The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose.

CenA is a bacterial cellulase (beta-1,4-glucanase) comprised of a globular catalytic domain joined to an extended cellulose-binding domain (CBD) by a short linker peptide. The adsorption of CenA and its two isolated domains to crystalline cellulose was analyzed. CenA and CBD.PTCenA' (the CBD plus linker) adsorbed rapidly to cellulose at 30 degrees C, and no net desorption of protein was observed during the following 16.7 h. There was no detectable adsorption of the catalytic domain. Scatchard plots of adsorption data for CenA and for CBD.PTCenA were nonlinear (concave upward). The adsorption of CenA and CBD.PTCenA exceeded 7 and 8 mumol/g cellulose, respectively, but saturation was not attained at the highest total protein concentrations employed. A new model for adsorption was developed to describe the interaction of a large ligand (protein) with a lattice of overlapping potential binding sites (cellobiose residues). A relative equilibrium association constant (Kr) of 40.5 and 45.3 liter.g cellulose-1 was estimated for CenA and CBD.PTCenA, respectively, according to this model. A similar Kr value (33.3 liter.g-1) was also obtained for Cex, a Cellulomonas fimi enzyme which contains a related CBD but which hydrolyzes both beta 1,4-xylosidic and beta-1,4-glucosidic bonds. It was estimated that the CBD occupies approximately 39 cellobiose residues on the cellulose surface.     

Effects of adsorption properties and mechanical agitation of two detergent cellulases towards cotton cellulose

Abstract

The impacts of two hybrid cloned commercial cellulases designed for detergency on cotton fibres were compared. HiCel45 has a family 45 catalytic domain and a fungal cellulose binding module (CBM) from the fungus Humicola insolens. BaCel5 has a family 5 catalytic domain and a fungal CBM from Bacillus spp. BaCel5 bound irreversibly to cellulose under the buffer conditions tested while HiCel45 was found to bind reversibly to cellulose because it showed low adsorption. BaCel5 seems to yield more activity towards cotton than HiCel45 under mild stirring conditions, but under strong mechanical agitation both enzymes produce similar amount of sugars. HiCel45 had a more progressive production of residual reducing ends on the fabric than BaCel5. These studies seem to indicate that HiCel45 is a more cooperative enzyme with detergent processes where high mechanical agitation is needed.     

A novel, small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast

A method is presented for the isolation of genes encoding hydrolytic enzymes without any knowledge of the corresponding proteins. cDNA made from the organism of interest is cloned into a yeast vector to construct an expression library in the yeast Saccharomyces cerevisiae. Colonies producing hydrolytic enzymes are screened by activity plate assays. In this work, we constructed a yeast expression library from the filamentous fungus Trichoderma reesel and isolated a new β-1,4-endoglucanase gene on plates containing β-glucan. This gene, eg15, codes for a previously unknown small protein of 242 amino acids. Despite its small size, the protein contains two conservative domains found in Trichoderma cellulases, namely the cellulose-binding domain (CBD) and the iinker region that connects the CBD to the catalytic core domain. Molecular modelling of the EGV CBD revealed some interesting structural differences compared to the CBD of the major celluiase CBHI from T. reesei. The catalytic core of EGV is unusually small for a ceiiulase and represents a new family of ceilulases (Family K) and of glycosyl hydrolases (Famlly 45) together with the endoglucanase B of Pseudomonas fluorescens and the endoglucanase V of Humicola insolens on the basis of hydrophobic ciuster anaiysis.     

Adsorption mode of exo- and endo-cellulases from Irpex lacteus (Polyporus tulipiferae) on cellulose with different crystallinities.

The adsorption mode of two highly purified cellulases, exo- and endo-type cellulases, from Irpex lacteus (Polyporus tulipiferae) was investigated by using pure cellulosic materials with different crystallinity as substrates. Adsorption of the two enzymes on the substrates was found to fit the Langmuir-type adsorption isotherm. Maximum amount of adsorbed enzyme obtained from the Langmuir plots showed an inverse correlation to the crystallinity of the substrate with both enzymes, and this value of endo-type cellulase was less dependent on the degree of crystallinity of substrates than that of exo-type cellulase, whose isotherms reached saturation in the range of low enzyme concentrations. The two enzymes showed relatively high affinities for all the substrates and their affinities increased with increasing crystallinity, but this tendency was less marked with endo-type cellulase than with exo-type one. In addition, large negative values of free energy change were observed on the adsorption of both enzymes, and the values became more negative with increasing crystallinity. Consequently, both cellulases showed high adsorption on crystalline cellulose and the adsorption process became smoother with increasing crystallinity. The adsorption of the two types of cellulases was endothermic with an increase in entropy, especially for amorphous cellulose, suggesting the occurrence of water release from the substrates during enzyme adsorption. In addition, the changes in thermodynamic parameters (delta H, delta S, and delta G) in adsorption of exo-type cellulase were larger than in that of endo-type enzyme.     

Unusual sequence organization in CenB, an inverting endoglucanase from Cellulomonas fimi.

The nucleotide sequence of the cenB gene was determined and used to deduce the amino acid sequence of endoglucanase B (CenB) of Cellulomonas fimi. CenB comprises 1,012 amino acids and has a molecular weight of 105,905. The polypeptide is divided by so-called linker sequences rich in proline and hydroxyamino acids into five domains: a catalytic domain of 607 amino acids at the N terminus, followed by three repeats of 98 amino acids each which are greater than 60% identical, and a C-terminal domain of 101 amino acids which is 50% identical to the cellulose-binding domains of C. fimi cellulases Cex and CenA. A deletion mutant of the cenB gene encodes a polypeptide lacking the C-terminal 333 amino acids of CenB. The truncated polypeptide is catalytically active and, like intact CenB, binds to cellulose, suggesting that CenB has a second cellulose-binding site. The sequence of amino acids 1 to 461 of CenB is 35% identical, with a further 15% similarity, to that of a cellulase from avocado, which places CenB in cellulase family E. CenB releases mostly cellobiose and cellotetraose from cellohexaose. Like CenA, CenB hydrolyzes the beta-1,4-glucosidic bond with inversion of the anomeric configuration. The pH optimum for CenB is 8.5, and that for CenA is 7.5.     

Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulase engd from Clostridium cellulovorans

Clostridium cellulovorans produces a cellulase complex (cellulosome) as well as noncellulosomal cellulases. In this study, we determined a factor that affected the solubility of the cellulosomal cellulase EngB and the noncellulosomal EngD when they were expressed in Escherichia coli. The catalytic domains of EngB and EngD formed inclusion bodies when expressed in E. coli. On the other hand, both catalytic domains containing the C-terminal cellulose-binding domain (CBD) of EngD were expressed in soluble form. Fusion with the CBD of EngD also helped increased the solubility of cellulosomal cellulase EngL upon expression in E. coli. These results indicate that the CBD of EngD plays an important role in the soluble expression of the catalytic domains of EngB, EngL, and EngD. The possible mechanisms of solubilization by fusion of the catalytic domain with the CBD from EngD are discussed.     

Removal of lignin and reuse of cellulases for continuous saccharification of lignocelluloses

Method of the removal of lignin and reuse of cellulases for a continuous saccharification of lignocelluloses were investigated. Only lignin could be separated from hydrolysates by differences in the settling velocity; it was removed from the saccharification process by flocculation with chitosan without loss of cellulases. The ultra-filtration membrane PM10 (Amicon) could be used for recovery of cellulases, but the membrane UH-1 (Toyo Roshi) was better for this purpose, because no cellulases leaked from the membrane, and the amount of cellulase adsorbed to the membrane was less. The cellulases were inactivated by vigorous agitation of the solution in an ultra-filtration device. The loss of cellulase activity by such agitation increased with agitation time, but could be controlled by recovery at a low speed of agitation, so the cellulases could be reused.     

Visualization of enzymatic hydrolysis of cellulose using AFM phase imaging

Complete cellulase, an endoglucanase (EGV) with cellulose-binding domain (CBD) and a mutant endoglucanase without CBD (EGI) were utilized for the hydrolysis of a fully bleached reed Kraft pulp sample. The changes of microfibrils on the fiber surface were examined with tapping mode atomic force microscopy (TM–AFM) phase imaging. The results indicated that complete cellulase could either peel the fibrillar bundles along the microfibrils (peeling) or cut microfibrils into short length across the length direction (cutting) during the process. After 24 h treatment, most orientated microfibrils on the cellulose fiber surface were degraded into fragments by the complete cellulase. Incubation with endoglucanase (EGV or EGI) also caused peeling action. But no significant size reduction of microfibrils length was observed, which was probably due to the absence of cellobiohydrolase. The AFM phase imaging clearly revealed that individual EGV particles were adsorbed onto the surface of a cellulose fiber and may be bound to several microfibrils.     

Identification of tandemly repeated type VI cellulose-binding domains in an endoglucanase from the aerobic soil bacterium Cellvibrio mixtus

Cellulose-binding domains (CBD) play a pivotal role during plant cell wall hydrolysis by cellulases and xylanases from aerobic soil bacteria. Recently we␣have reported the molecular characterisation of a single-domain endoglucanase from Cellvibrio mixtus, suggesting that some cellulases produced by this aerobic bacterium preferentially hydrolyse soluble cellulosic substrates. Here we describe the complete nucleotide sequence of a second cellulase gene, celB, from the soil bacterium C. mixtus. It revealed an open reading frame of 1863 bp that encoded a polypeptide, defined as cellulase B (CelB), with a predicted M _r of 66 039. CelB contained a glycosyl hydrolase family 5 catalytic domain at its N terminus followed by two repeated domains, which exhibited sequence identity with type VI CBD previously found in xylanases. Full-length CelB bound to cellulose while catalytically active truncated cellulase derivatives were unable to bind the polysaccharide, confirming that CelB is a modular enzyme and that the type VI CBD homologues were functional. Analysis of the biochemical properties of CelB revealed that the enzyme hydrolyses a range of cellulosic substrates, although it was unable to depolymerise Avicel. We propose that type VI CBD, usually found in xylanases, provide an additional mechanism by which cellulases can accumulate on the surface of the plant cell wall, although they do not potentiate cellulase activity directly. These results demonstrate that C. mixtus, in common with other aerobic bacteria, is able to produce cellulases that are directed to the hydrolysis of cellulose in its natural environment, the plant cell wall. Received: 6 October 1997 / Received revision: 22 December 1997 / Accepted: 2 January 1998     

Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I.

Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.     

Molecular cloning, sequencing, and expression of a novel multidomain mannanase gene from Thermoanaerobacterium polysaccharolyticum

The manA gene of Thermoanaerobacterium polysaccharolyticum was cloned in Escherichia coli. The open reading frame of manA is composed of 3,291 bases and codes for a preprotein of 1,097 amino acids with an estimated molecular mass of 119,627 Da. The start codon is preceded by a strong putative ribosome binding site (TAAGGCGGTG) and a putative -35 (TTCGC) and -10 (TAAAAT) promoter sequence. The ManA of T. polysaccharolyticum is a modular protein. Sequence comparison and biochemical analyses demonstrate the presence of an N-terminal leader peptide, and three other domains in the following order: a putative mannanase-cellulase catalytic domain, cellulose binding domains 1 (CBD1) and CBD2, and a surface-layer-like protein region (SLH-1, SLH-2, and SLH-3). The CBD domains show no sequence homology to any cellulose binding domain yet reported, hence suggesting a novel CBD. The duplicated CBDs, which lack a disulfide bridge, exhibit 69% identity, and their deletion resulted in both failure to bind to cellulose and an apparent loss of carboxymethyl cellulase and mannanase activities. At the C-terminal region of the gene are three repeats of 59, 67, and 56 amino acids which are homologous to conserved sequences found in the S-layer-associated regions within the xylanases and cellulases of thermophilic members of the Bacillus-Clostridium cluster. The ManA of T. polysaccharolyticum, besides being an extremely active enzyme, is the only mannanase gene cloned which shows this domain structure.     

DNA sequences of three beta-1,4-endoglucanase genes from Thermomonospora fusca.

The DNA sequences of the Thermomonospora fusca genes encoding cellulases E2 and E5 and the N-terminal end of E4 were determined. Each sequence contains an identical 14-bp inverted repeat upstream of the initiation codon. There were no significant homologies between the coding regions of the three genes. The E2 gene is 73% identical to the celA gene from Microbispora bispora, but this was the only homology found with other cellulase genes. E2 belongs to a family of cellulases that includes celA from M. bispora, cenA from Cellulomonas fimi, casA from an alkalophilic Streptomyces strain, and cellobiohydrolase II from Trichoderma reesei. E4 shows 44% identity to an avocado cellulase, while E5 belongs to the Bacillus cellulase family. There were strong similarities between the amino acid sequences of the E2 and E5 cellulose binding domains, and these regions also showed homology with C. fimi and Pseudomonas fluorescens cellulose binding domains.     

Biochemistry and genetics of actinomycete cellulases.

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T. curvata". The T. fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.     

Use of cellulases and recombinant cellulose binding domains for refining TCF kraft pulp

The modular endoglucanase Cel9B from Paenibacillus barcinonensis is a highly efficient biocatalyst, which expedites pulp refining and reduces the associated energy costs as a result. In this work, we set out to identify the specific structural domain or domains responsible for the action of this enzyme on cellulose fibre surfaces with a view to facilitating the development of new cellulases for optimum biorefining. Using the recombinant enzymes GH9–CBD3c, Fn3–CBD3b, and CBD3b, which are truncated forms of Cel9B, allowed us to assess the individual effects of the catalytic, cellulose binding, and fibronectin‐like domains of the enzyme on the refining of TCF kraft pulp from Eucalyptus globulus. Based on the physico‐mechanical properties obtained, the truncated form containing the catalytic domain (GH9–CBD3c) has a strong effect on fibre morphology. Comparing its effect with that of the whole cellulase (Cel9B) revealed that the truncated enzyme contributes to increasing paper strength through improved tensile strength and burst strength and also that the truncated form is more effective than the whole enzyme in improving tear resistance. Therefore, the catalytic domain of Cel9B has biorefining action on pulp. Although cellulose binding domains (CBDs) are less efficient toward pulp refining, evidence obtained in this work suggests that CBD3b alters fibre surfaces and influences paper properties as a result. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Large-Scale Analyses of Glycosylation in Cellulases

Cellulases are important glycosyl hydrolases (GHs) that hydrolyze cellulose polymers into smaller oligosaccharides by breaking the cellulose β (1→4) bonds,and they are widely used to produce cellulosic ethanol from the plant biomass.N-linked and O-linked glycosylations were proposed to impact the catalytic efficiency,cellulose binding affinity and the stability of cellulases based on observations of individual cellulases.As far as we know,there has not been any systematic analysis of the distributions of N-...     

Novel cellulose-binding-domain protein in Phytophthora is cell wall localized

Cellulose binding domains (CBD) in the carbohydrate binding module family 1 (CBM1) are structurally conserved regions generally linked to catalytic regions of cellulolytic enzymes. While widespread amongst saprophytic fungi that subsist on plant cell wall polysaccharides, they are absent amongst most plant pathogenic fungal cellulases. A genome wide survey for CBM1 was performed on the highly destructive plant pathogen Phytophthora infestans, a fungal-like Stramenopile, to determine if it harbored cellulolytic enzymes with CBM1. Only five genes were found to encode CBM1, and none were associated with catalytic domains. Surveys of other genomes indicated that the CBM1-containing proteins, lacking other domains, represent a unique group of proteins largely confined to the Stramenopiles. Immunolocalization of one of these proteins, CBD1, indicated that it is embedded in the hyphal cell wall. Proteins with CBM1 domains can have plant host elicitor activity, but tests with Agrobacterium-mediated in planta expression and synthetic peptide infiltration failed to identify plant hypersensitive elicitation with CBD1. A structural basis for differential elicitor activity is proposed.     

A unique chitinase with dual active sites and triple substrate binding sites from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85 degrees C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA.     

Effect of Urea on the Enzymatic Hydrolysis of Lignocellulosic Substrate and Its Mechanism

Effect of hydrogen bond breaker (urea) addition on the enzymatic hydrolysis of Avicel and eucalyptus pretreated by dilute acid (Eu-DA) was investigated. Urea enhanced the enzymatic hydrolysis of Eu-DA at 50 or 30 °C when the concentration of urea was below 60 g/L, while it inhibited the hydrolysis of Avicel. Low concentration urea (<?240 g/L) had little effect on the cellulase spatial structure and its activity. But it decreased cellulase binding to cellulose surface to inhibit the cellulose hydrolysis. Meanwhile, urea obviously prevented the adsorption of cellobiohydrolase I (CBHI) on the lignin in spite of little effect on the adsorption of β-glucosidase (BGL) and two endoglucanases (EGIII and EGV) on lignin. It was proposed that urea enhanced the enzymatic efficiency of Eu-DA by decreasing the cellulase adsorption on lignin surface.     

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification

Background

Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.

Results

In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion^a (AFEX?-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.

Conclusions

Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.