Membrane-based recovery of glucose from enzymatic hydrolysis of ionic liquid pretreated cellulose.

In this work, a membrane-based downstream process for the recovery of glucose from cellulose hydrolysis is described and evaluated. The cellulose is pretreated with the ionic liquid 1,3-dimethyl-imidazolium dimethylphosphate to reduce its crystallinity. After enzymatic conversion of cellulose to glucose the hydrolysate is filtered with an ultrafiltration membrane to remove residual particulates and enzymes. Nanofiltration is applied to purify the glucose from molecular intermediates, such as cellobiose originating from the hydrolysis reaction. Finally, the ionic liquid is removed from the hydrolysate via electrodialysis. Technically, these process steps are feasible. An economic analysis of the process reveals that the selling price of glucose from this production process is about 2.75 €/kg which is too high as compared to the current market price.

Enhanced enzymatic cellulose degradation by cellobiohydrolases via product removal.

Product inhibition by cellobiose decreases the rate of enzymatic cellulose degradation. The optimal reaction conditions for two Emericella (Aspergillus) nidulans-derived cellobiohydrolases I and II produced in Pichia pastoris were identified as CBHI: 52 °C, pH 4.5-6.5, and CBHII: 46 °C, pH 4.8. The optimum in a mixture of the two was 50 °C, pH 4.9. An almost fourfold increase in enzymatic hydrolysis yield was achieved with intermittent product removal of cellobiose with membrane filtration (2 kDa cut-off): The conversion of cotton cellulose after 72 h was ~19 % by weight, whereas the conversion in the parallel batch reaction was only ~5 % by weight. Also, a synergistic effect, achieving ~27 % substrate conversion, was obtained by addition of endo-1,4-ß-D-glucanase. The synergistic effect was only obtained with product removal. By using pure, monoactive enzymes, the work illustrates the profound gains achievable by intermittent product removal during cellulose hydrolysis.

Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785.

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of ß-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 °C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 °C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.

Production of antifungal cellobiose lipids by Trichosporon porosum.

The yeast Trichosporon porosum suppresses growth of ascomycetes and basidiomycetes belonging to 52 genera. It is due to secretion of a thermostable fungicidal agent. The suppression was maximal at pH 3.5-4.0. Fungicidal preparation obtained from the culture broth was shown to be a mixture of cellobiosides of dihydrodecane acid with different degree of acetylation of cellobiose residue. The preparation caused the death of Candida albicans and Filobasidiella neoformans cells in the concentrations of 0.2 and 0.03 mM, respectively.

A multistage process to enhance cellobiose production from cellulosic materials.

Cellobiose, a disaccharide, is a valuable product that can be obtained from cellulose hydrolysis. In this study, a simple methodology is presented to enhance the production and improve the selectivity of cellobiose during enzymatic hydrolysis of cellulose. The approach consisted of a multistage removal of filtrate via vacuum filtration and resuspension of the retentate. By this process, the remaining solid was further hydrolyzed without additional enzyme loading. Compared to the continuous hydrolysis process, the production of cellobiose increased by 45%. Increased selectivity of cellobiose is due to the loss of beta-glucosidases in the filtrate, while enhanced productivity is likely due to mitigated product inhibition.

[Fungicidal activity of cellobiose lipids from cultural fluid of yeast Cryptococcus humicola and Pseudozyma fusiformata].

Cellobiose lipids of yeast fungi Cryptococcus humicola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.

Insertional mutagenesis of a fungal biocontrol agent led to discovery of a rare cellobiose lipid with antifungal activity.

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2',4'-diacetoxy-5'-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa.

Occurrence and localization of apocarotenoids in arbuscular mycorrhizal plant roots.

The core structure of the yellow pigment from arbuscular mycorrhizal (AM) maize roots contains the apocarotenoids mycorradicin (an acyclic C14 polyene) and blumenol C cellobioside (a C13 cyclohexenone diglucoside). The pigment seems to be a mixture of different esterification products of these apocarotenoids. It is insoluble in water and accumulates as hydrophobic droplets in the vacuoles of root cortical cells. Screening 58 species from 36 different plant families, we detected mycorradicin in mycorrhizal roots of all Liliopsida analyzed and of a considerable number of Rosopsida, but also species were found in which mycorradicin was undetectable in mycorrhizal roots. Kinetic experiments and microscopic analyses indicate that accumulation of the yellow pigment is correlated with the concomitant degradation of arbuscules and the extensive plastid network covering these haustorium-like fungal structures. The role of the apocarotenoids in mycorrhizal roots is still unknown. The potential C40 carotenoid precursors, however, are more likely to be of functional importance in the development and functioning of arbuscules.

Resin selection and single-step production and recovery of lactic acid from pretreated wood.

Four ion-exchange resins (Amberlite IRA 900, IRA 400, IRA 96, and IRA 67) were employed for lactic acid recovery from simultaneous saccharification and fermentation (SSF) media. The best resins (Amberlite IRA 900 and IRA 400) were assayed for capacity, regenerant consumption, percentage of lactic acid recovery, and product concentration. Almost quantitative lactic acid recoveries at constant capacities were achieved in four sequential loading/regeneration cycles. A strong-base resin (Amberlite IRA 400) was selected for intermittent lactic acid separation in a typical SSF process, in which pretreated wood was saccharified by cellulases in the presence of Lactobacillus delbrueckii. The dynamics of lactic acid generation and lactic acid recovery were established.

Antioxidant effect of two flavonoids from the bark of Ficus bengalensis Linn in hyperlipidemic rats.

Two flavonoid compounds, viz. 5,7-dimethyl ether of leucopelargonidin 3-0-alpha-L rhamnoside and 5,3'-dimethyl ether of leucocyanidin 3-0-alpha-D galactosyl cellobioside obtained from the bark of F. bengalensis were evaluated for their antioxidant action in hyperlipidemic rats. The results were compared with the activity of a structurally similar flavonoid, quercetin, a known antioxidant. The Ficus compounds showed significant antioxidant effects which may be attributed to their polyphenolic nature. The methylation of two hydrozyl groups in the Ficus flavonoids might have slowed down their antioxidant action as compared to quercetin.