Laccase production and simultaneous decolorization of synthetic dyes in unique inexpensive medium by new isolates of white rot fungus

Morphological and biochemical analysis of the newly isolated white rot fungal (WRF-1) strain has ability to secrete laccase in the economical medium consisted of synthetic dyes, groundnut shell (GNS) and cyanobacterial biomass (algal bloom) under submerged shaking condition at pH 5.0 and 30 °C ± 2 °C temperature. WRF-1 strain was found to decolorize synthetic dyes efficiently at pH 5.0 and 30 °C ± 2 °C temperature. The laccase activity of strain was purified to homogeneity by chromatography with yield up to 70%. The molecular mass of laccase was found to be 70 kDa by SDS-PAGE and isoelectric point was 4.8. Biotransformation of the dyes was followed spectrophotometrically and dyes were found to decolorize completely after 6 days of fermentation. LC-MS studies were used to decipher the degradation profile of synthetic dyes by WRF-1. Indigo carmine gets degraded to isatin sulfonic acid and 4-amino-3-methylbenzenesulphonic acid whereas methyl orange degraded metabolites were identified as p-N,N′-dimethylamine phenyldiazine and p-hydroxybenzene sulfonic acid. Thus the study would give a road map for the production and application of laccase enzyme on a larger scale using low cost substrate.     

Pre-steady state kinetic studies on the microsecond time scale of the laccase from Trametes versicolor

Laccase from Trametes versicolor reduces dioxygen to water. The enzyme is used in green chemistry applications such as the selective oxidation of alcohols in the presence of a suitable mediator (TEMPO) or in biofuel cells. We studied the catalytic mechanism of the enzyme by the stopped-flow and our newly developed rapid-mixing rapid sampling method, which has an experimental dead time of 75 ± 15 μs. Equilibrium and kinetic analyses yielded a reduction potential of 717 ± 5 mV for Type 1 copper center. EPR and low-temperature UV-Vis spectroscopy indicate that oxidation of the blue copper center and OO bond splitting occur within 100 μs, without detectable formation of a peroxide intermediate. These results indicate a rapid internal electron transfer between the various copper centers (>25.000/s) and rapid binding of O₂ (k_on > 5 × 10⁷ M⁻¹ s⁻¹). Mechanistic aspects of the catalytic cycle are shortly discussed.     

Decolorization of reactive dyes by a thermostable laccase produced by Ganoderma lucidum in solid state culture

Dye decolorizing potential of the white rot fungus Ganoderma lucidum KMK2 was demonstrated for recalcitrant textile dyes. G. lucidum produced laccase as the dominant lignolytic enzyme during solid state fermentation (SSF) of wheat bran (WB), a natural lignocellulosic substrate. Crude enzyme shows excellent decolorization activity to anthraquinone dye Remazol Brilliant Blue R (RBBR) without redox mediator whereas diazo dye Remazol Black-5 (RB-5) requires a redox mediator. Polyacrylamide gel electrophoresis (PAGE) of crude enzyme confirms that the laccase enzyme was the major enzyme involved in decolorization of either dyes. Native and SDS-PAGE indicates that the presence of single laccase with molecular weight of 43 kDa. N-Hydroxybenzotriazole (HBT) at a concentration of 1 mM was found as the best redox mediator. RB-5 (50 mg l^−l) was decolorized by 62% and 77.4% within 1 and 2 h, respectively by the crude laccase (25 U ml⁻¹). RBBR (50 mg l^−l) was decolorized by 90% within 20 h, however, it was more efficient in presence of HBT showing 92% decolorization within 2 h. Crude laccase showed high thermostability and maximum decolorization activity at 60 °C and pH 4.0. The decolorization was completely inhibited by the laccase inhibitor sodium azide (0.5 mM). Enzyme inactivation method is a good method which averts the undesirable color formation in the reaction mixture after decolorization. High thermostability and efficient decolorization suggest that this crude enzyme could be effectively used to decolorize the synthetic dyes from effluents.     

Effect of inducers and culturing processes on laccase synthesis in Phanerochaete chrysosporium NCIM 1197 and the constitutive expression of laccase isozymes

Phanerochaete chrysosporium NCIM 1197 constitutively secretes considerable level of extracellular enzyme laccase in defined growth medium. Effect of several inducers on laccase production was attempted and found that copper sulphate alone at 30 mM concentration accelerate the laccase production at 3.5-fold increase compared to control. Solid-state fermentation using wheat bran as substrate exhibit, maximum laccase activity of 48.89 ± 1.82 U/L on day 5, whereas it was only 30.21 ± 1.66 and 22.56 ± 1.22 U/L, respectively, in batch fermentation in a laboratory scale bioreactor and in static liquid culture on the same day. The molecular weight of partially purified laccase was found to be 62 kDa. The presence of isoforms as evidenced through Native PAGE reveals that, supplementation of inducers only accelerates the laccase production and are not at all involved in the isoform expressions.     

In vitro culture of Trichogramma pretiosum on an artificial medium

The composition of an artificial medium and environmental conditions are described for the in vitro rearing of the egg parasite Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae). The medium was composed of defined amounts of protein, carbohydrates, lipid, salts, and vitamins, but also contained up to 40% insect hemolymph. The hemolymph was necessary to induce pupation. T. pretiosum eggs were obtained by dissection of Heliothis virescens (F.) (Lepidoptera: Noctuidae) eggs. In vitro reared T. pretiosum were similar in size to H. virescens reared T. pretiosum, and females were fecund.

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Résumé Les oeufs de Trichogramma pretiosum ont été obtenus par dissection d'oeufs d'Heliothis virescens. T. pretiosum Riley (Hymenoptère, Trichogrammatidae) a été élevé avec succès sur un substrat synthétique. Outre des quantités définies de protéines, glucides, lipides, éléments minéraux et vitamines, la ration contenait aussi jusqu'à 40% d'hémolymphe de Manduca sexta. L'hémolymphe était nécessaire pour induire la nymphose. En plus de la nourriture, les conditions d'environnement sont apparues extrêmement importantes pour élever T. pretiosum dans des conditions satisfaisantes. Le contrôle de l'humidité relative, en particulier, était le facteur le plus important. Les adultes produits au cours de cette étude étaient d'apparence normale; ils se sont accouplés sans problèmes, les femelles étaient fécondes et leur taille ne différait pas de celle d'individus élevés sur H. virescens.

     

Kinetics and key enzyme activities of phenanthrene degradation by Pseudomonas mendocina

Phenanthrene degradation by Pseudomonas mendocina CGMCC 1.766, a new phenanthrene-degrading strain, was investigated in this work. When cells were grown on 20, 50, 100 and 200 mg l⁻¹ of phenanthrene, the doubling time was 18.3, 19.8, 21.0 and 20.3 h and the growth yield during exponential phase was 242, 271, 221 and 206 mg protein (g phenanthrene)⁻¹, respectively. High level accumulation of the intermediate metabolite 1-hydroxy-2-naphthoic acid (1H2N) up to ≈94% of its theoretical yield was observed. Dynamic profiles of the activities of two key enzymes, i.e. polycyclic aromatic hydrocarbon (PAH) dioxygenase (PDO) and catechol-2,3-oxygenase (C23O), during the biodegradation were revealed and the results suggest a delicate mechanism in the regulation of these phenanthrene-degrading enzymes in this strain.     

Advantages in using non-isothermal bioreactors in bioremediation of water polluted by phenol by means of immobilized laccase from Rhus vernicifera

Laccase from Rhus vernicifera was immobilized on a polypropylene membrane chemically modified with chromic acid. Ethylenediamine and glutaraldehyde were used as spacer and bifunctional coupling agent, respectively. Phenol was used as substrate.To know how the immobilization procedures affected the enzyme reaction rate the catalytic behavior of soluble and insoluble laccase was studied under isothermal conditions as a function of pH, temperature and substrate concentration. From these studies, two main singularities emerged: (i) the narrower pH–activity profile of the soluble enzyme in comparison to that of the insoluble counterpart and (ii) the increase in pH and thermal stability of the insoluble enzyme.The laccase catalytic behavior was also studied in a non-isothermal bioreactor as a function of substrate concentration and size of the applied transmembrane temperature difference. It was found that, under non-isothermal conditions and keeping constant the average temperature of the bioreactor, the enzyme reaction rate linearly increased with the increase of the temperature difference.     

Enzymatic and spectroscopic studies on the activation or inhibition effects by substituted phenolic compounds in the oxidation of aryldiamines and catechols catalyzed by Rhus vernicifera laccase

The effect of various phenolic compounds on the activity of Rhus vernicifera laccase (Lc) has been evaluated using two different substrates, N,N-dimethyl-p-phenylenediamine and p-tert-butylcatechol. The observed effect strongly depends on the phenol employed and involves either a moderate activation, by halophenols, or inhibition, by acidic phenols. The collective data are consistent with an open active site in Lc, which is capable of accommodating more than one substrate or phenol molecule. According to NMR relaxation experiments, a phenol molecule binds at an average distance from type 1 Cu of about 6 Å, while evidence from electron paramagnetic resonance (EPR) experiments shows that binding of another phenol molecule induces a change, and probably occurs close to, the type 2/type 3 cluster. The effect of phenolic compounds on Lc reactivity is related to a modification of the substrate affinity for the enzyme. This affinity can either be increased, probably through π-stacking or other types of interactions, or decreased, due to competition for the same site. In addition, the alteration induced in the trinuclear copper cluster has a marked effect on the enzyme reactivity. The inhibition observed with acidic phenols is probably due to the protonation of an enzyme intermediate produced at the trinuclear site, e.g. the peroxy intermediate, that causes the release of hydrogen peroxide and prevents the reaction of this intermediate with the substrate.     

Kinetic studies on the Rhizomucor miehei lipase catalyzed esterification reaction of oleic acid with 1-butanol in a biphasic system

The kinetics of the esterification of oleic acid with 1-butanol catalyzed by free Rhizomucor miehei lipase in a biphasic system was studied in a batch reactor. The reaction appeared to proceed via a Ping Pong bi–bi mechanism with 1-butanol inhibition. The kinetic constants of the model were determined from experiments at 30 °C with initial concentrations of oleic acid and 1-butanol in the organic phase and 0.05–0.2 g L⁻¹ enzyme in the aqueous phase. The model was used to simulate the batch concentration profiles of the product as well as the initial reaction rates. Agreement of the model with both the batch concentration profiles (average error of 7.2%) and the initial reaction rate per experiment (average error of 16.0%) was good.     

In-vitro culture of Plasmodium falciparum: Utility of modified (RPNI) medium for drug-sensitivity studies using SYBR Green I assay

Studies were carried out to establish the potential of RPNI medium for drug-sensitivity studies using the MSF assay. The drug sensitivity of standard anti-malarials was compared using both the (³H) Hypoxanthine incorporation assay and the MSF assay. The media supplements used during the study have been human serum, FBS and ALBUMAX-II. Drug sensitivity of two parasite lines, adapted to grow separately in conventional as well as in RPNI medium was compared to observe the effect of RPNI medium on functional characteristics of the parasite. The results revealed identical IC₅₀ values of standard anti-malarials obtained by both the (³H) Hypoxanthine incorporation assay and the MSF assay and no untoward effect of FBS and ALBUMAX-II could be noticed on the chemo-sensitivity of standard anti-malarials. Apart from this the chemo-sensitive response of parasite line adapted to grow in RPNI medium was observed to be intact. These findings showed that RPNI medium has potential to be used for chemo-sensitivity studies and the MSF assay being more convenient was observed to be most suitable assay for bio evaluation of new molecules.     

Structural and developmental studies of Chlamydomyzium oviparasiticum from Rhabditis nematodes and in culture

The oomycete (Peronosporomycete) Chlamydomyzium oviparasiticum, previously recorded as a parasite of rotifer eggs, was found infecting Rhabditis nematodes in a sample of rotting garden compost. For the first time C. oviparasiticum was cultured in liquid media, which enabled more detailed studies of zoospore behaviour and facilitated the use of confocal microscopy. Rhabditis nematodes were successfully re-infected from liquid-cultured inoculum. Light (including video) microscopy and transmission electron microscopy were used to document details of thallus development, zoospore release and resting spore morphology to enable comparison with other oomycete species. This species showed several significant saprolegnialian characters such as the ‘achlyoid’ pattern of spore formation, centrifugal cleavage and structured encystment vesicles. In contrast, spore release into a transient vesicle was a peronosporalean characteristic. The thick-walled resting spores showed relatively poor cytoplasmic preservation and had a thick multi-layered wall. It was still not possible to unequivocably decide whether these were chlamydospores or parthenogenically formed oospores. The phylogenetic significance of these observations is discussed.     

Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80–150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 °C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 °C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K_M was 42 mM, and V_max was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence.     

不同海拔对福建戴云山黄山松林土壤微生物生物量和土壤酶活性的影响

温度、水分等多种环境因子随海拔梯度发生变化,会直接或间接影响土壤微生物生物量、群落结构以及土壤酶活性。然而关于中亚热带地区山地森林生态系统土壤酶活性变化响应的研究还是相对匮乏。戴云山山脉是中国最大的黄山松种质基因基地,本研究以中亚热带戴云山1300 m (L)、1450 m(M)、1600 m(H)的黄山松(Pinus taiwanensis)为研究对象,探究不同海拔下土壤微生物生物量和土壤酶活性如何变化及驱动土壤酶活性变化的主要的环境因子。结果表明:海拔梯度对淀积层(B)土壤的酶活性影响整体较小,在淋溶层(A)土壤中,随海拔升高,纤维素水解酶(β-葡糖苷酶(βG)、纤维素水解酶(CBH))显著降低,因此使土壤可溶性碳(DOC)和微生物生物量碳(MBC)随海拔升高有下降趋势。尽管酸性磷酸酶活性(ACP)随海拔升高显著增加,然而有效磷(AP)无显著变化。此外随海拔升高,微生物生物量氮(MBN)、微生物生物量磷(MBP)显著降低。冗余分析(RDA)结果发现,MBP和碳/氮比(C/N)是影响A层中土壤酶活性变化最重要的因子,而在B层中,土壤含水量(WC)和MBP对土壤酶活性起主要作用。研究表明,磷限制的亚热带地区,无机磷很容易被铁铝固定,MBP可以对土壤中的有效磷进行补充,成为影响本地区酶活性的主要因素。随海拔海拔降低,土壤有机碳、氮分解相关酶活性较高,从而加速了土壤碳、氮周转。因此,探究不同海拔梯度酶活性变化为预测中亚热带亚山地森林生态系统土壤碳、氮、磷养分循环提供了重要的理论依据,也为戴云山自然保护区黄山松林管理提供一定的科学依据。     

Copper tolerance of the biomass crops Elephant grass (Pennisetum purpureum Schumach), Vetiver grass (Vetiveria zizanioides) and the upland reed (Phragmites australis) in soil culture

Pot trials were conducted to study the influence of copper (Cu) on the growth and biomass of Elephant grass (EG, Pennisetum purpureum Schumach), Vetiver grass (VG, Vetiveria zizanioides) and the upland reed (UR, Phragmites australis). Cu toxicity in EG, VG and UR was positively correlated with the total and bioavailable Cu concentrations in the soil. Based on the EC₅₀, dry weights, Cu contents, chlorophyll contents and photosynthesis rates, the Cu tolerance of the three species followed the trend EG > VG > UR. There were no significant differences in the unit calorific values among the different plants, though the total calorific values of EG were higher than those of VG and UR due to its higher biomass. The addition of KH₂PO₄ to the soil decreased the bioavailability of Cu and the Cu uptake by plants. EG could therefore be a good candidate for growth on Cu-contaminated soils, especially those improved by phosphate.     

Occurrence and toxicity of an Anabaena bloom in a tropical reservoir (Southeast Brazil)

Potentially toxic cyanobacterial blooms are becoming common in the Brazilian reservoirs in all regions of the country. During October 2004, a dense bloom of cyanobacteria occurred in the Monjolinho Reservoir (São Carlos, São Paulo State, Brazil) and a significant amount of cyanobacterial material accumulated on the water surface. Phytoplankton analysis showed that the main species in this bloom were Anabaena circinalis and Anabaena spiroides. Cladoceran (Ceriodaphnia dubia and Ceriodaphnia silvestrii) and mouse bioassays were performed to detect toxic products in extracts of the natural samples collected at the three different dates during in short period. To prepare the extracts, freeze-dried cells were dispersed in distilled water and subjected to repeated freeze/thaw cycles and sonication and centrifuging processes. Crude extracts were toxic both to cladocerans (LC₅₀ 94–406 mg freeze-dried cells L⁻¹) and mice (indicative LD₅₀ 297–445 mg freeze-dried cells kg⁻¹) and the toxicity of the bloom increased for cladocerans during the occurrence of the bloom. Toxin analysis by ELISA revealed that microcystin (MC) was found in the water of the reservoir (concentrations ranging from 28 to 45 μg L⁻¹). In addition, microcystin was also found in freeze-dried cyanobacteria cells with concentrations ranging from 138 to 223 μg g⁻¹. On the other hand, neurotoxins (saxitoxin and gonyautoxin) were not detected in any of the natural samples by HPLC. Signs of toxicity in mice did not indicate whether the bloom samples were predominantly hepatotoxic or neurotoxic. It is known that natural Anabaena blooms can contain other toxic compounds besides microcystins and neurotoxins such as lipopolysaccharides or other toxins not identified or known. Methods of detecting cyanotoxins used in this study were insufficient to clarify the toxicological features of Anabaena bloom and indicated that other methods should be investigated.     

Production of quinoprotein d-glucose dehydrogenase in the culture medium of Acinetobacter calcoaceticus

On addition of low concentrations (0.005%) of Triton X-100 to a mineral medium supplemented with 0.5% heptadecane, a marked stimulation of growth rate was observed for Acinetobacter calcoaceticus strains able to grow on alkanes while appreciable amounts of soluble quinoprotein d-glucose dehydrogenase [d-glucose: (pyrroloquinoline-quinone) 1-oxidoreductase, EC 1.1.99.17] were found in the culture medium. At higher Triton X-100 concentrations (0.04%), still larger amounts of d-glucose dehydrogenase and also cytoplasmic enzyme activities appeared in the culture medium. Although combinations of other carbon sources plus non-ionic detergents also produced these enzymes in the medium, the combination of heptadecane and Triton X-100 gave higher levels and had a stabilizing effect on d-glucose dehydrogenase. Therefore, by using this combination and culturing within certain pH limits, a stable enzyme solution, having already a high specific activity, is produced while the cell harvesting and disruption steps can be circumvented. The results indicate that d-glucose dehydrogenase in this organism is a periplasmic enzyme, coupled to a cytochrome b.     

氮沉降对中亚热带米槠天然林微生物生物量及酶活性的影响

氮沉降对土壤微生物的扰动可能会影响土壤的养分循环,然而关于中亚热带天然林土壤微生物及酶活性对氮沉降的响应鲜有报道。通过3 a的氮沉降模拟实验,研究中亚热带米槠天然林土壤的理化性质、土壤微生物量及土壤酶活性的响应。结果表明:氮沉降并未引起土壤的有机碳和总氮显著性变化;高氮(80 kg N hm~(-2)a~(-1))处理下,土壤p H下降,出现酸化现象;低氮(40 kg N hm~(-2)a~(-1))处理促进淋溶层(A层)中土壤纤维素分解酶(β-葡萄糖苷酶和纤维素水解酶)和木质素分解酶(多酚氧化酶和过氧化物酶)活性升高,同时促进土壤微生物生物量碳、氮的积累。冗余分析(RDA)表示,可溶性有机碳(DOC)是驱动A层土壤酶活性的重要环境因子;而在淀积层(B层),这4种酶活性并未发生显著性差异。施氮处理后,A、B层中土壤的酸性磷酸酶活性增加(P0.05)。研究表明:低水平氮沉降增加了土壤微生物生物量碳氮含量以及土壤有机碳分解相关酶活性,从而加速了土壤碳周转;这为未来氮沉降增长背景下,探索中亚热带天然林土壤碳源汇问题提供了依据。     

氮添加对戴云山黄山松林土壤有机氮解聚酶活性的影响及其调控因素

解聚作用是控制土壤有机氮矿化和氮素有效性供应的关键,然而氮沉降对亚热带森林土壤有机氮解聚作用的影响机制尚不明确。以福建戴云山黄山松林为研究对象,设置对照（CT）、低氮（LN）和高氮（HN）3个氮添加水平,进行为期2年的氮沉降模拟试验。通过分析土壤化学性质、微生物生物量和土壤8种有机氮解聚酶活性的变化,探究土壤有机氮解聚作用响应氮沉降的机理过程。结果表明：短期氮添加显著增加0-10 cm和10-20 cm土层矿质氮含量,并显著增加了10-20 cm土层微生物生物量碳（MBC）的含量。同时,0-10 cm土壤锰过氧化物酶活性随氮添加量增加而显著提高,HN处理下土壤漆酶活性显著高于LN和CT;10-20 cm土壤的酸性蛋白酶、碱性蛋白酶、中性蛋白酶和漆酶活性均随氮添加量增加而显著提高,但是谷氨酰胺酶活性变化相反。冗余分析表明两个土层有机氮解聚酶活性影响因素不同,土壤硝态氮（NO₃^--N）是0-10 cm土层有机氮解聚酶活性的主要影响因素,而10-20 cm土层有机氮解聚酶活性由NO₃^--N和MBC共同影响。综上所述,亚热带黄山松林土壤不同有机氮解聚酶对氮添加的响应不一致,主要受土壤NO₃^--N和MBC调节。该研究有助于拓宽土壤氮循环对氮沉降的响应机理,同时对维持土壤有效氮含量和提高黄山松生态系统生产力具有重要意义。     

Detection of Campylobacter jejuni in poultry samples using an enzyme-linked immunoassay coupled with an enzyme electrode

An enzyme-linked immunoassay coupled with a tyrosinase modified enzyme electrode was used for rapid detection of Campylobacter jejuni. The immunomagnetic separation (IMS) method was investigated to achieve optimal isolation of C. jejuni cells. Eight types of beads with three different sizes and function groups were coated with anti-C. jejuni to isolate C. jejuni from the sample solution. Bead size and coating methods were found to be major factors that influenced the capture efficacy. Streptavidin-labeled beads (2.8 μm) provided the greatest capture ability. Three blocking reagents were tested to minimize non-specific binding. Bovine serum albumin (BSA) showed the best blocking capability. Two IMS formats were tested. Competitive immunoassay cut the detection time to 1.5 h, but the detection limit was relatively high (10⁶ CFU/ml). This system was evaluated using C. jejuni pure culture and poultry samples inoculated with C. jejuni. This detection method for C. jejuni could be completed within 2.5 h and had a detection limit of 2.1×10⁴ CFU/ml. No significant difference was found between pure culture samples and poultry samples (P>0.01). A linear relationship was found between C. jejuni cell numbers and the peak current ratio in a range of 10²–10⁷ CFU/ml (R²=0.94).