Search

Currently, the valorization of agroindustrial waste is of great interest. Moringa oleifera is a multipurpose tree whose softwood residues could be used as raw material for low-cost cellulase production. The aim of this study was to isolate, identify, and characterize microorganisms with cellulolytic activity in different carbon sources. We isolated and purified 42 microorganisms from M. oleifera biomass. Fungi presenting the largest hydrolytic halos in carboxymethylcellulose as a substrate were molecularly identified as Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) and Cladosporium cladosporioides (FC2). The ability of these fungal strains to break down cellulose was assessed in a submerged fermentation using either amorphous CMC or crystalline form (Avicel). P. funiculosum and C. cladosporioides displayed similar endoglucanase (606U/l) and exoglucanase (205U/l) activities in the Avicel-containing medium, whereas F. verticillioides showed the highest level of β-glucosidase activity (664U/l) in the carboxymethylcellulose medium. In addition, the effect of three culture media (A, B, and C) on cellulase production was evaluated in P. funiculosum using moringa straw as a carbon source. The results showed a volumetric productivity improvement of cellulases that was 2.77-, 8.26-, and 2.30-fold higher for endoglucanase, exoglucanase and β-glucosidase, respectively when medium C containing moringa straw was used as a carbon source. The enzymatic extracts produced by these fungi have biotechnological potential especially for second-generation bioethanol production (2G) from moringa straw. This is the first report on the use of M. oleifera biomass to induce the production of various cellulases in P. funiculosum., (Copyright © 2019 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

A psychrotolerant yeast strain Mrakia robertii A2-3 isolated from cryoconites of Hamtah glacier, Himalaya, India was investigated for the production of cold-tolerant endoglucanase. Optimum endoglucanase production was found at 15°C with an initial pH of 5.5, and potent inducers were 1% wt/vol of xylose and KNO 3 and 0.1% wt/vol of NaCl. Under optimum conditions, the enzyme production was 1.81-fold higher than the unoptimized conditions. Crude enzyme was partially purified by ammonium sulfate precipitation followed by dialysis. The enzyme was purified to 2.53-fold and the yield was 6.03% with specific activity of 17.38 U/mg and molecular weight ~57 kDa. The K m and V max values of the partially purified enzyme were found to be 1.57 mg/ml and 142.85 U/mg, respectively. The characterization study revealed that the best temperature was 15°C for activity and stability. Furthermore, the enzyme showed the highest activity at pH 11.0 and was stable at pH 6.0. Fe 2+ , Mn 2+ , Na 2+ , Cu 2+ , Co 2+ , Ca 2+ proved to be activators of endoglucanase. Ethylenediamine tetraacetic acid showed very low effect on the enzyme activity whereas it was active with Tween-80 and sodium deoxycholate. The present study successfully produced a cold-active endoglucanase with novel properties making it promising as a biocatalyst for industrial processes., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Salt stable cellulases are implicated in detritic food webs of marine invertebrates for their role in the degradation of cellulosic material. A haloarchaeon, Haloferax sulfurifontis GUMFAZ2 producing cellulase was successfully isolated from marine Haliclona sp., a sponge inhabiting the rocky intertidal region of Anjuna, Goa. The culture produced extracellular xylanase-free cellulase with a maximum activity of 11.7 U/ml, using carboxymethylcellulose-Na (CMC-Na), as a sole source of carbon in 3.5 M NaCl containing medium, pH 7 at 40°C and produced cellobiose and glucose, detectable by thin-layer chromatography. Nondenaturing polyacrylamide gel electrophoresis of the crude enzyme, revealed a single protein band of 19.6 kDa which on zymographic analysis exhibited cellulase activity while corresponding sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a molecular weight of 46 kDa. Unlike conventional cellulases, this enzyme is active in presence of 5 M NaCl and does not have accompanying xylanase activity, hence can be considered as xylanase-free cellulase. Such enzymes from haloarchaea offer great potential for biotechnological application because of their stability at high salinity and is therefore worth pursuing., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To enhance purification yield of the carboxymethylcellulase (CMCase) of P. aquimaris LBH-10, E. coli BL21/LBH-10 was constructed to produce the six histidine-tagged CMCase (CMCase with a His-tag). The purification yield of the CMCase with a His-tag produced by E. coli BL21/LBH-10 was 44.4%. The molecular weight of the CMCase with a His-tag was determined as 56 kDa. Its K m and V max were 7.4 g/L and 70.9 g/L min, respectively. The CMCase with a His-tag hydrolyzed avicel, carboxymethylcellulose (CMC), filter paper, pullulan, and xylan but did not hydrolyze cellobiose and p-nitrophenyl-β-D-glucopyranoside. The optimal temperature for reaction was 50 °C and more than 75% of its original activity was maintained at broad temperatures ranging from 20 to 70 °C after 24 h. The optimal pH was 4.0 and more than 60% of its original activity was maintained at pH ranging from 4.0 to 7.0. The activity of the CMCase with a His-tag was enhanced by CoCl 2 , KCl, PbCl 2 , RbCl 2 , and SrCl 2 until the concentration of 100 mM, but inhibited by EDTA, HgCl 2 , MnCl 2 , and NiCl 2 . The characteristics of the CMCase with a His-tag produced by E. coli BL21/LBH-10 were little different from the CMCase without a His-tag, which seemed to resulted from the conformational change in the structure due to a His-tag. The purification yield of the CMCase with a His-tag using affinity chromatography from the cell broth after cell breakdown was proven to be more economic than that from the supernatant with its low concentration of cellulase.

Detailed molecular mechanisms underpinning enzymatic reactions are still a central problem in biochemistry. The need for active site flexibility to sustain catalytic activity constitutes a notion of wide acceptance, although its direct influence remains to be fully understood. With the aim of studying the relationship between structural dynamics and enzyme catalysis, the cellulase Cel5A from Bacillus agaradherans is used as a model for in silico comparative analysis with mesophilic and psychrophilic counterparts. Structural features that determine flexibility are related to kinetic and thermodynamic parameters of catalysis. As a result, three specific positions in the vicinity of the active site of Cel5A are selected for protein engineering via site-directed mutagenesis. Three Cel5A variants are generated, N141L, A137Y and I102A/A137Y, showing a concomitant increase in the catalytic activity at low temperatures and a decrease in activation energy and activation enthalpy, similar to cold-active enzymes. These results are interpreted in structural terms by molecular dynamics simulations, showing that disrupting a hydrogen bond network in the vicinity of the active site increases local flexibility. These results provide a structural framework for explaining the changes in thermodynamic parameters observed between homologous enzymes with varying temperature adaptations., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Main Conclusion: PtrGH9A7, a poplar β-type endo-1,4-β-glucanase gene induced by auxin, promotes both plant growth and lateral root development by enhancing cell expansion. Endo-1,4-β-glucanase (EGase) family genes function in multiple aspects of plant growth and development. Our previous study found that PtrCel9A6, a poplar EGase gene of the β subfamily, is specifically expressed in xylem tissue and is involved in the cellulose biosynthesis required for secondary cell wall formation (Yu et al. in Mol Plant 6:1904-1917, 2013). To further explore the functions and regulatory mechanism of β-subfamily EGases, we cloned and characterized another poplar β-type EGase gene PtrGH9A7, a close homolog of PtrCel9A6. In contrast to PtrCel9A6, PtrGH9A7 is predominantly expressed in parenchyma tissues of the above-ground part; in roots, PtrGH9A7 expression is specifically restricted to lateral root primordia at all stages from initiation to emergence and is strongly induced by auxin application. Heterologous overexpression of PtrGH9A7 promotes plant growth by enhancing cell expansion, suggesting a conserved role for β-type EGases in 1,4-β-glucan chains remodeling, which is required for cell wall loosening. Moreover, the overexpression of PtrGH9A7 significantly increases lateral root number, which might result from improved lateral root primordium development due to enhanced cell expansion. Taken together, these results demonstrate that this β-type EGase induced by auxin signaling has a novel role in promoting lateral root formation as well as in enhancing plant growth.

Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to degradation, necessitating high dosages of cellulase to digest, thereby impeding the economic feasibility of cellulosic biofuels. Several recombinant cellulolytic yeast strains have been developed to reduce the cost of enzyme addition, but few of these strains are able to efficiently degrade crystalline cellulose due to their low cellulolytic activities. Here, by combining the cellulase ratio optimization with a novel screening strategy, we successfully improved the cellulolytic activity of a Saccharomyces cerevisiae strain displaying four different synergistic cellulases on the cell surface. The optimized strain exhibited an ethanol yield from Avicel of 57% of the theoretical maximum, and a 60% increase of ethanol titer from rice straw. To our knowledge, this work is the first optimization of the degradation of crystalline cellulose by tuning the cellulase ratio in a cellulase cell-surface display system. This work provides key insights in engineering the cellulase cocktail in a consolidated bioprocessing yeast strain. Biotechnol. Bioeng. 2017;114: 1201-1207. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Cellulose synthesis in bacteria is a complex process involving the concerted action of several enzymes whose genes are often organized in operons. This process influences many fundamental physiological aspects such as bacteria and host interaction, biofilm formation, among others. Although it might sound contradictory, the participation of cellulose-degrading enzymes is critical to this process. The presence of endoglucanases from family 8 of glycosyl hydrolases (GH8) in bacterial cellulose synthase (Bcs) complex has been described in different bacteria, including the model organism Komagataeibacter xylinus; however, their role in this process is not completely understood. In this study, we describe the biochemical characterization and three-dimensional structure of a novel GH8 member from Raoultella ornithinolytica, named AfmE1, which was previously identified by our group from the metagenomic analysis of the giant snail Achatina fulica. Our results demonstrated that AfmE1 is an endo-β-1,4-glucanase, with maximum activity in acidic to neutral pH over a wide temperature range. This enzyme cleaves cello-oligosaccharides with a degree of polymerization ≥ 5 and presents six glucosyl-binding subsites. The structural comparison of AfmE1 with other GH8 endoglucanases showed significant structural dissimilarities in the catalytic cleft, particularly in the subsite +3, which correlate with different functional mechanisms, such as the recognition of substrate molecules having different arrangements and crystallinities. Together, these findings provide new insights into molecular and structural features of evolutionarily conserved endoglucanases from the bacterial cellulose biosynthetic machinery.

Pretreatment of lignocellulosic biomass has been taken up as a global challenge as it comprises a large renewable source of fermentable sugars. In this study, effect of electron beam irradiation (EBI) on a hybrid grass variety investigated as a biomass pretreatment method. Dry biomass samples after characterization were exposed to EBI doses of 0, 75, 150 and 250 kGy. The pretreated biomass samples were enzymatically hydrolyzed using Trichoderma reesei ATCC 26921 cellulase for 144 h. The enzyme loadings were 15 and 30 FPU/g of biomass. The structural changes and degree of crystallinity of the pretreated biomass were studied by FTIR, XRD and SEM analyses. The lignocellulosic biomass sample showed 12.0% extractives, 36.9% cellulose, 28.4% hemicellulose, 11.9% lignin and 8.6% ash. Significant improvements in the reducing sugar and glucose yields were observed in the hydrolysate of EBI pretreated biomass compared to the control. In 250 kGy exposed samples 79% of the final reducing sugar yield was released within 48 h of hydrolysis at an enzyme loading rate of 30FPU/g of biomass. The IR crystallinity index calculated from the FTIR data and degree of crystallinity (XRD) decreased in the EBI treated samples. A significant negative correlation was observed between degree of crystallinity and the glucose yield from enzymatic hydrolysis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pneumocystis jirovecii pneumonia is an opportunistic fungal infection that causes severe respiratory impairment in immunocompromised patients. The viability of Pneumocystis organisms is dependent on the cyst cell wall, a structural feature that is regulated by essential cell wall-associated enzymes. The formation of the glucan-rich cystic wall has been previously characterized, but glucan degradation in the organism-specifically, degradation during trophic excystment-is not yet fully understood. Most studies of basic Pneumocystis biology have been conducted in Pneumocystis carinii or Pneumocystis murina, the varieties of this genus that infect rats and mice, respectively. Furthermore, all known treatments for P. jirovecii were initially discovered through studies of P. carinii. Accordingly, in this study, we have identified a P. carinii beta-1,3-endoglucanase gene (PCEng2) that is demonstrated to play a significant role in cell wall regulation. The cDNA sequence contained a 2.2-kb open reading frame with conserved amino acid domains homologous to similar fungal glycosyl hydrolases (GH family 81). The gene transcript showed up-regulation in cystic isolates, and the expressed protein was detected within both cyst and trophic forms. Complementation assays in Eng2-deleted Saccharomyces cerevisiae strains showed restoration of the cell wall separation defect during proliferation, demonstrating the importance of PCEng2 protein. during fungal growth. These findings suggest that regulation of cyst cell wall beta-glucans is a fundamental process during completion of the Pneumocystis life cycle.

To identify factors that influence cytoskeletal organization we screened for Arabidopsis (Arabidopsis thaliana) mutants that show hypersensitivity to the microtubule destabilizing drug oryzalin. We cloned the genes corresponding to two of the 131 mutant lines obtained. The genes encoded mutant alleles of PROCUSTE1 and KORRIGAN, which both encode proteins that have previously been implicated in cellulose synthesis. Analysis of microtubules in the mutants revealed that both mutants have altered orientation of root cortical microtubules. Similarly, isoxaben, an inhibitor of cellulose synthesis, also altered the orientation of cortical microtubules while exogenous cellulose degradation did not. Thus, our results substantiate that proteins involved in cell wall biosynthesis influence cytoskeletal organization and indicate that this influence on cortical microtubule stability and orientation is correlated with cellulose synthesis rather than the integrity of the cell wall.

The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long, extended and flexible linker region (LR) containing three loops closed by three disulfide bridges. To evaluate the possible role of this region in cold adaptation, the LR was sequentially shortened by protein engineering, successively deleting one and two loops of this module, whereas the last disulfide bridge was also suppressed by replacing the last two cysteine residue by two alanine residues. The kinetic and thermodynamic properties of the mutants were compared with those of the full-length enzyme, and also with those of the cold-adapted CM alone and with those of the homologous mesophilic enzyme, Cel5A, from Erwinia chrysanthemi. The thermostability of the mutated enzymes as well as their relative flexibility were evaluated by differential scanning calorimetry and fluorescence quenching respectively. The topology of the structure of the shortest mutant was determined by SAXS (small-angle X-ray scattering). The data indicate that the sequential shortening of the LR induces a regular decrease of the specific activity towards macromolecular substrates, reduces the relative flexibility and concomitantly increases the thermostability of the shortened enzymes. This demonstrates that the long LR of the full-length enzyme favours the catalytic efficiency at low and moderate temperatures by rendering the structure not only less compact, but also less stable, and plays a crucial role in the adaptation to cold of this cellulolytic enzyme.

In this study, the applicability of a "fed-batch" strategy, that is, sequential loading of substrate or substrate plus enzymes during enzymatic hydrolysis was evaluated for hydrolysis of steam-pretreated barley straw. The specific aims were to achieve hydrolysis of high substrate levels, low viscosity during hydrolysis, and high glucose concentrations. An enzyme system comprising Celluclast and Novozyme 188, a commercial cellulase product derived from Trichoderma reesei and a beta-glucosidase derived from Aspergillus niger, respectively, was used for the enzymatic hydrolysis. The highest final glucose concentration, 78 g/l, after 72 h of reaction, was obtained with an initial, full substrate loading of 15% dry matter weight/weight (w/w DM). Conversely, the glucose yields, in grams per gram of DM, were highest at lower substrate concentrations, with the highest glucose yield being 0.53 g/g DM for the reaction with a substrate loading of 5% w/w DM after 72 h. The reactions subjected to gradual loading of substrate or substrate plus enzymes to increase the substrate levels from 5 to 15% w/w DM, consistently provided lower concentrations of glucose after 72 h of reaction; however, the initial rates of conversion varied in the different reactions. Rapid cellulose degradation was accompanied by rapid decreases in viscosity before addition of extra substrate, but when extra substrate or substrate plus enzymes were added, the viscosities of the slurries increased and the hydrolytic efficiencies decreased temporarily.

A novel gene (Ba-ega) of Bacillus sp. AC-1, encoding an endoglucanase (Ba-EGA), was cloned and expressed in Escherichia coli. Ba-ega, containing a 1,980-bp open reading frame (ORF), encoded a protein of 659 amino acids and had a molecular mass of 74.87 kDa. Ba-EGA was a modular enzyme composed of a family-9 glycosyl hydrolase catalytic module (CM9) and a family-3 carbohydrate-binding module (CBM3). To investigate the functions of the CBM3 and CM9, a number of truncated derivatives of Ba-EGA were constructed, and all were active. The catalytic module (rCM9) alone was less stable at high temperature than the recombinant Ba-EGA (rBa-EGA). The temperature stability for the complex of rCM9 and rCBM3 was still lower than rBa-EGA, but higher than rCM9 alone. These observations indicated the existence of a non-covalent interaction between CM9 and CBM3 that might strengthen the stability of CM9. However, this interaction is not strong enough to mimic the protective effect of the CBM in the wild-type enzyme.

The Saccharomyces cerevisiae galactokinase ScGal1, a key enzyme for D-galactose metabolism, catalyzes the conversion of D-galactose to D-galactose 1-phosphate, whereas its catalytically inactive paralogue, ScGal3, activates the transcription of the GAL pathway genes. In Kluyveromyces lactis the transcriptional inducer function and the galactokinase activity are encoded by a single bifunctional KlGal1. Here, we investigated the cellular function of the single galactokinase GAL1 in the multicellular ascomycete Hypocrea jecorina (=Trichoderma reesei) in the induction of the gal genes and of the galactokinase-dependent induction of the cellulase genes by lactose (1,4-O-beta-D-galactopyranosyl-D-glucose). A comparison of the transcriptional response of a strain deleted in the gal1 gene (no putative transcriptional inducer and no galactokinase activity), a strain expressing a catalytically inactive GAL1 version (no galactokinase activity but a putative inducer function), and a strain expressing the Escherichia coli galK (no putative transcriptional inducer but galactokinase activity) showed that, in contrast to the two yeasts, both the GAL1 protein and the galactokinase activity are fully dispensable for induction of the Leloir pathway gene gal7 by D-galactose and that only the galactokinase activity is required for cellulase induction by lactose. The data document a fundamental difference in the mechanisms by which yeasts and multicellular fungi respond to the presence of D-galactose, showing that the Gal1/Gal3-Gal4-Gal80-dependent regulatory circuit does not operate in multicellular fungi.

A critical structural feature of many microbial endo-beta-1,4-glucanases (EGases, or cellulases) is a carbohydrate binding module (CBM), which is required for effective crystalline cellulose degradation. However, CBMs are absent from plant EGases that have been biochemically characterized to date, and accordingly, plant EGases are not generally thought to have the capacity to degrade crystalline cellulose. We report the biochemical characterization of a tomato EGase, Solanum lycopersicum Cel8 (SlCel9C1), with a distinct C-terminal noncatalytic module that represents a previously uncharacterized family of CBMs. In vitro binding studies demonstrated that this module indeed binds to crystalline cellulose and can similarly bind as part of a recombinant chimeric fusion protein containing an EGase catalytic domain from the bacterium Thermobifida fusca. Site-directed mutagenesis studies show that tryptophans 559 and 573 play a role in crystalline cellulose binding. The SlCel9C1 CBM, which represents a new CBM family (CBM49), is a defining feature of a new structural subclass (Class C) of plant EGases, with members present throughout the plant kingdom. In addition, the SlCel9C1 catalytic domain was shown to hydrolyze artificial cellulosic polymers, cellulose oligosaccharides, and a variety of plant cell wall polysaccharides.

Protein secretion is a complex process that can be modulated by folding factors in the endoplasmic reticulum (ER), such as calnexin, a highly-conserved molecular chaperone involved in quality control. In Schizosaccharomyces pombe, calnexin (Cnx1p) is essential for cell viability. The calnexin/Cnx1p determinants required for viability have been mapped within the last 123 residues of its C-terminus. To better understand the role(s) of calnexin/Cnx1p in secretion, we screened for cnx1 mutants 'super-secreting' cellulase. We identified ss14_cnx1, a mutant secreting 10-fold higher levels of the glycoprotein cellulase than the wild-type strain. While cellulase did not interact with ss14_Cnx1p, the ratio of secreted activity/quantity for this enzyme was not affected, suggesting that the quality control of folding in the ER was adequate in the mutant strain. Surprisingly, the ss14_Cnx1p mutant is composed of the 160 N-terminal amino acids of the mature molecule, thus this mutant defines a novel calnexin/Cnx1p region supporting Sz. pombe viability. Interestingly, like viable mutants spanning the last 52 aa of calnexin/Cnx1p, the 160 N-terminal residues encoded by ss14_cnx1 also forms a complex with the essential BiP chaperone. These results reveal the so far unidentified importance of the N-terminal region of calnexin/Cnx1p.

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity--4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a beta-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.

Anaerobic fungi possess high cellulolytic activities, which are organised in high molecular mass (HMM) complexes. Besides catalytic modules, the cellulolytic enzyme components of these complexes contain non-catalytic modules, known as dockerins, that play a key role in complex assembly. Screening of a genomic and a cDNA library of two Piromyces species resulted in the isolation of two clones containing inserts of 5.5 kb (Piromyces sp. E2) and 1.5 kb (Piromyces equi). Both clones contained the complete coding region of a glycoside hydrolase (GH) from family 6, consisting of a 20 amino acid signal peptide, a 76 (sp. E2)/81 (P. equi) amino acid stretch comprising two fungal non-catalytic docking domains (NCDDs), a 24 (sp. E2)/16 (P. equi) amino acid linker, and a 369 amino acid catalytic module. Homology modelling of the catalytic module strongly suggests that the Piromyces enzymes will be processive cellobiohydrolases. The catalytic residues and all nearby residues are conserved. The reaction is thus expected to proceed via a classical single-displacement (inverting) mechanism that is characteristic of this family of GHs. The enzyme, defined as Cel6A, encoded by the full-length Piromyces E2 sequence was expressed in Escherichia coli. The recombinant protein expressed had a molecular mass of 55 kDa and showed activity against Avicel, supporting the observed relationship of the sequence to those of known cellobiohydrolases. Affinity-purified cellulosomes of Piromyces sp. E2 were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. A major band was detected with the molecular weight of Cel6A. A tryptic fingerprint of this protein confirmed its identity.

The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in E. coli; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of dockerins found in R. flavefaciens plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative R. flavefaciens bands, including an approximately 110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in R. flavefaciens.

Abstract. An endo-beta-1,4-glucanase (EG) was purified from the hindgut of an Australian mound-building termite, Coptotermes lacteus. The hindgut extract had a peak separate from those for extracts obtained from the salivary glands and the midgut based on sephacryl S-200 gel chromatography, and also demonstrated an origin different from the endogenous EGs of the termite itself. The recovery was further purified by SDS-PAGE, and its N-terminal amino acid sequence analyzed. This showed high homology to EGs from glycoside hydrolase family (GHF) 7. PCR-based cloning methods were applied to the hindgut contents of C. lacteus and individual protozoan symbionts from C formosanus. cDNAs encoding putative EGs homologous to GHF7 members were then identified. The functionality of one of the putative proteins was confirmed by its expression in Escherichia coli.

This is a summary of the recent work on some glycosidases of sand flies and their Leishmania parasites. Glycosidases catalyze the hydrolysis of complex sugar subunits of polysaccharides into simple sugars. Leishmania major parasites secrete chitinase and N-acetylglucosaminase, which enables them to survive in the gut of the sand fly and are important in facilitating their transmission by the phlebotomine sand fly Phlebotomus papatasi. These enzymes are found in a wide range of trypanosomatids and the gene locus is highly conserved. The sand flies feed on plants and the ingested tissues may contain cellulose particles that the sand flies are unable to digest. Cellulolytic enzymes are secreted by L. major promastigotes and this may help to break down cellulose in infected flies and sustain their growth. Starch is a main photosynthesis product that is stored in leaves. Starch grains have been found in the midguts of field caught sand flies and alpha-amylase, the specific enzyme for starch, has been found in the salivary glands and other organs of Lutzomyia longipalpis and P. papatasi. Alpha-amylase and alpha-glucosidase are expressed by L. major promastigotes and alpha-glucosidase is secreted by several trypanosomatid genera, but not by all those examined. Primers originally designed to amplify P. papatasi amylase DNA sequences, by polymerase chain reaction (PCR), also amplified DNA from all Old World Leishmania species, indicating that the gene is highly conserved between sand flies and these parasites.

A comprehensive experimental study of substrate inhibition in cellulose hydrolysis based on a well defined system is presented. The hydrolysis of bacterial cellulose by synergistically operating binary mixtures of cellobiohydrolase I from Trichoderma reesei and five different endoglucanases as well as their catalytic domains displays a characteristic substrate inhibition. This inhibition phenomenon is shown to require the two-domain structure of an intact cellobiohydrolase. The experimental data were in accordance with a mechanism where cellobiohydrolases previously bound to the cellulose by means of their cellulose binding domains are able to find chain ends by lateral diffusion. An increased substrate concentration at a fixed enzyme load will also increase the average diffusion distance/time needed for cellobiohydrolases to reach new chain ends created by endoglucanases, resulting in an apparent substrate inhibition of the synergistic action. The connection between the binding properties and the substrate inhibition is encouraging with respect to molecular engineering of the binding domain for optimal performance in biotechnological processes.

A temperature-sensitive, elongation-deficient mutant of Arabidopsis thaliana was isolated. At the non-permissive temperature of 31 degrees C, the mutation impaired tissue elongation; otherwise, tissue development was normal. Hypocotyl cells that had established cell walls at 21 degrees C under light-dark cycles ceased elongation and swelled when the mutant was shifted to 31 degrees C and darkness, indicating that the affected gene is essential for cell elongation. Analysis of the cell walls of mutant plants grown at 31 degrees C revealed that the cellulose content was reduced to 40% and the pectin content was increased to 162% of the corresponding values for the wild type grown at the same temperature. The increased amounts of pectin in the mutant were bound tightly to cellulose microfibrils. No change in the content of hemicellulose was apparent in the 31 degrees C-adapted mutant. Field emission-scanning electron microscopy suggested that the structure of cellulose bundles was affected by the mutation; X-ray diffraction, however, revealed no change in the crystallite size of cellulose microfibrils. The regeneration of cellulose microfibrils from naked mutant protoplasts was substantially delayed at 31 degrees C. The recessive mutation was mapped to chromosome V, and map-based cloning identified it as a single G-->A transition (resulting in a Gly(429)-->Arg substitution) in KORRIGAN, which encodes a putative membrane-bound endo-1,4-beta-glucanase. These results demonstrate that the product of this gene is required for cellulose synthesis.

A beta-1,4-endoglucanase named MI-ENG1, homologous to the family 5 glycoside hydrolases, was previously isolated from the plant parasitic root-knot nematode Meloidogyne incognita. We describe here the detection of the enzyme in the nematode homogenate and secretion and its complete biochemical characterization. This study is the first comparison of the enzymatic properties of an animal glycoside hydrolase with plant and microbial enzymes. MI-ENG1 shares many enzymatic properties with known endoglucanases from plants, free-living or rumen-associated microorganisms and phytopathogens. In spite of the presence of a cellulose-binding domain at the C-terminus, the ability of MI-ENG1 to bind cellulose could not be demonstrated, whatever the experimental conditions used. The biochemical characterization of the enzyme is a first step towards the understanding of the molecular events taking place during the plant-nematode interaction.

Cellulose-producing Acetobacter xylinum has been known to secrete a cellulose-hydrolyzing beta-1,4-endoglucanase (CMCax). When antibodies to recombinant CMCax were added to the culture medium, the formation of cellulose fiber was severely inhibited. Western blot analysis using the antibody showed that this enzyme was secreted into the medium even by a cellulose non-producing strain (Cel-). These results indicate that beta-1,4-endoglucanase in the medium plays a critical role in the formation of cellulose fiber by the microorganism.

Thermomonospora fusca E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), an internal family IIIc cellulose binding domain (CBD), a fibronectinlike domain, and a family II CBD. Constructs containing the CD alone (E4-51), the CD plus the family IIIc CBD (E4-68), and the CD plus the fibronectinlike domain plus the family II CBD (E4-74) were made by using recombinant DNA techniques. The activities of each purified protein on bacterial microcrystalline cellulose (BMCC), filter paper, swollen cellulose, and carboxymethyl cellulose were measured. Only the whole enzyme, E4-90, could reach the target digestion of 4.5% on filter paper. Removal of the internal family IIIc CBD (E4-51 and E4-74) decreased activity markedly on every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% of the activity on BMCC even though it did not bind. A low-activity mutant of one of the catalytic bases, E4-68 (Asp55Cys), did bind to BMCC, although E4-51 (Asp55Cys) did not. The ratios of soluble to insoluble reducing sugar produced after filter paper hydrolysis by E4-90, E4-68, E4-74, and E4-51 were 6.9, 3.5, 1.3, and 0.6, respectively, indicating that the family IIIc CBD is important for E4 processivity.

Two cDNA clones encoding endo-beta-1,4-glucanases (EGases) were isolated from a radiata pine (Pinus radiata) cDNA library prepared from immature female strobili. The cDNAs PrCel1 (inus adiata cellulase ) and PrCel2 encode proteins 509 and 515 amino acids in length, respectively, including putative signal peptides. Both proteins contain domains conserved in plant and bacterial EGases. The proteins PRCEL1 and PRCEL2 showed strong similarity to each other (76% amino acid identity), and higher similarity to TPP18 (73 and 67%, respectively), an EGase cloned from tomato (Lycopersicon esculentum) pistils, than to any other reported EGases. Northern-blot analyses indicated that both genes displayed a similar pattern of expression. The only significant difference was in the level of expression. In situ hybridizations were used to demonstrate that, within differentiating pine reproductive structures, PrCel1 expression was greatest in microsporangia in pollen strobili and near the developing ovule in the seed strobili. Expression was also found in vegetative tissues, especially in regions experiencing cell elongation, such as the elongating region of root tips. Both proteins have an ability to degrade carboxymethylcellulose in vitro. Genomic-blot analysis indicated the presence of a family of EGase genes in the radiata pine genome, and that PrCel1 and PrCel2 are transcribed from distinct one-copy genes.

The widespread presence of cellulose-binding domains in cellulases from aerobic bacteria and fungi suggests the existence of a strong selective pressure for the retention of these non-catalytic modules. The complete nucleotide sequence of the cellulase gene, celA, from the aerobic soil bacterium Cellvibrio mixtus, was determined. It revealed an open reading frame of 1089 bp that encoded a polypeptide, defined as cellulase A (CelA), of M(r) 41,548. CelA displayed features characteristic of an endo-beta-1,4-glucanase, rapidly decreasing the viscosity of the substrate while releasing only moderate amounts of reducing sugar. Deletion studies in celA revealed that removal of 78 nucleotides from the 5' end or 75 from the 3' end of the gene led to the complete loss of cellulase activity of the encoded polypeptides. The deduced primary structure of CelA revealed an N-terminal signal peptide followed by a region that exhibited significant identity with the catalytic domains of cellulases belonging to glycosyl hydrolase family 5. These data suggest that CelA is a single-domain endoglucanase with no distinct non-catalytic cellulose-binding domain. Analysis of the biochemical properties of CelA revealed that the enzyme hydrolyses a range of soluble cellulosic substrates, but was inactive against Avicel, xylan or any other hemicellulose. CelA was resistant to proteolytic inactivation by pancreatic proteinases and surprisingly, in view of its mesophylic origin, was shown to be thermostable. The significance of these findings in relation to the role of single-domain cellulases in plant cell wall hydrolysis by aerobic microorganisms is discussed.

The relative contributions of four major cellulases of Trichoderma reesei (1,4-beta-D-glucan cellobiohydrolase I [CBH I], CBH II, endo-1,4-beta-D-glucanase I [EG I], and EG II) to the generation of the cellulase inducer from cellulose were studied with isogenic strains in which the corresponding genes (cbh1, cbh2, egl1, and egl2) had been deleted by insertion of the Aspergillus nidulans amdS marker gene. During growth on lactose (a soluble carbon source provoking cellulase gene expression), these strains showed no significant alterations in their ability to express the respective other cellulase genes, with the exception of the strain containing delta cbh1, which exhibited an increased steady-state level of cbh2 mRNA. On crystalline cellulose as the only carbon source, however, significant differences were apparent: strains in which cbh2 and egl2, respectively, had been deleted showed no expression of the other cellulase genes, whereas strains carrying the cbh1 or egl1 deletion showed these transcripts. The delta cbh1-containing strain also showed enhanced cbh2 mRNA levels under these conditions. A strain in which both cbh1 and cbh2 had been deleted, however, was unable to initiate growth on cellulose. Addition of 2 mM sophorose, a putative inducer of cellulase gene expression, to such cultures induced the transcription of egl1 and egl2 and restored the ability to grow on cellulose. We conclude that CBH II and EG II are of major importance for the efficient formation of the inducer from cellulose in T. reesei and that removal of both cellobiohydrolases renders T. reesei unable to attack crystalline cellulose.

The anaerobic rumen fungus Piromonas communis, when cultured on cotton fibre as the carbon source, produces an extracellular cellulase that is capable of solubilizing "crystalline" hydrogen-bond-ordered cellulose, in the form of the cotton fibre, at a rate that is greater than that of any other cellulases reported in the literature hitherto. The cell-free culture fluid is also very rich in xylan-degrading enzymes. The activity towards crystalline cellulose resides in a high-molecular-mass (approximately 700-1000 kDa) component (so-called crystalline-cellulose-solubilizing component, CCSC) that comprises endo (1-->4)-beta-D-glucanase (carboxymethylcellulase), beta-D-glucosidase and another enzyme that appears to be important for the breakdown of hydrogen-bond-ordered cellulose. The CCSC is associated with only a small amount of the endo(1-->4)-beta-D-glucanase (1.9%), beta-D-glucosidase (0.7%) and protein (0.5%) found in the crude cell-free cellulase preparation. The CCSC, unlike the bulk of the endo(1-->4)-beta-D-glucanase and beta-D-glucosidase, is very strongly absorbed on the microcrystalline cellulose, Avicel.

The celV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 bp and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of CelV1 was highly similar to that of CelV of another E. c. subsp. carotovora strain SCR1193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. CelV1 mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.

Using electroncytochemical and biochemical methods, differences between the cytochemical reaction intensity and activity of the cellulosolytic enzymes in Funaria hygrometrica moss cells grown for 30 days in the horizontal clinostat (2 rev/min) and in control have been studied. It has been shown that on clinostating the precipitate amount and size increases with the cellulase activity enhancement in the periplasmic space and protonema cell walls, when compared to control. Using biochemical methods it has been found that the activity of both endo-1,4-beta-glucanase and exo-1,4-beta-glucanase was higher under these conditions. A decrease of cellulose total content, its crystalline form, and pectic substances as well as an increase of hemicellulose content have been revealed in the clinostated material compared to control. Data obtained are discussed regarding the possible mechanism of cellulase activation and synthesis inhibition and cellulose crystallization in plant cell walls at clinostating.

A cellulase gene (endA) was isolated from a library of Ruminococcus flavefaciens strain 17 DNA fragments inserted in pUC13. The endA product showed activity against acid-swollen cellulose, carboxymethyl-cellulose, lichenan, cellopentaose and cellotetraose, but showed no activity against cellotriose or binding to avicel. Nucleotide sequencing indicated an encoded product of 455 amino acids which showed significant sequence similarity (ranging from 56% to 61%) with three endoglucanases from Ruminococcus albus, and with Clostridium thermocellum endoglucanase E. Little relatedness was found with a cellodextrinase previously isolated from R. flavefaciens FD1.

An endo-type semi-alkaline cellulase (CMCase I) produced by an alkalophilic Streptomyces strain has an extraordinarily long leader peptide of about 70 amino acids (aa), which can be grouped into four distinct regions, an NH2-terminal region (13 aa), an Arg-cluster region (13 aa), a hydrophobic region (23 aa), and an Ala/Pro-repeat region (12 aa). For identification of the function of each part of the leader peptide for secretion of the enzyme, mutation in the leader peptide were generated by site-directed mutagenesis using the cloned gene, and the mutant genes were expressed in a cellulase-negative mutant strain, Streptomyces lividans HN1. Although all the alterations in the leader peptide, except for one case, decreased secretion to various extents, in S. lividans, the following conclusions were obtained. Comparison of the intra- and extra-cellular enzyme activities of the mutants suggested that the Arg-cluster region was essential in secretion of the cellulase. Deletion of 8 amino acids rich in threonine and proline in the NH2-terminal region enhanced the secretion to a small extent. Deletion of the Ala/Pro repeat region had almost no effect on secretion.

Six endoglucanases (Endo I; II; III; IV; V; VI), three exoglucanases (Exo I; II; III) and a beta-glucosidase (beta-gluc I) were isolated from a commercial cellulase preparation derived from Trichoderma viride, using gel filtration on Bio-Gel, anion exchange on DEAE-Bio-Gel A, cation exchange on SE-Sephadex and affinity chromatography on crystalline cellulose. Molecular masses were determined by polyacrylamide gel electrophoresis. One group of endoglucanases (Endo I, Endo II and Endo IV) with Mr of 50 000, 45 000 and 23 500 were more random in their attack on carboxymethylcellulose than another group (Endo III, Endo V and Endo VI) showing Mr of 58 000, 57 000 and 53 000 respectively. Endo III was identified as a new type of endoglucanase with relatively high activity on crystalline cellulose and moderate activity on carboxymethylcellulose. Exo II and Exo III with Mr of 60 500 and 62 000 respectively showed distinct adsorption affinities on a column of crystalline cellulose and could be eluted by a pH gradient to alkaline regions. These enzymes were cellobiohydrolases as judged by high-pressure liquid chromatography of the products obtained from incubation with H3PO4-swollen cellulose. It was concluded that these exoglucanases are primarily active on newly generated chain ends. Exo I was essentially another type of exoglucanase which in the first instance was able to split off a cellobiose molecule from a chain end and then hydrolyse this molecule in a second step to two glucose units beta-Gluc I was a new type of aryl-beta-D-glucosidase which had no activity on cellobiose. The enzyme had a Mr of 76 000 and was moderately active on CM-cellulose, crystalline cellulose and xylan and highly active on p-nitrophenyl-beta-D-glucose and p-nitrophenyl-beta-D-xylose.

Experiments concerning tumor transformation of plant cells or protoplasts need complete knowledge of the effects of media components. Here inhibiting effects of cellulase Onozuka, glucose, sucrose and mannitol (up to 0.3M), of tissue culture media, and of the antibiotic aureomycin on the tumor initiation process in Kalanchoe leaves are reported. Also the influence of these substances on the growth of the tumor inducing Agrobacterium tumefaciens is studied. The results lead to the design of conditions which should be favourable to in vitro transformation.