Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose,as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome

The cellulosome is a supramolecular multienzyme complex formed by species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Cellulosomal enzymes bound to the scaffoldin protein act synergistically to degrade crystalline cellulose. However, there have been few attempts to reconstitute intact cellulosomes due to the difficulty of heterologously expressing full-length scaffoldin proteins. We describe the synthesis of a full-length scaffoldin protein containing nine cohesin modules, CipA; its deletion derivative containing two cohesin modules, ΔCipA; and three major cellulosomal cellulases, Cel48S, Cel8A, and Cel9K, of the Clostridium thermocellum cellulosome. The proteins were synthesized using a wheat germ cell-free protein synthesis system, and the purified proteins were used to reconstitute cellulosomes. Analysis of the cellulosome assembly using size exclusion chromatography suggested that the dockerin module of the enzymes stoichiometrically bound to the cohesin modules of the scaffoldin protein. The activity profile of the reconstituted cellulosomes indicated that cellulosomes assembled at a CipA/enzyme molar ratio of 1/9 (cohesin/dockerin = 1/1) and showed maximum synergy (4-fold synergy) for the degradation of crystalline substrate and ∼2.4-fold-higher synergy for its degradation than minicellulosomes assembled at a ΔCipA/enzyme molar ratio of 1/2 (cohesin/dockerin = 1/1). These results suggest that the binding of more enzyme molecules on a single scaffoldin protein results in higher synergy for the degradation of crystalline cellulose and that the stoichiometric assembly of the cellulosome, without excess or insufficient enzyme, is crucial for generating maximum synergy for the degradation of crystalline cellulose.     

Purification and Characterization of endo-(1→4)-β-xylanase fromGeotrichum candidum 3C

A method of purification of endo-( 1 → 4)-β-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid ofGeotrichum candidum 3C, grown for three days, is described. The enzyme, purified 23-fold, had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as the substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30–45°C for 1 h. With xylan from birch wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50°C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10,12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.     

Small Angle X-ray Scattering Analysis of Clostridium thermocellum Cellulosome N-terminal Complexes Reveals a Highly Dynamic Structure

Clostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.     

Isolation of α-Glucuronidase Enzyme from a Rumen Metagenomic Library

α-Glucuronidase enzymes play an essential role in the full enzymatic hydrolysis of hemicellulose. Up to this point, all genes encoding α-glucuronidase enzymes have been cloned from individual, pure culture strains. Using a high-throughput screening strategy, we have isolated the first α-glucuronidase gene (rum630-AG) from a mixed population of microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The RUM630-AG enzyme had optimum activity at pH 6.5 and 40 °C. When birchwood xylan was used as substrate, the RUM630-AG functioned synergistically with an endoxylanase enzyme to hydrolyze the substrate.     

Co-operative actions and degradation analysis of purified xylan-degrading enzymes from Thermomonospora fusca BD25 on oat-spelt xylan

AIMS: To determine and quantify the products from the degradation of xylan by a range of purified xylan-degrading enzymes, endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase produced extracellularly by Thermomonospora fusca BD25. METHODS AND RESULTS: The amounts of reducing sugars released from oat-spelt xylan by the actions of endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase were equal to 28.1, 4.6 and 7% hydrolysis (as xylose equivalents) of the substrate used, respectively. However, addition of beta-xylosidase and alpha-l-arabinofuranosidase preparation to endoxylanase significantly enhanced (70 and 20% respectively) the action of endoxylanase on the substrate. The combination of purified endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase preparations produced a greater sugar yield (58.6% hydrolysis) and enhanced the total reducing sugar yield by around 50%. The main xylooligosaccharide products released using the action of endoxylanase alone on oat-spelt xylan were identified as xylobiose and xylopentose. alpha-l-Arabinofuranosidase was able to release arabinose and xylobiose from oat-spelt xylan. In the presence of all three purified enzymes the hydrolysis products of oat-spelt xylan were mainly xylose, arabinose and substituted xylotetrose with lesser amount of substituted xylotriose. CONCLUSIONS: The addition of the beta-xylosidase and alpha-l-arabinofuranosidase enzymes to purified xylanases more than doubled the degradation of xylan from 28 to 58% of the total substrate with xylose and arabinose being the major sugars produced. SIGNIFICANCE AND IMPACT OF THE STUDY: The results highlight the role of xylan de-branching enzymes in the degradation of xylan and suggest that the use of enzyme cocktails may significantly improve the hydrolysis of xylan in industrial processes.     

Development and Biotechnological Application of a Novel Endoxylanase Family GH10 Identified from Sugarcane Soil Metagenome

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (β/α)₈-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production.     

Purification and characteristic of endo-(1--4)-beta-xylanase from Geotrichum candidum 3C

A method of purification of endo-(1-->4)-beta-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid of Geotrichum candidum 3C, grown for three days, is described. The enzyme purified 23-fold had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30-45 degrees C for 1 h. With xylan from beach wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50 degrees C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10, 12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.     

Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

Background

Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed.

Results

CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass.

Conclusion

We have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.

     

Beta-xylosidase activity of a GH3 glucosidase/xylosidase from yak rumen metagenome promotes the enzymatic degradation of hemicellulosic xylans

Aims: To characterize the duel activities of a glycosyl hydrolase family 3 β‐glucosidase/xylosidase from rumen bacterial metagenome and to investigate the capabilities of its β‐d ‐xylosidase activities for saccharification of hemicellulosic xylans. Methods and Results: A β‐glucosidase/xylosidase gene RuBGX1 was cloned from yak (Bos grunniens) rumen using the metagenomic technology. Recombinant RuBGX1, expressed in Escherichia coli, demonstrated high hydrolytic activities on both p‐nitrophenyl‐β‐d ‐glucopyranoside (pNP‐Glc) and p‐nitrophenyl‐β‐d ‐xylopyranoside (pNP‐Xyl) substrates. Analysis of the kinetic properties indicated that RuBGX1 had a lower affinity for pNP‐Glc substrate as the K_m was 0·164 mmol l^?1 for pNP‐Glc and 0·03 mmol l^?1 for pNP‐Xyl at pH 6·0 and 50°C, respectively. The capabilities of RuBGX1 β‐xylosidase for hydrolysis of xylooligosaccharide substrates were further investigated using an endoxylanase‐coupled assay. Hydrolysis time courses illustrated that a significant increase (about 50%) in the reducing sugars, including xylobiose, xylotriose and xylotetraose, was achieved by supplementing endoxylanase with RuBGX1. Enzymatic product analysis using high‐performance anion‐exchange chromatography‐pulsed amperometric detection showed that RuBGX1 could release xyloses from intermediate xylooligosaccharides produced by endoxylanase. Conclusions: The RuBGX1 shows β‐glucosidase activity in hydrolysis of cello‐oligosaccharides; meanwhile, it has β‐xylosidase activity and functions synergistically with endoxylanase to promote the degradation of hemicellulosic xylans. Significance and Impact of the study: This was the first to report the β‐xylosidase activity of family 3 β‐glucosidase/xylosidase functioned in the degradation of hemicellulosic xylans. The bifunctional β‐glucosidase/xylosidase property of RuBGX1 can be used in simultaneous saccharification of cellulose and xylan into fermentable glucose and xylose.     

Characterization of Xylan Utilization and Discovery of a New Endoxylanase in Thermoanaerobacterium saccharolyticum through Targeted Gene Deletions

The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C₅ and C₆ sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of β-xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type. This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum.     

Structural features of the Nostoc punctiforme debranching enzyme reveal the basis of its mechanism and substrate specificity

The debranching enzyme Nostoc punctiforme debranching enzyme (NPDE) from the cyanobacterium Nostoc punctiforme (PCC73102) hydrolyzes the α‐1,6 glycosidic linkages of malto‐oligosaccharides. Despite its high homology to cyclodextrin/pullulan (CD/PUL)‐hydrolyzing enzymes from glycosyl hydrolase 13 family (GH‐13), NPDE exhibits a unique catalytic preference for longer malto‐oligosaccharides (>G8), performing hydrolysis without the transgylcosylation or CD‐hydrolyzing activities of other GH‐13 enzymes. To investigate the molecular basis for the property of NPDE, we determined the structure of NPDE at 2.37‐Å resolution. NPDE lacks the typical N‐terminal domain of other CD/PUL‐hydrolyzing enzymes and forms an elongated dimer in a head‐to‐head configuration. The unique orientation of residues 25–55 in NPDE yields an extended substrate binding groove from the catalytic center to the dimeric interface. The substrate binding groove with a lengthy cavity beyond the ?1 subsite exhibits a suitable architecture for binding longer malto‐oligosaccharides (>G8). These structural results may provide a molecular basis for the substrate specificity and catalytic function of this cyanobacterial enzyme, distinguishing it from the classical neopullulanases and CD/PUL‐hydrolyzing enzymes. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Comparative characterization of deletion derivatives of the modular xylanase XynA of Thermotoga maritima

The modular Xylanase XynA from Thermotoga maritima consists of five domains (A1-A2-B-C1-C2). Two similar N-terminal domains (A1-A2-) are family 22 carbohydrate-binding modules (CBMs), followed by the catalytic domain (-B-) belonging to glycoside hydrolase family 10, and the C-terminal domains (-C1-C2), which are members of family 9 of CBMs. The gradual deletion of the non-catalytic domains resulted in deletion derivatives (XynAΔC; XynAΔA1C and XynAΔNC) with increased maximum activities (V _max) at 75°C, pH 6.2. Furthermore, these deletions led to a shift of the optimal NaCl concentration for xylan hydrolysis from 0.25 (XynA) to 0.5 M (XynAΔNC). In the presence of the family 22 CBMs, the catalytic domain retained more activity in the acidic range of the pH spectrum than without these domains. In addition to the deletion derivatives of XynA, the N-terminal domains A1 and A2 were produced recombinantly, purified, and investigated in binding studies. For soluble xylan preparations, linear β-1,4-glucans and mixed-linkage β-1,3-1,4-glucans, only the A2 domain mediated binding, not the A1 domain, in accordance with previous observations. The XynA deletion enzymes lacking the C domains displayed low affinity also to hydroxyethylcellulose and carboxymethylcellulose. With insoluble oat spelt xylan and birchwood xylan as the binding substrates, the highest affinity was observed with XynAΔC and the lowest affinity with XynAΔNC. Although the domain A1 did not bind to soluble xylan preparations, the insoluble oat spelt xylan-binding data suggest that this domain does play a role in substrate binding in that it improves the binding to insoluble xylans.     

<Emphasis Type="Italic">Thermobifida fusca</Emphasis> exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A

Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein–protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes’ CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.     

Structure,activity, and stability of metagenome‐derived glycoside hydrolase family 9 endoglucanase with an N‐terminal Ig‐like domain

A metagenome‐derived glycoside hydrolase family 9 enzyme with an N‐terminal immunoglobulin‐like (Ig‐like) domain, leaf‐branch compost (LC)‐CelG, was characterized and its crystal structure was determined. LC‐CelG did not hydrolyze p‐nitrophenyl cellobioside but hydrolyzed CM‐cellulose, indicating that it is endoglucanase. LC‐CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5–9. Its denaturation temperature was 81.4°C, indicating that LC‐CelG is a thermostable enzyme. The structure of LC‐CelG resembles those of CelD from Clostridium thermocellum (CtCelD), Cel9A from Alicyclobacillus acidocaldarius (AaCel9A), and cellobiohydrolase CbhA from C. thermocellum (CtCbhA), which show relatively low (29–31%) amino acid sequence identities to LC‐CelG. Three acidic active site residues are conserved as Asp194, Asp197, and Glu558 in LC‐CelG. Ten of the thirteen residues that form the substrate binding pocket of AaCel9A are conserved in LC‐CelG. Removal of the Ig‐like domain reduced the activity and stability of LC‐CelG by 100‐fold and 6.3°C, respectively. Removal of the Gln40‐ and Asp99‐mediated interactions between the Ig‐like and catalytic domains destabilized LC‐CelG by 5.0°C without significantly affecting its activity. These results suggest that the Ig‐like domain contributes to the stabilization of LC‐CelG mainly due to the Gln40‐ and Asp99‐mediated interactions. Because the LC‐CelG derivative lacking the Ig‐like domain accumulated in Escherichia coli cells mostly in an insoluble form and this derivative accumulated in a soluble form exhibited very weak activity, the Ig‐like domain may be required to make the conformation of the active site functional and prevent aggregation of the catalytic domain.     

Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module

The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE–CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE–CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.     

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

Background

Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture.

Results

Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins.

Conclusions

The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.

     

Growth and production of xylanolytic enzymes by the extreme thermophilic anaerobic bacterium Thermotoga thermarum

 Cultivation of the extreme thermophilic anaerobic bacterium Thermotoga thermarum at 77°C on xylan was accompanied by the formation of heat-stable endoxylanase (136U/l), β-xylosidase (44U/l) and α-arabinofuranosidase (10U/l). These enzymes were mainly associated with the cells and could not be released by detergent treatment {0.1–1.0mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS)}. Endoxylanases with a molecular weight of 40, 83 and 100kDa were induced when xylan or xylose were used as substrates for growth. In the presence of other sugars like glucose, maltose, arabinose or starch, low concentrations of the low-molecular-weight endoxylanase (40kDa) was detected. Xylose was found to be the best substrate for the induction of β-xylosidase and α-arabinofuranosidase but not for growth. Cultivation of T. thermarum in a dialysis batch fermentor resulted in a significant increase in cell concentration and enzyme level. A total cell count of 1.3×10⁹ cells/ml and 202U/l of endoxylanase were measured when partially soluble birchwood xylan was used as the carbon source. The use of insoluble beechwood xylan as the substrate caused the elevation of the maximal cell concentration and enzyme level up to 2.0×10⁹ cells/ml and 540U/l, respectively. Received: 14 September 1995/Received revision: 15 December 1995/Accepted: 18 December 1995     

Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate‐binding module (CBM)–cyan fluorescent protein (CFP)

In enzymatic saccharification of lignocellulosics, the access of the enzymes to exposed cellulose surfaces is a key initial step in triggering hydrolysis. However, knowledge of the structure–hydrolyzability relationship of the pretreated biomass is still limited. Here we used fluorescent‐labeled recombinant carbohydrate‐binding modules (CBMs) from Clostridium josui as specific markers for crystalline cellulose (CjCBM3) and non‐crystalline cellulose (CjCBM28) to analyze the complex surfaces of wood tissues pretreated with NaOH, NaOH–Na₂S (kraft pulping), hydrothermolysis, ball‐milling, and organosolvolysis. Japanese cedar wood, one of the most recalcitrant softwood species was selected for the analysis. The binding analysis clarified the linear dependency of the exposure of crystalline and non‐crystalline cellulose surfaces for enzymatic saccharification yield by the organosolv and kraft delignification processes. Ball‐milling for 5–30 min increased saccharification yield up to 77%, but adsorption by the CjCBM–cyan fluorescent proteins (CFPs) was below 5%. Adsorption of CjCBM–CFPs on the hydrothermolysis pulp were less than half of those for organosolvolysis pulp, in coincidence with low saccharification yields. For all the pretreated wood, crystallinity index was not directly correlated with the overall saccharification yield. Fluorescent microscopy revealed that CjCBM3–CFP and CjCBM28–CFP were site‐specifically adsorbed on external fibrous structures and ruptured or distorted fiber surfaces. The assay system with CBM–CFPs is a powerful measure to estimate the initiation sites of hydrolysis and saccharification yields from chemically delignified wood pulps. Biotechnol. Bioeng. 2010; 105: 499–508. © 2009 Wiley Periodicals, Inc.     

Enhanced direct ethanol production by cofactor optimization of cell surface‐displayed xylose isomerase in yeast

Xylose isomerase (XylC) from Clostridium cellulovorans can simultaneously perform isomerization and fermentation of d ‐xylose, the main component of lignocellulosic biomass, and is an attractive candidate enzyme. In this study, we optimized a specified metal cation in a previously established Saccharomyces cerevisiae strain displaying XylC. We investigated the effect of each metal cation on the catalytic function of the XylC‐displaying S. cerevisiae. Results showed that the divalent cobalt cations (Co²⁺) especially enhanced the activity by 46‐fold. Co²⁺ also contributed to d ‐xylose fermentation, which resulted in improving ethanol yields and xylose consumption rates by 6.0‐ and 2.7‐fold, respectively. Utility of the extracellular xylose isomerization system was exhibited in the presence of mixed sugar. XylC‐displaying yeast showed the faster d ‐xylose uptake than the yeast producing XI intracellularly. Furthermore, direct xylan saccharification and fermentation was performed by unique yeast co‐culture system. A xylan‐degrading yeast strain was established by displaying two kinds of xylanases; endo‐1,4‐β‐xylanase (Xyn11B) from Saccharophagus degradans, and β‐xylosidase (XlnD) from Aspergillus niger. The yeast co‐culture system enabled fine‐tuning of the initial ratios of the displayed enzymes (Xyn11B:XlnD:XylC) by adjusting the inoculation ratios of Xylanases (Xyn11B and XlnD)‐displaying yeast and XylC‐displaying yeast. When the enzymes were inoculated at the ratio of 1:1:2 (1.39 × 10¹³: 1.39 × 10¹³: 2.78 × 10¹³ molecules), 6.0 g/L ethanol was produced from xylan. Thus, the cofactor optimization and the yeast co‐culture system developed in this study could expand the prospect of biofuels production from lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1068–1076, 2017     

Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85.

Two different endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8), designated 1 and 2, have been purified by column chromatography to apparent homogeneity from the nonsedimentable extracellular culture fluid of the strictly anaerobic, ruminal bacterium Fibrobacter succinogenes S85 grown on crystalline cellulose. Endoxylanases 1 and 2 were shown to be basic proteins of 53.7 and 66.0 kDa, respectively, with different pH and temperature optima, as well as different substrate hydrolysis characteristics. The Km and Vmax values with water-soluble oat spelts xylan as substrate were 2.6 mg ml-1 and 33.6 mumol min-1 mg-1 for endoxylanase 1 and 1.3 mg ml-1 and 118 mumol min-1 mg-1 for endoxylanase 2. Endoxylanase 1, but not endoxylanase 2, released arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, but not from arabinan, arabinogalactan, or aryl-alpha-L-arabinofuranosides. With an extended hydrolysis time, endoxylanase 1 released 62.5 and 50% of the available arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, respectively. Endoxylanase 1 released arabinose directly from the xylan backbone, and this preceded hydrolysis of the xylan to xylooligosaccharides. Endoxylanase 2 showed significant activity against carboxymethyl cellulose but was unable to substantially hydrolyze acid-swollen cellulose. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on water-soluble xylan and xylooligosaccharides. Because of their unique hydrolytic properties, endoxylanases 1 and 2 appear to have strategic roles in plant cell wall digestion by F. succinogenes in vivo.