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A β-1,3-1,4 glucanase gene of Paenibacillus sp. X4, bglc8H, was cloned and characterized. BGlc8H was predicted to be a protein of 409 amino acid residues, including a signal peptide of 31 amino acids. The mature enzyme was predicted to have 378 amino acid residues; its [corrected] molecular mass and pI were estimated as 41,561 Da and 7.61, respectively. BGlc8H belongs to glycoside hydrolase family 8 (GH8). Site-directed mutants of Glu95 and Asp156 of BGlc8H showed a near-complete loss of activity, indicating that they are catalytically-active residues. Unlike other GH8 members, BGlc8H had broad substrate specificity and hydrolyzed barley-β-D-glucanchitosancarboxymethyl-celluloseand lichenan. BGlc8H had a lower ratio of lichenase/barley-β-D-glucanase activities compared to GH16 enzymes. BGlc8H was optimally active at pH 5 and 50 °C, except for barley-β-D-glucanase (40 °C) and chitosanase (pH 7) activities. BGlc8H hydrolyzed cello-oligosaccharides (G3-G6) to G3 and G2 but not to G1. Ca(2+) increased the activity and thermostability of BGlc8H for lichenan suggesting its use for the saccharification of cellulosic biomass.

Enhanced yields of different lignocellulases were obtained under statistically-optimized parameters using Pholiota adiposa. The kcat value (4,261 s−1) of purified xylanase under standard assay conditions was the highest value ever reported. On covalent immobilization of the crude xylanase preparation onto functionalized silicon oxide nanoparticles, 66 % of the loaded enzyme was retained on the particle. Immobilized enzyme gave 45 % higher concentrations of xylooligosaccharides compared to the free enzyme. After 17 cycles, the immobilized enzyme retained 97 % of the original activity, demonstrating its prospects for the synthesis of xylooligosaccharides in industrial applications.

Methylan polysaccharide derivatives were prepared by dialkylaminoalkylation and reductive amination followed by quaternization. Their antitumor activity was investigated and a relationship between structure and activity is suggested. For quaternized DEAE-methylan at only 75 μg ml−1, tumor cell proliferation was suppressed by 58–84% in three cell lines tested in the order Colo

Mutagenesis of Erwinia rhapontici was performed to enhance the production of isomaltulose from sucrose. A mutant strain, BN 68089, was obtained through a screening process involving automated and miniaturized cultivation in Bioscreen C. This high-throughput, miniaturized screening system was optimized to identify the mutant strain, which had a conversion yield (90%) and productivity (194 g l−1 h−1). The BN 68089 mutant cells were immobilized in sodium alginate and when operated in a packed bed reactor gave a yield of 89% and a productivity of 144 g l−1 h−1 of at 30 °C, the optimal temperature. Immobilized BN 68089 cells exhibited 8% and 15% higher yield and productivity, respectively, than those of the wild-type strain.

Supplementation with CaCl2⋅2H2O (40 mg l−1) or CuSO4⋅5H2O (10 mg l−1) improved vancomycin production by Amycolatopsis orientalis by 12 and 11%, respectively; used in combination, they acted synergistically. Ca2+ decreased the intracellular concentration of vancomycin 36%, whereas Cu2+ increased the intracellular activity of TDP-glucose:aglycosylvancomycin glucosyltransferase 3 times more than control. Ca2+ probably works by altering the permeability of cells to vancomycin, whereas Cu2+ increases the activity of an enzyme responsible for vancomycin biosynthesis.

A novel microorganism was isolated which is able to produce mannitol when grown in the presence of fructose and glucose as carbon sources. In flask culture in a medium containing 150 g fructose l−1, it yielded 67 g mannitol l−1 after 168 h. In fed-batch culture with 3–12% (w/v) fructose, production reached a maximum of 209 g mannitol l−1 after 200 h, corresponding to an 83% yield and a 1.03 g l−1 h−1 productivity. The isolated strain was identified as Candida magnoliae based on identical sequences in the D1/D2 domain of its 26S rDNA and a similar carbon source utilization pattern with C. magnoliae reference strains.

Of the vitamins tested, inositol was the most effective for erythritol production. To increase erythritol production by Torula sp., inositol and a related compound, phytic acid (myoinositol hexaphosphate), were added to the culture media. Erythritol production in the presence of phytic acid was greater than that in the presence of inositol, due to the synergistic effects of phosphate and inositol. Supplementation with phosphate and inositol increased cell growth, erythritol production, and the activity of erythrose reductase in cells. Inositol was a more effective stimulator of cell growth and erythritol production than was phosphate.

The production of erythritol and the erythritol yield from glucose by Torula sp. were improved, in increasing order, by supplementing with 10 mg MnSO4 ⋅ 4H2O l−1, 2 mg CuSO4 ⋅ 5H2O l−1, and both 10 mg MnSO4 ⋅ 4H2O l−1 and 2 mg CuSO4 ⋅ 5H2O l−1. Mn2+ decreased the intracellular concentration of erythritol, whereas Cu2+ increased the activity of erythrose reductase in cells. These results suggest that Mn2+ altered the permeability of cells, whereas Cu2+ increased the activity of erythrose reductase in cells.

A β-1,3-1,4 glucanase gene of Paenibacillus sp. X4, bglc8H, was cloned and characterized. BGlc8H was predicted to be a protein of 409 amino acid residues, including a signal peptide of 31 amino acids. The mature enzyme was predicted to have 378 amino acid residues; ITS molecular mass and pI were estimated as 41,561 Da and 7.61, respectively. BGlc8H belongs to glycoside hydrolase family 8 (GH8). Site-directed mutants of Glu95 and Asp156 of BGlc8H showed a near-complete loss of activity, indicating that they are catalytically-active residues. Unlike other GH8 members, BGlc8H had broad substrate specificity and hydrolyzed barley-β-D-glucan > chitosan > carboxymethyl-cellulose > and lichenan. BGlc8H had a lower ratio of lichenase/barley-β-d-glucanase activities compared to GH16 enzymes. BGlc8H was optimally active at pH 5 and 50 °C, except for barley-β-d-glucanase (40 °C) and chitosanase (pH 7) activities. BGlc8H hydrolyzed cello-oligosaccharides (G3–G6) to G3 and G2 but not to G1. Ca2+ increased the activity and thermostability of BGlc8H for lichenan suggesting its use for the saccharification of cellulosic biomass.

Enhanced yields of different lignocellulases were obtained under statistically-optimized parameters using Pholiota adiposa. The k (cat) value (4,261 s(-1)) of purified xylanase under standard assay conditions was the highest value ever reported. On covalent immobilization of the crude xylanase preparation onto functionalized silicon oxide nanoparticles, 66 % of the loaded enzyme was retained on the particle. Immobilized enzyme gave 45 % higher concentrations of xylooligosaccharides compared to the free enzyme. After 17 cycles, the immobilized enzyme retained 97 % of the original activity, demonstrating its prospects for the synthesis of xylooligosaccharides in industrial applications.

A recombinant putative beta-galactosidase from Thermoplasma acidophilum was purified as a single 57 kDa band of 82 U mg(-1). The molecular mass of the native enzyme was 114 kDa as a dimer. Maximum activity was observed at pH 6.0 and 90 degrees C. The enzyme was unstable below pH 6.0: at pH 6 its half-life at 75 degrees C was 28 days but at pH 4.5 was only 13 h. Catalytic efficiencies decreased as p-nitrophenyl(pNP)-beta-D-fucopyranoside (1067) > pNP-beta-D-glucopyranoside (381) > pNP-beta-D-galactopyranoside (18) > pNP-beta-D-mannopyranoside (11 s(-1) mM(-1)), indicating that the enzyme was a beta-glycosidase.

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted D-psicose into D-allose but it did not convert D-xylose, L-rhamnose, D-altrose or D-galactose. The production of D-allose by RpiB was maximal at pH 7.5 and 65 degrees C for 30 min. The half-lives of the enzyme at 50 degrees C and 65 degrees C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50 degrees C, 165 g D-allose l(-1 ) was produced without by-products from 500 g D-psicose l(-1) after 6 h.

Supplementation with CaCl(2).2H(2)O (50 mg l(-1)) or CuSO(4).5H(2)O (10 mg l(-1)) improved mannitol production by Candida magnoliae by 14.5 and 18.6% (25 and 32 g/L), respectively. When used in combination, they acted synergistically: Ca(2+) decreased the intracellular concentration of mannitol 30%, whereas Cu(2+ )increased the intracellular activity of mannitol dehydrogenase 1.6-times more than control. Ca(2+) probably works by altering the permeability of cells to mannitol, whereas, Cu(2+) increases the activity of an enzyme responsible for mannitol biosynthesis.

Mutagenesis of Erwinia rhapontici was performed to enhance the production of isomaltulose from sucrose. A mutant strain, BN 68089, was obtained through a screening process involving automated and miniaturized cultivation in Bioscreen C. This high-throughput, miniaturized screening system was optimized to identify the mutant strain, which had a conversion yield (90%) and productivity (194 g l(-1) h(-1)). The BN 68089 mutant cells were immobilized in sodium alginate and when operated in a packed bed reactor gave a yield of 89% and a productivity of 144 g l(-1) h(-1) of at 30 degrees C, the optimal temperature. Immobilized BN 68089 cells exhibited 8% and 15% higher yield and productivity, respectively, than those of the wild-type strain.