Showing papers in "Enzyme and Microbial Technology in 2002"

Abstract: Direct electron transfer from different Shewanella putrefaciens strains to an electrode was examined using cyclic voltammetry and a fuel cell type electrochemical cell. Both methods determine the electrochemical activity of the bacterium without any electrochemical mediators. In the cyclic voltammetric studies, anaerobically grown cells of Shewanella putrefaciens MR-1, IR-1, and SR-21 showed electrochemical activities, but no activities were observed in aerobically grown Shewanella putrefaciens cells nor in aerobically and anaerobically grown E. coli cell suspensions. The electrochemical activities measured by the cyclic voltammetric method were closely related to the electric potential and current generation capacities in the microbial fuel cell system. Cytochromes localized to the outer membrane are believed to facilitate the direct electron transfer to the electrode from the intact bacterial cells. The concentration of the electron donor in the anode compartment determined the current generation capacity and potential development in the microbial fuel cell. When the high concentration of the bacteria (0.47 g dry cell weight/liter) and an electrode that has large surface area (apparent area: 50 cm2) were used, relatively high Coulombic yield (over 3 C for 12 h) was obtained from the bacteria.

Abstract: Lignocellulose is a potential substrate for ethanol production. However, high cellulose conversion requires high enzyme loading, which makes the process less economically feasible. Addition of surfactants to enzymatic hydrolysis of lignocellulose increases the conversion of cellulose into soluble sugars. The mechanism is not known for the increase of lignocellulose hydrolysis by surfactant addition, therefore, experiments were designed to explore mechanisms of surfactant effects. A number of surfactants were screened for their ability to improve enzymatic hydrolysis of steam-pretreated spruce (SPS). Non-ionic surfactants were found to be the most effective. Studies of adsorption of the dominating cellulase of Trichoderma reesei, Cel7A (CBHI), during hydrolysis showed that the anionic and non-ionic surfactants reduced enzyme adsorption to the lignocellulose substrate. The approximate reduction of enzyme adsorption was from 90% adsorbed enzyme to 80% with surfactant addition. Cellulase stability in the presence of surfactants was studied by activity and fluorescence measurements. Surfactants were shown to have only a weak effect on cellulase temperature stability. Our conclusions from studies of lignocellulose and delignified substrates are that the improved conversion of lignocellulose with surfactant can be explained by the reduction of the unproductive enzyme adsorption to the lignin part of the substrate. This is due to hydrophobic interaction of surfactant with lignin on the lignocellulose surface, which releases unspecifically bound enzyme. A new approach with mixed charged and non-ionic surfactants has been introduced to further improve the positive effect of the surfactant addition.

Abstract: Manganese peroxidase (MnP) is the most common lignin-modifying peroxidase produced by almost all wood-colonizing basidiomycetes causing white-rot and various soil-colonizing litter-decomposing fungi. Multiple forms of this glycosylated heme protein with molecular weights normally at 40 to 50 kDa are secreted by ligninolytic fungi into their microenvironment. There, MnP preferentially oxidizes manganese(II) ions (Mn2+), always present in wood and soils, into highly reactive Mn3+, which is stabilized by fungal chelators such as oxalic acid. Chelated Mn3+ in turn acts as low-molecular weight, diffusible redox-mediator that attacks phenolic lignin structures resulting in the formation of instable free radicals that tend to disintegrate spontaneously. MnP is capable of oxidizing and depolymerizing natural and synthetic lignins as well as entire lignocelluloses (milled straw or wood, pulp) in cell-free systems (in vitro). In vitro depolymerization is enhanced in the presence of co-oxidants such as thiols (e.g. glutathione) or unsaturated fatty acids and their derivatives (e.g. Tween 80). The review summarizes and discusses different approaches to prove lignin decomposition in vitro and lists, in addition, other recalcitrant substances oxidizible by MnP.

Abstract: This review summarizes all the research efforts that have been spent to immobilize laccase and tyrosinase for various applications, including synthetic and analytical purposes, bioremediation, wastewater treatment, and must and wine stabilization. All immobilization procedures used in these areas are discussed. Considerations on the efficacy of immobilized copper oxidases and products, in addition to their kinetic parameters are also discussed. The available data indicate that the immobilization of laccase into cationic polymer cross-linked with epichlorohydrin appears to be a promising procedure for industrial applications. The development of laccase and tyrosinase-based biosensors to monitor a wide range of compounds appears to be at a mature stage of technology.

Abstract: Three peroxidases involved in lignin degradation are produced by white-rot fungi. Lignin peroxidase (LiP) is characterized by oxidation of high redox-potential aromatic compounds (including veratryl alcohol) whereas manganese peroxidase (MnP) requires Mn2+ to complete the catalytic cycle and forms Mn3+ chelates acting as diffusing oxidizers. Pleurotus and Bjerkandera versatile peroxidase (VP) is able to oxidize Mn2+ as well as non-phenolic aromatic compounds, phenols and dyes. Phanerochaete chrysosporium has two gene families including ten LiP-type and three MnP-type genes coding different isoenzymes expressed during secondary metabolism. Two VP genes have been recently cloned from Pleurotus eryngii. Phanerochaete chrysosporium MnP and P. eryngii VP are induced by H2O2, being Mn2+ involved in regulation of their transcript levels. At least eighteen more ligninolytic peroxidase genes have been cloned from other white-rot fungi. Protein sequence comparison reveals that typical MnP from P. chrysosporium and two other fungi (showing a longer C-terminal tail) are separated from other ligninolytic peroxidases, which form two main groups including P. chrysosporium LiP and Pleurotus peroxidases respectively. LiP and MnP crystal structures and VP theoretical molecular models are available. The high redox potential of ligninolytic peroxidases seems related to the distance between heme iron and proximal histidine, and the ability of MnP to oxidize Mn2+ is due to a Mn-binding site formed by three acidic residues near the internal heme propionate. Pleurotus eryngii VP show higher sequence and structural affinities with P. chrysosporium LiP than MnP, but includes a Mn-binding site accounting for its ability to oxidize Mn2+. The functionality of this site was demonstrated by site-directed mutagenesis of MnP and VP. All fungal peroxidases, which exhibit similar topology (11–12 helices) and folding, also include binding sites for two structural Ca2+. Veratryl alcohol was first modeled near LiP heme, but evidence for oxidation at the protein surface via a long-range electron transfer pathway has accumulated. Chemical and site-directed mutagenesis modification confirmed that an exposed tryptophan is involved in veratryl alcohol oxidation however, multiple sites could be responsible for oxidation of different aromatic substrates and dyes by these peroxidases.

Abstract: Although many wood decay basidiomycetes secrete oxidative and hydrolytic enzymes that participate in lignocellulose biodedgradation, it is generally recognized now that these enzymes cannot penetrate sound wood, and that fungi must employ smaller agents to initiate decay. Reactive oxygen species (ROS) are likely candidates, and evidence is accumulating that some wood decay fungi produce these oxidants. For example, we have observed that, when they grow on wood, white rot and brown rot basidiomycetes degrade polyethylene glycol, a recalcitrant polymer that resists attack by species other than nonspecific, extracellular oxidants. By contrast, xylariaceous ascomycetes do not degrade polyethylene glycol, and therefore do not produce these oxidants when they degrade wood. The ROS that basidiomycetes employ remain unidentified in most cases, but the hydroxyl radical (·OH) is a likely participant, because there is a well-established pathway for its production via the Fenton reaction (H 2 O 2 + Fe 2+ + H + → H 2 O + Fe 3+ + ·OH). The two best documented fungal pathways for ·OH production involve the extracellular reduction of Fe 3+ and O 2 by cellobiose dehydrogenase or by secreted hydroquinones. Peroxyl (ROO·) and hydroperoxyl (·OOH) radicals, although they are weaker oxidants, may also be involved in wood decay because they are produced via reactions of ·OH and by some fungal enzymes.

Abstract: The white-rot fungus Trametes pubescens MB 89 is an excellent producer of the industrially important enzyme laccase. Extracellular laccase formation can be considerably stimulated by the addition of Cu(II) in the millimolar range to a simple, glucose-based culture medium. When using glucose, a typically repressing substrate, as the main carbon source, significant laccase formation by T. pubescens only started when glucose was completely consumed from the culture medium. In addition, the nitrogen source employed had an important effect on laccase synthesis. When using an optimized medium containing glucose (40 g l−1), peptone from meat (10 g l−1), and MgSO4·7H2O and stimulating enzyme formation by the addition of 2.0 mM Cu, maximal laccase activities obtained in a batch cultivation were approximately 330 U ml−1. Under these conditions it was not necessary to add aromatic compounds that are routinely used as inducers of laccase in fungi. By applying a fed-batch strategy, in which a glucose solution was fed continuously to a cultivation of T. pubescens so that the glucose concentration in the medium never exceeded a certain low, critical value, glucose repression could be avoided and production of laccase was almost doubled as compared to the batch cultivation (740 U ml−1).

Abstract: The biosorption of three commonly used reactive dyes, from aqueous solutions by oven-dried Rhizopus arrhizus biomass was studied in a batch system with respect to pH, initial dye concentration and initial metal ion concentration. The biomass exhibited maximum dye uptake at pH 2 due to its positively charged nature at acidic pH and the anionic nature of the reactive dyes. Reactive orange 16 dye was adsorbed most effectively to a maximum of approximately 200 mg/g. The presence of high levels of cadmium did not significantly impair the adsorption capacity of the biomass. Dye removal from a multicomponent solution of all three dyes was also achieved. Rhizopus biomass was found to exhibit superior removal properties than activated charcoal.

Abstract: Sugarcane bagasse was pre-treated by steam explosion at 205 and 215degreesC and hydrolysed with cellulolytic enzymes. The hydrolysates were subjected to enzymatic detoxification by treatment with the phenoloxidase laccase and to chemical detoxification by overliming. Approximately 80% of the phenolic compounds were specifically removed by the laccase treatment. Overliming partially removed the phenolic compounds, but also other fermentation inhibitors such as acetic acid, furfural and 5-hydroxy-methyl-furfural. The hydrolysates were fermented with the recombinant xylose-utilising Saccharomyces cerevisiae laboratory strain TMB 3001, a CEN.PK derivative with over-expressed xylulokinase activity and expressing the xylose reductase and xylitol dehydrogenase of Pichia stipitis, and the S. cerevisiae strain ATCC 9658 1, isolated from a spent sulphite liquor fermentation plant. The fermentative performance of the lab strain in undetoxified hydrolysate was better than the performance of the industrial strain. An almost two-fold increase of the specific productivity of the strain TMB 3001 in the detoxified hydrolysates compared to the undetoxified hydrolysates was observed. The ethanol yield in the fermentation of the hydrolysate detoxified by overliming was 0.18 g/g dry bagasse, whereas it reached only 0.13 g/g dry bagasse in the undetoxified hydrolysate. Partial xylose utilisation with low xylitol formation was observed. (Less)

Abstract: A process for simultaneous efficient production of bioactive ganoderic acid (GA) and Ganoderma polysaccharide by fed-batch fermentation of Ganoderma lucidum was developed. Effects of carbon source and initial sugar concentration on the production of GA and polysaccharide were studied at first. Sucrose as a carbon source was suitable for the extracellular polysaccharide (EPS) production although the cells could not grow well. Lactose was beneficial for the cell growth and production of GA and intracellular polysaccharide (IPS). When the initial lactose concentration exceeded 35 g/l, the GA accumulation was decreased. The GA production was remarkably improved by pulse feeding of lactose, when its residual concentration was between 10 and 5 g/l. The fed-batch fermentation process in shake flasks could be successfully reproduced in stirred bioreactors (STR) with dissolved oxygen (DO) controlled between 20 and 35% of air saturation during fermentation. The maximum cell density reached 21.89±0.35 and 20.95±0.01 g DW/l in STR and flasks, respectively. The production of EPS reached 0.87±0.05 (STR) and 0.75±0.05 g/l (flask), and the total production of IPS reached 2.49±0.07 (STR) and 2.40±0.05 g/l (flask). The total production of GA reached 367.1±17.4 and 297.9±13.7 mg/l for STR and flask, respectively, and their corresponding productivity was 29.6±0.5 and 14.3±0.1 mg/l per day. Not only the cell density but also the content, production and productivity of GA and IPS obtained in this work are the highest reported in agitated fermentation of G. lucidum.

Abstract: Relatively large (0.19 m column diameter, 2 m tall, 0.06 m3 working volume) outdoor bubble column and airlift bioreactors (a split-cylinder and a draft-tube airlift device) were compared for monoseptic fed-batch culture of the microalga Phaeodactylum tricornutum. The three photobioreactors produced similar biomass versus time profiles and final biomass concentration (∼4 kg m−3). The maximum specific growth rate observed within a daily illuminated period in the exponential growth phase, had a value of 0.08 h−1 on the third day of culture. Because of night-time losses of biomass, the specific growth rate averaged over the 4-days of exponential phase was 0.021 h−1 for the three reactors. The biomass in the vertical column reactors did not experience photoinhibition under conditions (photosynthetically active daily averaged irradiance value of 1150±52 μE m−2 s−1) that are known to cause photoinhibition in conventional thin-tube horizontal loop reactors. Because of good gas-liquid mass transfer, the dissolved oxygen concentration in the reactors at peak photosynthesis remained

Abstract: To compete in nature against other forms of life, microorganisms possess regulatory mechanisms which control production of their metabolites, thus, protecting against overproduction and excretion of these primary and secondary metabolites into the environment. To effect such an economical form of life, they possess regulatory mechanisms which control production of these metabolites and protect against overproduction and excretion into the environment of excess concentrations. In the field of industrial fermentation, the opposite concept prevails. Fermentation microbiologists search for a rare overproducing strain in nature, then further deregulate the microorganism so that it overproduces huge quantities of a desired commercially important product such as a metabolite or an enzyme. Deregulation is brought about by nutritional as well as classical and molecular genetic manipulations to bypass and/or remove negative regulatory mechanisms and to enhance positive regulatory mechanisms. These mechanisms include induction, nutritional regulation by sources of carbon, nitrogen and phosphorus, and feedback control. The controls and their modification by biotechnologists are the subjects of this review.

Abstract: Dietary transitions have resulted in generally higher meat intakes. Intensive feeding of animals is a rather inefficient way of producing dietary protein and it has also a variety of undesirable environmental and health impacts. Partial substitution of meat protein by plant proteins incorporated in ground meats and processed meat products would help to moderate these impacts.

Abstract: The study of enzymatic transesterification of high oleic sunflower oil with butanol by immobilised Lipozyme® was realised in n-hexane and in a solvent-free system. A simplified model, based on the Ping Pong Bi Bi with alcohol competitive inhibition mechanism, was proposed to describe transesterification kinetics. The solvent-free reaction medium was characterised by higher initial velocities and a 6-times volumic productivity but at the thermodynamic equilibrium only about 60% of the potential oleic acid is converted into ester, against 95% in n-hexane. The resulting product is a mixture of ester, monoglyceride, diglyceride and triglyceride with lubricant and surfactant properties and its composition depends on the initial substrate ratio.

Abstract: One hundred and fifteen fungi from different physio-ecological groups were compared for their capacity to decolourize two structurally different dyes in agar plates. We found that the azo dye, Acid Red 183, was much more resistant to decolourization by the examined strains in both solid and liquid cultures. Among the tested fungi, 69 strains showed decolourization of the anthraquinonic dye, Basic Blue 22, within 5–14 days, and only 16 strains were able to decolourize the azo dye, Acid Red 183, within 21 days. Furthermore, the potential of selected strains for decolourization of dyes was examined with regard to their extracellular oxidative factors both enzymatic and non-enzymatic. In static aqueous culture, the three selected fungi ( Bjerkandera fumosa , Kuehneromyces mutabilis , and Stropharia rugoso-annulata ) formed fungal mats, which did not decolourize any dye beyond some mycelial sorption. In comparison to the static cultures, the agitated cultures (180 rpm) removed 75 to 100% of the colour of Basic Blue 22 and 20 to 100% of Acid Red 183 colour.

Abstract: Intracellular oxalate decarboxylase (ODC, EC 4.1.1.2) activity was screened in the mycelium of 12 white rot fungi. ODC activity was detected in the mycelial extracts of Dichomitus squalens, Phanerochaete sanguinea, Trametes ochracea, and Trametes versicolor (strain R/7) after addition of 5-mM oxalic acid to the liquid culture medium. In D. squalens, intracellular ODC activity increased six-fold with addition of oxalic acid. Production of extracellular organic acids by the four ODC-positive fungi was followed in liquid cultures and in solid state cultures of spruce wood chips by using HPLC and capillary zone electrophoresis (CZE). The four ODC-positive fungi secreted oxalic acid both in liquid and solid state cultures showing different production patterns until the end of growth (31 days). Upon cultivation on solid spruce wood chips, manganese peroxidase (MnP) activity peaked simultaneously in these fungi with the accumulation of extracellular oxalic acid. In addition to oxalic acid, glyoxylic and formic acids were detected in the cultures of D. squalens.

Abstract: This study investigates the anaerobic treatability of Reactive Black 5 in an anaerobic/aerobic sequential process Laboratory scale upflow anaerobic sludge blanket (UASB) reactor/completely stirred tank reactors (CSTR) were operated at different organic loadings and hydraulic retention times (HRT) The effects of shock organic loading on the chemical oxygen demand (COD), color removal, and methane gas production efficiencies were investigated in UASB reactor The effects of both sludge retention time (SRT) and food to mass ( F / M ) ratio on the color and COD removal efficiencies were also investigated in aerobic reactor The reactive dye used in this study contains the groups NN, SO 3 , SO and is in use in the some textile industries in Turkey The studies were carried out in continuous mode and the effluent of the UASB reactor was used as feed for the CSTR reactor COD removal efficiencies decreased from 56 to 27% with increases in COD loadings from 5 to 25 kg COD m −3 per day in the anaerobic UASB reactor The color removals were 92 and 87%, respectively, for aforementioned organic loadings The methane percentages were found to be 76 and 60% at organic loading rates of 249 and 148 kg COD m −3 per day, respectively, in UASB reactor COD removal efficiencies of 28, 42, and 90% were obtained at SRTs of 17, 57 and 11 days in the aerobic CSTR reactor Optimum sludge retention time was 11 days in aerobic reactor A total of 67 and 28% COD removal efficiencies were obtained at F / M ratios varying between 005 and 017 and between 030 and 14 kg COD kg −1 MLSS per day A 90–95% color and 40–60% COD removal efficiencies were obtained depending on the applied organic loadings in the UASB reactor The remaining COD was removed with a treatment efficiency of 85–90% in the aerobic CSTR reactor

Abstract: Lipase from Candida rugosa (CRL) has been purified and immobilized by using different immobilization protocols: interfacial adsorption on hydrophobic supports, ionic adsorption on PEI-coated supports, and covalent immobilization (on glutaraldehyde supports). This gave enzyme immobilized with different orientations and microenvironments. The catalytic properties (activity, specificity, and enantioselectivity) of the different derivatives have been found to be dramatically different. Very significant changes on activity with different substrates were found. For example, interfacially adsorbed derivative was the most active using simple substrates (ethyl butyrate) while PEI derivative was the most active hydrolysing ionic substrates (2-phenyl-2-butyroylacetic acid at pH 7) or methyl mandelate. The E value also depends strongly on the derivative and the conditions employed. Thus, the interfacially absorbed enzyme varied its enanatioselectivity (toward S isomer) from 1.6 to 85 in the hydrolysis of ( R , S )-2-phenyl-2-butyroylacetic acid when the pH value varied from 7 to 5. However, the glutaraldehyde derivative presented a high enantioselectivity ( E =400) toward R isomer (the inverse E value compared to the previous derivative) at both pH conditions. Polyethyleneimine (PEI) derivative presented a slight enantiopreference toward the S isomer. Thus, using different derivatives, it has been possible to obtain both pure enantiomers from the ester or the product. Similar changes in the E values were obtained in the hydrolysis of methyl mandelate though here always there was a enantiopreference for the S isomer. Using this substrate, the best derivative was the PEI derivative at pH 5 ( E =300), while the glutaraldehyde one presented an E value of only 10.

Abstract: Amylolytic enzymes (α-amylase and glucoamylase) from Thermomyces lanuginosus ATCC 34626 were purified to electrophoretic homogeneity. The molecular mass of purified α-amylase and glucoamylase were 61 and 75 kDa, respectively. Their p I values were calculated to be 3.5–3.6 and 4.1–4.3. The amylolytic enzymes from T. lanuginosus exhibit pH optima in the range 4.6–6.6 in the case of α-amylase and 4.4–5.6 in the case of glucoamylase. Both purified enzymes have temperature optima at 70 °C. Zn 2+ ions strongly inhibit both enzyme activities. Mn 2+ and Fe 2+ ions are activators in the case of glucoamylase; Ca 2+ and Ba 2+ are activators in the case of α-amylase. With half-life times longer than 1 day at 60 °C both enzymes prove to be thermostable in the pH range 4.5–8.5. The amylolytic enzymes from T . lanuginosus loose activities rapidly when incubated at temperature higher than 80 °C or at pH lower than 4.0. Both enzymes are found to be glycosylated; 8.5% carbohydrate in the case of α-amylase and 3.3% in the case of glucoamylase. The K m and V max of α-amylase on soluble starch were 0.68 mg/ml and 45.19 U/mg, respectively. The K m values of glucoamylase on maltose, maltotriose, maltotetraose, maltopentose and soluble starch were 6.5, 3.5, 2.1, 1.1 mM and 0.8 mg/ml, respectively. The first 37 residues of N-terminal of the purified α-amylase of T. lanuginosus ATCC 34626 were sequenced. Almost complete homology with the α-amylase from Aspergillus oryzae and Emericella nidulans was observed.

Abstract: Properties of tyrosinase (EC 1.14.18.1) from mushroom, immobilized by entrapment in polyacrylamide, alginate and gelatin gels were examined. Maximum activity immobilization yield of 88% was obtained in gelatin followed by 67 and 57% in Cu-alginate and polyacrylamide gels, respectively. The enzyme entrapped in alginate and polyacrylamide gels exhibited broader pH activity profile while in the case of gelatin a shift of pH optimum toward alkaline side, as compared to the soluble enzyme, was observed. The temperature optima for the soluble enzyme was 20°C and it shifted to 35 and 40°C after entrapment of the enzyme in alginate and gelatin gels, respectively. The enzyme embodied in gelatin showed greater storage stability as well as thermal stability at 40°C compared to the other preparations. It was demonstrated that the immobilized enzyme could be used repeatedly, after intermittent storage, for production of L-DOPA (3,4-dihydroxyphenylalanine). Cu-alginate entrapped tryosinase was found to be superior in this regard. The enzyme in gelatin gels retained about 30% of its initial activity after 8 cycles of use. The results indicate a possibility of employing gel entrapped tyrosinase from mushrooms for construction of bioreactors for production of L-DOPA.

Abstract: Biologic decolorization of textile dyestuff Everzol Turquoise Blue G, a phthalocyanine type reactive dyestuff, by white-rot fungi Coriolus versicolor MUCL was studied in a rotating biological contactor (RBC). The effects of different operating parameters such as, disc type, rotational speed (10–40 rpm), glucose (5–10 g/L) and dyestuff concentration (50–500 mg/liter) on the decolorization performance of white rot fungi were investigated. The system was operated in repeated- batch mode with 48 h hydraulic retention time. The environmental conditions were adjusted to T = 28°C and pH = 4.5–5.0. TOC, glucose and dyestuff concentrations were determined throughout the experiments. Three different disc types; plastic, metal mesh covered plastic discs and metal mesh discs were used and the plastic disc was found to be the most suitable one. The highest decolorization efficiency (80%) was obtained with a rotational speed of 30 rpm. Minimum glucose concentration for 77% decolorization efficiency was 5 g/L. Decolorization efficiency was around 80% for 50–200 mg/liter initial dyestuff concentrations and decreased to 33% for 500 mg dyestuff/liter.

Abstract: Oxidoreductases can be applied for bonding of fiberboards, particle boards, paper boards and kraft-liner boards. In this work we report on pilot-scale production of laccase bonded fiberboards made from fibers of beech ( Fagus sylvatica ). Dioxane extractable lignin from fibers and boards are isolated and the molecular mass estimated by gel permeation chromatography. The strength properties of the enzyme bonded boards are comparable to boards bonded by an urea–formaldehyde adhesive, whereas the dimensional stability properties of the enzyme bonded boards are not at the same level. Wax treatment of the fibers, in order to improve dimensional stability of boards, is not compatible with the enzyme treatment. Cross-linking of the lignin can be observed in enzyme treated fibers and boards. Hot pressing of enzyme treated fibers results in a substantial cross-linking of lignin in boards. The enzymatic bonding effect may be caused by covalent bonds between fibers or an adhesive effect of polymerized loosely associated lignin. Laccase catalyzed bonding requires higher pressing temperatures and longer pressing times, and the concept may not be economically feasible as it is. However, it shows promise and possibilities in the use of oxidative enzymes for industrial bonding and modification of lignin.

Abstract: The efficiency of different preparations of lipases was evaluated in organic solvents. Lipases from Humicola lanuginosa, Candida rugosa, Rhizomucor miehei and Pseudomonas cepacia were adsorbed onto the surfactant sorbitan monostearate (Span 60) and the specific activities were compared in hexane to crude powder (used straight from the bottle) and lipase freeze-dried from buffer solution. Lipases adsorbed on the surfactant were "activated" 1.9- to 150-fold compared to the crude lipase. The solubility of the lipase-surfactant preparation in the reaction media was extremely low and the preparation contained aggregates of micrometer size. In further comparisons lipase from H. lanuginosa was freeze-dried in the presence of KCl, crown ethers, immobilised by entrapment into a sol-gel and immobilised on porous polypropylene support (Accurel EP-100). Addition of potassium chloride before freeze-drying of the lipase increased the activity up to 46-fold compared to crude powder. The additive probably worked as an immobilisation matrix for the lipase. When 18-crown-6 was added to the lipase before freeze-drying a 40-fold increase in activity was achieved. In this case a low amount of additive (0.4 mg crown ether/g protein) was needed for activation indicating that specific interactions were involved in the activation. In order to obtain maximal activity, immobilisation on Accurel EP-100 and entrapment into a sol-gel were the best methods to use. The activities were 400- and 320-fold better than that of crude powder. The high activities obtained were due to an improved dispersion of the catalyst in the organic media. The protein loading that could be used when the lipase was adsorbed onto Accurel EP-100 was much higher than what could be used when the lipase was entrapped into a sol-gel. This makes the adsorption technique the best for practical applications. (C) 2002 Elsevier Science Inc. All rights reserved. (Less)

Abstract: There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enatioselective manner. Chiral intermediates and fine chemicals are in high demand both from pharmaceutical and agrochemical industries for the preparation bulk drug substances and agricultural products. In this review article, microbial/enzymatic processes have been described for the synthesis of chiral intermediates for anticancer drugs, antiviral agents, β3-receptor agonists, antihypertensive drugs, melatonin receptor agonists, anticholesterol drugs, and anti-Alzheimer’s drugs.

Abstract: The enzyme complement of a selection of commercial food-grade peptidase and lipase preparations was investigated. Each preparation was assayed for protein content, proteinase activity at pH 5.5 and 7.0 at 37 °C using azocasein and semi-quantitatively assayed for lipase, peptidase, proteinase, phosphatase and glycosidase activity by the API-ZYM system. Each peptidase preparation was also assayed for various endo-, carboxy-, amino- and di-peptidase activities at pH 5.5 and 7.0 at 37 °C, using chromogenic or fluorogenic substrates, while each lipase preparation was assayed for esterase and lipase activity at pH 7.0 at 37 °C using p-nitrophenol substrates. All enzyme preparations were found to contain enzyme activities in addition to their stated main activity. According to the API-ZYM system the peptidase preparations contained varying levels of lipase, proteinase, peptidase, phosphatase and glycosidase activity, with the lipase preparations containing lipase, phosphatase and glycosidase activity. Only two peptidase and two lipase preparations contained significant amounts of proteinase activity as measured by azocasein. The peptidase and lipase activities of the preparations appeared to be dependent upon source. Most peptidase preparations had significantly more activity at pH 7.0 than at 5.5.

Abstract: Bioaccumulation of the reactive textile dyes, Remazol Blue, Reactive Black and Reactive Red, by the yeast species Candida tropicalis growing in molasses medium was studied in a batch system as a function of initial pH and initial dye concentration. The optimum pH value for bioaccumulation was determined as 3.0 for all the dyes tested. The maximum specific bioaccumulation capacity of C. tropicalis was 111.9 mg g−1 for Remazol Blue, 101.9 mg g−1 for Reactive Black and 79.3 mg g−1 for Reactive Red at approximately 700 mg l−1 initial dye concentration. Higher bioaccumulation percentages were observed at lower concentrations of all the dyes. In general, the increase in dye concentration inhibited the growth of yeast and caused a long lag period. Remazol Blue dye gave a considerably higher dye bioaccumulation percentage by the yeast among the dyes tested.

Abstract: Response surface methodology (RSM) was employed to study the effect of culture medium on the production of lovastatin in mixed solid-liquid state (or submerged) cultures by Monascus ruber. The maximal lovastatin yield (131 mg/L, average of three repeats) appeared at the region where the respective concentrations of rice powder, peptone, glycerin, and glucose were around 34.4 g/L, 10.8 g/L, 26.4 ml/L, and 129.2 g/L, respectively. The optimized medium resulted in a significant increase of lovastatin yield, as compared with that obtained by the fermentation of many other M. ruber species.

Abstract: A new wood-degrading fungus Trichophyton rubrum LKY-7 secretes a high level of laccase in a glucose-peptone liquid medium. The production of laccase by the fungus was barely induced by 2,5-xylidine. The laccase has been purified to homogeneity through three chromatography steps in an overall yield of 40%. The molecular mass of the purified laccase was about 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE). The purified laccase had the distinct blue color and the basic spectroscopic features of a typical blue laccase: two absorption maxima at 278 and 610 nm and a shoulder at 338 nm. The N-terminus of the laccase has been sequenced, revealing high homology to laccases from wood-degrading white-rot fungi such as Ceriporiopsis subvermispora, but little similarity to laccases from non-wood-degrading fungi such as Agaricus bisporus and Cryptococcus neoformans. The enzyme had a low redox potential of ∼0.5 V, yet it was one of the most active laccases in oxidizing a series of representative substrates/mediators. Compared with other fungal laccases, the laccase has a very low Km value with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a substrate and a very high Km value with violuric acid as a substrate. The laccase had the isoelectric point of 4.0. The laccase had very acidic optimal pH values (3–4), although it was more stable at neutral pH than at acidic pH. The laccase oxidized hydroquinone faster than catechol and pyrogallol. The oxidation of tyrosine by the laccase was not detectable under the reaction conditions. The laccase was strongly inhibited by sodium azide and sodium fluoride.

Abstract: New extracellular endoglucanases, designated RCE1 and RCE2, produced by Rhizopus oryzae isolated from the soil, were purified to apparent homogeneity from the culture supernatant. The molecular mass of RCE1 and that of RCE2 were found to be 41 kDa and 61 kDa, respectively. The N-terminal amino acid sequences of RCE1 and RCE2 showed high homology with those of the family I cellulose-binding domains. Internal amino acid sequences of RCE1 and RCE2 showed homology with that of the catalytic domain of EGV from Humicola insolens belonging to family 45 endoglucanase. The cellooligosaccharide hydrolysis patterns of RCE1 and RCE2 were similar to that of EGV from H. insolens. These results indicate that RCE1 and RCE2 are family 45 endoglucanases having a cellulose binding domain at their N-terminus. RCE1 and RCE2 hydrolyzed carboxymethylcellulose (CMC), insoluble cellooligosaccharide (G33), cellohexaose, and cellopenpaose, but not Avicel, xylan, galactan, arabinan, mannan, or laminarin. The CMCase activity of both enzymes was inhibited by Cu 2+ , Zn 2+ , Co 2+ , and Pb 2+ . The optimum pH for the CMCase activity of both enzymes was found to be between pH value 5.0 and 6.0, and the optimum temperature was 55°C, the lowest among the family 45 endoglucanases. These results indicate that RCE1 and RCE2 represent a new type of endoglucanases having the lowest optimum temperature among the family 45 endoglucanases.

Abstract: An examination of the rheological properties of activated sludges from three sewage treatment works and a digested sludge from a dual mesophilic-thermophilic anaerobic digester (TAD-MAD) showed that all the sludges exhibited yield stresses the magnitude of which varied with the suspended solids’ concentration and the type of treatment which they had received. In particular, the TAD-MAD sludge had a yield stress which was an order of magnitude lower than the activated sludges at comparable solids’ concentrations. Treatment of the sludges with ultrasound reduced the values of the yield stress and this effect was irreversible. The surface charge carried by the sludge particles and the bound water of the sludge were both found to be related to the yield stress. The bound water content of the activated sludge samples was related to the filtration characteristics, as measured by the capillary suction time method. However, the TAD-MAD sludges were found to have poor filtration characteristics. Microscopic examinations showed that the TAD-MAD sludges contained a predominance of small particles (