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Herein, we cloned and expressed an endo-β-1,4-glucanase gene (celA1805) from Bacillus subtilis B111 in Escherichia coli. The recombinant celA1805 contains a glycosyl hydrolase (GH) family 8 domain and shared 76.8% identity with endo-1,4-β-glucanase from Bacillus sp. KSM-330. Results showed that the optimal pH and temperature of celA1805 were 6.0 and 50°C, respectively, and it was stable at pH 3-9 and temperature ≤50°C. Metal ions slightly affected enzyme activity, but chemical agents generally inhibited enzyme activity. Moreover, celA1805 showed a wide substrate specificity to CMC, barley β-glucan, lichenin, chitosan, PASC and avicel. The Km and Vmax values of celA1805 were 1.78 mg/ml and 50.09 μmol/min/mg. When incubated with cellooligosaccharides ranging from cellotriose to cellopentose, celA1805 mainly hydrolyzed cellotetrose (G4) and cellopentose (G5) to cellose (G2) and cellotriose (G3), but hardly hydrolyzed cellotriose. The concentrations of reducing sugars saccharified by celA1805 from wheat straw, rape straw, rice straw, peanut straw, and corn straw were increased by 0.21, 0.51, 0.26, 0.36, and 0.66 mg/ml, respectively. The results obtained in this study suggest potential applications of celA1805 in biomass saccharification.

Lactobacillus amylovorus are known to exist in the intestinal flora of healthy cattle or pigs. The L. amylovorus strain 1394N20 was isolated from the feces of the Hanwoo calf (Bos taurus coreanae). The genome of strain 1394N20 consists of a single circular chromosome (2,176,326 bp) with overall guanine + cytosine content of 37.8 mol%. Moreover, 2,281 protein-coding sequences, 15 rRNAs, and 65 tRNAs genes were identified in the chromosome based on the results of annotation. The bacterium has a gene encoding endoglucanase, an enzyme that hydrolyzes the 1,4-β-D-glycosidic linkages in cellulose, hemicellulose, lichenin, and cereal β-D-glucans. Genomic sequencing of L. amylovorus strain 1394N20 reveals the immense potential of the strain as a probiotic with nutrient digestibility.

Lichenin from thalli of Flavocetraria cucullata and pectin from vegetative mass of Amaranthus retroflexus were isolated. Their monomeric compositions were established. The polysaccharides lichenin and pectin were shown to promote removal of cadmium ions from mice upon chronic intoxication. Increased levels of lipid peroxidation, elevated contents of total low-molecular-mass antioxidants (LMAO), and activation of superoxide dismutase in red blood cells were not observed in experimental groups of mice that received aqueous solutions (1 and 2%) of pectin and lichenin relative to a control. Administration of 1% solutions of lichenin and pectin was shown to be most effective upon chronic intoxication with cadmium(II) chloride. The malondialdehyde content and total LMAO remained at the level of the intact group. This may be due to the fact that 2% solutions of pectin and lichenin had higher viscosities that reduced their in vivo bioavailability in mice.

Lichenin from the thalli of Flavocetraria cucullata and pectin from the vegetative mass of Amaranthus retroflexus were isolated and their monomeric compositions were established. It was established that lichenin and pectin polysaccharides favored effective removal of cadmium ions from mice organism upon chronic intoxication. In the experimental groups of mice that received 1 and 2% aqueous solutions of pectin and lichenin, there were no increase in the level of lipid peroxidation, in the content of low molecular weight antioxidants, and in the activity of superoxide dismutase in red blood cells relative to the control. Upon chronic intoxication with cadmium (II) chloride, the most effective treatment was the introduction of 1% solutions of lichenine and pectin, for which the content of malondialdehyde and the amount of low molecular weight antioxidants remained on the level of intact group. This may be due to the fact that 2% solutions of pectin and lichenin have a higher viscosity, which reduces their bioavailability in the body of mice.

The isolation, heterologous gene expression, and characteristics of a new β-glucanase from Bacillus pumilus is described. The bgl1 gene from the Bacillus pumilus Bg57 VKPM В-13195 strain, which consists of 729 nucleotides, encodes a secreted endo-β-l,3(4)-D-glucanase (EC 3.2.1.6) containing 214 amino acids and 28 residues of the putative signal peptide in the N-terminal area. The nucleotide sequence of bgl1 and amino acid sequence of the mature Bgl protein have the highest homology (89 and 95%, respectively) with the sequences for β-l,3-l,4-glucanase from Bacillus licheniformis. A fragment of the gene encoding the mature protein was expressed in Pichia pastoris. The purified recombinant enzyme of Bgl was active towards barley β-glucan and lichenin. With barley β-glucan, the optimal pH and temperature were shown to be 6.0 and 50°C, respectively; Km and Vmax were 2.2 mg/mL and 3036.4 μmol/(min mg), respectively. The recombinant protein Bgl showed a high specific activity, thermal stability, and resistance to digestive enzymes. Other characteristics of the recombinant protein, including pH stability and sensitivity to metal ions and chemical reagents, were also determined.

β-1,3-1,4-Glucanases are a type of hydrolytic enzymes capable of catalyzing the strict cleavage of β-1,4 glycosidic bonds adjacent to β-1,3 linkages in β-D-glucans and have exhibited great potential in food and feed industrials. In this study, a novel glycoside hydrolase (GH) family 12 β-1,3-1,4-glucanase (CtGlu12A) from the thermophilic fungus Chaetomium sp. CQ31 was identified and biochemically characterized. CtGlu12A was most active at pH 7.5 and 65 °C, respectively, and exhibited a high specific activity of 999.9 U mg−1 towards lichenin. It maintained more than 80% of its initial activity in a wide pH range of 5.0–11.0, and up to 60 °C after incubation at 55 °C for 60 min. Moreover, the crystal structures of CtGlu12A with gentiobiose and tetrasccharide were resolved. CtGlu12A had a β-jellyroll fold, and performed retaining mechanism with two glutamic acids severing as the catalytic residues. In the complex structure, cellobiose molecule showed two binding modes, occupying subsites −2 to −1 and subsites + 1 to + 2, respectively. The concave cleft made mixed β-1,3-1,4-glucan substrates maintain a bent conformation to fit into the active site. Overall, this study is not only helpful for the understanding of the substrate-binding model and catalytic mechanism of GH 12 β-1,3-1,4-glucanases, but also provides a basis for further enzymatic engineering of β-1,3-1,4-glucanases.

The formation of a glucan/chitin/glycoprotein cell wall matrix is vital for fungal survival, growth, and morphogenesis. The cell wall proteins are important cell wall components and function in adhesion, signal transduction, and as cell wall structural elements. In this report we demonstrate that Neurospora crassa GH72 glucan transferases function to crosslink cell wall glycoproteins into the cell wall. With an in vitro assay, we show that the glucan transferases are able to attach lichenin, a cell wall glucan with a repeating β-1,4-glucose-β-1,4-glucose-β-1,3-glucose structure, to cell wall glycoproteins. We propose that the pathway for attachment of lichenin to the glycoprotein has four steps. First, N-linked oligosaccharides present on the glycoproteins are modified by the addition of a galactomannan. As part of our report we have characterized the structure of the galactomannan, which consists of an α-1,6-mannose backbone with galactofuranose side chains. In the second step, the galactomannan is processed by members of the GH76 α-1,6-mannanases. In the third step, the glucan transferases cleave the lichenin and create substrate-enzyme intermediates. In the final step, the transferases transfer the lichenin to the processed galactomannan. We demonstrate that the N. crassa glucan transferases have demonstrate specificity for the processed galactomannan and for lichenin. The energy from the cleaved glycosidic bond in lichenin is retained in the substrate-enzyme intermediate and used to create a new glycosidic bond between the lichenin and the processed galactomannan. The pathway effectively crosslinks glycoproteins into the fungal cell wall.

Lichens are symbiotic photosynthesizing organisms whose thalli are formed by fungi and algae/cyanobacteria. Among the factors that contribute to the ability of a lichen to tolerate harsh environmental conditions is the presence of unique secondary metabolites, such as lichen acids (e.g., usnic and gyrophoric acids), catechins, polysaccharides (including lichenin), and the dark pigment melanin. Melanin is a highly polymeric pigment containing phenolic and indole groups. Information about genes encoding enzymes responsible for melanin synthesis in lichens is very limited. The main aim of the present research was to characterize genes of oxidoreductases involved in melanin synthesis in lichens. It was found that synthesis of eu- and allomelanins in lichens is supported by the activity of genes homologous to those in Ascomycota. Corresponding proteins can be oligomeric or form multidomain structures, and the interplay of these enzymes is a prerequisite for the complex process of melanin synthesis in lichens.

This review discusses the wealth of information available for the N. crassa cell wall. The basic organization and structure of the cell wall is presented and how the wall changes during the N. crassa life cycle is discussed. Over forty cell wall glycoproteins have been identified by proteomic analyses. Genetic and biochemical studies have identified many of the key enzymes needed for cell wall biogenesis, and the roles these enzymes play in cell wall biogenesis are discussed. The review includes a discussion of how the major cell wall components (chitin, β-1,3-glucan, mixed β-1,3-/ β-1,4- glucans, glycoproteins, and melanin) are synthesized and incorporated into the cell wall. We present a four-step model for how cell wall glycoproteins are covalently incorporated into the cell wall. In N. crassa, the covalent incorporation of cell wall glycoproteins into the wall occurs through a glycosidic linkage between lichenin (a mixed β-1,3-/β-1,4- glucan) and a “processed” galactomannan that has been attached to the glycoprotein N-linked oligosaccharides. The first step is the addition of the galactomannan to the N-linked oligosaccharide. Mutants affected in galactomannan formation are unable to incorporate glycoproteins into their cell walls. The second step is carried out by the enzymes from the GH76 family of α-1,6-mannanases, which cleave the galactomannan to generate a processed galactomannan. The model suggests that the third and fourth steps are carried out by members of the GH72 family of glucanosyltransferases. In the third step the glucanosyltransferases cleave lichenin and generate enzyme/substrate intermediates in which the lichenin is covalently attached to the active site of the glucanosyltransferases. In the final step, the glucanosyltransferases attach the lichenin onto the processed galactomannans, which creates new glycosidic bonds and effectively incorporates the glycoproteins into the cross-linked cell wall glucan/chitin matrix.

A novel endo-β-1,3-1,4-glucanase gene (PbBglu16A) was cloned from Paenibacillus barengoltzii and heterogeneously expressed in Escherichia coli. The recombinant β-1,3-1,4-glucanase (PbBglu16A) was purified to homogeneity with a recovery yield of 78.6% and a specific activity of 431.8Umg-1. The molecular mass of PbBglu16A was estimated to be 44.0kDa by SDS-PAGE. The optimal pH and temperature of PbBglu16A were 6.0 and 55°C, respectively. The enzyme was stable within pH 3.5-9.0 and up to 55°C. PbBglu16A exhibited high substrate specificity towards barley β-glucan, oat β-glucan and lichenin. PbBglu16A showed an endo-type cleavage pattern and hydrolyzed endogenous enzyme-deactivated oat bran into β-gluco-oligosaccharides with a yield of 7.0%, which mainly consisted of trioligosaccharide and tetraoligosaccharide. Further, PbBglu16A could promote mashing with a reduced filtration time (14.0%) and viscosity (3.4%). Thus, PbBglu16A might be a promising candidate for the production of β-gluco-oligosaccharides and in brewing industry.

An exoglucanase (Exg-D) from the glycoside hydrolase family 5 subfamily 38 (GH5_38) was heterologously expressed and structurally and biochemically characterised at a molecular level for its application in alkyl glycoside synthesis. The purified Exg-D existed in both dimeric and monomeric forms in solution, which showed highest activity on mixed-linked &beta, glucan (88.0 and 86.7 U/mg protein, respectively) and lichenin (24.5 and 23.7 U/mg protein, respectively). They displayed a broad optimum pH range from 5.5 to 7 and a temperature optimum from 40 to 60 &deg, C. Kinetic studies demonstrated that Exg-D had a higher affinity towards &beta, glucan, with a Km of 7.9 mg/mL and a kcat of 117.2 s&minus, 1, compared to lichenin which had a Km of 21.5 mg/mL and a kcat of 70.0 s&minus, 1. The circular dichroism profile of Exg-D showed that its secondary structure consisted of 11% &alpha, helices, 36% &beta, strands and 53% coils. Exg-D performed transglycosylation using p-nitrophenyl cellobioside as a glycosyl donor and several primary alcohols as acceptors to produce methyl-, ethyl- and propyl-cellobiosides. These products were identified and quantified via thin-layer chromatography (TLC) and liquid chromatography&ndash, mass spectrometry (LC-MS). We concluded that Exg-D is a novel and promising oligomeric glycoside hydrolase for the one-step synthesis of alkyl glycosides with more than one monosaccharide unit.

A new cellulase (TaCel45) of glycoside hydrolase family 45 was identified in the thermophilic fungus Thielavia arenaria XZ7 and was successfully expressed in Pichia pastoris. The specific activities of TaCel45 towards lichenin, sodium carboxymethylcellulose (CMC-Na), and barley β-glucan were 769, 498, and 486 U/mg protein, respectively, which are higher than the values for all other reported GH45 cellulases. TaCel45 had maximum activity at pH 5.0–6.0 and 60–65 °C with barley β-glucan and CMC-Na as substrates and had a melting temperature (Tm) of 68.4 °C. However, TaCel45 exhibited extraordinary thermostability at 90 and 100 °C, retaining more than 70 and 45% of its activity after a 1-h incubation, respectively. Seven mutants (C11S, C12S, C16S, C31S, C171S, C193S, and C203S) were then constructed to investigate the effects of each disulfide bond on the structure, activity, and stability of TaCel45. As a result, six disulfide bonds (C11-C136, C16-C87, C31-C57, C88-C203, C90-C193, and C160-Cy171) were found to be indispensable for the folding, secretion, and activity of TaCel45, while C12-C48 was critical for thermal adaptation and refolding. The mutant C12S showed decreased optimal temperature and Tm values of 50 and 60.2 °C, respectively, and retained less than 50% of the thermal refolding ability of the wild type. Overall, this study demonstrated that disulfide bonds play a vital role in the folding and refolding capability and thermostability of this GH45 cellulase.

Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using Nf EG12A from Neosartorya fischeri P1 and EG (PDB 1KS4 ) from Aspergillus niger . Acidophilic and thermophilic Nf EG12A had the highest catalytic efficiency ( k cat / K m , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into Nf EG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency ( k cat / K m ) of these enzymes (in descending order, Nf EG12A [100%], Nf EG12A-FN [140%], and Nf EG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate ( k cat ). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis. IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the −1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, Nf EG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate ( k cat ). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.

A 1245-bp endoglucanase gene of glycoside hydrolase (GH) family 7, egl7A , was cloned from the acidothermophilic fungus Talaromyces emersonii CBS394.64 and successfully expressed in Pichia pastoris . Sequence alignments indicated that Egl7A had highest identity of 62.7% at the amino acid level with the functionally characterized endoglucanase from Aspergillus terreus NIH2624. Purified recombinant Egl7A exhibited the maximum activity at pH 4.5 and 70 °C, retained stable over the pH range of 2.0–12.0 and at 65 °C, and was strongly resistant to acidic and neutral proteases, most metal ions and SDS. The enzyme exhibited the highest specific activity reported so far (11,299 U mg −1 ) when using barley β-glucan as the substrate. Egl7A exhibited broad substrate specificity, including barley β-glucan, lichenin, CMC-Na, and xylan and had capacity to cleave cellopentaose and cellohexaose into smaller units rapidly. Under simulated mashing conditions, addition of Egl7A reduced the mash viscosity by 12.40%; when combined with a GH10 xylanase, more viscosity reduction (27.75%) was observed, which is significantly higher than that of the commercial enzyme Ultraflo XL (17.91%). All these properties make Egl7A attractive for potential applications in the feed and brewing industries.

Two isolates of aerobic, budding, pink-pigmented bacteria, designated strains PX4T and PT1, were isolated from a boreal Sphagnum peat bog and a forested tundra wetland. Cells of these strains were non-motile spheres that occurred singly or in short chains. Novel isolates were capable of growth at pH values between 3.5 and 6.5 (optimum at pH 5.0–5.5) and at temperatures between 6 and 30 °C (optimum at 15–25 °C). Most sugars and a number of polysaccharides including pectin, xylan, lichenin and Phytagel were used as growth substrates. The major fatty acids were C16 : 0, C18 : 1ω9 and C18 : 0; the major polar lipids were phosphocholine and trimethylornithine. The quinone was menaquinone-6, and the G+C content of the DNA was 66 mol%. Strains PX4T and PT1 were members of the order Planctomycetales and displayed 93–94 % 16S rRNA gene sequence similarity to Aquisphaera giovannonii, 91–92 % to species of the genus Singulisphaera and 90–91 % to Isosphaera pallida. The two novel strains, however, differed from members of these genera by cell morphology, substrate utilization pattern and a number of physiological characteristics. Based on these data, the novel isolates should be considered as representing a novel genus and species of planctomycetes, for which the name Paludisphaera borealis gen. nov., sp. nov., is proposed. The type strain is PX4T ( = DSM 28747T = VKM B-2904T). We also suggest the establishment of a novel family, Isosphaeraceae fam. nov., to accommodate stalk-free planctomycetes with spherical cells, which can be assembled in short chains, long filaments or shapeless aggregates. This family includes the genera Isosphaera, Aquisphaera, Singulisphaera and Paludisphaera.

The cDNA encoding β-1,3(4)-glucanase, named PsBg16A, from Paecilomyces sp. FLH30 was cloned, sequenced, and over expressed in Pichia pastoris, with a yield of about 61,754 U mL⁻¹ in a 5-L fermentor. PsBg16A has an open reading frame of 951 bp encoding 316 amino acids, and the deduced amino acid sequence of PsBg16A revealed that it belongs to glycoside hydrolase family 16. The purified recombinant PsBg16A had a pH optimum at 7.0 and a temperature optimum at 70 °C, and randomly hydrolyzed barley β-glucan, lichenin, and laminarin, suggesting that it is a typical endo-1,3(4)-β-glucanase (EC 3.2.1.6) with broad substrate specificity for β-glucans.

The 1,3(4)-beta-D-glucanases of glycoside hydrolase family 16 provide useful examples of versatile yet specific protein-carbohydrate interactions. In the present study, we report the X-ray structures of the 1,3(4)-beta-D-glucanase Phanerochaete chrysosporium Laminarinase 16A in complex with beta-glucan products from laminarin (1.6 A) and lichenin (1.1 A) hydrolysis. The G6G3G3G glucan, in complex with the enzyme, showed a beta-1,6 branch in the acceptor site. The G4G3G ligand-protein complex showed that there was no room for a beta-1,6 branch in the -1 or -2 subsites; furthermore, the distorted residue in the -1 subsite and the glucose in the -2 subsite required a beta-1,3 bond between them. These are the first X-ray crystal structures of any 1,3(4)-beta-D-glucanase in complex with glucan products. They provide details of both substrate and product binding in support of earlier enzymatic evidence.

An endoglucanase (1, 4-b-D glucan glucanohy- drolase, EC 3.2.1.4) which was catalytically more active and exhibited higher affinity towards barley b-glucan, xyloglu- can and lichenin as compared to carboxymethylcellulose (CMC) was purified from Aspergillus terreus strain AN1 following ion-exchange and hydrophobic interaction chro- matography and gel filtration. The purified enzyme (40-fold) that apparently lacked a cellulose-binding domain showed a specific activity of 60 lmol mg -1 protein -1 against CMC. The purified enzyme had a molecular weight of 78 and 80 KDa as indicated by sodium dodecyl sulphate-poly- acrylamide gel electrophoresis and gel filtration, respectively, and a pI of 3.5. The enzyme was optimally active at temperature 60C and pH 4.0, and was stable over a broad range of pH (3.0-5.0) at 50C. The endoglucanase activity was positively modulated in the presence of Cu 2? , Mg 2? ,C a 2? ,N a ? , DTT and mercaptoethanol. Endoglu- canase exhibited maximal turn over number (Kcat) and catalytic efficiency (Kcat/km) of 19.11 9 10 5 min -1 and 29.7 9 10 5 mM -1 min -1 against barley b-glucan as sub- strate, respectively. Hydrolysis of CMC and barley b-glucan liberated cellobiose, cellotriose, cellotetraose and detectable amount of glucose. The hydrolysis of xyloglucan, how- ever, apparently yielded positional isomers of cellobiose, cellotriose and cellotetraose as well as larger

Several water-soluble cellulose derivatives have been subjected to degradation with a cellulase preparation from Streptomyces sp. QM B814. Structural analysis of the enzymolysis products is described, particularly in relation to carboxymethylcellulose. For comparison, corresponding derivatives of lichenin, a soluble β-glucan structurally related to cellulose, have been examined with the use of this enzyme. The results indicate that the distribution of substituents in the cellulose derivatives is not uniform and that accessibility is important in determining the pattern of derivatization. Both carboxymethyl-and hydro-xyethylcellulose appear to be attacked by the cellulase in a similar fashion, and the two kinds of substituent groups are equally effective in limiting hydrolysis by this enzyme.

A gene encoding cellulase belonging to the glycosyl hydrolase family 9 along with its native promoter was isolated from Bacillus licheniformis F11, cloned in Escherichia coli DH5 α and subcloned by transconjugation to Bacillus megaterium MS941. Functionality of the encoded protein was proven both in heterologous hosts, E. coli and B. megaterium . In the latter, the gene product was found in the extracellular fraction expressing a high specific activity; whereas in E. coli the protein was not secreted into the medium, and rather, showed a lower specific activity. The optimum temperature of the recombinant enzyme expressed in the hosts range from 65-75 oC; whereas the optimum pH is 6. The recombinant enzyme was stable between 50-60 oC and in a broad pH range (pH 5 - 9). Addition of Ca 2+ and Fe 3+ enhanced the enzyme activity, whereas EDTA and Cu 2+ had the opposite effect. Lichenin, rather than carboxyl methyl cellulose, is the preferred substrate.

Summary Aromatic organic compounds that are present in the environment can have toxic effects or provide carbon sources for bacteria. We report here the global response of Bacillus subtilis 168 to phenol and catechol using proteome and transcriptome analyses. Phenol induced the HrcA, s B and CtsR heat-shock regulons as well as the Spx disulfide stress regulon. Catechol caused the activation of the HrcA and CtsR heat-shock regulons and a thiol-specific oxidative stress response involving the Spx, PerR and FurR regulons but no induction of the s B regulon. The most surprising result was that several catabolite-controlled genes are derepressed by catechol, even if glucose is taken up under these conditions. This derepression of the carbon catabolite control was dependent on the glucose concentration in the medium, as glucose excess increased the derepression of the CcpA-dependent lichenin utilization licBCAH operon and the ribose metabolism rbsRKDACB operon by catechol. Growth and viability experiments with catechol as sole carbon source suggested that B. subtilis is not able to utilize catechol as a carbon-energy source. In addition, the microarray results revealed the very strong induction of the yfiDE operon by catechol of which the yfiE gene shares similarities to glyoxalases/bleomycin resistance proteins/extradiol dioxygenases. Using recombinant His 6 -YfiE Bs we demonstrate that YfiE shows catechol-2,3-dioxygenase activity in the presence of catechol as the metabolite 2-hydroxymuconic semialdehyde was measured. Furthermore, both genes of the yfiDE operon are essential for the growth and viability of B. subtilis in the presence of catechol. Thus, our studies revealed that the catechol-2,3-dioxygenase YfiE is the key enzyme of a meta cleavage pathway in B. subtilis involved in the catabolism of catechol.

Effect of environmental factors on production of lichenin, a bacteriocin-like compound produced by Bacillus licheniformis 26L-10/3RA isolated from buffalo rumen was studied. Lichenin represents the first anaerobiosis-specific expression of broad-spectrum antibacterial compound effective only under anaerobic conditions. Production of lichenin by B. licheniformis 26L-10/3RA was found to be very high at 39 degrees C in L-10 medium supplemented with 0.5% glucose and 20% (w/v) inert thermocol beads. Lichenin production was highest at pH 6.8 after 72-96h of incubation. Our study also indicated that Lichenin is not a plasmid-linked characteristic and is encoded by chromosomal DNA. Results obtained can be used in large-scale production of Lichenin for potential application in manipulating rumen function intended for improving productivity of the ruminants.

SUMMARY Digestive juice from the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes exhibited total cellulase activity and activities of two cellulase enzymes; endo-β-1,4-glucanase and β-1,4-glucosidase. These enzymes hydrolysed native cellulose to glucose. The digestive juice of both species also contained laminarinase,licheninase and xylanase, which hydrolysed laminarin, lichenin and xylan,respectively, to component sugars. The pH optima of β-1,4-glucosidase,endo-β-1,4-glucanase and total cellulase from G. natalis were 4–5.5, 5.5 and 5.5–7, respectively. In the digestive juice from D. hirtipes, the corresponding values were 4–7, 5.5–7 and 4–9, respectively. The pH of the digestive juice was 6.69±0.03 for G. natalis and 6.03±0.04 for D. hirtipes and it is likely that the cellulases operate near maximally in vivo. In G. natalis, total cellulase activity and endo-β-1,4-glucanase activity were higher than in D. hirtipes, and the former species can thus hydrolyse cellulose more rapidly. β-1,4-glucosidase from G. natalis was inhibited less by glucono-d-lactone(Ki=11.12 mmol l-1) than was theβ-1,4-glucosidase from D. hirtipes (Ki=4.53 mmol l-1). The greater resistance to inhibition by theβ-1,4-glucosidase from G. natalis may contribute to the efficiency of the cellulase system in vivo by counteracting the effects of product inhibition and possibly dietary tannins. The activity ofβ-1,4-glucosidase in the digestive juice of D. hirtipes was higher than that of G. natalis.

Three laminarinases (LAM, LIC 1, and LIC 2) and two cellulases (CEL 1 and CEL 2) were purified to homogeneity from Periplaneta americana midguts. These beta-glucanases are secreted by salivary glands, stabilized by calcium ions, and have pH optima around 6. LAM (46 kDa) is active only on laminarin, native or with oxidized ends, and so it is an endo-beta-1,3-glucanase (EC 3.2.1.39). It processively releases mainly glucose from laminarin and shows lytic activity on fungal cells. LIC 1 (25 kDa) is an endo-beta-1,3(4)-glucanase (EC 3.2.1.6.), because it cleaves internal bonds on both laminarin and lichenin. LIC 1 lyses fungal cells and apparently have high affinity for sequences of cellotetraoses linked by beta-1,3 links, releasing cellotetraose from lichenin. The reaction catalyzed by LIC 1 is not in rapid equilibrium, as suggested by activity-pH data. These data also showed that a group in LIC 1 with pK=4.9 is necessary for substrate binding. LIC 2 (23 kDa) seems to be similar to LIC 1. The laminarinases are inactivated by carbodiimide, suggesting the presence of a carboxyl group involved in catalysis. LAM and LIC 2 are inhibited by excess laminarin as substrate. CEL 1 (72 kDa) and CEL 2 (73 kDa) quickly decrease the molecular weight of lichenin used as substrate. Therefore, they are endo-beta-1,4-glucanases (EC 3.2.1.4). Both CEL 1 and CEL 2 are also active on crystalline cellulose. The specificities of P. americana beta-glucanases agree with the omnivorous detritus-feeding habit of this insect, as they are able to attack plant (CEL 1, CEL 2, LIC 1 and LIC 2) and fungal (LIC 1 and LAM) cell walls.

Summary In the Kerguelen islands, two epigeic earthworms, one native Microscolex kerguelensis (Lankester 1879) and one introduced Dendrodrilus rubidus tenuis (Eisen 1874) are assumed to occupy the same ecological niche, i.e. to use the same food substrates. To test this hypothesis, the digestive capacities of the two species were compared using natural and synthetic glycosidic substrates. Both species hydrolysed starch, maltose, laminarin and N-acetylglucosamine, suggesting the ability to digest roots, yeasts, brown algae and fungi in soil. The main degradation of carbohydrates took place in the foregut, except in D. rubidus tenuis where some polysaccharides (laminarin, lichenin, xylan, cellulose and CMC) were mostly hydrolysed in the anterior tract. The degradation of carbohydrates mainly took place in the foregut and/or in the midgut, but in D. rubidus tenuis the hydrolysis of some polysaccharides (laminarin, cellulose and CMC) occurred in both the anterior tract and the foregut. The earthworms did not degrade lactose, sucrose and cellobiose; this result could be related to the weakness of the cellulasic and xylanasic activities. Hence, the full enzyme set generally found for the degradation of cellulose was not present in these epigeic earthworms.

Summary • Thalli of Cetraria islandica (Icelandic moss) and Evernia prunastri (oak moss) contain considerable amounts of the linear (1→3), (1→4)-β-glucan lichenin (lichenan), which different proportions of linkage groups in either species. • The immunocytochemical location of lichenan within the thallus is reported using a monoclonal antibody (barley anti-(1→3), (1→4)-β-glucan antibody) and low-temperature scanning electron microscopy (LTSEM) techniques. • The antibody labelled ultrathin sections of C. islandica, but not of E. prunastri. In C. islandica, lichenin was located in the extracellular matrix of the peripheral cortex and in a thick outer wall layer of medullary hyphae. LTSEM of fully hydrated and desiccated thalli of C. islandica showed that both the cortical extracellular matrix and the outer wall layer of medullary hyphae shrink dramatically during drought stress, indicating that these are major sites of water storage. A mycobiont-derived, hydrophilic wall surface layer prevents the thallus interior from becoming water-logged at full hydration. • The LTSEM observations and immunocytochemical data strongly suggest that lichenin is a structural compound, rather than a storage product, with important functions in thallus water relations.

Analysis of a carboxymethyl-cellulase clone isolated from the cDNA library of the ruminal fungus, Piromyces rhizinflata 2301, revealed that the clone encoded a polypeptide containing a cellulase catalytic domain, designated CelAcd. CelAcd was flanked by a 28-amino acid linker peptide at the N-terminus and linked to a dockerin domain by a 7-amino acid linker at the C-terminus. CelAcd showed homology with the family 5 of glycosyl hydrolases. CelAcd plus the linker peptides at both termini (designated CelcdN'C') was expressed in Escherichia coli and the purified enzyme was characterized. The feature of particular interest of CelcdN'C' was its bifunctional endo- and exo-glucanase activity, demonstrated by its ability to hydrolyse carboxymethyl cellulose (CMC) and pNP-beta-D-cellobioside. Furthermore, CelcdN'C' exhibited relatively high ability to degrade both microcrystalline Avicel and filter paper. CelcdN'C' also showed activity against barley beta-glucan, Lichenin and oat spelt xylan. The optimal activity conditions for CelcdN'C' with CMC as the substrate were pH 5.5 and 50 degrees C. Fifty percent of the enzyme activity was lost when CelcdN'C' was treated at 55 degrees C for 10 min. CelcdN'C' retained more than 10% enzyme activity after being heated under 90 degrees C for 10 min. Deletion of the N-terminal flanking linker of CelcdN'C' (the resulting protein was designated CelcdC') did not alter the enzymatic function of the catalytic domain. However, the thermal stability of CelcdC' was dramatically reduced. We conclude that the N-terminal flanking linker of CelAcd stabilizes the enzyme protein.

The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBD CelK ) was expressed in Escherichia coli and purified. It binds to acid-swollen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with capacities of 16.03 and 3.95 μmol/g of cellulose and relative affinities ( K r ) of 2.33 and 9.87 liters/g, respectively. The CBD CelK is the first representative of family IV CBDs to exhibit an affinity for BMCC. The CBD CelK also binds to the soluble polysaccharides lichenin, glucomannan, and barley β-glucan, which are substrates for CelK. It does not bind to xylan, galactomannan, and carboxymethyl cellulose. The CBD CelK contains 1 mol of calcium per mol. The CBD CelK has three thiol groups and one disulfide, reduction of which results in total loss of cellulose-binding ability. To reveal amino acid residues important for biological function of the domain and to investigate the role of calcium in the CBD CelK four highly conserved aromatic residues (Trp 56 , Trp 94 , Tyr 111 , and Tyr 136 ) and Asp 192 were mutated into alanines, giving the mutants W56A, W94A, Y111A, Y136A, and D192A. In addition 14 N-terminal amino acids were deleted, giving the CBD-N CelK . The CBD-N CelK and D192A retained binding parameters close to that of the intact CBD CelK , W56A and W94A totally lost the ability to bind to cellulose, Y136A bound to both ASC and BMCC but with significantly reduced binding capacity and K r and Y111A bound weakly to ASC and did not bind to BMCC. Mutations of the aromatic residues in the CBD CelK led to structural changes revealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation. Calcium content was drastically decreased in D192A. The results suggest that Asp192 is in the calcium-binding site of the CBD CelK and that calcium does not affect binding to cellulose. The 14 amino acids from the N terminus of the CBD CelK are not important for binding. Tyr136, corresponding to Cellulomonas fimi CenC CBD N1 Y85, located near the binding cleft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBD CelK correctly folded.

Two endoglucanase cDNAs, designated cel5A and cel45A, were isolated from a cDNA library of the anaerobic fungus Piromyces equi. Sequence analysis revealed that cel5A has an open reading frame of 5142 bp and encodes a 1714 amino acid modular enzyme, Cel5A, with a molecular mass of 194847 Da. Cel5A consists of four catalytic domains homologous to family-5 glycosyl hydrolases, two C-terminal dockerins and one N-terminal dockerin. This is the first report of a complete gene containing tandem repeats of family-5 catalytic domains. The cDNA cel45A has an open reading frame of 1233 bp and encodes a 410 amino acid modular enzyme, Cel45A, with a molecular mass of 44380 Da. The catalytic domain, located at the C terminus, is homologous to the family-45 glycosyl hydrolases. Cel45A is the first family-45 enzyme to be described in an anaerobe. The presence of dockerins at the N and C termini of Cel5A and at the N terminus of Cel45A implies that both enzymes are part of the high-molecular-mass cellulose-degrading complex produced by Piromyces equi. The catalytic domain nearest the C terminus of Cel5A and the catalytic domain of Cel45A were hyperexpressed as thioredoxin fusion proteins, Trx-Cel5A′ and Trx-Cel45A′, and subjected to biochemical analysis. Trx-Cel5A′ has a broad substrate range, showing activity against carboxymethylcellulose, acid-swollen cellulose, barley β-glucan, lichenin, carob galactomannan,p-nitrophenyl β-D-cellobiopyranoside and xylan. Trx-Cel45A′ is active against carboxymethylcellulose, acid-swollen cellulose and the mixed linkage glucans, barley β-glucan and lichenin.

The enzymatic hydrolysis of polysaccharides by the 1, 3(4)-beta-glucanase (LamR) from Rhodothermus marinus has been explored. The enzyme cleaves the 1,3-beta-linkages of 3-O-substituted glucose units in 1,3-beta-glucans such as laminarin and curdlan, and also the 1,4-beta-linkages of 3-O-substituted beta-glucose in beta-glucans such as lichenin and 1,3-1, 4-beta-glucan from the cell walls of barley endosperm. The polysaccharide substrates (laminarin, curdlan and barley beta-glucan) were characterised using NMR spectroscopy. The reaction of LamR with its substrates was followed by recording one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectra at suitable time intervals after addition of the enzyme. It is shown that hydrolysis occurs with retention of the anomeric configuration and that LamR performs transglycosylation to generate both 1, 3-beta-glycosidic and 1,4-beta glycosidic linkages. The transglycosylation results in, e.g. formation of the trisaccharide 4-O-glucosyl-laminaribiose from exclusively 1,3-beta-oligoglucosides. When barley 1,3-1,4-beta-glucan was incubated with LamR the beta-1, 4-linkages of 3-O-substituted beta-glycosyl residues were rapidly hydrolysed. Simultaneously de novo formation of 1,3-beta-glycosidic linkages was observed which, however, were cleaved during prolonged incubations. It is shown that a laminaribiosyl unit is the minimum requirement for formation of an enzyme-substrate complex and subsequent hydrolysis/transglycosylation.

Summary We have purified a β-1,3-endoglucanase (EC 3.2.1.39) from nonembryogenic Citrus sinensis (L.) Osbeck cv. Valencia callus to electrophoretic homogeneity by means of pH precipitation and ion exchange chromatography. The protein has an apparent Mr of 32,000, a pl > pH 10 and is serologically similar to a potato leaf glucanase induced by Phytophthora infestans infection. The enzyme hydrolyzes laminarin (Lam- inaria digitata) optimally at pH 5 and 50 °C. The enzyme will hydrolyze pachyman and laminarin extensively and yeast glucan slightly, but does not hydrolyze lichenin, barley glucan, cellulose, or starch. Product characterization by thin-layer chromatography indicates that the enzyme is an endohydrolase. The protein is N-terminal blocked, however, partial internal amino acid sequence analysis revealed that the peptide shared homology with a number of β-1,3-endoglucanases. Antibody to the purified protein was raised in a rabbit and used to screen an amplified cDNA library prepared from Citrus sinensis (L.) Osbeck cv. Valen- cia callus. A positive clone (pBGVC-1) containing a 1,249 by insert was isolated. A full length sequence of the clone was obtained and it contained a 1,229 by open reading frame starting at nucleotide 20. Sequence analysis indicated that the clone is homologous to other 0-1,3-endoglucanase genes. The predicted amino acid sequence was homologous with other 0-1,3-glucanases, contained both N- and C-terminal signal sequences, the glycosyl hydrolase family 17 signature sequence, and the sequence identical to the peptide that was sequenced from the purified protein.

Carbohydrase activities in the xylophagous Panesthia cribrata and the omnivorous Periplaneta americana were localised in the foregut and midgut and are endogenous. Levels of activities involved in the hydrolysis of cellulose and hemicellulose were higher in P. cribrata , while those directed against starch and sucrose, by comparison, were elevated in P. americana . Two endo- β -1,4-glucanase (EC 3.2.1.4) and two endo- β -1,4-xylanase (EC 3.2.1.8) components and additional components active against laminarin or lichenin, were secreted in the gut of P. cribrata . Two major β -glycosidase components, β -GD1 and β -GD2, were also secreted. β -GD1 was a β -fucosidase (EC 3.2.1.38), a β -mannosidase (EC 3.2.1.25) and a β -glucosidase (EC 3.2.1.21), which rapidly hydrolysed cellobiose, laminaribiose, and lactose. β -Galactosidase (EC 3.2.1.23) activity may also be partially associated with β -GD1. β -GD2 was a β -xylosidase (EC 3.2.1.37), an α -L-arabinosidase (EC 3.2.1.55) and a β -glucosidase, but was less active against β -linked disaccharides than was β -GD1. One β -N-acetylhexosaminidase (EC 3.2.1.52) and three α -galactosidase (EC 3.2.1.22) components were also secreted by P. cribrata . Two of the α -galactosidases may be associated with trehalase (EC 3.2.1.28) activity. A single sucrose α -glucosidase (EC 3.2.1.48) component hydrolysed sucrose, maltose and other α -linked disaccharides. β -fructofuranosidase (EC 3.2.1.26) activity is not secreted by P. cribrata .

Sixteen chemicals from different groups of known resistance inducers were tested as a soil drench in a humosoil : sand mix infested with an isolate of Rhizoctonia solani AG-4. Among these, 5-nitrosalicylic acid (2 mM aqueous solution), o-acetylsalicylic acid (2 mM), 2,6-dichloroisonicotinic acid (0.25 mM), 2-aminoisobutyric acid (2 mM) and lichenin (2 mM) controlled pre-emergence damping-off and post-emergence seedling mortality of bean cv. Dufrix, but gave little or weak and variable disease control in cucumber cv. Delikatess. Two-component mixtures of these five chemicals controlled the disease effectively in bean, mixtures containing 2-aminoisobutyric acid being the most effective, with seedling stands of up to 94% in infested soil. As none of these five chemicals (≤ 2 mM) reduced mycelial growth of R. solani on PDA plates, induced resistance in seedling tissues is implied in their disease control. Control was augmented in both hosts when any one of the five inducers was applied in a mixture with a cell suspension (1 × 109 cells mL−1) of an antagonistic fluorescent strain of Pseudomonas fluorescens; these mixtures showed additive effects and provided much better disease control in bean than in cucumber. The free-radical scavengers (antioxidants) ascorbic acid, benzoic acid, gluconic acid lactone and thiourea inhibited R. solani growth in vitro and efficiently controlled both pre-emergence damping-off and post-emergence seedling mortality of bean; ascorbic acid and benzoic acid also offered significant protection in cucumber. However, disease control values were reduced substantially when inducers were applied with antioxidants, because of strong antagonistic interactions in most of these mixtures. Probable mechanisms of disease control by 5-nitrosalicylic acid, o-acetylsalicylic acid, 2,6-dichloroisonicotinic acid, 2-aminoisobutyric acid and lichenin and their interactions with P. fluorescens and antioxidants are discussed.

A 971-bp cDNA, designated licA, was obtained from a library of Orpinomyces sp. strain PC-2 constructed in Escherichia coli. It had an open reading frame of 738 nucleotides encoding LicA (1,3-1,4-beta-D-glucanase; lichenase) (EC 3.2.1.73) of 245 amino acids with a calculated molecular mass of 27,929 Da. The deduced amino acid sequence had high homology with bacterial beta-glucanases, particularly in the central regions and toward the C-terminal halves of bacterial enzymes. LicA had no homology with plant beta-glucanases. The genomic DNA region coding for LicA was devoid of introns. More than 95% of the recombinant beta-glucanase produced in E. coli cells was found in the culture medium and periplasmic space. A N-terminal signal peptide of 29 amino residues was cleaved from the enzyme secreted from Orpinomyces, whereas 21 amino acid residues of the signal peptide were removed when the enzyme was produced by E. coli. The beta-glucanase produced by E. coli was purified from the culture medium. It had a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gels. The Km and Vmax values with lichenin as the substrate at pH 6.0 and 40 degrees C were 0.75 mg/ml and 3,790 micromol/min/mg, respectively. With barley beta-glucan as the substrate, the corresponding values were 0.91 mg/ml and 5,320 micromol/min/mg. This enzyme did not hydrolyze laminarin, carboxymethylcellulose, pustulan, or xylan. The main products of lichenin and barley beta-glucan hydrolysis were triose and tetraose. LicA represented the first 1,3-1,4-beta-D-glucanase reported from fungi. The results presented suggest that licA of Orpinomyces had a bacterial origin.

Free-living pathogenic amoebae Acanthamoeba spp. and Balamuthia mandrillaris are causative agents of important diseases of human: rarely occurring but highly fatal granulomatous amoebic encephalitis (both) and keratitis (Acanthamoeba). One of the reasons for the problematic therapy is differentiation into highly resistant cysts often found in affected tissues. In our study we have found that correct encystation in Acanthamoeba requires apart from others, the presence of functioning Golgi apparatus transporting the cyst wall material to the cell surface; glycogen phosphorylase degrading glycogen into glucose which seems to be further used for cellulose synthesis and two non-constitutive cellulose synthases. Acanthamoeba cellulose synthases seem not to be inhibited by known herbicides. In the cyst wall of acanthamoebae we detected cellulose, -mannan, and -1, 3-1, 4-linked glucan [lichenin or mixed-linkage glucan (MLG)]. Cellulose is present in the inner (endocyst) and the outer (exocyst) layers of the cyst wall, whereas-mannan and MLG are found in the endocyst. In a protozoan organism, MLG was detected for the first time. The MLG of Acanthamoeba has a similar composition to that found in barley with high amount of cellobiosyl and cellotriosyl followed by cellotetraosyl units. In contrast, with...

A gene library of a newly isolated Cellulomonas sp. strain was constructed in Escherichia coli and clones were screened for endoglucanase activity using dye-labelled carboxymethylcellulose. Seventeen clones were isolated that carried DNA inserts coding for endoglucanase enzymes. Of the 17 clones, one carrying the gene cegA, was further characterized. The recombinant endoglucanase was purified by FPLC. The endoglucanase was active against carboxymethylcellulose, lichenin and also degraded crystalline cellulose and birchwood xylan. The molecular mass of the enzyme (36 kDa), and its pH (7.4) and temperature (35 degrees C) optima were determined.

A novel (1-->3)-beta-D-glucan-binding protein (T-GBP) has been purified from the amoebocyte lysate of the Japanese horseshoe crab, Tachypleus tridentatus. It is a basic protein (pI 9.2) which appears to be a homotetramer composed of subunits with an apparent mol wt of 168000 and with an amino-terminal sequence (20 residues) KSGFILTAPKSLTLGRNNRL. T-GBP exerted an inhibitory effect on the (1-->3)-beta-D-glucan-initiated coagulation cascade reconstituted with purified preparations of factor G and the proclotting enzyme from the lysate. The binding of (1-->3)-beta-D-glucans to T-GBP was evaluated by measuring the residual amidolytic activity of the clotting enzyme, the product of the coagulation cascade, using Boc-Leu-Gly-Arg-4-nitroanilide as the chromogenic substrate. The binding specificity of a wide range of (1-->3)-beta-D-glucans and other polysaccharides towards T-GBP was expressed by the relative inhibition (%) of the activation of factor G, the first protease zymogen in the pathway, which is activated by binding to (1-->3)-beta-D-glucans. T-GBP was found to have a high affinity for linear (1-->3)-beta-D-glucans, e.g. pachyman, curdlan, and paramylon. It was able to bind to (1-->3)-beta-D-glucans with side-chain branches and mixed linkage such as schizophyllan, lentinan, laminarins, yeast beta-D-glucan, and (1-->3),(1-->4)-beta-D-glucans such as lichenin and barley beta-D-glucan. Binding of pachyman to T-GBP was demonstrated by an enzyme-linked immunosorbent assay using a specific antibody (rabbit IgG) raised against T-GBP.

Two β-1,3-glucanase isozymes (EC 3.2.1.39) GI and GII were purified from fresh Hevea latex using ionexchange and affinity chromatography. The two isozymes, as determined by SDS-PAGE, are monomeric proteins of M r ca 32 000 and 35 000, respectively. Specificity studies indicate that the two isozymes require relatively long runs of contiguous β-1,3- d -glucosidic linkages with a low degree of glucosyl substitution, such as in CM-pachyman and laminarin. The enzymes hydrolyse neither the β-1,4- d -glucosidic linkages of lichenin and barley glucan nor the β-1,6- d -glucosidic linkages of yeast glucan and pustulan. Kinetic analyses with laminarin as substrate indicate apparent K m values of 1.25 mg ml −1 (GI) and 1.33 mg ml −1 (GII), and V max of 2.86 nkat (GI) and 2.65 nkat (GII). The pH optima of the two isozymes are 4.5 (GI) and 5.0 (GII). Both isozymes are relatively heat-stable and retain full activity up to 60°. GII may be the glucanase isoform which plays an important role in response to fungal infection by rubber trees.

A water-soluble semisynthetic polysaccharide, γ-propoxy-sulfo-lichenin (PSL), was prepared by reaction of propansultone with lichenin, a natural occurring β-1.3/1.4-linked glucan originating from Cetraria sp. PSL represents a class of mixed-linked β-glucans with long and hydrophilic side chains in position C-6 of the glucan backbone. PSL with a degree of substitution of 0.8 and an average molecular weight of 250 kDa exhibited a strong antitumor activity in doses of 25 and 5 mg/kg against solid sarcoma 180 (100% resp. 82% tumor inhibition). The antitumor activity of PSL was shown to be dependent on the dimension of the molecule: the higher the average molecular weight, the higher was the inhibition rate obtained in the antitumor assay. No antitumor effect was observed by using a pretreatment of animals prior to transplantation of sarcoma 180. On syngenic DBA/2-MC.SC1 fibrosarcoma PSL inhibited tumor growth by about 88% at a concentration of 25 mg/kg. PSL failed to exhibit any direct cytotoxic effects on hormone-independent MDA-MB 231 mammacarcinoma. For PSL an indirect antitumor effect via modulation of the host mediate immune defence is postulated.

Ruminococcus albus 8 is a fibrolytic ruminal bacterium capable of utilization of various plant cell wall polysaccharides. A bioinformatic analysis of a partial genome sequence of R. albus revealed several putative enzymes likely to hydrolyze glucans, including lichenin, a mixed-linkage polysaccharide of glucose linked together in β-1,3 and β-1,4 glycosidic bonds. In the present study, we demonstrate the capacity of four glycoside hydrolases (GHs), derived from R. albus , to hydrolyze lichenin. Two of the genes encoded GH family 5 enzymes (Ra0453 and Ra2830), one gene encoded a GH family 16 enzyme (Ra0505), and the last gene encoded a GH family 3 enzyme (Ra1595). Each gene was expressed in Escherichia coli , and the recombinant protein was purified to near homogeneity. Upon screening on a wide range of substrates, Ra0453, Ra2830, and Ra0505 displayed different hydrolytic properties, as they released unique product profiles. The Ra1595 protein, predicted to function as a β-glucosidase, preferred cleavage of a nonreducing end glucose when linked by a β-1,3 glycosidic bond to the next glucose residue. The major product of Ra0505 hydrolysis of lichenin was predicted to be a glucotriose that was degraded only by Ra0453 to glucose and cellobiose. Most importantly, the four enzymes functioned synergistically to hydrolyze lichenin to glucose, cellobiose, and cellotriose. This lichenin-degrading enzyme mix should be of utility as an additive to feeds administered to monogastric animals, especially those high in fiber.

A new gene, RuCelA, encoding a bifunctional xylanase/endoglucanase, was cloned from a metagenomic library of yak rumen microorganisms. RuCelA showed activity against xylan and carboxymethylcellulose (CMC), suggesting bifunctional xylanase/endoglucanase activity. The optimal conditions for xylanase and endoglucanase activities were 65°C, pH 7.0 and 50°C, pH 5.0, respectively. In addition, the presence of Co(+) and Co(2+) can greatly improve RuCelA's endoglucanase activity, while inhibits its xylanase activity. Further examination of substrate preference showed a higher activity against barley glucan and lichenin than against xylan and CMC. Using xylan and barley glucan as substrates, RuCelA displayed obvious synergistic effects with β-1,4-xylosidase and β-1,4-glucosidase. Generation of soluble oligosaccharides from lignocellulose is the key step in bioethanol production, and it is greatly notable that RuCelA can produce xylo-oligosaccharides and cello-oligosaccharides in the continuous saccharification of pretreated rice straw, which can be further degraded into fermentable sugars. Therefore, the bifunctional RuCelA distinguishes itself as an ideal candidate for industrial applications.

THE constitution and properties of pectic substances have been intensively studied1. Pectins, when reacting with the bifunctional formaldehyde under acidic conditions, have been found to form three-dimensional networks. This process takes place much more slowly than the corresponding reaction with polysaccharides containing primary hydroxyl groups (for example, lichenin). An accelerated reaction of pectic substances with formaldehyde may be obtained by using the monoglycol ester of pectic acid (I) instead of the natural methyl ester. The glycol ester is easily produced2 by treating pectic acid with ethylene oxide. A greater reactivity with formaldehyde, even under neutral conditions, would be expected upon the introduction of primary amino-groups into the pectic acid molecule.

A great deal of experimental evidence has accumulated in the past several decades, suggesting that polysaccharides have wide bioactivities. Cladonia furcata polysaccharide, CFP-2, a water-soluble lichenin with a mean M r 7.6×10 4 , was first obtained by 0.25 M NaOH solution extraction, ethanol precipitation, DEAE-cellulose, and Sephadex G-200 column chromatography. Gas chromatography of acid hydrolyzate of CFP-2 suggested that it was composed of d -glucose, d -galactose, and d -mannose in the molar ratios of 8:1:1. Periodate oxidation, Smith degradation, IR, and NMR spectroscopy analysis revealed that CFP-2 had a backbone consisting of α-(1→3) and α-(1→4)-linked d -glucopyranosyl residues substituted at O -6 with β-(1→6)-linked d -galactopyranosyl residue and α-(1→6)-linked d -mannopyranosyl residue. CFP-2 was able to reduce viability of cultured HL-60 and K562 cells. The antiproliferative properties of CFP-2 appeared to be attributable to its induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by Western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 remained largely unchanged, but the Bax, Fas, and FasL expression showed up-regulation. Moreover, the telomerase activity analyzed by TRAP-ELISA assay in HL-60 cells treated with CFP-2 decreased as compared with the untreated control cells. These results suggest that CFP-2 could have a possible cancer therapeutic potential.