Relationship between production of 3-indoleacetic acid and peroxidase-laccase activities depending on the culture periods in Funalia trogii (Trametes trogii)

The relationship between production of 3-indoleacetic acid (IAA) and peroxidase and laccase activity was investigated in white-rot fungusFunalia trogii (Trametes trogii). F. trogii produced IAA and peroxidase and laccase as both primary metabolite and secondary metabolite; the levels of IAA may be influenced by peroxidase and laccase. A correlation exists between the levels of IAA and peroxidase-laccase activity.     

Highly stable laccase from repeated-batch culture of Funalia trogii ATCC 200800

The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55–90°C, which was maximal at 80–95°C. It was highly stable within the temperature intervals 20–50°C. The half life of the enzyme was about 2 h and 5 min at 60°C and 70°C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0–9.0. NaN₃ and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.     

硬毛粗盖孔菌过氧化氢酶家族基因鉴定及表达分析

硬毛粗盖孔菌是一种结实能力强的耐热真菌,为了探究其过氧化氢酶(CAT)基因家族的基本特征和功能,分别对不同交配型单核体Ct001_29和Ct001_31基因组的CAT基因家族进行了鉴定,并分析了不同CAT家族基因在不同温度条件下培养的菌丝、原基和子实体中的表达特征。硬毛粗盖孔菌基因组上有3个CAT基因(Ctcat1-Ctcat3),编码510-743个氨基酸;Ctcat1、Ctcat2和Ctcat3的等位基因之间的结构及序列相对保守,但编码区也存在少量SNP变异(6-14个)。在25 ℃培养菌丝和35 ℃培养菌丝、原基及子实体中,35 ℃菌丝的CAT酶活最高,为278 U/mg蛋白,原基期CAT酶活最低,为4 U/mg蛋白。Ctcat2的表达丰度显著高于Ctcat1和Ctcat3,Ctcat1和Ctcat3具有相似的表达模式,在35 ℃菌丝、原基和子实体中均上调表达;而Ctcat2具有相反的表达模式,在35 ℃菌丝、原基和子实体中均下调表达。此外,Ctcat2在原基时期、Ctcat3在25 ℃菌丝和35 ℃菌丝中具有偏好表达,Ct29cat2和Ct29cat3的表达量均高于各自的等位基因。本研究所发现的CAT等位基因表达偏好为进一步揭示大型真菌CAT的基因功能奠定了基础。     

Decolourisation of the textile dye Astrazon Red FBL by Funalia trogii pellets.

The effects of various conditions such as initial pH, dye concentrations, amount of pellet, temperature and agitation on decolourising activity of Funalia trogii were investigated. These, except initial pH, were all found to be important for dye decolourising activity of F. trogii. The decolourisation of the dye involved adsorption of the dye compound by fungal pellets at the initial stage, followed by the decolourisation through microbial metabolism. Heat-killed pellets were also tested for their ability to decolourise Astrazon Red dye. These pellets adsorbed the dye and 55% decolourisation was obtained in 24 h. But at the second cycle there was only 24% decolourisation. Our observation showed that Astrazon Red dye decolourisation by heat-killed pellets was mainly due to biosorption. The longevity of the decolourisation activity of F. trogii pellets was also investigated in repeated batch mode. Variations in the amount of pellet increased % decolourisation and stability of pellets.     

硬毛粗盖孔菌子实体提取物抑制H_(22)荷瘤小鼠肿瘤活性研究

本文对硬毛粗盖孔菌子实体不同提取物的抗肿瘤活性进行筛选。通过建立H_(22)荷瘤小鼠肿瘤移植模型,研究硬毛粗盖孔菌子实体的石油醚提取物、二氯甲烷提取物、乙酸乙酯提取物、甲醇提取物以及水提取物等5种提取物的高、中、低剂量组对H_(22)荷瘤小鼠的抑瘤作用,并对其H_(22)荷瘤小鼠的抑瘤率、体质量、胸腺指数、脾脏指数、肝脏指数、肾脏指数进行了考察。对小鼠肿瘤、脾脏、肾脏进行了组织病理学检查,对血清中的细胞因子IL‐2、IL‐4、IFN‐γ及TNF‐α的含量进行测定。结果表明:各组分均有抑瘤效果,其中乙酸乙酯提取物中剂量组(1 000mg/kg)抑瘤效果最佳,与阴性对照组比较有极显著差异(P0.01),并且这组的H_(22)荷瘤小鼠血清中白介素‐2的含量显著增加,同时肿瘤细胞通过HE染色后,可观察到出现坏死面积增大,与抑制肿瘤活性具有相关性。     

Effect of cold storage on tissue and cellular glutathione

One of the mechanisms thought to cause injury in preserved organs is the formation of oxygen free radicals. The cell is protected from oxidative stress by many defense mechanisms. A major defense mechanism involves glutathione and glutathione-dependent enzymes. During organ preservation by simple cold storage the loss of glutathione may sensitize the organ to free radical damage after transplantation. In this study we show that glutathione is depleted from the rabbit liver, kidney, and heart cold-stored (5 degrees C) for up to 72 h in the UW solution without glutathione. In the first 24 h kidney glutathione decreased to 84 +/- 3% of control values, liver glutathione decreased to 49 +/- 3% of control values, and heart glutathione decreased to 73 +/- 3% of control values. After 48 h of storage the kidney and liver lost an additional 30 and 20%, respectively, whereas heart glutathione changed very little. By 72 h all three organs had lost more than 50% of the glutathione found in freshly obtained tissue. To determine if glutathione added to the UW solution can effectively prevent this loss of glutathione during preservation, hepatocytes were cold-stored for up to 72 h in a preservation solution with and without glutathione. We found that adding glutathione to the preservation solution slowed the rate of loss of glutathione from the cells. These data suggest that at hypothermia the cell may be permeable to GSH. Methods to suppress the loss of glutathione during preservation of organs may be an important factor in suppressing oxygen free radical injury.     

Effect of S-nitrosothiols on cellular glutathione and reactive protein sulfhydryls

S-Nitrosothiols may cause many of the biological effects of NO and cellular effects have been attributed to S-nitrosylation of reactive protein sulfhydryls. This report examines the effect of S-nitrosothiols on the low-molecular-weight thiols and protein thiols in NIH/3T3 cells. A low concentration of S-nitrosocysteine increased the cysteine content of the cells, with no evidence of either low-molecular-weight thiol or protein S-nitrosylation. Millimolar amounts of S-nitrosocysteine produced S-nitrosoglutathione (GSNO), cysteinyl glutathione, cysteine, and glutathione disulfide. Large amounts of protein S-nitrosylation and lesser amounts of protein S-glutathiolation and S-cysteylation were also observed. GSNO and S-nitroso-N-acetylpenicillamine (SNAP) were much less effective than S-nitrosocysteine, but a combination of cysteine and GSNO produced S-nitrosocysteine-like effects. In cultured hepatocytes, millimolar S-nitrosocysteine was significantly less effective since the cells contained three times more glutathione than NIH/3T3 cells. Results suggest that S-nitrosocysteine enters cells intact, and low concentrations do not significantly increase cellular pools of S-nitrosothiol or S-nitrosylated protein. Millimolar concentrations of S-nitrosocysteine generate S-nitrosylated, S-glutathiolated, and S-cysteylated proteins, as well as a variety of low-molecular-weight disulfides and S-nitrosothiols.     

Biosorption of Copper(II) by Live and Dried Biomass of the White Rot Fungi Phanerochaete chrysosporium and Funalia trogii

Biosorption is an innovative and alternative technology to remove heavy metal pollutants from aqueous solution using live, inactive and dead biomasses such as algae, bacteria and fungi. In this study, live and dried biomass of Phanerochaete chrysosporium and Funalia trogii was applied as heavy metal adsorbent material. Biosorption of copper(II) cations in aqueous solution by live and dried biomass of Phanerochaete chrysosporium and Funalia trogii was investigated to study the effects of initial heavy metal concentration, pH, temperature, contact time, agitation rate and amount of fungus. Copper(II) was taken up quickly by fungal biomass (live or dried) during the first 15 min and the most important factor which affected the copper adsorption by live and dried biomass was the pH value. An initial pH of around 5.0 allowed for an optimum adsorption performance. Live biomass of two white rot fungi showed a high copper adsorption capacity compared with dried biomass. Copper(II) uptake was found to be independent of temperature in the range of 20–45 °C. The initial metal ion concentration (10–300 mg/L) significantly influenced the biosorption capacity of these fungi. The results indicate that a biosorption as high as 40–60 % by live and dried biomass can be obtained under optimum conditions.     

Effect of endotoxin on the rat colon glutathione level

The effect of endotoxin on the colon glutathione level was studied in male rats. Endotoxin (Escherichia coli) from 25 ug to 100 ug/100g body weight was administered intravenously. The Glutathione level was measured 16 hours after endotoxin was given. Results showed that endotoxin significantly enhanced the colon glutathione concentration as measured by 5,5'-dithiobis (2-nitrobenzoic acid). The increase which ranged from 11% to 50% was dose dependent. At an endotoxin dose of 1000 ug/100g body weight, colon glutathione level was found to be enhanced from 2 hours up to 48 hours. In contrast, the duodenum and jejunum glutathione levels were found to be significantly reduced. The increase in the colon glutathione level may have a protective effect against oxidative damage to the colon.     

Effect of pineal indoles on activities of the antioxidant defense enzymes superoxide dismutase, catalase, and glutathione reductase, and levels of reduced and oxidized glutathione in rat tissues.

Male Sprague-Dawley rats were randomly divided into four groups. Two of the groups received a single intraperitoneal injection of melatonin and 5-methoxytryptamine (5 mg/kg body weight), respectively, at 9 PM. One group received an intraperitoneal injection of 5-methoxytryptophol (5 mg/kg body weight) at 9 AM. The remaining group received alcoholic saline (vehicle) and served as the control. All rats were sacrificed 90 min after injection and the livers, kidneys, and brains were dissected. The activities of superoxide dismutase, catalase, and glutathione reductase in the organs were measured. It was found that both melatonin and 5-methoxytryptamine were approximately equipotent in enhancing the activities of superoxide dismutase and glutathione reductase in the kidney and liver, while 5-methoxytryptophol displayed a weaker effect. Both melatonin and 5-methoxytryptamine augmented the level of reduced glutathione in the kidney and liver, while 5-methoxytryptophol did so only in the kidney. All three pineal indoles increased the activity of superoxide dismutase and lowered the ratio of oxidized to reduced glutathione in the brain.     

Influence of growth conditions on the production of xylanolytic enzymes by Trametes trogii

Various nitrogen and carbon sources were examined as inducers of the production of endoxylanase and -xylosidase by Trametes trogii. T. trogii grown on xylan plus crystalline cellulose provided supernatants with the highest enzymatic activities. Organic nitrogen sources (especially asparagine and casamino acids) were the best for enzyme production. The increase in xylan concentration stimulated endoxylanase production whereas significant differences were not attained in -xylosidase production with more than 5g xylan/l in the culture medium. pH 4.0 was optimal for endoxylanase production, while -xylosidase production was maximum at pH 5.5. Temperatures in the range of 23–28°C stimulated enzyme production. The endoxylanase activity in the crude culture filtrate was greatest at 50°C and pH around 5.0. The optimum pH and temperature for -xylosidase activity were 5.5 and 50°C respectively.     

Adsorption with biodegradation for decolorization of reactive black 5 by Funalia trogii 200800 on a fly ash-chitosan medium in a fluidized bed bioreactor-kinetic model and reactor performance

A non-steady-state mathematical model system for the kinetics of adsorption and biodegradation of reactive black 5 (RB5) by Funalia trogii (F. trogii) ATCC 200800 biofilm on fly ash-chitosan bead in the fluidized bed process was derived. The mechanisms in the model system included adsorption by fly ash-chitosan beads, biodegradation by F. trogii cells and mass transport diffusion. Batch kinetic tests were independently performed to determine surface diffusivity of RB5, adsorption parameters for RB5 and biokinetic parameters of F. trogii ATCC 200800. A column test was conducted using a continuous-flow fluidized bed reactor with a recycling pump to approximate a completely-mixed flow reactor for model verification. The experimental results indicated that F. trogii biofilm bioregenerated the fly ash-chitosan beads after attached F. trogii has grown significantly. The removal efficiency of RB5 was about 95 % when RB5 concentration in the effluent was approximately 0.34 mg/L at a steady-state condition. The concentration of suspended F. trogii cells reached up to about 1.74 mg/L while the thickness of attached F. trogii cells was estimated to be 80 μm at a steady-state condition by model prediction. The comparisons of experimental data and model prediction show that the model system for adsorption and biodegradation of RB5 can predict the experimental results well. The approaches of experiments and mathematical modeling in this study can be applied to design a full-scale fluidized bed process to treat reactive dye in textile wastewater.     

Biodegradation of olive oil mill wastewaterby Coriolus versicolor and Funalia trogii:effects of agitation,initial COD concentration,inoculum size and immobilization

The biodegradation of olive oil mill wastewater (OOMW) by Coriolus versicolor and Funalia trogii was investigated. Initial COD concentration, agitation and inoculum size were all found to be significant for biodegradation. Adding glucose, sulphate or nitrogen had no effect on biodegradation. During growth in optimum conditions, C.versicolor removed approximately 63% COD, 90% phenol and 65% colour within 6 days and F. trogii removed approximately 70% COD, 93% phenol and 81% colour of the OOMW used. The fungi also excreted large amounts of extracellular laccase into the medium. High biodegradation yields were also obtained by fungi immobilized in calcium alginate gels.     

Copper induction of lignin-modifying enzymes in the white-rot fungus Trametes trogii

Trametes trogii, a white rot basidiomycete involved in wood decay worldwide, produces several ligninolytic enzymes, laccase being the dominant one, with higher titers than those reported for most other white rot fungi studied up to date. The effect of copper on in vitro production of extracellular ligninolytic activities was studied. CuSO(4)·5H(2)O concentrations from 1.6 μM to 1.5 mM were tested in a synthetic medium with glucose 20 g/L and asparagine 3 g/L. The addition of copper (up to 1 mM) did not affect growth but strongly stimulated ligninolytic enzyme production; faster decolorization of the polymeric dye Poly R-478 was observed as well. Maximal production of manganese peroxidase, laccase, and glyoxal oxidase [1.28 U/mL, 93.8 U/mL (with a specific activity of 720 U/mg protein), and 0.46 U/mL respectively] was attained with 1 mM CuSO(4)·5H(2)O. However, higher copper concentrations inhibited growth and notably decreased manganese peroxidase production, although they did not affect laccase secretion. Laccase activity in the culture filtrate was maximal at 50 C and pH 3.4, and the enzyme was completely stable at pH 4.4 and above, and at 30 C for up to 5 d. Denaturing polyacrylamide gel electrophoresis of extracellular culture fluids showed two laccase activity bands (mol wt 38 and 60 kDa respectively). The pattern of isoenzyme production was not affected by medium composition but differed with culture age.     

Differential decolorization of textile dyes in mixtures and the joint effect of laccase and cellobiose dehydrogenase activities present in extracellular extracts from Funalia trogii

The largest part of the bio-decolorization investigations have been performed to date on a single dye without exploring the behavior in complex mixtures as the real dyeing baths. Therefore, mixtures of dyes belonging to azo and anthraquinonic classes, chosen among the most utilized in textile wool dyeing, were employed for comparative enzymatic decolorization studies using the extracellular extracts from the white rot fungus Funalia trogii, to understand how the concomitant presence of more than one dye could influence their degradation course and yield.Fungal extracts containing laccase activity only were capable to partially decolorize dyes mixtures from the different classes analyzed. The deconvolution of the decolorization with time allowed to monitor the degradation of the single dyes in the mixtures evidencing a time dependent differential decolorization not observed for the singles alone. Some dyes in the blend were in fact decolorized only when the most easily converted dyes were largely transformed. These experiments would allow to help the dyeing factories in the selection of the most readily degraded dyes.Since F. trogii grown on different media and activators shows diverse levels of expression of the redox enzymes laccase and cellobiose dehydrogenase (CDH), the dyes mixtures recalcitrant to decolorization by laccase activity alone, were subjected to the combined action of extracts containing laccase and CDH. The use of CDH, in support to the activity of laccase, resulted in substantial decolorization increases (>84%) for all the refractory dyes mixtures.     

Biodegradation of olive oil mill wastewaterby Coriolus versicolor and Funalia trogii:effects of agitation, initial COD concentration, inoculum size and immobilization

The biodegradation of olive oil mill wastewater (OOMW) by Coriolus versicolor and Funalia trogii was investigated. Initial COD concentration, agitation and inoculum size were all found to be significant for biodegradation. Adding glucose, sulphate or nitrogen had no effect on biodegradation. During growth in optimum conditions, C.versicolor removed approximately 63% COD, 90% phenol and 65% colour within 6 days and F. trogii removed approximately 70% COD, 93% phenol and 81% colour of the OOMW used. The fungi also excreted large amounts of extracellular laccase into the medium. High biodegradation yields were also obtained by fungi immobilized in calcium alginate gels.