecular weights of 96,000, 98,000, and 114,000. On further fractionation with a microcrystalline cellulose column, the 114,000-molecular-weight (beta)-glucosidase, which had 82% of the (beta)-glucosidase activity, was bound to cellulose. The (beta)-glucosidases with molecular weights of 96,000 and 98,000 did not bind to cellulose. The cellulose-bound (beta)-glucosidase was eluted completely from the cellulose matrix with water. Cellulose-bound (beta)-glucosidase catalyzed p-nitrophenylglucoside hydrolysis, suggesting that the catalytic site is not involved in cellulose binding. When the cellulose-binding form was incubated with papain for 20 h, no decrease in the enzyme activity was observed; however, approximately 74% of the papain-treated glucosidase did not bind to microcrystalline cellulose. SDS-PAGE analysis of the nonbinding glucosidase produced by papain indicated the presence of three bands with molecular weights in the range of 95,000 to 97,000. On the basis of these results, we propose that the low-molecular-weight (96,000 and 98,000) non-cellulose-binding (beta)-glucosidase forms are most probably formed from the higher-molecular-weight (114,000) cellulose-binding (beta)-glucosidase via extracellular proteolytic hydrolysis. Also, it appears that the extracellular (beta)-glucosidase from P. chrysosporium might be organized into two domains, a cellulose-binding domain and a catalytic domain. Kinetic characterization of the cellulose-binding form is also presented.     

Kinetics of cellobiose hydrolysis using cellobiase composites from Ttrichoderma reesei and Aspergillus niger

The enzymatic hydrolysis of cellulose to glucose involves the formation of cellobiose as an intermediate. It has been found necessary(1) to add cellobiase from Aspergillus niger (NOVO) to the cellobiase component of Trichoderma reesei mutant Rut C-30 (Natick) cellulase enzymes in order to obtain after 48 h complete conversion of the cellobiose formed in the enzymatic hydrolysis of biomass. This study of the cellobiase activity of these two enzyme sources was undertaken as a first step in the formation of a kinetic model for cellulose hydrolysis that can be used in process design. In order to cover the full range of cellobiose concentrations, it was necessary to develop separate kinetic parameters for high- and low-concentration ranges of cellobiose for the enzymes from each organism. Competitive glucose inhibition was observed with the enzymes from both organisms. Substrate inhibition was observed only with the A. niger enzymes.     

Enzymatic characterization of ancestral/group-IV clade xyloglucan endotransglycosylase/hydrolase enzymes reveals broad substrate specificities

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan–xyloglucan donor/acceptor substrate activities. To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the ‘ancestral’ XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link β-glucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.     

Different effects of carbohydrate binding modules on the viscoelasticity of nanocellulose gels

Many cellulose degrading and modifying enzymes have distinct parts called carbohydrate binding modules (CBMs). The CBMs have been shown to increase the concentration of enzymes on the insoluble substrate and thereby enhance catalytic activity. It has been suggested that CBMs also have a role in disrupting or dispersing the insoluble cellulose substrate, but dispute remains and explicit evidence of such a mechanism is lacking. We produced the isolated CBMs from two major cellulases (Cel6A and Cel7A) from Trichoderma reesei as recombinant proteins in Escherichia coli. We then studied the viscoelastic properties of native unmodified cellulose nanofibrils (CNF) in combination with the highly purified CBMs to detect possible functional effects of the CBMs on the CNF. The two CBMs showed clearly different effects on the viscoelastic properties of CNF. The difference in effects is noteworthy, yet it was not possible to conclude for example disruptive effects. We discuss here the alternative explanations for viscoelastic effects on CNF caused by CBMs, including the effect of ionic cosolutes.     

DNA polymerases from non stimulated and phytohemagglutinin stimulated normal human lymphocytes

The presence and some properties of DNA polymerases isolated from normal human lymphocytes, non stimulated and stimulated by phytohemagglutinin, are described. In the non stimulated lymphocytes two cytoplasmic DNA polymerases are found, one eluting from DEAE cellulose at 0.07 M NaCl (CI_n) and the other at 0.13 M NaCl (CII_n). In the nuclear soluble fraction only one enzyme activity is found (NI_n) which does not adsorb to DEAE cellulose. In the cytoplasm of stimulated lymphocytes only one enzyme activity is detected (CI_s) which elutes from DEAE cellulose at 0.12 M NaCl. The nuclear soluble fraction contains two activities, NI_s, which does not adsorb to DEAE cellulose, and NII_s, which elutes from DEAE cellulose at 0.07 M NaCl. Some properties of the different enzymes are described which indicate that NI_n and NI_s enzymes are clearly different from the others.     

Enzyme mechanisms involved in cellulose hydrolysis by the rot fungus Sporotrichum pulverulentum

This article presents a review of the enzyme mechanisms involved in degradation of cellulose by the white-rot fungus Sporotrichum poulverulentum. The hydrolytic enzymes involved include: (1) five endo-1,4-β-glucanases; (2) one exo-1,4-β-glucanase, and (3) one or several 1,4-β-glucosidases. A recently discovered oxidative enzyme of importance in in vitro cellulose degradation seems to be a cellobiose oxidase. An oxidoreductase, cellobiose:quinone oxidoreductase, is of importance both in cellulose and in lignin degradation. Regulatory mechanisms of the extracellular enzyme activities, such as monosugar levels causing catabolite repression of the endoglucanases, have also been investigated. The enzymes used by S. pulverulentum in cellulose hydrolysis are compared to those used by Trichoderma viride. Very similar types of enzymes are used in both cases. However, no oxidative enzyme has so far been found to be involved in extracellular cellulose degradation in the case of T. viride. Recommendations for further research are given.     

Cellulolysis by Mucor pusillus

Culture filtrates of Mucor pusillus NRRL 2543 contained hydrolytic enzymes that attacked native cellulose, acid-swollen cellulose, carboxymethylcellulose, and cellobiose. The distribution profiles of cellulolytic and beta-glucosidase activities after gel filtration on Sephadex G-75 showed the presence of several active peaks. Glucose was the only product of hydrolysis when native cellulose was used as the substrate. Acid-swollen cellulose, when treated with cellulase free of beta-glucosidase activity, gave rise to glucose, cellobiose, and at least two higher molecular weight components which were also hydrolyzed in turn to cellobiose and glucose. The presence of a multiple cellulolytic enzyme system was indicated, the components of which may have specific roles in the degradation of cellulose.     

白蚁及共生微生物木质纤维素水解酶的种类

白蚁是热带生态系统重要的木质纤维素降解者。白蚁种类丰富,可分成高等白蚁和低等白蚁,食性也具有各自特点。白蚁自身可以产生纤维素酶,主要是GHF9的内切葡聚糖酶(EG),也有β-葡萄糖苷酶(GB)。低等白蚁共生的原虫中已发现丰富的纤维素酶基因,属于GHF5,7和45。同时还有其他相关功能基因,如木聚糖酶和果胶类物质水解酶。高等白蚁肠道中没有共生原虫。高等培菌白蚁可以利用共生蚁巢伞属真菌促进木质纤维素降解,真菌可以产生纤维素酶,果胶质水解酶类、木聚糖酶,同时还产生可能与木质素分解相关的一种漆酶,但是从分子水平,关于共生真菌纤维素水解酶的研究还较少。白蚁肠道已分离出许多具有木质纤维素降解能力的菌株,最近的研究也发现了大量细菌纤维素酶基因。白蚁-共生系统丰富的木质纤维素水解酶类为发展生物方法开发纤维素乙醇这一思路提供有价值的资源。     

Improved immobilization of fusion proteins via cellulose-binding domains

Cellulose-binding domains (CBDs) are structurally and functionally independent, noncatalytic modules found in many cellulose or hemicellulose degrading enzymes. Recent biotechnological applications of the CBDs include facilitated protein immobilization on cellulose supports. In some occasions there have been concerns about the stability of the CBD driven immobilization. Here we have studied the chromatographic behavior of variants of the Trichoderma reesei cellobiohydrolase I CBD belonging to family I. Both CBDs fused to antibody fragments and isolated CBDs were studied and compared. Tritium labeling by reductive methylation was used as a sensitive detection method. The fusion protein as well as the isolated CBD was found to leak from the column at a rate of 0.3-0.5% of the immobilized protein per column volume. However, the leakage could be overcome by using two CBDs instead of a single CBD for the immobilization. In this way leakage was reduced to less than 0.01% per column volume. The improved immobilization could also be seen as a decreased migration of the protein down the column in extended washes.     

Site-directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B.

Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.     

Recycle of enzymes and substrate following enzymatic hydrolysis of steam-pretreated aspenwood

The commercial production of chemicals and fuels from lignocellulosic residues by enzymatic means still requires considerable research on both the technical and economic aspects. Two technical problems that have been identified as requiring further research are the recycle of the enzymes used in hydrolysis and the reuse of the re calcitrant cellulose remaining after incomplete hydrolysis. Enzyme recycle is required to lower the cost of the enzymes, while the reuse of the spent cellulose will lower the feedstock cost. The conversion process studied was a combined enzymatic hydrolysis and fermentation (CHF) procedure that utilized the cellulolytic enzymes derived from the fungus Trichoderma harzianum E58 and the yeast Saccharomyces cerevisiae. The rate and extent of hydrolysis and ethanol production was monitored as was the activity and hydrolytic potential of the enzymes remaining in the filtrate after the hydrolysis period. When a commercial cellulose was used as the substrate for a routine 2-day CHF process, 60% of the original treated, water-extracted aspenwood was used as the substrate, only 13% of the original filter paper activity was detected after a similar procedure. The combination of 60% spent enzymes with 40% fresh enzymes resulted in the production of 30% less reducing sugars than the original enzyme mixture. Since 100% hydrolysis of the cellulose portion is seldom accomplished in an enzymatic hydrolysis pro cess, the residual cellulose was used as a substrate for the growth of T. harzianum E58 and production of celulolytic enzymes. The residue remaining after the CHF process was used as a substrate for the production of the cellulolytic enzymes. The production of enzymes from the residue of the Solka Floc hydrolysis was greater than the production of enzymes from the original Solka Floc.