Mechanism-based Modelling for Fitting the Double-exponential Progress Curves of Cellulase Reaction.

Enzymatic hydrolysis of cellulosic biomass is a complex process involving many factors, including multiple enzymes, heterogeneous substrates, and multi-step enzyme reactions. Cellulase researchers have conventionally used a double-exponential equation to fit the experimental time course of product formation, but no theoretical basis for this has been established. Here we present a mechanism-based equation that fits well the progress curves of cellulase reaction, incorporating the concepts of non-productive and productive binding on the cellulose surface and processivity.

Synthesis of a natural core substrate with lignin-xylan cross-linkage for unveiling the productive kinetic parameters of glucuronoyl esterase.

Lignin-carbohydrate complexes (LCCs) present a considerable hurdle to the economic utilization of lignocellulosic biomass. Glucuronoyl esterase (GE) is an LCC-degrading enzyme that catalyzes the cleavage of the cross-linkages between lignin and xylan in LCCs. Benzyl-d-glucuronate (Bn-GlcA), a commercially available substrate, is widely used to evaluate GE activity assays.

Thermotolerance Mechanism of Fungal GH6 Cellobiohydrolase. Part I. Characterization of Thermotolerant Mutant from the Basidiomycete .

Cellobiohydrolase (CBH), belonging to glycoside hydrolase family 6 (GH6), plays an essential role in cellulose saccharification, but its low thermotolerance presents a challenge in improving the reaction efficiency. Based on a report that chimeric CBH II (GH6) engineered to remove non-disulfide-bonded free Cys shows increased thermotolerance, we previously mutated the two free Cys residues to Ser in GH6 CBH from the basidiomycete (Cel6A) and obtained a thermotolerant double mutant, C240S/C393S (Yamaguchi ., J.

Thermotolerance Mechanism of Fungal GH6 Cellobiohydrolase. Part II. Structural Analysis of Thermotolerant Mutant from the Basidiomycete .

Glycoside hydrolase family 6 cellobiohydrolase (GH6 CBH) is a group of cellulases capable of hydrolyzing crystalline cellulose. However, the synergistic reaction of GH6 CBH with other cellulases is hindered by its relatively low thermotolerance. We previously obtained a thermotolerant double mutant, C240S/C393S, of GH6 CBH from the basidiomycete (Cel6A) by replacing the two free cysteine (Cys) residues, C240 and C393, with serine (Yamaguchi .

Lytic polysaccharide monooxygenase increases cellobiohydrolases activity by promoting decrystallization of cellulose surface.

Efficient depolymerization of crystalline cellulose requires cooperation between multiple cellulolytic enzymes. Through biochemical approaches, molecular dynamics (MD) simulation, and single-molecule observations using high-speed atomic force microscopy (HS-AFM), we quantify and track synergistic activity for cellobiohydrolases (CBHs) with a lytic polysaccharide monooxygenase (LPMO) from . Increasing concentrations of LPMO (AA9D) increased the activity of a glycoside hydrolase family 6 CBH, Cel6A, whereas the activity of a family 7 CBH (Cel7D) was enhanced only at lower concentrations of AA9D.

Acetylated Xylan Degradation by Glycoside Hydrolase Family 10 and 11 Xylanases from the White-rot Fungus .

Endo-type xylanases are key enzymes in microbial xylanolytic systems, and xylanases belonging to glycoside hydrolase (GH) families 10 or 11 are the major enzymes degrading xylan in nature. These enzymes have typically been characterized using xylan prepared by alkaline extraction, which removes acetyl sidechains from the substrate, and thus the effect of acetyl groups on xylan degradation remains unclear. Here, we compare the ability of GH10 and 11 xylanases, Xyn10A and Xyn11B, from the white-rot basidiomycete to degrade acetylated and deacetylated xylan from various plants.

Comparison of glycoside hydrolase family 3 β-xylosidases from basidiomycetes and ascomycetes reveals evolutionarily distinct xylan degradation systems.

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) β-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite.

Role of Tryptophan 38 in Loading Substrate Chain into the Active-site Tunnel of Cellobiohydrolase I from .

Cellobiohydrolase I from ( Cel7A) is one of the best-studied cellulases, exhibiting high activity towards crystalline cellulose. Tryptophan residues at subsites -7 and -4 (Trp40 and Trp38 respectively) are located at the entrance and middle of the tunnel-like active site of Cel7A, and are conserved among the GH family 7 cellobiohydrolases. Trp40 of Cel7A is important for the recruitment of cellulose chain ends on the substrate surface, but the role of Trp38 is less clear.

Thermostable Mutants of Glycoside Hydrolase Family 6 Cellobiohydrolase from the Basidiomycete .

Thermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) are a kind of cellulase. In general, CBHs belonging to glycoside hydrolase (GH) family 6 (Cel6) act synergistically with CBHs of GH family 7 (Cel7) and other carbohydrate-active enzymes during the degradation of cellulosic biomass.

Single Amino Acid Mutation of Pyranose 2-Oxidase Results in Increased Specificity for Diabetes Biomarker 1,5-Anhydro-D-Glucitol.

Pyranose 2-oxidases catalyze the oxidation of various pyranose sugars at the C2 position. However, their potential application for detecting sugars other than glucose in blood is hindered by relatively high activity towards glucose. In this study, in order to find a mutant enzyme with enhanced specificity for 1,5-anhydro-D-glucitol (1,5-AG), which is a biomarker for diabetes mellitus, we conducted site-directed mutagenesis of pyranose 2-oxidase from the basidiomycete ( POX).

Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-β-1,3-galactanase from .

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases.

Convergent evolution of processivity in bacterial and fungal cellulases.

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e.

Effect of C-6 Methylol Groups on Substrate Recognition of Glucose/Xylose Mixed Oligosaccharides by Cellobiose Dehydrogenase from the Basidiomycete .

Cellobiose dehydrogenase (CDH) is a flavocytochrome catalyzing oxidation of the reducing end of cellobiose and cellooligosaccharides, and has a key role in the degradation of cellulosic biomass by filamentous fungi. Here, we use a lineup of glucose/xylose-mixed β-1,4-linked disaccharides and trisaccharides, enzymatically synthesized by means of the reverse reaction of cellobiose phosphorylase and cellodextrin phosphorylase, to investigate the substrate recognition of CDH. We found that CDH utilizes β-D-xylopyranosyl-(1→4)-D-glucopyranose (Xyl-Glc) as an electron donor with similar and values to cellobiose.

Evaluation of Ammonia Pretreatment for Enzymatic Hydrolysis of Sugarcane Bagasse to Recover Xylooligosaccharides.

Sugarcane bagasse is a useful biomass resource. In the present study, we examined the efficacy of ammonia pretreatment for selective release of hemicellulose from bagasse. Pretreatment of bagasse with aqueous ammonia resulted in significant loss of xylan.

Crystal Structure of the Catalytic and Cytochrome Domains in a Eukaryotic Pyrroloquinoline Quinone-Dependent Dehydrogenase.

Pyrroloquinoline quinone (PQQ) was discovered as a redox cofactor of prokaryotic glucose dehydrogenases in the 1960s, and subsequent studies have demonstrated its importance not only in bacterial systems but also in higher organisms. We have previously reported a novel eukaryotic quinohemoprotein that exhibited PQQ-dependent catalytic activity in a eukaryote. The enzyme, pyranose dehydrogenase (PDH), from the filamentous fungus (PDH) of the Basidiomycete division, is composed of a catalytic PQQ-dependent domain classified as a member of the novel auxiliary activity family 12 (AA12), an AA8 cytochrome domain, and a family 1 carbohydrate-binding module (CBM1), as defined by the Carbohydrate-Active Enzymes (CAZy) database.

Bridging the Micro-Macro Gap between Single-Molecular Behavior and Bulk Hydrolysis Properties of Cellulase.

The microscopic kinetics of enzymes at the single-molecule level often deviate considerably from those expected from bulk biochemical experiments. Here, we propose a coarse-grained-model approach to bridge this gap, focusing on the unexpectedly slow bulk hydrolysis of crystalline cellulose by cellulase, which constitutes a major obstacle to mass production of biofuels and biochemicals. Building on our previous success in tracking the movements of single molecules of cellulase on crystalline cellulose, we develop a mathematical description of the collective motion and function of enzyme molecules hydrolyzing the surface of cellulose.

Protein components of water extracts from fruiting bodies of the reishi mushroom Ganoderma lucidum contribute to the production of functional molecules.

Background: Mushrooms have been widely considered as health foods as their extracts have anti-hypertensive and anti-tumor activities. After a thorough literature survey, we hypothesized that enzymes in mushroom extracts play an important role in synthesizing functional molecules. Therefore, in this study, proteins extracted from reishi mushroom (Ganoderma lucidum), which is used in oriental medicine, were identified by the proteomic approach, and appropriate extraction methods for improving angiotensin-converting enzyme (ACE) inhibitory activities were investigated.

Structural and thermodynamic insights into β-1,2-glucooligosaccharide capture by a solute-binding protein in .

β-1,2-Glucans are bacterial carbohydrates that exist in cyclic or linear forms and play an important role in infections and symbioses involving Gram-negative bacteria. Although several β-1,2-glucan-associated enzymes have been characterized, little is known about how β-1,2-glucan and its shorter oligosaccharides (Sop s) are captured and imported into the bacterial cell. Here, we report the biochemical and structural characteristics of the Sop -binding protein (SO-BP, Lin1841) associated with the ATP-binding cassette (ABC) transporter from the Gram-positive bacterium Calorimetric analysis revealed that SO-BP specifically binds to Sop s with a degree of polymerization of 3 or more, with values in the micromolar range.

Interdomain flip-flop motion visualized in flavocytochrome cellobiose dehydrogenase using high-speed atomic force microscopy during catalysis.

Cellobiose dehydrogenase (CDH) is a dual domain flavocytochrome, which consists of a dehydrogenase (DH) domain containing a flavin adenine dinucleotide and a cytochrome (CYT) domain containing -type heme. To directly visualize the dynamic domain motion of class-I CDH from (CDH) during catalysis using high-speed atomic force microscopy, the apo-form of CDH was anchored to a heme-immobilized flat gold surface that can specifically fix the orientation of the CYT domain. The two domains of CDH are found to be immobile in the absence of cellobiose, whereas the addition of cellobiose triggers an interdomain flip-flop motion involving domain-domain association and dissociation.

Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis.

Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from (AXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. AXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop.

Crystal structure of a family 6 cellobiohydrolase from the basidiomycete Phanerochaete chrysosporium.

Cellobiohydrolases belonging to glycoside hydrolase family 6 (CBH II, Cel6A) play key roles in the hydrolysis of crystalline cellulose. CBH II from the white-rot fungus Phanerochaete chrysosporium (PcCel6A) consists of a catalytic domain (CD) and a carbohydrate-binding module connected by a linker peptide, like other known fungal cellobiohydrolases. In the present study, the CD of PcCel6A was crystallized without ligands, and p-nitrophenyl β-D-cellotrioside (pNPG3) was soaked into the crystals.

Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris.

Background: Random mutagenesis is a powerful technique to obtain mutant proteins with different properties from the wild-type molecule. Error-prone PCR is often employed for random mutagenesis in bacterial protein expression systems, but has rarely been used in the methylotrophic yeast Pichia pastoris system, despite its significant advantages, mainly because large (μg-level) amounts of plasmids are required for transformation.

Results: We developed a quick and easy technique for random mutagenesis in P.

Single-molecule Imaging Analysis of Binding, Processive Movement, and Dissociation of Cellobiohydrolase Trichoderma reesei Cel6A and Its Domains on Crystalline Cellulose.

Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose I The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose.

The use of neutron scattering to determine the functional structure of glycoside hydrolase.

Neutron diffraction provides different information from X-ray diffraction, because neutrons are scattered by atomic nuclei, whereas X-rays are scattered by electrons. One of the key advantages of neutron crystallography is the ability to visualize hydrogen and deuterium atoms, making it possible to observe the protonation state of amino acid residues, hydrogen bonds, networks of water molecules and proton relay pathways in enzymes. But, because of technical difficulties, less than 100 enzyme structures have been evaluated by neutron crystallography to date.

pH-dependent electron transfer reaction and direct bioelectrocatalysis of the quinohemoprotein pyranose dehydrogenase.

A pyranose dehydrogenase from Coprinopsis cinerea (CcPDH) is an extracellular quinohemoeprotein, which consists a b-type cytochrome domain, a pyrroloquinoline-quinone (PQQ) domain, and a family 1-type carbohydrate-binding module. The electron transfer reaction of CcPDH was studied using some electron acceptors and a carbon electrode at various pH levels. Phenazine methosulfate (PMS) reacted directly at the PQQ domain, whereas cytochrome c (cyt c) reacted via the cytochrome domain of intact CcPDH.