The modular cellulase CelZ of the thermophilic bacterium Clostridium stercorarium contains a thermostabilizing domain

The non-catalytic region of the Clostridium stercorarium cellulase CelZ (Avicelase I) comprises two protein segments (C and C′) grouped into different subfamilies of cellulose-binding domain (CBD) family III. The C-terminally located family IIIb domain C was identified as a true cellulose-binding domain responsible for anchoring the CelZ enzyme to cellulose. The family IIIc domain C′ immediately adjacent to the catalytic domain was unable to mediate binding to cellulose. A deletion study revealed a lack of independence of this pair of domains: almost the entire C′ domain was required to maintain the catalytic activity and the thermostability of the enzyme.     

High Speed Atomic Force Microscopy Visualizes Processive Movement of Trichoderma reesei Cellobiohydrolase I on Crystalline Cellulose

Fungal cellobiohydrolases act at liquid-solid interfaces. They have the ability to hydrolyze cellulose chains of a crystalline substrate because of their two-domain structure, i.e. cellulose-binding domain and catalytic domain, and unique active site architecture. However, the details of the action of the two domains on crystalline cellulose are still unclear. Here, we present real time observations of Trichoderma reesei (Tr) cellobiohydrolase I (Cel7A) molecules sliding on crystalline cellulose, obtained with a high speed atomic force microscope. The average velocity of the sliding movement on crystalline cellulose was 3.5 nm/s, and interestingly, the catalytic domain without the cellulose-binding domain moved with a velocity similar to that of the intact TrCel7A enzyme. However, no sliding of a catalytically inactive enzyme (mutant E212Q) or a variant lacking tryptophan at the entrance of the active site tunnel (mutant W40A) could be detected. This indicates that, besides the hydrolysis of glycosidic bonds, the loading of a cellulose chain into the active site tunnel is also essential for the enzyme movement.     

Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates

Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.     

海栖热袍菌内切葡聚糖酶Cel12B与木聚糖酶XynA CBD结构域融合基因的构建、表达及融合酶性质分析

海栖热袍菌内切葡聚糖酶Cel12B是极耐热胞外酶,氨基酸序列分析表明不含有纤维素结合结构域(CBD),对结晶纤维素无活性,但同样菌种来源的木聚糖酶XynA有催化结构域和纤维素结合结构城。用同样极耐热酶CBD区域和Cel12B融合构建重组质粒pET-20b-Cel12B-CBD,经诱导表达后,对结晶纤维素有活性,酶学特性研究表明:最适反应温度为100℃、最适pH为5.8、在pH4.5~7.0时酶活力稳定,90℃保温2h仍有87%的酶活。     

Cloning and Sequence Analysis of Endoglucanase Genes from an Industrial Fungus,Aspergillus kawachii

Three endoglucanase genes (cel5A, cel5B, and cel61A) were cloned from an industrial fungus, Aspergillus kawachii. Yeasts transformed with these cDNAs showed endoglucanase activity in medium. Cel5A and Cel61A contained a type 1 cellulose-binding domain (CBD1) at the C-terminus of the enzyme. The putative catalytic regions of Cel5A and Cel5B showed homology with various endoglucanases belonging glycosyl hydrolase family 5 (GH5). Cel5B showed high homology with Cel5A in catalytic region, but it lacked CBD1 and linker. The cel5A contained four introns, whereas cel5B contained five introns. The putative catalytic region of Cel61A showed homology with enzymes belonging to GH61. The cel61A contained no introns.     

Dynamic Interaction of Trichoderma reesei Cellobiohydrolases Cel6A and Cel7A and Cellulose at Equilibrium and during Hydrolysis

The binding of cellobiohydrolases to cellulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding domains interact with cellulose and with each other and how binding changes during hydrolysis is still needed. In this study we used tritium labeling by reductive methylation to monitor binding of the two Trichoderma reesei cellobiohydrolases, Cel6A and Cel7A (formerly CBHII and CBHI), and their catalytic domains. Measuring hydrolysis by high-performance liquid chromatography and measuring binding by scintillation counting allowed us to correlate activity and binding as a function of the extent of degradation. These experiments showed that the density of bound protein increased with both Cel6A and Cel7A as hydrolysis proceeded, in such a way that the adsorption points moved off the initial binding isotherms. We also compared the affinities of the cellulose-binding domains and the catalytic domains to the affinities of the intact proteins and found that in each case the affinity of the enzyme was determined by the linkage between the catalytic and cellulose-binding domains. Desorption of Cel6A by dilution of the sample showed hysteresis (60 to 70% reversible); in contrast, desorption of Cel7A did not show hysteresis and was more than 90% reversible. These findings showed that the two enzymes differ with respect to the reversibility of binding.     

Structural,Biochemical, and Computational Characterization of the Glycoside Hydrolase Family 7 Cellobiohydrolase of the Tree-killing Fungus Heterobasidion irregulare

Root rot fungi of the Heterobasidion annosum complex are the most damaging pathogens in temperate forests, and the recently sequenced Heterobasidion irregulare genome revealed over 280 carbohydrate-active enzymes. Here, H. irregulare was grown on biomass, and the most abundant protein in the culture filtrate was identified as the only family 7 glycoside hydrolase in the genome, which consists of a single catalytic domain, lacking a linker and carbohydrate-binding module. The enzyme, HirCel7A, was characterized biochemically to determine the optimal conditions for activity. HirCel7A was crystallized and the structure, refined at 1.7 Å resolution, confirms that HirCel7A is a cellobiohydrolase rather than an endoglucanase, with a cellulose-binding tunnel that is more closed than Phanerochaete chrysosporium Cel7D and more open than Hypocrea jecorina Cel7A, suggesting intermediate enzyme properties. Molecular simulations were conducted to ascertain differences in enzyme-ligand interactions, ligand solvation, and loop flexibility between the family 7 glycoside hydrolase cellobiohydrolases from H. irregulare, H. jecorina, and P. chrysosporium. The structural comparisons and simulations suggest significant differences in enzyme-ligand interactions at the tunnel entrance in the −7 to −4 binding sites and suggest that a tyrosine residue at the tunnel entrance of HirCel7A may serve as an additional ligand-binding site. Additionally, the loops over the active site in H. jecorina Cel7A are more closed than loops in the other two enzymes, which has implications for the degree of processivity, endo-initiation, and substrate dissociation. Overall, this study highlights molecular level features important to understanding this biologically and industrially important family of glycoside hydrolases.     

Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile

Cel5A is a highly active endoglucanase from Thermoanaerobacter tengcongensis MB4, displaying an optimal temperature range between 75 and 80 °C. After three rounds of error-prone PCR and screening of 4700 mutants, five variants of Cel5A with improved activities were identified by Congo Red based screening method. Compared with the wild type, the best variants 3F6 and C3-13 display 135 ± 6% and 193 ± 8% of the wild type specific activity for the substrate carboxymethyl cellulose (CMC), besides improvements in the relative expression level in Escherichia coli system. Remarkable are especially the improvements in activities at reduced temperatures (50% of maximum activity at 50 °C and about 45 °C respectively, while 65 °C for the wild type). Molecular Dynamics simulations performed on the 3F6 and C3-13 variants show a decreased number of intra-Cel5A hydrogen bonds compared to the wild type, implying a more flexible protein skeleton which correlates well to the higher catalytic activity at lower temperatures. To investigate functions of each individual amino acid position site-directed (saturation) mutagenesis were generated and screened. Amino acid positions Val249 and Ile321 were found to be crucial for improving activity and residue Ile13 (encoded by rare codon AUA) yields an improved expression level in E. coli.     

Changes in the activity of the multifunctional β-glycosyl hydrolase (Cel44C-Man26A) from Paenibacillus polymyxa by removal of the C-terminal region to minimum size

Paenibacillus polymyxa GS01 secretes Cel44C-Man26A as a multifunctional enzyme with cellulase, xylanase, lichenase, and mannanase activities. Cel44C-Man26A consists of 1,352 amino acids in which present a catalytic domain (CD) of the glycosyl hydrolase family 44 (GH44), fibronectin domain type 3 (Fn3), catalytic domain of glycosyl hydrolase family 26 (GH26), and a cellulose-binding module type 3 (CBM3). A truncated Cel44C-Man26A protein, consisting of 549 amino acid residues, reacted as a multifunctional mature enzyme despite the absence of the 10 amino acids containing GH44, Fn3, GH26, and CBM3. However, the multifunctional activity was not found in the mature Cel44C-Man26A protein truncated to less than 548 amino acids. The truncated Cel44C-Man26A proteins showed the optimum pH for the lichenase activity was pH 7.0, pH 6.0 for the xylanase and mannanase, and pH 5.0 for the cellulase. The truncated Cel44C-Man26A proteins exhibited enzymatic activity 40–120% higher than the full-length Cel44C.     

Engineering a family 9 processive endoglucanase from Paenibacillus barcinonensis displaying a novel architecture

Cel9B from Paenibacillus barcinonensis is a modular endoglucanase with a novel molecular architecture among family 9 enzymes that comprises a catalytic domain (GH9), a family 3c cellulose-binding domain (CBM3c), a fibronectin III-like domain repeat (Fn3_1,2), and a C-terminal family 3b cellulose-binding domain (CBM3b). A series of truncated derivatives of endoglucanase Cel9B have been constructed and characterized. Deletion of CBM3c produced a notable reduction in hydrolytic activity, while it did not affect the cellulose-binding properties as CBM3c did not show the ability to bind to cellulose. On the contrary, CBM3b exhibited binding to cellulose. The truncated forms devoid of CBM3b lost cellulose-binding ability and showed a reduced activity on crystalline cellulose, although activity on amorphous celluloses was not affected. Endoglucanase Cel9B produced only a small ratio of insoluble products from filter paper, while most of the reducing ends produced by the enzyme were released as soluble sugars (91%), indicating that it is a processive enzyme. Processivity of Cel9B resides in traits contained in the tandem of domains GH9–CBM3c, although the slightly reduced processivity of truncated form GH9–CBM3c suggests a minor contribution of domains Fn3_1,2 or CBM3b, not contained in it, on processivity of endoglucanase Cel9B.     

A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains

A bacterial strain Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). The cel44C-man26A gene was cloned from this endophytic strain. This 4,056-bp gene encodes for a 1,352-aa protein which, based on BLAST search homologies, contains a glycosyl hydrolase family 44 (GH44) catalytic domain, a fibronectin domain type 3, a glycosyl hydrolase family 26 (GH26) catalytic domain, and a cellulose-binding module type 3. The multifunctional enzyme domain GH44 possesses cellulase, xylanase, and lichenase activities, while the enzyme domain GH26 possesses mannanase activity. The Cel44C enzyme expressed in and purified from Escherichia coli has an optimum pH of 7.0 for cellulase and lichenase activities, but is at an optimum pH of 5.0 for xylanase and mannanase activities. The optimum temperature for enzymatic activity was 50°C for all substrates. No detectable enzymatic activity was detected for the Cel44C-Man26A mutants E91A and E222A. These results suggest that the amino acid residues Glu₉₁ and Glu₂₂₂ may play an important role in the glycosyl hydrolases activity of Cel44C-Man26A.     

Molecular simulations provide new insights into the role of the accessory immunoglobulin-like domain of Cel9A

Cel9A from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius belongs to the subfamily E1 of family 9 glycoside hydrolases, many members of which have an N-terminal Ig-like domain followed by the catalytic domain. The Ig-like domain is not directly involved in either carbohydrate binding or biocatalysis; however, deletion of the Ig-domain promotes loss of enzymatic activity. We have investigated the functional role of the Ig-like domain using molecular dynamics simulations. Our simulations indicate that residues within the Ig-like domain are dynamically correlated with residues in the carbohydrate-binding pocket and with key catalytic residues of Cel9A. Free energy perturbation simulations indicate that the Ig-like domain stabilizes the catalytic domain and may be responsible for the enhanced thermostability of Cel9A.     

The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144

The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD_KZ), originating from Pseudomonas aeruginosa bacteriophage ?KZ, has been examined using a fusion protein of PBD_KZ and green fluorescent protein (PBD_KZ-GFP). A fluorescence recovery after photobleaching analysis of bound PBD_KZ-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 × 10⁷ M⁻¹ for the PBD_KZ-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD_KZ-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.     

Construction and characterization of different fusion proteins between cellulases and β-glucosidase to improve glucose production and thermostability

A β-glucosidase from Clostridium cellulovorans (CcBG) was fused with one of three different types of cellulases from Clostridium thermocellum, including a cellulosomal endoglucanase CelD (CtCD), a cellulosomal exoglucanase CBHA (CtCA) and a non-cellulosomal endoglucanase Cel9I (CtC9I). Six bifunctional enzymes were constructed with either β-glucosidase or cellulase in the upstream. CtCD-CcBG showed the favorable specific activities on phosphoric acid swollen cellulose (PASC), an amorphous cellulose, with more glucose production (2 folds) and less cellobiose accumulation (3 folds) when compared with mixture of the single enzymes. Moreover, CtCD-CcBG had significantly improved thermal stability with a melting temperature (T_m) of 10.9 °C higher than that of CcBG (54.5 °C) based on the CD unfolding experiments. This bifunctional enzyme is thus useful in industrial application to convert cellulose to glucose.     

Enzyme-linked enzyme-binding assay for Pin1 WW domain ligands

Peptidyl prolyl cis-trans isomerase (PPIase) interacting with NIMA-1 (Pin1) catalyzes the cis-trans isomerization of pSer/pThr-Pro amide bonds. Pin1 is a two-domain protein that represents a promising target for the treatment of cancer. Both domains of Pin1 bind the pSer/pThr-Pro motif; PPIase enzymatic activity occurs in the catalytic domain, and the WW domain acts as a recognition module for the pSer/pThr-Pro motif. An assay we call an enzyme-linked enzyme-binding assay (ELEBA) was developed to measure the K_d of ligands that bind selectively to the WW domain. A ligand specific for the WW domain of Pin1 was covalently immobilized in a 96-well plate. Commercially available Pin1 conjugated to horseradish peroxidase was used for chemiluminescent detection of ligands that block the association of the WW domain with immobilized ligand. The peptide ligands were derived from the cell cycle regulatory phosphatase, Cdc25c, residues 45-50. The K_d values for Fmoc-VPRpTPVGGGK-NH₂ and Ac-VPRpTPV-NH₂ were determined to be 36 ± 4 and 110 ± 30 μM, respectively. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain, even in the presence of the catalytic domain. This method may be applied to any dual specificity, multidomain protein.     

Insights into Exo- and Endoglucanase Activities of Family 6 Glycoside Hydrolases from Podospora anserina

The ascomycete Podospora anserina is a coprophilous fungus that grows at late stages on droppings of herbivores. Its genome encodes a large diversity of carbohydrate-active enzymes. Among them, four genes encode glycoside hydrolases from family 6 (GH6), the members of which comprise putative endoglucanases and exoglucanases, some of them exerting important functions for biomass degradation in fungi. Therefore, this family was selected for functional analysis. Three of the enzymes, P. anserina Cel6A (PaCel6A), PaCel6B, and PaCel6C, were functionally expressed in the yeast Pichia pastoris. All three GH6 enzymes hydrolyzed crystalline and amorphous cellulose but were inactive on hydroxyethyl cellulose, mannan, galactomannan, xyloglucan, arabinoxylan, arabinan, xylan, and pectin. PaCel6A had a catalytic efficiency on cellotetraose comparable to that of Trichoderma reesei Cel6A (TrCel6A), but PaCel6B and PaCel6C were clearly less efficient. PaCel6A was the enzyme with the highest stability at 45°C, while PaCel6C was the least stable enzyme, losing more than 50% of its activity after incubation at temperatures above 30°C for 24 h. In contrast to TrCel6A, all three studied P. anserina GH6 cellulases were stable over a wide range of pHs and conserved high activity at pH values of up to 9. Each enzyme displayed a distinct substrate and product profile, highlighting different modes of action, with PaCel6A being the enzyme most similar to TrCel6A. PaCel6B was the only enzyme with higher specific activity on carboxymethylcellulose (CMC) than on Avicel and showed lower processivity than the others. Structural modeling predicts an open catalytic cleft, suggesting that PaCel6B is an endoglucanase.     

Biochemical characterization of a 27 kDa 1,3-β-d-glucanase from Trichoderma asperellum induced by cell wall of Rhizoctonia solani

Trichoderma asperellum produces two extracellular 1,3-β-d-glucanase upon induction with cell walls from Rhizoctonia solani. A minor 1,3-β-d-glucanase was purified to homogeneity by ion exchange chromatography on Q-Sepharose and gel filtration on Sephacryl S-100. A typical procedure provided 13.8-fold purification with 70% yield. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 27 kDa. The enzyme exhibited optimum catalytic activity at pH 3.6 and 45 °C. It was thermostable at 40 °C, and retained 75% activity after 60 min at 45 °C. The K_m and V_max values for 1,3-β-d-glucanase, using laminarin as substrate, were 0.323 mg ml⁻¹ and 0.315 U min⁻¹, respectively. The enzyme was strongly inhibited by Hg²⁺ and SDS. The enzyme was only active toward glucans containing β-1,3-linkages. Peptide sequences showed similarity with two endo-1,3(4)-β-d-glucanases from Aspergillus fumigatus Af293when compared against GenBank non-redundant database.     

Thermostable single domain antibody–maltose binding protein fusion for Bacillus anthracis spore protein BclA detection

We constructed a genetic fusion of a single domain antibody (sdAb) with the thermal stable maltose binding protein from the thermophile Pyrococcus furiosus (PfuMBP). Produced in the Escherichia coli cytoplasm with high yield, it proved to be a rugged and effective immunoreagent. The sdAb–A5 binds BclA, a Bacillus anthracis spore protein, with high affinity (K_D ∼ 50 pM). MBPs, including the thermostable PfuMBP, have been demonstrated to be excellent folding chaperones, improving production of many recombinant proteins. A three-step purification of E. coli shake flask cultures of PfuMBP–sdAb gave a yield of approximately 100 mg/L highly purified product. The PfuMBP remained stable up to 120 °C, whereas the sdAb–A5 portion unfolded at approximately 68 to 70 °C but could refold to regain activity. This fusion construct was stable to heating at 1 mg/ml for 1 h at 70 °C, retaining nearly 100% of its binding activity; nearly one-quarter (24%) activity remained after 1 h at 90 °C. The PfuMBP–sdAb construct also provides a stable and effective method to coat gold nanoparticles. Most important, the construct was found to provide enhanced detection of B. anthracis Sterne strain (34F2) spores relative to the sdAb–A5 both as a capture reagent and as a detection reagent.     

Gelation of high-methoxy pectin by enzymic de-esterification in the presence of calcium ions: a preliminary evaluation

Cohesive gels have been obtained by de-esterification of 1.0 wt % high-methoxy citrus pectin (degree of esterification ≈ 68%) in the presence of Ca²⁺ cations, using a commercial preparation (NovoShape) of fungal methyl esterase cloned from Aspergillus aculeatus. A convenient rate of network formation (gelation within ∼30 min) was achieved at an enzyme concentration of 0.2 PEU/g pectin. At a Ca²⁺-concentration of 40 mM and incubation temperature of 20 °C, severe syneresis (>7% of sample mass) was observed, but release of fluid decreased with decreasing concentration of Ca²⁺ and increasing temperature of incubation, becoming undetectable for 10 mM Ca²⁺ at 30 °C. Under these conditions, progressive development of solid-like character (storage modulus, G′) was observed during 160 min of enzymic de-esterification, and the mechanical spectrum recorded at the end of the incubation period had the form typical of a biopolymer gel. On subsequent heating to 70 °C, dissociation of the gel network (sigmoidal reduction in G′ and G″) was observed. At or above the midpoint temperature of this melting process (∼50 °C), there was no indication of gel formation on enzymic de-esterification (at 50 or 60 °C). At lower temperatures (20, 30 and 40 °C), the rate of gelation (assessed visually) showed no systematic increase as the incubation temperature was increased towards the temperature-optimum of the enzyme (∼50 °C). This unexpected behaviour is attributed to competition between faster de-esterification and slower formation of Ca²⁺-induced ‘egg-box’ junctions.     

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

The genome of the thermophilic bacterium Caldicellulosiruptor bescii encodes three multimodular enzymes with identical C-terminal domain organizations containing two consecutive CBM3b modules and one glycoside hydrolase (GH) family 48 (GH48) catalytic module. However, the three proteins differ much in their N termini. Among these proteins, CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) with a GH9/CBM3c binary partner in the N terminus has been shown to use a novel strategy to degrade crystalline cellulose, which leads to its outstanding cellulose-cleaving activity. Here we show that C. bescii Xyn10C (CbXyn10C), the N-terminal GH10 domain from CbXyn10C/Cel48B, can also degrade crystalline cellulose, in addition to heterogeneous xylans and barley β-glucan. The data from substrate competition assays, mutational studies, molecular modeling, and docking point analyses point to the existence of only one catalytic center in the bifunctional xylanase/β-glucanase. The specific activities of the recombinant CbXyn10C on Avicel and filter paper were comparable to those of GH9/CBM3c of the robust CelA expressed in Escherichia coli. Appending one or two cellulose-binding CBM3bs enhanced the activities of CbXyn10C in degrading crystalline celluloses, which were again comparable to those of the GH9/CBM3c-CBM3b-CBM3b truncation mutant of CelA. Since CbXyn10C/Cel48B and CelA have similar domain organizations and high sequence homology, the endocellulase activity observed in CbXyn10C leads us to speculate that CbXyn10C/Cel48B may use the same strategy that CelA uses to hydrolyze crystalline cellulose, thus helping the excellent crystalline cellulose degrader C. bescii acquire energy from the environment. In addition, we also demonstrate that CbXyn10C may be an interesting candidate enzyme for biotechnology due to its versatility in hydrolyzing multiple substrates with different glycosidic linkages.