Robert F H Dekker

Abstract: The class of oligosaccharides possessing biological activities is important to human health. The awareness and the adequacy of the diet are necessary to ensure that all the benefits of these bioactive carbohydrates are present. Oligosaccharides present unique physicochemical and biological properties that can be exploited commercially for specific applications in foods, cosmetics and pharmacology. Advances in technology used in the production of these carbohydrate oligomers are necessary for their development and inclusion, as functional products in foods (nutriceuticals), or for therapeutic applications as medicinals. Oligosaccharides can be produced through a number of different methods (physical, chemical and enzymatic catalysed reactions) from their parent polysaccharides or glycoconjugates, as well as through specific chemical synthesis and enzyme-catalysed transglycosylation reactions. More recently, solid-phase synthesis has allowed the synthesis of complex carbohydrate sugar chains at scaled-up levels of preparation. The use of enzymes in preference to physical and chemical processes in obtaining oligosaccharides has become an attractive approach through the advent of biomolecular protein engineering technology. This has resulted in new mutant enzymes as biocatalysts for the synthesis of complex oligosaccharides. This book presents an overview on different pathways leading to the production of bioactive oligosaccharides for biotechnological applications. Mostly, these carbohydrate oligomers constitute a nutritional source of �fiber� (prebiotic) that is beneficial to bacterial growth in the lower distal part of the human intestinal tract promoting health, and general well-being. Oligosaccharides, like some of their polysaccharide counterparts, can induce innate immune responses, and this unique property has led to potential applications for their commercialization as immunoceuticals.

Abstract: Commercial oil-yielding seeds (castor, coconut, neem, peanut, pongamia, rubber and sesame) were collected from different places in the state of Tamil Nadu (India) from which 1279 endophytic fungi were isolated. The oil-bearing seeds exhibited rich fungal diversity. High Shannon-Index H0 was observed with pongamia seeds (2.847) while a low Index occurred for coconut kernel-associated mycoflora (1.018). Maximum Colonization Frequency (%) was observed for Lasiodiplodia theobromae (176). Dominance Index (expressed in terms of the Simpson�s Index D) was high (0.581) for coconut kernel-associated fungi, and low for pongamia seed-borne fungi. Species Richness (Chao) of the fungal isolates was high (47.09) in the case of neem seeds, and low (16.6) for peanut seeds. All 1279 fungal isolates were screened for lipolytic activity employing a zymogram method using Tween-20 in agar. Forty isolates showed strong lipolytic activity, and were morphologically identified as belonging to 19 taxa (Alternaria, Aspergillus, Chalaropsis, Cladosporium, Colletotrichum, Curvularia, Drechslera, Fusarium, Lasiodiplodia, Mucor, Penicillium, Pestalotiopsis, Phoma, Phomopsis, Phyllosticta, Rhizopus, Sclerotinia, Stachybotrys and Trichoderma). These isolates also exhibited amylolytic, proteolytic and cellulolytic activities. Five fungal isolates (Aspergillus niger, Chalaropsis thielavioides, Colletotrichum gloeosporioides, Lasiodiplodia theobromae and Phoma glomerata) exhibited highest lipase activities, and the best producer was Lasiodiplodia theobromae (108 U/mL), which was characterized by genomic sequence analysis of the ITS region of 18S rDNA.

Abstract: Lasiodiplodan, an exopolysaccharide of the (1,6)-β-D-glucan type, is produced by Lasiodiplodia theobromae MMPI when grown under submerged culture on glucose. The objective of this study was to evaluate lasiodiplodan production by examining the effects of carbon (glucose, fructose, maltose, sucrose), and nitrogen sources (KNO3, (NH4)2 SO4, urea, yeast extract, peptone), its production in shake flasks compared to a stirred-tank bioreactor, and to study the rheology of lasiodiplodan, and lasiodiplodan�s anti-proliferative effect on breast cancer MCF-7 cells. Although glucose (2.05±0.05 g L-1), maltose (2.08±0.04 g L-1) and yeast extract (2.46±0.06 g L-1) produced highest amounts of lasiodiplodan, urea as N source resulted in more lasiodiplodan per unit biomass than yeast extract (0.74±0.006 vs. 0.22±0.008 g g-1). A comparison of the fermentative parameters of L. theobromae MMPI in shake-flasks and a stirred-tank bioreactor at 120h on glucose as carbon source showed maximum lasiodiplodan production in agitated flasks (7.01±0.07 g L-1) with a specific yield of 0.25±0.57 g g-1 and a volumetric productivity of 0.06±0.001 g L-1 h-1. A factorial 22 statistical design developed to evaluate the effect of glucose concentration (20 to 60 g L-1) and impeller speed (100 to 200 rpm) on lasiodiplodan production in the bioreactor showed highest production (6.32 g L-1) at 72h. Lasiodiplodan presented pseudoplastic behaviour, and the apparent viscosity increased at 60°C in the presence of CaCl2. Anti-proliferative activity of lasiodiplodan was demonstrated in MCF-7 cells, which was time and dose dependent with an IC50 of 100 μg lasiodiplodan mL-1.

Abstract: The exopolysaccharide botryosphaeran (EPSGLC; a (1�3)(1�6)-β-D-glucan from Botryosphaeria rhodina MAMB-05) was sulfonated to produce a water-soluble fraction (EPSGLC-S) using pyridine and chlorosulfonic acid in formamid. This procedure was then repeated twice to produce another fraction (EPSGLC-RS) with a higher degree of substitution (DS, 1.64). The purity of each botryosphaeran sample (unsulfonated and sulfonated) was assessed by gel filtration chromatography (Sepharose CL-4B), where each polysaccharide was eluted as a single symmetrical peak. The structures of the sulfonated and re-sulfonated botryosphaerans were investigated using ultraviolet-visible (UV-Vis), Fourier-transform infrared (FT-IR), and 13C nuclear magnetic resonance (13C NMR) spectroscopies. EPSGLC and EPSGLC-RS were also assayed for anticoagulation activity, and EPSGLC-RS was identified as an anticoagulant.

Abstract: Differential scanning calorimetry (DSC), thermogravimetry (TG) and Fouriertransform infra-red spectroscopy (FT-IR) analyses were performed to investigate changes in the physico-chemical properties of botryosphaerans, a family of exopolysaccharides (EPS) produced by the fungus Botryosphaeria rhodina MAMB-05 grown on glucose (EPSGLC), sucrose (EPSSUC) and fructose (EPSFRU). A slight endothermic transition and small mass loss attributable to the removal of water of hydration were observed in the DSC and TG analyses, respectively, for the three EPS samples. The FT-IR spectra confirmed no structural changes occurred during thermal treatment. Viscometry was utilized to obtain information on the rheological behaviour of the EPS in aqueous solutions. The Power Law and Cross Equations determined the natural pseudoplastic characteristics of the EPS. Comparatively, results obtained for EPS produced when B. rhodina MAMB-05 was grown on each of the three carbohydrate sources demonstrated similar apparent viscosity values for EPSGLC and EPSSUC, while EPSFRU displayed the lowest apparent viscosity of the three botryosphaerans, suggesting a higher degree of ramification and lower Mw. EPSGLC and EPSSUC possessed similar degrees of ramification. The slight differences found in their viscosities can be explained by the differences in the type of branching among the three botryosphaerans, thus varying the strength of intermolecular interactions and consequently, consistency and viscosity. The physico-chemical studies of botryosphaerans represent the originality of this work, and the knowledge of these properties is an important criterion for potential applications.

Abstract: Botryosphaeran, a water-soluble exopolysaccharide of the β-(1,3;1,6)-D-glucan type that has been isolated from the culture medium of Botryosphaeria rhodina MAMB-05 grown in submerged fermentation using glucose as the sole carbon source, was previously demonstrated to be non-genotoxic in peripheral blood and bone marrow, and exhibited strong anticlastogenic activity. In the present study, the effects of botryosphaeran were investigated in streptozotocin-induced diabetic rats as well as in high-fat diet-fed hyperlipidemic Wistar rats. The plasma glucose level was reduced by 52% in the diabetic group of rats after administration of 12 mg botryosphaeran/kg body weight of the rats (b.w.)/day by gavage over 15 days. A reduction in the median ration intake was accompanied by an increase in the median body weight gain, as well as the efficiency of food conversion. These results demonstrate that botryosphaeran has protective effects by reducing the symptoms of cachexia in diabetes mellitus. Botryosphaeran administered by gavage at a concentration of 12 mg botryosphaeran/kg b.w./day over 15 days also reduced the plasma levels of total cholesterol and low density lipoprotein-cholesterol by 18% and 27%, respectively, in hyperlipidemic rats. Based on these findings, we conclude that botryosphaeran possesses hypoglycemic and hypocholesterolemic properties in conditions of diabetes mellitus and hyperlipidemia, respectively, and may be used as an oral anti-diabetic agent.

Abstract: Botryosphaeria rhodina MAMB-05 produced β-1,3-glucanases and botryosphaeran when grown on glucose, while Trichoderma harzianum Rifai only produced the enzyme. A comparison of long-term cultivation (300 h) by Botryosphaeria rhodina demonstrated a correlation between the formation of botryosphaeran (48 h) and its consumption (after 108 h), and de-repression of β-1,3-glucanase synthesis when glucose was depleted from the nutrient medium, whereas for Trichoderma harzianum enzyme production commenced during exponential growth. Growth profiles and levels of β-1,3-glucanases produced by both fungi on botryosphaeran also differed, as well as the production of β-1,3-glucanases and β-1,6-glucanases on glucose, lactose, laminarin, botryosphaeran, lasiodiplodan, curdlan, Brewer�s yeast powder and lyophilized fungal mycelium, which were dependent upon the carbon source used. A statistical mixture-design used to optimize β-1,3-glucanase production by both fungi evaluated botryosphaeran, glucose and lactose concentrations as variables. For Botryosphaeria rhodina, glucose and lactose promoted enzyme production at the same levels (2.30 U mL-1), whereas botryosphaeran added to these substrates exerted a synergic effect favorable for β-glucanase production by Trichoderma harzianum (4.25 U mL-1).

Abstract: Beta-glucosidase production by Debaryomyces pseudopolymorphus UCLM-NS7A using a simple nutrient medium containing cellobiose was evaluated under several biochemical and physiological parameters in submerged fermentation. Enzyme induction was also examined using different carbon and nitrogen sources. Cellobiose and ammonium nitrate were the best C and N sources to enhance beta-glucosidase production. The addition of NaCl, MgSO4, yeast extract, ethanol and Tween 80 to the nutrient medium before inoculation was also compared. A factorial design to optimize enzyme production was developed using four variables that most influenced beta-glucosidase production and data analyzed by the response surface method. Optimal conditions to produce beta-glucosidase in shake-flasks were 1.25% cellobiose, 0.05% Tween 80, 0.4% NH4NO3 over 72 hours. In another factorial design to further increase enzyme production, a lab fermenter using prior-determined shake-flask optimized conditions resulted in higher beta-glucosidase titres at 72 hours, pH controlled at 6.25 and agitation of 200 rpm.

Abstract: The present study is the first describing the sequencing of a fragment of the copper-oxidase domain of a laccase gene in the family Botryosphaeriaceae. The aim of this work was to assess the degree of genetic and evolutionary relationships of a laccase gene from Botryosphaeria rhodina MAMB-05 with other ascomycete and basidiomycete laccase genes. A 38-amino acid sequence of the copper-oxidase domain from several different fungi, insects, a plant and a bacterial species were retrieved from GenBank and aligned. Phylogenetic analyses were performed using Neighbour-joinning, Maximum Parsimony, and Bayesian inference methods, and clustered the organisms studied into five gene clades: fungi (ascomycetes and basidiomycetes), insects, plant and bacterium. Also, the topologies showed that fungal laccases of the ascomycetes and basidiomycetes are clearly separated into 2 distinct clusters. This evidence indicated B. rhodina MAMB-05 and other closely-related ascomycetes as a new biological resource given the biotechnological potential of their laccase genes.

Abstract: Botryosphaeran (EPSFRU), an exopolysaccharide of the beta-(1,3;1,6)-D-glucan type with 31 % branching at C-6, is produced by the fungus Botryosphaeria rhodina MAMB-05 when grown on fructose as carbon source. Botryosphaeran was derivatized by sulfonation to induce anticoagulant activity. The effectiveness of the sulfonation reaction by chlorosulfonic acid in pyridine was monitored by the degree of substitution and FT-IR analysis of the sulfonated EPSFRU (once sulfonated, EPSFRU SULF; and resulfonated, EPSFRU RESULF). Activated partial thromboplastin time (APTT) and thrombin time (TT) tests of EPSFRU RESULF indicated significant in vitro anticoagulant activity that was dose-dependent. EPSFRU did not inhibit any of the coagulation tests.

Abstract: The ascomyceteous fungus Botryosphaeria rhodina MAMB-05 secretes a β (1 � 3; 1 � 6)-D-glucan named botryosphaeran, with approximately 22% of branching, into liquid culture media. A comparison of the production of botryosphaeran in three different carbon sources showed higher yields in sucrose, followed by glucose and fructose. The difference in chemical structure of the polymers obtained under the three conditions was related to the degree of branching, and the aqueous solutions of this family of biopolymers behaved as gels, even at low concentration (1 to 6 g/L). The Herschel-Bulkley rheological model was used to represent the experimental data of shear stress versus deformation rate, and the results showed that the polysaccharides presented Non-Newtonian behaviour with the characteristics of pseudoplastic solutions. Thixotropic behaviour, as confirmed by the hysteresis curves of the polysaccharide solutions (6 g/L), was shown by the solutions obtained from the three different carbon sources. These findings suggest that this family of botryosphaerans have physicochemical properties suitable for use in commercial applications.

Abstract: Nine isolates of Botryosphaeria spp. were screened for lipases when cultivated on eight different plant seed oils and glycerol, and all produced lipases. Botryosphaeria ribis EC-01 produced highest lipase titres on soybean oil and glycerol, while eight isolates of Botryosphaeria rhodina produced significantly lower enzyme titres. B. ribis EC-01 produced lipase when grown on different fatty acids, surfactants, carbohydrates and triacylglycerols, with highest enzyme titres produced on Triton X-100-emulsified stearic (316.7 U/mL), palmitic (283.5 U/mL) and oleic (247.4 U/mg) acids, and soybean oil (105.6 U/mL), as well as castor oil (191.2 U/mg); an enhancement of 9-fold over soybean oil-grown cultures. Glycerol was also a good substrate for lipase production. The crude lipase extract was optimally active at pH 8.0 and 55 ºC, stable between 30 and 55 ºC and pH 1 to 10, and tolerant to 50 % (v/v) glycerol, methanol and ethanol. The crude lipase showed affinity for substrates of short, average and long-chain fatty acids (different esters of p-nitrophenol and triacylglycerols). Zymograms developed with 4-methylumbelliferylbutyrate showed two bands of lipolytic activity at 45 and 15 kDa. This is the first report on the production of lipases by Botryosphaeria ribis grown on these different carbon sources.

Abstract: Botryosphaeran, a (1,3;1,6)-β-D-glucan produced by Botryosphaeria rhodina MAMB-05, was found to be present in a triple helix conformation from helix-coil transition studies using Congo Red. The triple helix conformation was disrupted at increasing alkali concentrations. Conformational changes were also observed using phenanthrene as a fluorescent probe, and the fluorescence intensity decreased 80 % in the presence of dimethyl sulfoxide. The results confirmed the triple helix conformation of botryosphaeran, an important property manifesting biological response modifying activity.

Abstract: Four exopolysaccharides (EPS) obtained from Botryosphaeria rhodina strains isolated from rotting tropical fruit (graviola, mango, pinha and orange) grown on sucrose were purified on Sepharose CL-4B. Total acid hydrolysis of each EPS yielded only glucose. Data from methylation analysis and 13C NMR spectroscopy indicated that the EPS from the graviola isolate consisted of a main chain of glucopyranosyl (1,3) linkages substituted at O-6 as shown in the putative structure below: The EPS of the other fungal isolates consisted of a linear chain of (1,6)-linked glucopyranosyl residues of the following structure: FTIR spectra showed one band at 891 cm-1, and 13C NMR spectroscopy showed that all glucosidic linkages were of the beta-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. beta-(1,6)-DGlucans produced as exocellular polysaccharides by fungi are uncommon.

Abstract: Three D-glucans were isolated from the mycelium of the fungus Botryosphaeria rhodina MAMB-05 by sequential extraction with hot water and hot aqueous KOH (2 % w/v) followed by ethanol precipitation. Following their purification by gel permeation chromatography on Sepharose CL-4B, the structural characteristics of the D-glucans were determined by FT-IR and 13C NMR spectroscopy, and after methylation, by GC-MS. The hot-water extract produced a fraction designated Q1A that was a β(1,6)-D-glucan. The alkaline extract when subjected to repeated freeze-thawing yielded two fractions: K1P (insoluble) that comprised a β(1,3)-D-glucan with β-D-glucose branches at C-6 with the structure, and K1SA (soluble) consisting of a backbone chain comprising alpha(1,4)-linked D-glucopyranosyl residues substituted at O-6 with alpha-D-glucosyl residues as outlined:

Abstract: Orange bagasse comprising pulp tissues, rind, and seeds, constitutes a major industrial food waste arising from processing oranges for juice, and represents a fermentation feedstock for the production of enzymes. Botryosphaeria rhodina MAMB-05 grown on essential oils-extracted orange bagasse in submerged (SmF) and solid-state fermentation (SSF) with and without added nutrients produced pectinase and laccase. Highest enzyme titres (pectinase, 32 U ml-1; laccase, 46 U ml-1) occurred in SSF without added nutrients, indicating nutrient sufficiency of orange bagasse at a solids concentration of 16 % (w v-1) to sustain growth and high enzyme titres. Orange essential oil extract added to nutrient medium containing 1 % glucose in SmF strongly inhibited fungal growth with consequent lower laccase and pectinase activities. The results demonstrate the need to remove the essential oils fraction before citrus waste can be successfully used as a fermentation substrate for enzyme production.

Abstract: PURIFICATION AND CHARACTERIZATION OF A GENTIOHEXAOSE OLIGOSACCHARIDE OBTAINED FROM BOTRYOSPHAERAN BY PARTIAL ACID HYDROLYSIS. A hexa-oligosaccharide was obtained by partial-acid hydrolysis from botryosphaeran, an exopolysaccharide (EPS) of the β-(1�3;1�6)-D-glucan type, produced by the ascomyceteous fungus Botryosphaeria rhodina MAMB-05. The oligosaccharide was purified by chromatography on Sephadex G-15 and charcoal-Celite and the analyses followed by HPAEC/PAD. The structure of the oligosaccharide was determined by NMR and mass spectrometry, and showed that it consisted of six β-D-glucopyranosyl units O-6 substituted (gentiohexaose).

Abstract: Biopolymers such as exopolysaccharides (EPS) are produced by microbial species and possess unusual properties known to modify biological responses, among them antimutagenicity and immunomodulation. Botryosphaeran, a newly described fungal (1,3;1,6)-beta-D-glucan produced by Botryosphaeria rhodina MAMB-05, was administered by gavage to mice at three doses (7.5, 15 and 30 mg/kg b.w./day) over 15 days, and found to be non-genotoxic by the micronucleus test in peripheral blood and bone marrow. Botryosphaeran administered at doses of 15 and 30 mg EPS/kg b.w. decreased significantly (p <0.001) the clastogenic effect of cyclophosphamide-induced micronucleus formation resulting in a reduction of the frequency of micronucleated cells of 78 and 82 % in polychromatic erythrocytes of bone marrow, and reticulocytes in peripheral blood, respectively. The protective effect was dose-dependent, and strong anticlastogenic activity was exerted at low EPS doses. Variance analysis (ANOVA) showed no significant differences (p <0.05) among the median body weights of the groups of mice treated with botryosphaeran during experiments evaluating genotoxic and protective activities of botryosphaeran. This is the first report on the biological activity attributed to botryosphaeran.

Abstract: The physiological requirements for enhancing the production of laccases by the ascomycete, Botryosphaeria rhodina MAMB-05, in submerged cultivation was examined under non-induced and induced (veratryl alcohol, VA) conditions. Under non-induced conditions (- VA), the initial pH, C:N ratio and inorganic N source did not appear to influence laccase production, but the effects of Tween 80, soybean oil and copper, significantly increased laccase production, while proline and urea suppressed laccase formation. Tween 60 could serve as a sole carbon source for laccase production. Under induced conditions of fungal growth with VA as the laccase inducer, factors such as the influence of inoculum type, the time-point of addition of inducer, initial pH, C:N ratio, and the type of N source, all influenced the production of laccases, however, proline and urea did not suppress laccase formation. Each of these physiological conditions also affected biomass production differently. The nutritional conditions examined for B. rhodina MAMB-05 are discussed in relation to their influence on fungal growth and laccase production.

Abstract: The addition of soybean oil and Tween 80 was evaluated with the objective of increasing the production of botryosphaeran, an exopolysaccharide (EPS) of the (1�3);1�6)-β-D-glucan type produced by the fungus Botryosphaeria rhodina MAMB-05. Factorial design and analysis by response surface methodology was developed to select the main factors that would affect and enhance EPS production. The optimized culture conditions were: 40 g l-1 glucose with 10 ml l-1 soybean oil, and 4.5 ml l-1 Tween 80, during 72 h cultivation at 28 �C (180 rpm) and initial pH 5.7. The predicted result for botryosphaeran production was 8.22 ± 1.36 g l-1, and that for the observed was 7.74 ± 0.13 g l-1. The partial characterization of botryosphaeran produced under the optimized conditions showed one type of polysaccharide with β-glycosidic linkages containing mainly glucose as monosaccharide.

Abstract: Nine isolates of Botryosphaeria spp. were evaluated for their growth and the production of cell wall-lytic enzymes (laccase, pectinase and beta-1,3-glucanase) when grown on basal medium in the absence and presence of the laccase inducer, veratryl alcohol (VA). The genetic relationship among the nine isolates collected from different host plants was determined by RAPD analyses. ITS sequence analysis showed eight closely related isolates classified as Botryosphaeria rhodina, and one isolate classified as Botryosphaeria ribis. RAPD analysis resolved the isolates into three main clusters based upon levels of laccase and beta-1,3-glucanase activity. There appears to be no correlation between pectinase production and genetic diversity among the nine isolates. However, the strain characterized as B. ribis, positioned out of the main cluster, was found to be the highest producer of pectinases in the presence of VA.

Abstract: Botryosphaeria species are plurivorous phytopathogenic fungi colonizing a wide range of host plants of agricultural, forestry, ecological and economic importance. Species identification in the Botryosphaeria genus is based mainly on morphological characteristics of the anamorphs in combination with sequence analysis from the Internal Transcribed Spacer (ITS) region. The purpose of this study was to determine a minimal DNA sequence of the ITS region which could be applied for the identification of Botryosphaeria species. A total of 23 entries from the ITS sequences of 13 Botryosphaeria species, and several fragment lengths obtained from these entries, were compared with those already deposited in public databases using the BLASTn search tool. The secure identification of 11 of the 13 studied species was possible from the start of the ITS1 and the end of the ITS2 region using fragments up to 200 bp. These results were similar to those obtained with the complete ITS1/5.8S/ITS2 region. Phylogenetic trees based upon the neighbor-joining method showed similar topology when generated by complete ITS sequence and compared with those obtained with 200bp sequences. The results in this study showed that for identification of the greater number of the Botryosphaeria species, a simple sequence read with 200 bp obtained from the beginning of the ITS1 region, or from the end of the ITS2 region should be sufficient. This procedure makes the identification process more rapid and easier, and reduces the assembly efforts.

Abstract: An aryl-beta-D-glucosidase-encoding gene (bglH) of an endophytic Bacillus pumilus strain (CL16) was cloned from a shotgun genomic library constructed in Escherichia coli. The nucleotide sequence of the entire cloned fragment (2484 bp) was determined and characterized. An incomplete ORF of 534 bp (ORF1) designed bglP and a complete ORF of 1419 bp (ORF2) designed bglH, located in the fragment, are organized in an operon. The protein deduced from 1419 bp (ORF2) has 472 amino acid residues without a characteristic signal peptide sequence, suggesting that the enzyme is localized in the cytoplasm. The amino acid sequence deduced from the bglH gene had high similarity with beta-glucosidases from the glycosyl hydrolase family 1. The over-expression of the B. pumilus bglH gene in E. coli showed a protein of 54 KDa whose identity was confirmed by mass spectrometry (MALDI-TOF).

Abstract: Botryosphaeran, a (1�3;1�6)-β-D-glucan produced by Botryosphaeria rhodina, and laminarin were hydrolysed by two fungal β-glucanases predominantly of the 1,3-type produced by B. rhodina and Trichoderma harzianum Rifai grown on botryosphaeran as sole carbon source. Both β-glucanase preparations presented different modes of attack on botryosphaeran and laminarin. Laminarin was hydrolysed to the extent of ~50 % in 1 h and 100 % within 24 h, and its hydrolysis products were mainly glucose and gentiobiose, and lesser amounts of laminaribiose and oligosaccharides of DP 3-4 during the early stages of hydrolysis, while botryosphaeran yielded mainly glucose and gentiobiose with some trisaccharide, but no laminaribiose or tetrasaccharide when hydrolysed by the T. harzianum enzyme. By contrast, B. rhodina beta-1,3-glucanases produced predominantly glucose during all stages of botryosphaeran hydrolysis. Some physicochemical properties of the 1,3- and 1,6- beta-glucanases, and beta-glucosidases contained in the two fungal beta-glucanase preparations are also described for the first time.

Abstract: Two botryosphaerans, exopolysaccharides (EPS) secreted by the ascomyceteous fungus Botryosphaeria rhodina, when grown on sucrose and fructose as sole carbon sources, were structurally compared after their isolation from the culture medium. Both EPS were submitted to trypsin digestion, and eluted as a single peak on gel filtration. Total acid hydrolysis yielded only glucose, and data from methylation analysis and Smith degradation indicated that both EPS constituted a main chain of glucopyranosyl beta-(1,3) linkages substituted at O-6. The products obtained after partial acid hydrolysis demonstrated side chains consisting of glucosyl- and gentiobiosyl- linked beta-(1,6) residues. 13C-NMR spectroscopy studies showed that all glucosidic linkages were of the beta-configuration. The carbon source affected the side chain structures of botryosphaeran but not the main chain makeup. Sucrose produced less branching (21%) than fructose (31%).

Abstract: The herbicide, Scepter, whose active principle is imazaquin, is commonly used in soybean farming to combat wide-leaf weeds. The basidiomycete, Pleurotus ostreatus, and the ascomycete, Botryosphaeria rhodina, were evaluated for their growth and laccase production when cultured on basal media containing Scepter. Both fungi could grow on the herbicide when cultivated in solid and submerged liquid culture in the presence of Scepter at concentrations of 0 - 6 % (v/v) for P. ostreatus, and up to 0 - 50 % (v/v) for B. rhodina, and in each case produced laccases when assayed against ABTS [2,21-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] and 2,6-dimethoxyphenol. P. ostreatus could tolerate up to 6 % of Scepter before it became toxic to the fungus, while in the case of B. rhodina, 50 % (v/v) Scepter was the highest amount that supported grow, and laccase activity was detectable up to 25 % (v/v). An inverse relationship existed between the level of Scepter in the culture medium that supported fungal growth and laccase production. Analysis of the results showed that the fungi studied presented different behaviour towards Scepter in the culture environment.

Abstract: Botryosphaeria rhodina and Trichoderma harzianum Rifai were grown on botryosphaeran (an exopolysaccharide (EPS) of the β-1,3;1,6-D-glucan type produced by B. rhodina) as sole carbon source with the objective of producing beta-glucanases of the 1,3-type. Conditions for beta-1,3-glucanase production by T. harzianum were examined by a statistical response surface method, and showed maximal enzyme production at 5 days growth in media containing 1.5 g/l of EPS. Good agreement was obtained between the experimental values of β-1,3-glucanase activity and the corresponding values predicted by the mathematical model. The crude beta-1,3-glucanase preparations were active towards a number of different beta-1,3-glucans and beta-glucosides. The mycelium of B. rhodina also proved to be a good substrate for β-1,3-glucanase production by both fungal species.

Abstract: The influence of glucose concentration and other carbohydrates (monosaccharides: fructose, galactose, mannose; polyols: mannitol and sorbitol; disaccharides: lactose, sucrose and commercial sucrose; and industrial sugarcane molasses) were compared as sole carbon sources for the production of Botryosphaeran, an exopolysaccharide (EPS) produced by Botryosphaeria sp. The optimum glucose concentration for EPS production was 50 g l�1. With the exception of mannitol, the fungus produced EPS on all carbon sources studied, with highest yields occurring with sucrose followed by glucose. All EPS showed exclusively glucose after acid hydrolysis and monosaccharide analysis. FTIR spectroscopy demonstrated the presence of β-anomers indicating that all the EPS produced by Botryosphaeria sp. on the different carbon sources were essentially of the β-D-glucan type.

Abstract: Influence of Tween on constitutive and inducible laccase production by Botryosphaeria sp. Botryosphaeria sp. is a ligninolytic fungus that produces laccase. It was previously established that veratryl alcohol (VA) strongly induced laccase production in this ascomyceteous. It is well known that surfactants such as Tween-80 can increase the production of some fungal exo-enzymes. It has been reported that Tween-80 can also induce laccase production in basidiomycetes. In this work, the influence of Tweens 20, 40, 60 and 80 on the production of constitutive laccases (in the absence of VA) and inducible laccases (in the presence of VA) by Botryosphaeria sp. was evaluated, separately. Laccase activity was assayed using two putative laccase substrates: ABTS (PPO-I) and DMP (PPO-II). All of the Tweens examined increased the production of both constitutive and inducible laccase PPO-I, but did not affect the production of constitutive laccase PPO-II. Tweens 60 and 20 increased the production of inducible laccase PPO-II, while Tween 80 and 40 did not cause any effect.

Abstract: The influence of carbohydrates: glucose, fructose, galactose, galacturonic acid, xylose, lactose, sucrose, pectin and inulin, were evaluated as sole carbon source for the production of laccases by the ascomycete, Botryosphaeria sp. Veratryl alcohol, a laccase inducer, was added to culture media to study inducible laccase production on the same carbon sources. Inulinase and pectinase were also produced when Botryosphaeria sp. was grown on inulin, and galacturonic acid and pectin, respectively, and their levels were less in the presence of veratryl alcohol. Botryosphaeria sp. produced constitutive laccases on all carbon sources examined, and veratryl alcohol increased the laccase production on most of carbon sources studied except for inulin and galacturonic acid. Evidence is presented that Botryosphaeria sp. is also pectinolytic.

Abstract: The exopolysaccharide, Botryosphaeran, produced by the ligninolytic, ascomyceteous fungus Botryosphaeria sp., was isolated from the extracellular fluid by precipitation with ethanol, and purified by gel permeation chromatography to yield a carbohydrate-rich fraction (96%) composed mainly of glucose (98%). Infra-red and 13C NMR spectroscopy showed that all the glucosidic linkages were in the beta-configuration. Data from methylation analysis and Smith degradation indicated that Botryosphaeran was a (1 3)- beta-glucan with approx 22% side branching at C-6. The products obtained from partial acid hydrolysis demonstrated that the side branches consisted of single (1 6)-beta-linked glucosyl, and (1 6)-beta-linked gentiobiosyl residues.

Abstract: Four virulent strain isolates of the fungus, Bipolaris euphorbiae (previously identified as a Helminthosporium sp.), isolated from host plants in four states within Brazil were screened for the production of phytotoxins that promoted wilting and defoliation of the Brazilian weed, Euphorbia heterophylla, commonly found growing among soyabean crops. Only one isolate, B. euphorbiae Strain I (EUPH petropar from Mato Grosso state), produced phytotoxin in-vitro when grown in stationary culture for 7 d at 28 ºC on minimum salts medium supplemented with 1.5 % glucose as the sole carbon source. Phytotoxin was also produced when the fungal strain was grown on fructose, galactose, mannose, xylose and sucrose. The addition of nitrogen source (yeast extract, peptone or malt extract) to the culture medium did not influence phytotoxin production. The phytotoxin produced by Strain I was most active at pH 6.0, stable between pH 3-9, and was highly thermostable, remaining fully active when heated at 90 ºC for 1 h

Abstract: The concentration at which 50 % inhibition (I50) of fungal growth occurred for various lignin-related compounds was evaluated on Botryosphaeria sp. cultivated on agar medium. A general trend for inhibition of growth on lignin-related aromatic acids and phenolic compounds was based upon chemical structural features: benzoic acids containing di-methoxyl substituents on the aromatic ring were less inhibitory than the parent unsubstituted compound; p-phenolic benzoates containing a single methoxyl group were less inhibitory than those containing a di-methoxyl group; methoxylated derivatives of p-phenolic cinnamic acids were less inhibitory than the unsubstituted parent compound; but un-methylated hydroxyls on di-phenols allowed growth compared to the mono- and di-O-methylated derivatives, which were inhibitory. Botryosphaeria sp. grew on basal media containing up to 10 % (w v-1) lignosulfonate and kraft lignin in stationary culture. In submerged liquid cultivation, Botryosphaeria sp. grew on basal medium containing abietic acid, catechol, 4-chlorophenol, guaiacol, vanillyl alcohol, veratraldehyde and lignosulfonate, and in each case produced laccases active towards ABTS [2,21-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid). The production of laccases in each case implicated a possible role for their involvement in the biodegradation of aromatic compounds in Botryosphaeria sp. 4-Chlorophenol was the most toxic of the compounds examined.

Abstract: The oyster mushroom, Pleurotus ostreatus, cultivated in solid-state on sugarcane bagasse- wheat bran (5:1) medium in the presence of veratryl alcohol resulted in increased production of the fruiting body at earlier times compared to when the fungus was grown in the absence of veratryl alcohol. The results indicate a new physiological role for veratryl alcohol in stimulating fruiting body formation. Veratryl alcohol also stimulated laccase production during the mycelial growth stage. Evidence is also presented that laccases were involved in the physiological development of the fruiting body.

Abstract: A new physiological role for veratryl alcohol in fungi important in the biodegradation of the lignified plant cell wall is presented. Botryosphaeria sp., grown on starch, pectin, cellulose and xylan produced amylase, pectinase, cellulase, xylanase and laccase, whereas glucose and xylose repressed the synthesis of cellulase and xylanase, but not laccase. When cultured on these substrates in the presence of veratryl alcohol, laccases increased, but the levels of amylase, pectinase, cellulase and xylanase significantly decreased. Basal medium containing softwood kraft lignin in the presence of veratryl alcohol induced laccases above constitutive levels. Ethyl alcohol also stimulated laccase production.

Abstract: The relationship between three species of botryosphaeriaceous fungi, Botryosphaeria sp. isolate MAMB-5, Botryosphaeria ribis and Lasiodiplodia theobromae, were compared for the production of pycnidia and laccases. Laccases were produced both intra- and extra-cellularly when the fungi were cultivated on basal medium in the presence and absence of veratryl alcohol, with highest enzyme titres resulting for Botryosphaeria sp. MAMB-5. Electrophoretic examination of intracellular marker proteins (esterases and phosphatases) and laccases, indicated that the three species were genetically, distinctly different, although the laccase zymograms for the three fungi showed similarity. Conditions for the production of pycnidia occurred under continuous lighting at 28 °C, but differed among the three fungal species, and could be induced on artificial media (potato-dextrose and oat agar) under stress-induced conditions where the mycelium was stimulated by physical abrasion, and in the case of Botryosphaeria sp. isolate MAMB-5 on eucalypt woodchips. Evidence is presented that veratryl alcohol facilitated the secretion of intracellular-localised laccases into the extracellular medium.

Abstract: The ascomycete, Botryosphaeria sp, produced two extracellular constitutive laccases (PPO-I and PPO-II) active towards the substrates: 2, 2(1)-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) [ABTS], and 2,6-dimethoxyphenol (DMP), respectively. The production of both laccases increased when the fungal isolate was grown in the presence of veratryl alcohol, and resulted in optimal laccase production (100- and 25- fold, respectively) at 40 mM. The effect of aeration on growth and laccase production was studied in baffled flasks, and showed that aeration of the cultures increased the production of both enzymes 4-5 fold in the presence of veratryl alcohol. Both laccases were susceptible to inhibition by azide, acetate and chloride anions. Veratryl alcohol inhibited the laccase-catalyzed polymerization of DMP. Growing cultures of Botryosphaeria sp. produced an exopolysaccharide of the �-glucan type whose synthesis was depressed when grown in the presence of veratryl alcohol.

Abstract: A simple and economical method is described that allows rapid detection of laccase activity in chromatography column fractions during enzyme purification. Aliquots of column eluants are applied to filter paper coated with 2,2(1)-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing a numbered grid, and incubated at ambient temperature for 20 min. Indications of enzyme activity are simply observed by a colour change. This method avoids having to manually assay each fraction of a chromatographic run for enzyme activity

Abstract: Botryosphaeria sp. produced 2 laccases (PPO-I and PPO-II) constitutively, whose titres were enhanced by veratryl alcohol. The effect of veratryl alcohol and yeast extract concentration, time of cultivation, and agitation speed were evaluated by factorial analysis to select variables for optimizing production of laccases. Maximal laccase production was determined using a second-order central-composite design, and analysed by the response-surface method. Veratryl alcohol concentration and time of cultivation were the main factors increasing laccase production, while yeast extract had no influence within the range 0.2 to 2.0 % w/v. Response-surface analysis showed that 30.4 mM veratryl alcohol, for 4.5 days at 28 ºC and l80 rpm, were the optimal conditions to maximise PPO-I production, while conditions for maximal PPO-II production occurred within a range of 28-35 mM veratryl alcohol over a growth period of 4-5.5 days. The model predicted 5.6 U ml-1 for PPO-I, and 0.6-1.0 U ml-1 for PPO-II, which agreed with the experimentally observed results.

Abstract:

Abstract:

Abstract: Forty fungi isolated from diverse environments in Western Australia were screened for ligninolytic activity based on in vivo decolourization of the polymeric dye Poly R-478. Three isolates identified as Aspergillus, Botryosphaeria , and Coniochaeta species were selected for further studies. The Botryosphaeria and Coniochaeta isolates were found to produce laccase constitutively in submerged culture when grown on glucose or on ryegrass seed by solid state fermentation.. A comparison of the three isolates grown on glucose in the presence of 40 mmol l-1 3,4-dimethoxybenzyl (veratryl) alcohol showed that only the Botryosphaeria isolate produced laccase, and the laccase activity was 115 fold higher than on glucose alone.

Abstract:

Abstract:

Abstract: ABSTRACT A beta-glucosidase preparation derived from Aspergillus niger was immobilized onto a magnetic support and used in the enzymatic saccharification of a lignocellulosic material. The enzyme was immobilized onto polyethyleneimine-glutaraldehyde activated magnetite (PAM) and also onto titanium (IV) oxide (TiO2)-coated magnetite (TAM). Although > 80% of the protein applied was immobilized, only 15-27% of the enzyme activity was recovered after immobilization. The beta-glucosidase immobilized onto TiO2-coated magnetite suffered from enzyme being removed from the matrix under hydrolysis-use conditions, whereas the PAM enzyme remained attached to the matrix. The physicochemical properties of the immobilized beta-glucosidase preparations are described. Both immobilized beta-glucosidase preparations were capable of completely hydrolyzing cellobiose. Recycling of the immobilized enzymes (IME) resulted in reduced rates of hydrolysis with each recycling of the enzyme, although cellobiose was still capable of being completely hydrolyzed. The reduced hydrolysis performance was attributable to physical losses of tME during recovery and, in the case of TAM, enzyme loss from the matrix. Supplementing cellulase digests of steam-explosion pretreated Eucalyptus regnans pulps with immobilized beta-glucosidase resulted in enhanced hydrolysis. Cellulose-toglucose yields of 80% of theoretical predictions resulted within 24 h. The magnetically immobilized beta-glucosidase could easily be recovered from the lignocellulose solids suspension in a stirred batch reactor by applying a magnetic field. The recycled immobilized enzyme continued to convert cellobiose into glucose in 80% yields over a 24-h period. This is the first report of a magnetically immobilized beta-glucosidase preparation used in the enzymatic saccharification of a lignocellulosic material.

Abstract: ABSTRACT A magnetic immobilized lactase has been prepared using magnetite as the magnetic material. Magnetite was functionalized by treatment with polyethyleneimine and crosslinked with glutaraldehyde. Lactase was then covalenfly coupled to the activated magnetic matrix via the aldehyde groups. The conditions for optimal immobilization of enzyme are described. Eighty percent of the lactase activity was lost on immobilization and is thought to be owing to the orientation of enzyme binding to the matrix. The amount of protein coupled was 80% of that applied. The maximum lactase activity retained on the matrix following immobilization was 360 U/g matrix. The immobilized lactase showed optimal activity at pH 4.5 and 65 ~ The immob'flized lactase was more heat stable than the free enzyme, and retained 83% of its original activity after 14 d at 55 ~ Galactose competitively inhibited the immobilized lactase preparation (Ki 20 mM). The presence of high initial concentrations of galactose (10% w/v) did not prevent total hydrolysis of lactose. Glucose and calcium ions were activators of the immobilized enzyme. The immobilized enzyme hydrolyzed high concentrations of lactose (up to 25% w/v) to completion within 4--6 h in a stirred batch reactor at 55~C. There was no evidence of substrate inhibition at high substrate concentrations. The efficiency of hydrolysis of lactose by the immobilized lactase was better than that of the tree enzyme. The magnetic immobilized lactase was demonstrated to be suitable for use in the enzymatic hydrolysis of both pure, and cheese whey permeate, lactose.

Abstract:

Abstract: Enzymic saccharification of Eucalyptus regnans pulps pretreated by autohydrolysis-steam explosion resulted in low cellulose conversions into glucose when using trichodermal cellulase preparations. The reduced levels of glucose were attributable to the production of compounds during enzymic hydrolysis which were inhibitory to beta-D-glucosidase of Trichoderma reesei C-30 and in Meicelase, but not to the cellulases. Aspergillus niger beta-glucosidase was not inhibited, nor were beta- D-xylosidase(s) and 1,4-beta-D-xylanase(s) The inhibitory compound(s) could be extracted from the enzymic hydrolyzates with ethyl acetate. The ethyl acetate extractives inhibited beta-glucosidase in a competitive manner, and inhibitory action was not affected by pH. Addition of the inhibitory compound(s) to trichodermal cellulase digests of cellulose resulted in reduced glucose yields compared to a control. The inhibitory effects could be overcome when cellulase digests were supplemented with A. niger beta-glucosidase resulting in higher cellulose-to-glucose conversions. The inhibitory compound(s) were localized mainly in the heartwood of E. regnans. An inhibitor compound of this type has not hitherto been reported. The presence of inhibitory compound(s) in the autohydrolysis liquor fraction is also reported.

Abstract:

Abstract: Autohydrolysis explosion pretreatment of hardwood (Eucalyptus regnans) sawdust at 200°C and 6.9 MPa gas pressure (steam + nitrogen) for 5 min solubilized 85% of the total hemicellulose components and produced a pulp that was highly accessible to attack by cellulases from Trichoderma reesei C-30 and by a commercial preparation, Meicelase. The autohydrolysis liquor, representing 15% of the original weight of the sawdust on a solids basis, consisted mainly of xylose, xylose oligomers and minor amounts of galactose, mannose, arabinose, glucose and uronic acids. Enzymic hydrolysis of pretreated E. regnans pulps using Trichodermal cellulases resulted in saccharification yields of >SO% within 24 h from 10% (w/v) substrate slurries and 20 cellulase (FPU) units per g of pretreated pulp. The cellulose-to-glucose conversions were lower and this was attributable to the production of a compound(s) during enzymic hydrolysis that was inhibitory to the beta-glucosidase component, but not the cellulases, in the Trichodermal cellulase preparations. Enzymic digests supplemented with Novozym 188 beta-glucosidase showed >70% cellulose-to-glucose conversion within 24 h under similar conditions of hydrolysis. The inhibitor compound was not inhibitory to the Novozym 188 beta- glucosidases. Alkali-extracted autohydrolysis-exploded pulps were less susceptible to hydrolysis than unextracted pulps. Factors that influenced the extent of cellulose conversion into glucose such as enzyme-substrate and cellulase-to-beta-glucosidase ratios are also discussed.

Abstract: Structural breakdown of the lignin in autohydrolysis-exploded Pinus radiata and Eucalytus regnans and in sugarcane bagasse was examined using cross polarization-magic angle spinning CP/MAS) 13C NMR spectroscopy. The alkali- and acetone-insoluble (i.e. residual) lignins have undergone substantial cleave of the major (beta-0-4) interunit linkage and are most likely mixtures of partly depolymerized and repolymerised lignin fragments. Correlations between residual lignin structure, breakdown of the lignocellulose structure and enzymatic digestibility of the cellulosic carbohydrate are proposed.

Abstract: Softwood (Pinus radiata) and hardwood (Eucalyptus regnans) sawdust were pretreated by autohydrolysis at 200°C followed by explosive decompression. P. radiata pretreated pulps were poorly hydrolyzed by Trichoderma reesei C-30 cellulases compared to E. regnans pulps. Impregnation of P. radiata with 4.44% (w/w) SO, followed by autohydrolysis explosion resulted in increased enzymic saccharification of the pulps. Sulfur dioxide treatment resulted in more of the hemicellulose components (heteroxylan and glucomannan) being removed during autohydrolysis than when P. radiata was pretreated by autohydrolysis alone. Trichoderma reesei C-30 cellulase digests containing 10% (w/v) of the pretreated pulps and 20 FPU units/g pulp resulted in cellulose-to-glucose conversion yields of 32% with E. regnans, 16% with P. radiata and 27% with SO,-treated P. radiata within 24 h at 50°C (pH 5.0) and could be increased to 74%, 27% and 48%, respectively, when the cellulase digests were supplemented with an exogenous j3-glucosidase (Novozym 188). Cellulase, j3-glucosidase and xylanase production by T. reesei C-30 grown in shake culture (2SoC, pH 5.0) on 1% (w/v) of the pretreated pulps was highest when E. regnans and SO,-treated P. radiata were used as growth substrates, and enzyme yields compared with those when a purified a-cellulose (Solka floc) was used as growth substrate. Autohydrolysis-exploded P. radiata, however, proved to be a rather poor substrate for enzyme production. Typical FPU yields (as IU/g cellulose) were 53, P. radiata; 126, SO,-treated P. radiata; 136, Solka floc; and 165, E. regnans.

Abstract:

Abstract:

Abstract: A number of different yeasts are now recognized as being capable of fermenting the pentose sugar, Dxylose, into ethanol.'-3 The most prominent among these are Pachysolen tannophilus and several Candida species.%' DXylose is found principally in lignocellulosic materials where it occurs as the main constitutent of the hemicellulosic xylans (1,4-@~-heteroxylans). The utilization of the xylose component in these materials is thus important in the overall process economics of the bioconversion of lignocellulosic material into chemicals such as ethanol. With the exception of Candida XF-217,3 the conversion yields of xylose into ethanol for most yeasts were generally low (less than 70% of theoretical when grown on at least 50 g/L xylose). The low ethanol yields are attributable to a number of factors: 1) fermentation was not performed under conditions that maximize ethanol formation; 2) ethanol was not the major fermentation end-product, (e.g., acetic acid, xylitol, and arabinitol are also known p r o d ~ c t s ; ~ , ~ , ~ 3) ethanol toxicity; 4) ethanol is consumed concurrently as it is produced;' 5) ethanol is assimilated when the substrate becomes limitingfZ8 and 6) osmotic sensitivity to high substrate levels, i.e., substrate inhibition.* Attempts to increase ethanol yields of yeasts by adding exogenous lipids (e.g., oleic and linoleic acids, or ergosterol or its ester,'-'' lipid mixtures,*0"' or proteinlipid mixturesJ2) to nutrient medium have succeeded in improving ethanol yields and also in reducing fermentation times." These lipids, when added to the nutrient medium, were incorporated into the yeast's cellular membrane. The protective action of these lipids was to alleviate the inhibitory effect of ethanol which then allowed the cells to tolerate higher ethanol levels. This communication reports on improved ethanol yields arising from the fermentation of xylose by a Pachysolen tannophilus strain when grown semi-aerobically in the presence of exogenous-added lipids.

Abstract:

Abstract:

Abstract:

Abstract: Autohydrolysis of sunfiower seed hulls at 200°C for 5 min followed by explosive defibration solubilized >80% of the total hemicellulose (heteroxylan) and 85% of the pectic substances. The remaining residue, which consisted of cellulose (38%), lignin (45%) and residual heteroxylan (7%), was highly susceptible to hydrolysis by cellulases ~Trichodemna reesei C-30, Meicelase and Onozuka 3s). Enzymic digests containing 10% (w/v) substrate and 20 Fl?U/g pretreated hull resulted in sugar yields of >40% within 24h. This yield was increased to ca. 70% when digests were supplemented with exogenous beta-glucosidase. Themethod has potential use in the bioconversion of sunflower seed hulls into chemicals.

Abstract: D-Xylose was fermented to ethanol by a strain of Pachysolen tannophilus in yields greater than 0.3g ethanol per g xylose consumed. Ethanol production was influenced by xylose concentration and was at a maximum at 10%, w/v. Ethanol formation occurred at pH 2.75-2.50 but the yeast would not grow at this pH when the initial pH of the medium was less than 3.0. Ethanol was consumed by the yeast when the xy!ose concentration became limiting. ~- Arabinose, D-glucose, D-fructose, cellobiose, D-glucuronic acid, but not sucrose, were also fermented to ethanol by Pach~solen tannophilus. Kinetic studies on xylose fermentation established various parameters involved in growth, substrate utilization and ethanol formation when the yeast was fermenter grown.

Abstract:

Abstract:

Abstract:

Abstract: The phytopathogen Xanthomonas campestris when grown on several polysaccharide substrates, including galactomannan, produced beta-mannanases and carboxymethylcellulases which were present as cell wall-, or membrane-bound constituents, and also in the extracellular medium. The extracellular beta-mannanases are constitutive enzymes. Cell-bound /beta-mannanases could be partially solubilized by Triton X-100, but this treatment could not release cell-bound cellulase. This is the first report of the existence of a cell-bound beta- mannanase. Cell-free and bound beta-mannanases and carboxymethylcellulases were found to be of the endoenzyme type.

Abstract: Acetobacter xylinum is a Gram-negative bacterium well-known for its production of crystalline cellulose. It was demonstrated as early as 1958 [ 11, and subsequently confirmed [2-61, that membrane preparations from this organism catalyze the transfer of D-glucose from UDP-glucose to cellulose. In the original study by Glaser [I] cellulose was determined as the alkali-insoluble glucan fraction, the alkalisoluble material being discarded. Subsequent studies showed that there were also unidentified alkali-soluble transfer products [4-7] which have been discussed as intermediate polymers of cellulose biosynthesis [5,71. It is now shown that the major polymeric product formed in vitro from UDP-glucose is alkali-soluble and that glucose is transferred predominantly in a beta-1,2- rather than a beta-1,4-linkage. A brief abstract of some of these results has appeared [8].

Abstract: A cellular phenol-water extract of Acetobacter xylinum NRC 17007 was fractionated on Sepharose 4 B. The fraction eluting with the void volume consisted to about 95 ~ of glycogen-like material. The lipopolysaccharide fraction was of lower molecular weight and had the following composition (~, w/w): Mannose, 42; glucose, 7; galactose, 3.8; heptose, 2; 2-keto-3-deoxy-octonate, 1.2; glucosamine, 3.3; phosphate, 4.5; total fatty acids, 3.9. Among the fatty acids, 3-hydroxy-tetradecanoic acid was present, and 2-hydroxy- hexadecanoic acid predominated.

Abstract: Major comprehensive review on the enzymes that comprise the group called hemicellulases and includes: L-arabinanases, Galactanases, beta-Mananases, and beta-xylanases. There is some discussion too on the accessory enzymes involved in degradation of the 4 main categories that constitute the hemicelluloses of plants.

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract: Oligosaccharides present specific physicochemical and biological properties that can be exploited for specific applications in foods and pharmacology. They can be produced through a number of different physical, chemical and enzymatic catalysed reactions from their parent polysaccharides as well as through transglycosylation reactions. This chapter examines the pathways leading to the production of bioactive oligosaccharides that have biotechnological applications. These carbohydrate oligomers constitute a nutritional type of �fiber� that benefits the growth of bifidobacteria and lactobacilli in the colon promoting human health and well-being. The use of oligosaccharides to modify biological responses was recently reported, and this has included their effects as anti-inflammatory and anti-cholesterolaemic stimulating compounds. An overview of nutraceutical and biological functions of these carbohydrate fragments mainly for human health is also reported.

Abstract: Extreme environments, generally characterized by atypical temperatures, pH, pressure, salinity, toxicity and radiation levels, are inhabited by various microorganisms specifically adapted to these particular conditions. These microorganisms, called extremophiles, are of significant biotechnological importance as their enzymes (extremozymes) and biopolymers possess unique properties that offer insights into their biology and evolution. The enthusiastic search for novel extremophiles has largely been stimulated by the uniqueness of their survival mechanisms. This uniqueness can be transformed into valuable applications ranging from wastewater treatment to the diagnosis of infectious and genetic diseases. One adaptation strategy of particular importance to extremophiles is the production of extracellular polymeric substances (EPSs) that envelop the cell as a barrier protecting them against environmental extremes such as desiccation, temperature, pressure, salinity, acidity, heavy metals, and radiation. Due to their many interesting physicochemical and rheological properties, these biopolymers possess novel functionality that is generally superior to petrochemical-derived polymers in aspects that embrace biodegradability, and environmental and human compatibility. Consequently, biopolymers of extremophiles are widely used in foods, cosmetics, pharmaceutical products, textiles, detergents, adhesives, oil-recovery from wells, brewing and waste treatment processes. In this chapter, we present a brief overview of life under extreme environmental conditions. This is followed by a discussion of extremophilic microorganisms and their adaptation mechanisms, and specifically focuses on the production of EPSs and their ecological and physiological functions. The application areas of industrially important EPSs from various extremophilic producer strains are also mentioned.

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract:

Abstract: Botryosphaeran, a new fungal exopolysaccharide from the endophytic fungus Botryosphaeria rhodina MAMB-05, and algal laminarin were hydrolyzed by partially-fractionated enzymes of the β-glucanolytic complex from Trichoderma harzianum Rifai. β-Glucanase fractions (F-I and F-II) separated by gel permeation chromatography presented different modes of attack on botryosphaeran and laminarin. Botryosphaeran was hydrolyzed to the extent of 66% (F-I) and 98% (F-II) within 30 min, and its main hydrolysis products were gluco-oligosaccharides of DP�4, with lesser amounts of glucose, di- and tri-saccharides. The action of enzyme fractions I and II on laminarin resulted in 15% conversion to glucose, while the percentage of saccharification was radically different (70% for F-I and 25% for F-II). The different product arrays within the polysaccharide hydrolysates can be explained by the difference in the enzymes� specificities within each enzyme fraction, and the molecular structures of the polysaccharides and their complexity.

Abstract: Actinomycetes and fungi constitute groups of microorganisms for application in the bioremediation of recalcitrant xenobiotic compounds such as polyaromatic hydrocarbons, polychlorinated aromatic hydrocarbons including PCB's, and dioxins. Microbes active in composting and wood-decay a series of non-specific extracellular peroxidases that are involved in ligninolysis, but can also attack a broad range of aromatic and phenolic compounds as lignin is polyphenolic by nature (1). Decolorization of polymeric dyes has been proposed as a useful screening method for ligninolytic activity (2). The method is rapid and simple, enabling the handling of large numbers of microorganisms in screening programs. It has also been shown to be a good indicator of the initial transformation of xenobiotics mediated by the peroxidative activity of fungi (1). The objective of the work reported herein was to screen actinomycetes and fungi using the polymeric dye Poly R-478, and isolate those cultures which could have potential in bioremediation processes. The white-rot fungus, Phanerochaete chrysosporium, which is well-characterized in lignin degradation (3), was used as a reference ligninolytic fungus. Materials and Methods Microorganisms: The actinomycetes screened were isolated from decomposing pig manure, and included thermophiles (20 grown at 37°C, and 10 at 50°C), and mesophiles (20, grown at 28°C). Among the actinomycetes screened were Streptomyces, Thermonospora, Nocardiopsis, Micromonospora and Dactylosporangium species. Fungi were from the Murdoch University Plant Pathology culture collection and were isolated from decaying wood of Western Australia tree species. Some of them belong to the following genera: Coniochaeta, Pleurotus, Camarosporium, Humicola, Philophora, Botryosphaeria, Sordaria, Chaetomium, Philophora, Fusarium, Endothiella, Cytospora, Seimatosporium and Pestalotiopsis. Fungi were also isolated from the bark of trees and soils collected in the Perth metropolitan area. The ligninolytic fungus Phanerochaete chrysosporium (strain BKM-F-1767; ATCC 24725) was also screened. Culture medium and conditions: Actinomycetes were grown on solid medium according to Markin et. al.(4), using glucose as carbon source (0.3 and 1.0 % w/v) in the presence of 0.02 %(w/v) Poly R-478. Two controls were developed in parallel, one containing medium with Poly R-478 and no carbon source, the other with 1% glucose but without Poly R. Fungi were grown on minimum Vogel salts media containing glucose [1 % w/v] as carbon source, and 0.02 % (w/v) Poly R-478 were grown for 12 days at 28°C and shaken at 200 r.p.m. Cell-free filtrates were obtained by centrifugation (3000 rpm/15min). Results and Discussion Of the 50 actinomycete isolates screened, 6 (four mesophiles and two thermophiles), decolorized Poly R-478 on agar plates. Decolorization was observed in agar plates in which glucose was the carbon source after 6 days growth and increased until 12 days. Consequently enzymes involved in Poly R-478 decolorization result from secondary metabolism. This result is in accordance with enzymes involved in ligninolysis, which occurs when other readily biodegradable substrates are depleted from the media (3). Ten fungal isolates from the 40 wood-decay fungi screened were able to decolorize Poly R-478. The culture fluid of several isolates were selected and examined spectrophotometrically to determine spectral changes during decolorization. There were distinct spectral changes at 513 and 362 nm (see Figure 1); the absorption maxima for Poly R-478 (2), indicating that the culture filtrates from these isolates caused structural changes to Poly R-478. Two isolates (TR-2 and MAMB-4), although presenting higher absorbance values at 513 nm, were also chosen based on their spectral profile. The results for the culture filtrates from several isolates [(c) MU-154, (d) MAMB-5, (e) MAMB-4, and (f) TR-4] are presented in Figure 1, and are compared with that from Phanerochaete Chrysosporium (b), which showed very little change in the spectra of Poly R-478 when compared to a control (a) containing no culture filtrate. In the visible wavelength region, the level of decolorization was completely different for each microorganism. This could be interpreted as the reduction of the quinone groups present in Poly-R being catalysed by different enzymes (lignin peroxidase, Mn-peroxidase, laccase), which are implicated to be responsible for decolorization (1). MAMB-4 and TR-4 isolates presented higher absorption in the UV wavelength region and this is probably due to the formation of degradation products from Poly R-478. The implications of these findings will be discussed. References 1. Field, J.A.; Jong, E.; Feijoo-costa, G. and Bont, J.A.M. (1993). Tibtech: 11, 44-49. 2. Gold, MH, Glenn, JK, and Alic, M (1988). Meth. Enzymol. 161: 74-78. 3. Tien, M and Kirk, TK (1987). Crit. Rev. Microbiol. 15: 141-168. 4. Markin, JM, Crawford, DL and McCoy, E. (1974). TAPPI 57: 1051 5. Vogel, HJ. (1956). Genet. Bull. 13: 42-43.