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AIM: To evaluate the technological and safety properties of Lactobacillus futsaii CS3 and CS5 isolated from Thai fermented shrimp products (Kung-Som) in order to develop a valuable gamma-aminobutyric acid (GABA)-producing starter culture. METHODS AND RESULTS: Both strains showed a high GABA-producing ability (>8 mg ml(-1) ) in MRS broth containing 20 mg ml(-1) monosodium glutamate (MSG) for 120 h. They also exhibited inhibitory activity against foodborne pathogens and spoilage bacteria. Cell surface hydrophobicity and proteolytic activity were observed in both strains. Strain CS3 survived better under simulated gastrointestinal tract conditions with only 1·5 log-units cell decrease over 8 h. Both strains showed the ability to deconjugate taurocholate and taurodeoxycholate acid. Neither virulence genes nor biogenic amine production was detected. Strain CS3 exhibited susceptibility to all tested antibiotics with the exception of vancomycin, while strain CS5 showed resistance to vancomycin, ampicillin and chloramphenicol. CONCLUSIONS: Based on the results obtained, Lact. futsaii CS3 is very promising as a GABA-producing and potentially probiotic starter culture strain for applications in functional fermented foods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study focuses on the technological and safety characteristics of Lact. futsaii CS3 and CS5 including their high GABA-producing capacity for the first time. This provides a way of replacing chemical GABA by natural GABA using a GABA-producing starter culture candidate, at the same time offering the consumer new attractive food products.

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Cellobiose dehydrogenase from the ascomycete fungus Myriococcum thermophilum (MtCDH) was tested for the ability to generate bleaching species at a pH suitable for liquid detergents. The catalytic properties of MtCDH were investigated for a large variety of carbohydrate substrates using oxygen as an electron receptor. MtCDH produces H2O2 with all substrates tested (except fructose) but only in the presence of a chelant. Insoluble substrates like cellulose and cotton could as well be oxidized by MtCDH. To enhance the amount of cello-oligosaccharides in solution, different cellulases on cotton were used and in combination with MtCDH an increased H2O2 concentration could be measured. Additionally, the degradation of pure anthocyanins in solution (as model substrates for bleaching) was investigated in the absence and presence of a horseradish peroxidase. MtCDH was able to produce a sufficient amount of H2O2 to decolorize the anthocyanins within 2 h.

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Cellobiose dehydrogenase, the only currently known extracellular flavocytochrome, is formed not only by a number of wood-degrading but also by various phytopathogenic fungi. This inducible enzyme participates in early events of lignocellulose degradation, as investigated in several basidiomycete fungi at the transcriptional and translational level. However, its role in the ascomycete fungi is not yet obvious. Comprehensive sequence analysis of CDH-encoding genes and their translational products reveals significant sequence similarities along the entire sequences and also a common domain architecture. All known cellobiose dehydrogenases fall into two related subgroups. Class-I members are represented by sequences from basidiomycetes whereas class-II comprises longer, more complex sequences from ascomycete fungi. Cellobiose dehydrogenase is typically a monomeric protein consisting of two domains joined by a protease-sensitive linker region. Each larger (dehydrogenase) domain is flavin-associated while the smaller (cytochrome) domains are haem-binding. The latter shorter domains are unique sequence motifs for all currently known flavocytochromes. Each cytochrome domain of CDH can bind a single haem b as prosthetic group. The larger dehydrogenase domain belongs to the glucose-methanol-choline (GMC) oxidoreductase superfamily - a widespread flavoprotein evolutionary line. The larger domains can be further divided into a flavin-binding subdomain and a substrate-binding subdomain. In addition, the class-II (but not class-I) proteins can possess a short cellulose-binding module of type 1 at their C-termini. All the cellobiose dehydrogenases oxidise cellobiose, cellodextrins, and lactose to the corresponding lactones using a wide spectrum of different electron acceptors. Their flexible specificity serves as a base for the development of possible biotechnological applications.

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The extracellular cellobiose dehydrogenase (CDH) obtained from Chaetomium sp. INBI 2-26(-) has a molecular mass of 95 kDa and an isoelectric point of 5. This novel CDH is highly specific for the oxidation of cellobiose (K(m,app) 4.5 microM) and lactose (K(m,app) 56 microM). With 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) (cyt c(3+)) as electron acceptors, CDH was most active at pH 6. The turnover number of the enzyme for cellobiose, lactose, DCIP and cyt c(3+) was in the range of 9-14s(-1) at 20 degrees C and pH 6. The UV-visible spectrum revealed the flavohemoprotein nature of the enzyme. The cytochrome b domain of the enzyme was reduced by ascorbate, dithionite, as well as specifically by cellobiose in a wide range of pH. The apparent first order rate constants of the spontaneous re-oxidation of the reduced heme domain were estimated as 0.01 and 0.00039 s(-1) at pH 4.5 and 6.5, respectively. The half-inactivation time of CDH at pH 6 and 55 degrees C was ca. 100 min; the stability at pH 8 and, particularly, pH 4 was remarkably lower. Cellobiose stabilized the enzyme against thermal inactivation, whereas DCIP in turn sensitized the enzyme. The new enzyme revealed low affinity for crystalline cellulose, but was capable of binding onto H(3)PO(4)-swollen filter paper. The results show significant differences to already known CDHs and perspectives for several biotechnological applications, where CDH with maximal activity at neutral pH and high affinity for cellobiose and lactose night have some advantages.

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Cellobiose dehydrogenase (CDH) is an extracellular haemoflavoenzyme that is produced by a number of wood-degrading and phytopathogenic fungi and it has a proposed role in the early events of lignocellulose degradation and wood colonisation. In the presence of a suitable electron acceptor, e.g. 2,6-dichloro-indophenol, cytochrome c, or metal ions, CDH oxidises cellobiose to cellobionolactone. When screening 11 different Trametes spp. for the formation of CDH activity, all the strains investigated were found to secrete significant amounts of CDH when cultivated on a cellulose-containing medium. Amongst others, Trametes pubescens and Trametes villosa were identified as excellent, not-yet-described, producer strains of this enzyme activity that has various potential applications in biotechnology. CDH from both strains was purified to apparent homogeneity and subsequently characterised. Both monomeric enzymes have a molecular mass of approximately 90 kDa (gel filtration) and a pI value of 4.2-4.4. The best substrates are cellobiose and cellooligosaccharides; additionally, lactose, thiocellobiose, and xylobiose are efficiently oxidised. Glucose and maltose are poor substrates. The preferred substrate is cellobiose with a Km value of 0.21 mM and a kcat value of 22 s(-1) for CDH from T. pubescens; the corresponding values for the T. villosa enzyme are 0.21 mM and 24 s(-1), respectively. Both enzymes showed very high activity with one-electron acceptors such as ferricenium, ferricyanide, or the azino-bis-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical.

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The phytopathogenic fungus Sclerotium (Athelia) rolfsii CBS 191.62 is a very efficient producer of the hemoflavoprotein, cellobiose dehydrogenase (CDH), forming up to 225 mg l(-1) (15,000 units cytochrome c activity l(-1)) of this protein, which is of biotechnological interest for sensors, biocatalysis and bioremediation. Both cellulose as inducing substrate and the use of a rich medium containing increased concentrations of peptone from meat or suitable amino acids are important for attaining high CDH yields. CDH, containing a protease-sensitive linker region, can be cleaved by endogenous proteases into a catalytically active flavin fragment and an inactive heme domain. By using increased concentrations of peptone, or certain amino acids such as valine or leucine, or by adding exogenous protease inhibitors, this cleavage can be almost completely inhibited, so that more than 95% intact CDH is obtained under optimised culture conditions. When using non-inhibitory amino acids, e.g. glutamine or lysine, in the medium, more than 80% of the total cellobiose-oxidising activity can be attributed to the flavin fragment.

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AIMS: Evaluation of various Sclerotium spp. as producers of the biotechnologically attractive enzyme cellobiose dehydrogenase. METHODS AND RESULTS: All isolates of S. coffeicola, S. delphinii and S. rolfsii grown in shaken flasks on a cellulose-based medium produced appreciable amounts of the extracellular enzyme cellobiose dehydrogenase. CONCLUSIONS: Cellobiose dehydrogenase seems to play an important role in phytopathogenic Sclerotium spp.; a possible function could be either in the degradation of rigid lignocellulose or as a protective mechanism against toxic quinones. SIGNIFICANCE AND IMPACT OF THE STUDY: S. coffeicola and S. delphinii were identified as potent, not-yet-described producers of cellobiose dehydrogenase (CDH). The high levels of intact CDH produced by the different Sclerotium species should make them attractive producers for further studies and applications.

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The white-rot fungus Trametes pubescens MB 89 has been identified as an outstanding, although not-yet-described, producer of the industrially important enzyme laccase. Extracellular laccase formation could be greatly stimulated by the addition of Cu(II) to a simple, glucose-based culture medium. Using optimum Cu concentrations (1.5-2.0 mM), maximum values for laccase activity of approximately 65 U/ml were obtained. The synthesis of the laccase protein depended on the presence of Cu in the medium as shown by Western blot analysis. Copper had to be supplemented during the exponential phase of growth for its maximal effect; addition during the stationary phase, during which laccase activity is predominantly formed, resulted in markedly reduced laccase productivity. As was shown by X-ray microanalysis of T pubescens mycelia obtained from a laboratory fermentation, Cu was rapidly taken up by the fungal biomass. A possible explanation for this stimulatory effect of Cu on laccase biosynthesis could be a role for this enzyme activity in melanin synthesis. The stimulatory effect of Cu on laccase synthesis was also effective for several other basidiomycetes and hence could be used as a simple method to boost the production of this enzyme.

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Mycelial cultures of 76 strains of lignocellulose-degrading basidiomycete fungi were screened for the activity of pyranose dehydrogenase, a novel sugar oxidoreductase recently detected in Agaricus bisporus. Of these fungi, 37 strains belonging to seven phylogenetically related genera of mostly litter-decomposing Agaricales were positive for the dehydrogenase, based on activity assays towards D-glucose with 1,4-benzoquinone or ferricenium ion as electron acceptors, and on TLC/HPLC analyses of the reaction products. Lack of activity with O(2) as the oxidant, specificity for C-3 of D-glucose, and active extracellular secretion of the enzyme were used as criteria to differentiate pyranose dehydrogenase from pyranose 2-oxidase (EC 1.1.3.10), known to be produced by numerous wood-rotting fungi. Extracellular pyranose dehydrogenase from Macrolepiota rhacodes was heavily glycosylated. The enzyme was characterized as a 78-kDa flavoprotein under denaturing conditions and a 76-kDa native protein using gel filtration. This enzyme had a maximum extracellular activity of 4.1 U ml(-1) in 39-day liquid cultures. It exhibited broad selectivity for sugar substrates and oxidized D-glucose (K(m)=1.82) exclusively at C-3 to 3-dehydro-D-glucose (D-ribo-hexos-3-ulose), in contrast to pyranose dehydrogenases from Agaricus species, which acted at both C-3 and C-2 of D-glucose. The N-terminal sequence, AVVYRHPDEL, showed significant similarity with that reported for A. bisporus.

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We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are D-glucose, D-xylose, and L-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (k(cat)/K(m)), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H(2)O(2). An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.

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Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochrome c, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungus Sclerotium rolfsii (teleomorph: Athelia rolfsii) strain CBS 191.62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH from wood-rotting fungi, the intact, monomeric enzyme from S. rolfsii contains one heme b and one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pI value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, beta-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochrome c (equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical were the best electron acceptors, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Meldola's blue, and ferricyanide were also excellent acceptors. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochrome c. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.

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Pyranose dehydrogenase purified to homogeneity from the mycelia of the basidiomycete fungus Agaricus bisporus catalyzed the oxidation of D-xylose at C-2 to D-threo-pentos-2-ulose (2-keto-D-xylose) and successively at C-3 to D-glycero-pentos-2,3-diulose (2,3-diketo-D-xylose) using 1,4-benzoquinone as an electron acceptor. The sites of oxidation were deduced from the spectroscopic analysis (MS, NMR) of the N,N-diphenylhydrazone derivatives of the reaction products.

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Different mannanase preparations obtained from the filamentous fungus Sclerotium rolfsii were used for the hydrolysis of coffee mannan, thus reducing significantly the viscosity of coffee extracts. Mannan is the main polysaccharide component of these extracts and is responsible for their high viscosity, which negatively affects the technological processing of instant coffee. Coffee mannan was isolated from green defatted Arabica beans by delignification, acid wash and subsequent alkali extraction with a yield of 12.8%. Additionally, coffee extract polysaccharides were separated by alcohol precipitation and were found to form nearly half of the coffee extract dry weight. These isolated mannans as well as the mannan in the coffee extract were efficiently hydrolysed by the S. rolfsii mannanase, which resulted in significant viscosity reductions. Concurrently, the reducing sugar content increased continuously due to the release of various mannooligosaccharides including mannotetraose, mannotriose, and mannobiose. Both a partially purified, immobilised and a soluble, crude mannanase preparation were successfully employed for the degradation of coffee mannan.

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Recombinant beta-glycosidases from hyperthermophilic Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) have been characterized with regard to their potential use in lactose hydrolysis at about 70 degrees C or greater. Compared with SsbetaGly, CelB is approximately 15 times more stable against irreversible denaturation by heat, its operational half-life time at 80 degrees C and pH 5.5 being 22 days. The stability of CelB but not that of SsbetaGly is decreased 4-fold in the presence of 200 mM lactose at 80 degrees C. CelB displays a broader pH/activity profile than SsbetaGly, retaining at least 60% enzyme activity between pH 4 and 7. Both enzymes have a similar activation energy for lactose hydrolysis of approximately 75 kJ/mol (pH 5.5), and this is constant between 30 and 95 degrees C. D-Galactose is a weak competitive inhibitor against the release of D-glucose from lactose (Ki approximately 0.3 M), and at 80 degrees C the ratio of Ki, D-galactose to Km,lactose is 2.5 and 4.0 for CelB and SsbetaGly, respectively. SsbetaGly is activated up to 2-fold in the presence of D-glucose with respect to the maximum rate of glycosidic bond cleavage, measured with o-nitrophenyl beta-D-galactoside as the substrate. By contrast, CelB is competitively inhibited by D-glucose and has a Ki of 76 mM. The transfer of the galactosyl group from lactose to acceptors such as lactose or D-glucose rather than water is significant for both enzymes and depends on the initial lactose concentration as well as the time-dependent substrate/product ratio during batchwise lactose conversion. It is approximately 1.8 times higher for SsbetaGly, compared with CelB. Overall, CelB and SsbetaGly share their catalytic properties with much less thermostable beta-glycosidases and thus seem very suitable for lactose hydrolysis at >/=70 degrees C.

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The commonly used assay for measuring cellobiose dehydrogenase (CDH) activity, based on the reduction of dichlorophenol-indophenol (DCIP), has been adapted to measure this enzyme activity in the presence of laccase, which is often formed concurrently with CDH by a number of fungi. Laccase interferes with the assay by rapidly reoxidizing the reduced form of DCIP and can mask CDH activity completely. It can be conveniently and completely inhibited by 4 mM fluoride in the assay, while CDH activity is only slightly affected by the addition of this inhibitor. The modified assay enables the detection of low CDH activities even in the presence of very high excesses of laccase. It should be useful for screening culture supernatants of wood-degrading fungi for CDH since the assay is rapid and uses inexpensive and nontoxic reagents. Furthermore, it might be used for the detection of other enzyme activities which are assayed by following the reduction of quinones or analogue compounds such as DCIP.

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The NAD+-dependent xylitol dehydrogenase from the xylose-assimilating yeast Galactocandida mastotermitis has been purified in high yield (80%) and characterized. Xylitol dehydrogenase is a heteronuclear multimetal protein that forms homotetramers and contains 1 mol of Zn2+ ions and 6 mol of Mg2+ ions per mol of 37.4 kDa protomer. Treatment with chelating agents such as EDTA results in the removal of the Zn2+ ions with a concomitant loss of enzyme activity. The Mg2+ ions are not essential for activity and are removed by chelation or extensive dialysis without affecting the stability of the enzyme. Results of initial velocity studies at steady state for d-sorbitol oxidation and d-fructose reduction together with the characteristic patterns of product inhibition point to a compulsorily ordered Theorell-Chance mechanism of xylitol dehydrogenase in which coenzyme binds first and leaves last. At pH 7.5, the binding of NADH (Ki approximately 10 microM) is approx. 80-fold tighter than that of NAD+. Polyhydroxyalcohols require at least five carbon atoms to be good substrates of xylitol dehydrogenase, and the C-2 (S), C-3 (R) and C-4 (R) configuration is preferred. Therefore xylitol dehydrogenase shares structural and functional properties with medium-chain sorbitol dehydrogenases.

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Tetrameric, NADP-containing glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis catalyzes the oxidation of glucose into glucono-delta-lactone coupled to the reduction of fructose to sorbitol. GFOR is inactivated during substrate turnover in vitro, the long-term stability of the enzyme during conversions in biochemical reactors thereby being drastically reduced. The process of inactivation is triggered by structural transitions that are induced by the lactone product and involves aggregation as the ultimate cause of irreversible inactivation. Guanidinium hydrochloride-induced changes in the conformation of GFOR seem to be similar to those observed in the presence of lactone, and are manifested by incubation time-dependent increases in protein fluorescence and the solvent-exposed hydrophobic surface. The formation of high-order protein associates in solution in the presence of this denaturant proceeds from the native tetramer to a reversibly inactivated octamer and then to a dodecameric form that cannot be reactivated through spontaneous or assisted refolding. Therefore, stabilization of GFOR during turnover requires that the marked tendency of the enzyme to form aggregates is prevented efficiently. This goal has been accomplished in the presence of low urea concentrations (1.0 M), which led to a 10-fold increase in the half-life of GFOR under operational conditions.

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During a screening for the enzyme pyranose 2-oxidase (P2O) which has a great potential as a biocatalyst for carbohydrate transformations, Trametes multicolor was identified as a promising, not-yet-described producer of this particular enzyme activity. Furthermore, it was found in this screening that the enzyme frequently occurs in basidiomycetes. Intracellular P2O was produced in a growth-associated manner by T. multicolor during growth on various substrates, including mono-, oligo-, and polysaccharides. Highest levels of this enzyme activity were formed when lactose or whey were used as substrates. Peptones from casein and other casein hydrolysates were found to be the most favorable nitrogen sources for the formation of P2O. By applying an appropriate feeding strategy for the substrate lactose, which ensured an elevated concentration of the carbon source during the entire cultivation, levels of P2O activity obtained in laboratory fermentations, as well as the productivity of these bioprocess experiments, could be enhanced more than 2.5-fold.

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