Effects of Engineered Saccharomyces cerevisiae Fermenting Cellobiose through Low-Energy-Consuming Phosphorolytic Pathway in Simultaneous Saccharification and Fermentation.

Until recently, four types of cellobiose-fermenting Saccharomyces cerevisiae strains have been developed by introduction of a cellobiose metabolic pathway based on either intracellular ß-glucosidase (GH1-1) or cellobiose phosphorylase (CBP), along with either an energy-consuming active cellodextrin transporter (CDT-1) or a non-energy-consuming passive cellodextrin facilitator (CDT-2). In this study, the ethanol production performance of two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2 (N306I) with GH1-1 or CBP were compared with two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-1 (F213L) with GH1-1 or CBP in the simultaneous saccharification and fermentation (SSF) of cellulose under various conditions. It was found that, regardless of the SSF conditions, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the best ethanol production among the four strains. In addition, during SSF contaminated by lactic acid bacteria, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the highest ethanol production and the lowest lactate formation compared with those of other strains, such as the hydrolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-1 with GH1-1, and the glucose-fermenting S. cerevisiae with extracellular ß-glucosidase. These results suggest that the cellobiose-fermenting yeast strain exhibiting low energy consumption can enhance the efficiency of the SSF of cellulosic biomass.

An Aspergillus nidulans endo-ß-1,3-glucanase exhibited specific catalytic features and was used to prepare 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose with high antioxidant activity from barley ß-glucan and laminarin, respectively.

An endo-ß-1,3(4)-glucanase AnENG16A from Aspergillus nidulans shows distinctive catalytic features for hydrolysis of ß-glucans. AnENG16A hydrolyzed Eisenia bicyclis laminarin to mainly generate 3-O-ß-gentiobiosyl-d-glucose and hydrolyzed barley ß-glucan to mainly produce 3-O-ß-cellobiosyl-d-glucose. Using molecular exclusion chromatography, we isolated and purified 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose, respectively, from AnENG16A-hydrolysate of barley ß-glucan and E. bicyclis laminarin. Further study reveals that 3-O-ß-cellobiosyl-d-glucose had 8.99-fold higher antioxidant activity than barley ß-glucan and 3-O-ß-gentiobiosyl-d-glucose exhibited 43.0% higher antioxidant activity than E. bicyclis laminarin. Notably, 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose exhibited 148.9% and 116.0% higher antioxidant activity than laminaritriose, respectively, indicating that ß-1,4-linkage or -1,6-linkage at non-reducing end of ß-glucotrioses had enhancing effect on antioxidant activity compared to ß-1,3-linkage. Furthermore, 3-O-ß-cellobiosyl-d-glucose showed 237.9% higher antioxidant activity than cellotriose, and laminarin showed 5.06-fold higher antioxidant activity than barley ß-glucan, indicating that ß-1,4-linkage at reducing end of ß-glucans or oligosaccharides resulted in decrease of antioxidant activity compared to ß-1,3-linkage.

A reassessment of flocculosin-mediated biocontrol activity of Pseudozyma flocculosa through CRISPR/Cas9 gene editing.

Pseudozyma flocculosa is an epiphytic yeast with powerful antagonistic activity against powdery mildews. This activity has been associated with the production of a rare antifungal glycolipid, flocculosin. In spite of the discovery of a specific gene cluster for flocculosin synthesis, attempts to ascribe a functional role to the molecule have been hampered by the inability to efficiently transform P. flocculosa. In this study, two different approaches, target gene replacement by homologous recombination (HR) and CRISPR-Cas9 based genome-editing, were utilized to decipher the role of flocculosin in the biocontrol activity of P.flocculosa. It was possible to alter the production of flocculosin through edition of fat1 by HR, but such mutants displayed abnormal phenotypes and the inability to produce sporidia. Sequencing analyses revealed that transformation by HR led to multiple insertions in the genome explaining the pleiotrophic effects of the approach. On the other hand, CRISPR-Cas9 transformation yielded one mutant that was altered specifically in the proper synthesis of flocculosin. Notwithstanding the loss of flocculosin production, such mutant was phenotypically similar to the wild-type, and when tested for its biocontrol activity against powdery mildew, displayed the same efficacy. These results offer strong evidence that flocculosin-mediated antibiosis is not responsible for the mode of action of P. flocculosa and highlight the potential of CRISPR-Cas9 for functional studies of otherwise difficult-to-transform fungi such as P. flocculosa.

A novel bifunctional glucanase exhibiting high production of glucose and cellobiose from rumen bacterium.

Herbivores gastrointestinal microbiota is of tremendous interest for mining novel lignocellulosic enzymes for bioprocessing. We previously reported a set of potential carbohydrate-active enzymes from the metatranscriptome of the Hu sheep rumen microbiome. In this study, we isolated and heterologously expressed two novel glucanase genes, Cel5A-h38 and Cel5A-h49, finding that both recombinant enzymes showed the optimum temperatures of 50 °C. Substrate-specificity determination revealed that Cel5A-h38 was exclusively active in the presence of mixed-linked glucans, such as barley ß-glucan and Icelandic moss lichenan, whereas Cel5A-h49 (EC 3.2.1.4) exhibited a wider substrate spectrum. Surprisingly, Cel5A-h38 initially released only cellotriose from lichenan and further converted it into an equivalent amount of glucose and cellobiose, suggesting a dual-function as both endo-ß-1,3-1,4-glucanase (EC 3.2.1.73) and exo-cellobiohydrolase (EC 3.2.1.91). Additionally, we performed enzymatic hydrolysis of sheepgrass (Leymus chinensis) and rice (Orysa sativa) straw using Cel5A-h38, revealing liberation of 1.91 ± 0.30 mmol/mL and 2.03 ± 0.09 mmol/mL reducing sugars, respectively, including high concentrations of glucose and cellobiose. These results provided new insights into glucanase activity and lay a foundation for bioconversion of lignocellulosic biomass.

Cross-utilization of ß-galactosides and cellobiose in Geobacillus stearothermophilus.

Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-ß-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-ß-gal/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system.

Predominant secretion of cellobiohydrolases and endo-ß-1,4-glucanases in nutrient-limited medium by Aspergillus spp. isolated from subtropical field.

Biological degradation of cellulose from dead plants in nature and plant biomass from agricultural and food-industry waste is important for sustainable carbon recirculation. This study aimed at searching diverse cellulose-degrading systems of wild filamentous fungi and obtaining fungal lines useful for cellooligosaccharide production from agro-industrial wastes. Fungal lines with cellulolytic activity were screened and isolated from stacked rice straw and soil in subtropical fields. Among 13 isolated lines, in liquid culture with a nutrition-limited cellulose-containing medium, four lines of Aspergillus spp. secreted 50-60 kDa proteins as markedly dominant components and gave clear activity bands of possible endo-ß-1,4-glucanase in zymography. Mass spectroscopy (MS) analysis of the dominant components identified three endo-ß-1,4-glucanases (GH5, GH7 and GH12) and two cellobiohydrolases (GH6 and GH7). Cellulose degradation by the secreted proteins was analysed by LC-MS-based measurement of derivatized reducing sugars. The enzymes from the four Aspergillus spp. produced cellobiose from crystalline cellulose and cellotriose at a low level compared with cellobiose. Moreover, though smaller than that from crystalline cellulose, the enzymes of two representative lines degraded powdered rice straw and produced cellobiose. These fungal lines and enzymes would be effective for production of cellooligosaccharides as cellulose degradation-intermediates with added value other than glucose.

Biochemical characterization and low-resolution SAXS structure of two-domain endoglucanase BlCel9 from Bacillus licheniformis.

Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic efficiency at pH 7.0 and 60 °C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5-8.5) and temperatures (up to 50 °C) for extended periods of time (over 48 h). It exhibits the highest hydrolytic activity against phosphoric acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8 kDa as estimated from SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.

Expression and characterization of a glucose-tolerant ß-1,4-glucosidase with wide substrate specificity from Cytophaga hutchinsonii.

Cytophaga hutchinsonii is a gram-negative bacterium that can efficiently degrade crystalline cellulose by a novel strategy without cell-free cellulases or cellulosomes. Genomic analysis implied that C. hutchinsonii had endoglucanases and ß-glucosidases but no exoglucanases which could processively digest cellulose and produce cellobiose. In this study, BglA was functionally expressed in Escherichia coli and found to be a ß-glucosidase with wide substrate specificity. It can hydrolyze pNPG, pNPC, cellobiose, and cellodextrins. Moreover, unlike most ß-glucosidases whose activity greatly decreases with increasing length of the substrate chains, BglA has similar activity on cellobiose and larger cellodextrins. The K m values of BglA on cellobiose, cellotriose, and cellotetraose were calculated to be 4.8 × 10-2, 5.6 × 10-2, and 5.3 × 10-2 mol/l, respectively. These properties give BglA a great advantage to cooperate with endoglucanases in C. hutchinsonii in cellulose degradation. We proposed that C. hutchinsonii could utilize a simple cellulase system which consists of endoglucanases and ß-glucosidases to completely digest amorphous cellulose into glucose. Moreover, BglA was also found to be highly tolerant to glucose as it retained 40 % activity when the concentration of glucose was 100 times higher than that of the substrate, showing potential application in the bioenergy industry.

The Differential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Two ß -Glycoside Hydrolases.

Probiotics, prebiotics, and combinations thereof, that is, synbiotics, are known to exert beneficial health effects in humans; however interactions between pro- and prebiotics remain poorly understood at the molecular level. The present study describes changes in abundance of different proteins of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) when grown on the potential prebiotic cellobiose as compared to glucose. Cytosolic cell extract proteomes after harvest at late exponential phase of NCFM grown on cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis (2D-DIGE) in the acidic (pH 4-7) and the alkaline (pH 6-11) regions showing a total of 136 spots to change in abundance. Proteins were identified by MS or MS/MS from 81 of these spots representing 49 unique proteins and either increasing 1.5-13.9-fold or decreasing 1.5-7.8-fold in relative abundance. Many of these proteins were associated with energy metabolism, including the cellobiose related glycoside hydrolases phospho-ß-glucosidase (LBA0881) and phospho-ß-galactosidase II (LBA0726). The data provide insight into the utilization of the candidate prebiotic cellobiose by the probiotic bacterium NCFM. Several of the upregulated or downregulated identified proteins associated with utilization of cellobiose indicate the presence of carbon catabolite repression and regulation of enzymes involved in carbohydrate metabolism.

Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants.

Direct cellobiose production from cellulose by a genetically modified fungus-Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. crassa sextuple beta-glucosidase (bgl) gene deletion strains when grown on Avicel without exogenous cellulase addition. The sextuple bgl deletion strains expressing one of the three basally transcribed bgl genes are the best cellobiose producers. For most sextuple strains, the multiple bgl gene deletion has no negative effect on the production of other cellulases. The induction of major endoglucanases and exoglucanases on Avicel in most of the sextuple bgl deletions strains was as fast as or faster than that of the wild type, except for strain F4. The best cellobiose producing strain, F5, produced 7.7 g/L of cellobiose from 20 g/L of Avicel in four days and utilized the Avicel as fast as did the wild type (even in the presence of high cellobiose concentration). The cellobiose yield from cellulose was about 48.3%.

Enzymatic hydrolysis of ionic liquid-pretreated celluloses: contribution of CP-MAS 13C NMR and SEM.

The supramolecular structure of four model celluloses was altered prior to their enzymatic saccharification using two ionic liquid pretreatments: one with the commonly used 1-ethyl-3-methylimidazolium acetate ([Emim](+)[CH(3)COO](-)) and the other with the newly developed 1-ethyl-3-methylimidazolium methylphosphonate ([Emim](+)[MeO(H)PO(2)](-)). The estimation of crystallinity index (CrI) by solid state (13)C nuclear magnetic resonance for each untreated/pretreated celluloses was compared with the performances of their enzymatic hydrolysis. For α-cellulose, both pretreatments led to a significant decrease in CrI from 25% to 5% but had no effect on glucose yields. In contrast, The [Emim](+)[MeO(H)PO(2)](-) pretreatment on the long fibers of cellulose had no significant effect on the CrI although a conversion yield in glucose of 88% is obtained versus 32% without pretreatment. However, scanning electron microscopy analysis suggested a loss of fiber organization induced by both ionic liquid pretreatments leading to a larger accessibility by cellulases to the cellulose surface.

Identification of a biosynthesis gene cluster for flocculosin a cellobiose lipid produced by the biocontrol agent Pseudozyma flocculosa.

Flocculosin is an antifungal glycolipid produced by the biocontrol fungus Pseudozyma flocculosa. It consists of cellobiose, O-glycosidically linked to 3,15,16-trihydroxypalmitic acid. The sugar moiety is acylated with 2-hydroxy-octanoic acid and acetylated at two positions. Here we describe a gene cluster comprising 11 genes that are necessary for the biosynthesis of flocculosin. We compared the cluster with the biosynthesis gene cluster for the highly similar glycolipid ustilagic acid (UA) produced by the phytopathogenic fungus Ustilago maydis. In contrast to the cluster of U. maydis, the flocculosin biosynthesis cluster contains an additional gene encoding an acetyl-transferase and is lacking a gene homologous to the α-hydroxylase Ahd1 necessary for UA hydroxylation. The functions of three acyl/acetyl-transferase genes (Fat1, Fat2 and Fat3) including the additional acetyl-transferase were studied by complementing the corresponding U. maydis mutants. While P. flocculosa Fat1 and Fat3 are homologous to Uat1 in U. maydis, Fat2 shares 64% identity to Uat2, a protein involved in UA biosynthesis but with so far unknown function. By genetic and mass spectrometric analysis, we show that Uat2 and Fat2 are necessary for acetylation of the corresponding glycolipid. These results bring unique insights into the biocontrol properties of P. flocculosa and opportunities for enhancing its activity.

Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum.

Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming ß-glucosyl disaccharides with ß-(1→4)- regioselectivity from five monosaccharides as well as branched ß-glucosyl trisaccharides with ß-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict ß-(1→4)-regioselectivity and catalysed linear chain extension of the three ß-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two ß-glucosyl oligosaccharide product series to represent novel compounds, i.e. ß-D-glucopyranosyl-[(1→4)-ß-D-glucopyranosyl](n)-(1→2)-D-glucopyranose, and ß-D-glucopyranosyl-[(1→4)-ß-D-glucopyranosyl](n)-(1→3)-D-glucopyranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of ß-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α-d-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.

Use of cellulase inhibitors to produce cellobiose.

The economics driving biorefinery development requires high value-added products such as cellobiose for financial feasibility. This research describes a simple technology for increasing cellobiose yields during lignocellulosic hydrolysis. The yield of cellobiose produced during cellulose hydrolysis was maximized by modification of reaction conditions. The addition of an inhibitor from the group that includes glucose oxidase, gluconolactone, and gluconic acid during cellulase hydrolysis of cellulose increased the amount of cellobiose produced. The optimal conditions for cellobiose production were determined for four factors; reaction time, cellulase concentration, cellulose concentration, and inhibitor concentration using a Box-Behnken experimental design. Gluconolactone in the cellulase system resulted in the greatest production of cellobiose (31.2%) from cellulose. The yield of cellobiose was 23.7% with glucose oxidase, similar to 21.9% with gluconic acid.

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.

Identification of genes potentially involved in the biocontrol activity of Pseudozyma flocculosa.

Flocculosin is an antifungal cellobiose lipid linked to the biocontrol activity of Pseudozyma flocculosa and whose structure is very similar to that of ustilagic acid produced by Ustilago maydis. In this work, homologs of the U. maydis cyp1 gene, involved in the biosynthesis of ustilagic acid, were isolated and sequenced from P. flocculosa and P. fusiformata, the latter species being also known to produce ustilagic acid. Interestingly, no homologs were found in four other closely related Pseudozyma spp. from which no evidence of ustilagic acid production has ever been obtained, thus supporting the specificity of cyp1 with ustilagic acid synthesis. In addition, a homolog of the U. maydis uat1 gene involved in the acetylation of the molecule and located next to the cyp1 gene was partially sequenced from P. flocculosa. All three newly sequenced genes showed strong sequence similarity to their counterparts in U. maydis. Cyp1 expression was monitored in conditions that were either conducive or repressive to flocculosin production. Expression increased markedly (>100x) when P. flocculosa was inoculated in a growth medium conducive to flocculosin production but was rapidly downregulated in a repressive medium (in vitro) or on powdery mildew-infected cucumber leaves (in vivo). This suggests that the molecule was preferentially synthesized early in the process of searching for a growth substrate. This study provides the first identification of genes involved in the production of flocculosin, a molecule potentially associated with the biocontrol properties of P. flocculosa.

Synthesis of cellobiose from starch by the successive actions of two phosphorylases.

Cellobiose was enzymatically synthesized from starch using two phosphorylases. Under the presence of 1 M Pi inorganic phosphate), glucan phosphorylase converted 40% of glucose residues in the starch molecule into G1P (glucose-1-phosphate). By electrodialysis fitted with an ion exchange membrane having molecular weight cutoff of 100, Pi was effectively dialyzed out and G1P was recovered with 80% yield. G1P and glucose were incubated with cellobiose phosphorylase in the presence of magnesium acetate at an alkaline condition. Inorganic phosphate coformed with cellobiose was immediately removed as insoluble magnesium ammonium phosphate and 85% of added G1P was converted into cellobiose. On the whole, cellobiose was produced with 60% yield from G1P and, at least, 23.7% yield from starch.

Nutritional regulation and kinetics of flocculosin synthesis by Pseudozyma flocculosa.

This study sought to identify the factors and conditions that affected production of the antifungal glycolipid flocculosin by the biocontrol agent Pseudozyma flocculosa. For this purpose, different parameters known or reported to influence glycolipid release in fungi were tested. Concentration of the start-up inoculum was found to play an important role in flocculosin production, as the optimal level increased productivity by as much as tenfold. Carbon availability and nitrogen source (i.e., organic vs inorganic) both had a direct influence on the metabolism of P. flocculosa, leading to flocculosin synthesis. In general, if conditions were conducive for production of the glycolipid, carbon availability appeared to be the only limiting factor. On the other hand, if yeast extract was supplied as nitrogen source, fungal biomass was immediately stimulated to the detriment of flocculosin synthesis. Unlike other reports of glycolipid release by yeast-like fungi, inorganic nitrogen starvation did not trigger production of flocculosin. The relationship between the factors influencing flocculosin production in vitro and the conditions affecting the release of the molecule by P. flocculosa in its natural habitat appears to be linked to the availability of a suitable and plentiful food source for the biocontrol agent.

Biochemical characterization of cellulose-binding proteins (CBPA and CBPB) from the rumen cellulolytic bacterium Eubacterium cellulosolvens 5.

The cellulose-binding proteins, CBPA and CBPB, of rumen cellulolytic bacterium Eubacterium cellulosolvens 5 were biochemically characterized, and their properties were compared. Recombinant CBPA and CBPB were a typical 1,4-beta-endoglucanase. Both proteins bound to insoluble polysaccharides such as Avicel cellulose, acid swollen cellulose, lichenan, chitin, and oat spelt xylan. On the other hand, only recombinant CBPB bound to agarose and starch.

Hydrolyses and transglycosylations performed by purified alpha-D-glucosidase of the marine mollusc Aplysia fasciata.

The purification and characterisation of the alpha-glucosidase from the marine mollusc Aplysia fasciata are reported. Overall substrate specificity of the pure enzyme for both hydrolytic and transglycosylation reactions was studied. Remarkable characteristics of this enzyme are indicated by the results of the interesting survey of transglycosylation reactions reported: pyridoxine glucosylation, synthesis of chromophoric (pNP) di- and trisaccharides, glucosylation of cellobiose and sucrose. For these last two acceptors both the yields of reactions and the concentrations of products are comparable to those obtained using glycosyl transferases; in addition, synthesis of pyridoxine and chromophoric glycosides were still possible using a 1:1 ratio maltose:acceptor which is a very interesting characteristic from a synthetic point of view (effortless purification, productivity of each reaction batch, etc.).