Dephenolisation of olive mill wastewater using adapted Trametes versicolor

Olive mill wastewater (OMW) is an effluent of the olive oil extraction process. The large volumes involved, along with the high phenolic content and chemical oxygen demand, cause major environmental problems. The presence of phenolics limits the effectiveness of aerobic or anaerobic treatment of this wastewater. In most of the studies performed on OMW, the concentration of phenolics is reduced by diluting the OMW prior to biological treatment, which leads to an increase in waste volume. Therefore, the aim of this work was to investigate the possibility of reducing the phenolic content without dilution and without any addition of nutrients or pretreatment by using the white-rot fungi Trametes versicolor FPRL 28A INI. Through an adaptation process, the fungus was able to remove 78% of total phenolics in shake flask experiments and 39% in static culture using undiluted OMW medium. In continuously stirred tank reactor (CSTR) conditions, 70% of total phenolics removal was achieved. Analysis with GC–MS showed that all simple phenolics disappeared from the medium after the 8th day of cultivation at an 0.25 vvm aeration rate. The maximum activities of phenol degrading enzymes laccase and manganese peroxidase (MnP) obtained under these conditions were 762.14 ± 42.11 and 97.80 ± 8.11 U l^?1 respectively.     

Response surface analysis for enzymatic decolorization of Congo red by manganese peroxidase

The enzymatic decolorization process of manganese peroxidase (MnP) is a complex system, which is greatly affected by the concentrations of H₂O₂, Mn²⁺, dye and enzyme. This work aimed to study these factors and investigate the combined interactions between them by applying response surface methodology (RSM) for decolorization of Congo red with MnP from Schizophyllum sp. F17, meanwhile conventional one-factor-at-a-time analysis was carried out. Through the one-factor-at-a-time analysis the optimized H₂O₂, Mn²⁺, Congo red and MnP extract was 0.2 mM, 0.5 mM, 50 mg/l and 0.8 ml, respectively, and the maximum decolorization attained under such conditions was 24.2%. Response surface analysis was conducted through Box–Behnken design and a second-order polynomial model (R² = 0.8565) was generated to describe the combined effect and the interactions quantificationally. ANOVA analysis indicated that the interactions between H₂O₂ and MnP, between dye and MnP were significant; the optimum condition through RSM was found to be 0.35 mM H₂O₂, 0.5 mM Mn²⁺, 75 mg/l Congo red and 1.4 ml MnP extract, for maximum decolorization of 30.8%.     

Liquid state fermentation of apple pomace sludge for the production of ligninolytic enzymes and liberation of polyphenolic compounds

Ligninolytic enzyme production and polyphenolic compound extraction by liquid-state culture of Phanerochaete chrysosporium ATCC 24275 was investigated by employing apple pomace sludge and synthetic medium. Different physico-chemical and biological parameters namely viscosity, zeta potential and particle size, viability and enzyme production were investigated. The ligninolytic enzyme production was higher in apple pomace sludge (45 U/l of laccase, 220 U/l of MnP and 6.5 U/l of LiP) than in synthetic medium (17 U/l of laccase, 37 U/l of MnP and 6 U/l). These maximal activities were found during the stationary and decline phase. It was also found that enzyme production was strongly correlated with P. chrysoporium viability in both synthetic medium and apple pomace sludge. Moreover, physico-chemical parameters, such as particle size, zeta potential and viscosity were strongly correlated to the viability of P. chrysosporium and to the ligninolytic enzyme production. An increase in polyphenol content extracted by acetone (383–720 mg GAE/l) was observed during fermentation of apple pomace and it was found that the polyphenol content extracted by ethanol increased ～1.5 fold until 67 h of fermentation and later it decreased. It was found that antioxidant activity increased to 35% and eventually decreased based on the change in the polyphenol content.     

Broad spectrum anti-infective properties of benzisothiazolones and the parallels in their anti-bacterial and anti-fungal effects

Various mono- and bis-benzisothiazolone derivatives were synthesized and screened against different strains of bacteria and fungi in order to understand the effect of multiple electrophilic sulfur atoms and substitution pattern in the immediate vicinity of reactive sulfur. Staphyllococcus aureus-ATCC 7000699, MRSA and S. aureus-ATCC 29213 (Quality Control strain) were more susceptible to this class of compounds, and the most potent derivative 1.15 had MIC₅₀ of 0.4 μg/mL (cf. Gentamicin = 0.78 μg/mL). CLogP value, optimally in the range of 2.5–3.5, appeared to contribute more to the activity than the steric and electronic effects of groups attached at nitrogen. By and large, their anti-fungal activities also followed a similar trend with respect to the structure and CLogP values. The best potency of IC₅₀ = 0.1 μg/mL was shown by N-benzyl derivative (1.7) against Aspergillus fumigatus; it was also potent against Candida albicans, Cryptococcus neoformans, Sporothrix schenckii, and Candida parapsilosis with IC₅₀ values ranging from 0.4 to 1.3 μg/mL. Preliminary studies also showed that this class of compounds have the ability to target malaria parasite with IC₅₀ values in low micromolar range, and improvement of selectivity is possible through structure optimization.     

Purification and characterization of a novel laccase from the ascomycete Trichoderma atroviride: Application on bioremediation of phenolic compounds

The extracellular laccase produced by the ascomycete Trichoderma atroviride was purified and characterized and its ability to transform phenolic compounds was determined. The purified laccase had activity towards typical substrates of laccases including 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), dimethoxyphenol (2,6-DMP), syringaldazine and hydroquinone. The enzyme was a monomeric protein with an apparent molecular mass of 80 kDa and an isoelectric point of 3.5. The pH optima for the oxidation of ABTS and 2,6-DMP were 3 and 5, respectively, and the optimum temperature was 50 °C with 2,6-DMP. The laccase was stable at slightly acidic pH (4 and 5). It retained 80% of its activity after 4 h incubation at 40 °C. Under standard assay conditions, K_m values of the enzyme were 2.5 and 1.6 mM towards ABTS and 2,6-DMP, respectively. This enzyme was able to oxidize aromatic compounds present in industrial and agricultural wastewater, as catechol and o-cresol, although the transformation of chlorinated phenols required the presence of ABTS as mediator.     

Decolorization of textile azo dyes by aerobic bacterial consortium

The decolorization potential of two bacterial consortia developed from a textile wastewater treatment plant showed that among the two mixed bacterial culture SKB-II was the most efficient in decolorizing individual as well as mixture of dyes. At 1.3 g L^?1 starch supplementation in the basal medium by the end of 120 h decolorization of 80–96% of four out of the six individual azo dyes Congo red, Bordeaux, Ranocid Fast Blue and Blue BCC (10 mg L^?1) was noted. The culture exhibited good potential ability in decolorizing 50–60% of all the dyes (Congo red, Bordeaux, Ranocid Fast Blue and Blue BCC) when present as a mixture at 10 mg L^?1. The consortium SKB-II consisted of five different bacterial types identified by 16S rDNA sequence alignment as Bacillus vallismortis, Bacillus pumilus, Bacillus cereus, Bacillus subtilis and Bacillus megaterium which were further tested to decolorize dyes. The efficient ability of this developed consortium SKB-II to decolorize individual dyes and textile effluent using packed bed reactors is being carried out.     

Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization

Laccase from a tree legume, Leucaena leucocephala, was purified to homogeneity using a quick two-step procedure: alginate bead entrapment and celite adsorption chromatography. Laccase was purified 110.6-fold with an overall recovery of 51.0% and a specific activity of 58.5 units/mg. The purified laccase was found to be a heterodimer (∼220 kDa), containing two subunits of 100 and 120 kDa. The affinity of laccase was found to be highest for catechol and lowest for hydroquinone, however, highest K_cat and K_cat/K_m were obtained for hydroquinone. Purified laccase exhibited pH and temperature optima of 7.0 and 80 °C, respectively. Mn²⁺, Cd²⁺, Fe²⁺, Cu²⁺ and Na⁺ activated laccase while Ca²⁺ treatment increased laccase activity up to 3 mM, beyond which it inhibited laccase. Co²⁺, Hg²⁺, DTT, SDS and EDTA showed an inhibition of laccase activity. The Leucaena laccase was found to be fairly tolerant to organic solvents; upon exposure for 1 h individually to 50% (v/v) each of ethanol, DMF, DMSO and benzene, more than 50% of the activity was retained, while in the presence of 50% (v/v) each of methanol, isopropanol and chloroform, a 40% residual activity was observed. The purified laccase efficiently decolorized synthetic dyes such as indigocarmine and congo red in the absence of any redox mediator.     

Toxicity evaluation of parboiled rice effluent using sperm quality of zebrafish as bioindicator

This study aimed to assess the acute toxicity of raw and treated wastewater generated by the rice parboiling industry using zebrafish (Danio rerio) sperm quality as a bioindicator. Toxicity bioassays were conducted comparing physicochemical parameters of sperm quality for zebrafish at sublethal conditions (n = 150 fish, 50 per treatment). Acute toxicity was detected in all sperm quality parameters assessed for both raw and treated wastewater, when contrasted to the control (p < 0.05). For zebrafish exposed to raw effluent, negative correlations with parameters of sperm quality were observed for the concentration of iron, phosphorus and total suspended solids (p < 0.05). Salinity, the biochemical oxygen demand and the concentration of total suspended solids were negatively correlated with parameters of sperm quality for zebrafish exposed to treated effluent (p < 0.05). In comparison with the levels observed for the raw effluent, most physicochemical parameters of the treated effluent were reduced to levels within the limits required by the environmental legislation. Despite the physical and chemical parameters measured in the treated wastewater meeting environmental legislation thresholds, acute toxicity persisted. These results show that the sperm quality can be used as a bioindicator for wastewater toxicity and release of wastewater to surface water could affect the fertility of fishes.     

Purification and characterization of a new laccase from Shiraia sp.SUPER-H168

A new laccase from Shiraia sp.SUPER-H168 was purified by ion exchange column chromatography and gel permeation chromatography and the apparent molecular mass of this enzyme was 70.78 kDa, as determined by MALDI/TOF-MS. The optimum pH value of the purified laccase was 4, 6, 5.5 and 3 with 2,6-dimethoxyphenol (DMP), syringaldazine, guaiacol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as substrates, respectively. The optimum temperature of the purified laccase was 50 °C using DMP, syringaldazine and guaiacol as substrates, but 60 °C for ABTS. Inhibitors and metal ions of SDS, NaN₃, Ag⁺ and Fe³⁺ showed inhibition on enzyme activity of 10.22%, 7.86%, 8.13% and 67.50%, respectively. Fe²⁺ completely inhibited the purified laccase. The K_cat/K_m values of the purified laccase toward DMP, ABTS guaiacol and syringaldazine were 3.99 × 10⁶, 3.74 × 10⁷, 8.01 × 10⁴ and 2.35 × 10⁷ mol^?1 L S^?1, respectively. The N-terminal amino acid sequence of the purified laccase showed 36.4% similarity to Pleurotus ostrestus. Approximately 66% of the Acid Blue 129 (100 mg L^?1) was decolorized by 2.5 U of the purified laccase after a 120 min incubation at 50 °C. Acid Red 1 (20 mg L^?1) and Reactive Black 5 (50 mg L^?1) were decolorized by the purified laccase after the addition of Acid Blue 129 (100 mg L^?1).     

A novel membrane-surface liquid co-culture to improve the production of laccase from Ganoderma lucidum

In this work, a laccase producer, Ganoderma lucidum, was separated and identified according to its morphological characteristics and phylogenetic data. A 4000 U/l and 8500 U/l of laccase activity was obtained in 500 ml flask by submerged culture and biomembrane-surface liquid culture (BSLC), respectively. Furthermore, the novel biomembrane-surface liquid co-culture (BSLCc) was developed by adding Saccharomyces cerevisiae to reactor in order to shorten the fermentation period and improve laccase production. Laccase activity obtained by BSLCc, 23 000 U/l, is 5.8 and 2.7 times of that obtained by submerged culture and BSLC, respectively. In addition, laccase production by BSLCc was successfully scaled-up to 100 l reactor, and 38 000 U/l of laccase activity was obtained on day 8. The mechanism of overproducing laccase by BSLCc was investigated by metabolism pathway analysis of glucose. The results show glucose limitation in fermentation broth induces the secretion of laccase. The addition of S. cerevisiae, on one hand, leads to an earlier occurrence of glucose limitation state, and thus shortens the fermentation time; on the other hand, it also results in the appearance of a series of metabolites of the yeast including organic acids, ethanol, glycerol and so forth in fermentation broth, and both polyacrylamide gel electrophoresis analysis and enzyme activity detection of laccase show that these metabolites contribute to the improvement of laccase activity.     

Enhanced decolorization of Solar brilliant red 80 textile dye by an indigenous white rot fungus Schizophyllum commune IBL-06

An indigenously isolated white rot fungus, Schizophyllum commune IBL-06 was used to decolorize Solar brilliant red 80 direct dye in Kirk’s basal salts medium. In initial screening study, the maximum decolorization (84.8%) of Solar brilliant red 80 was achieved in 7 days shaking incubation period at pH 4.5 and 30 °C. Different physical and nutritional factors including pH, temperature and fungal inoculum density were statistically optimized through Completely Randomized Design (CRD), to enhance the efficiency of S. commune IBL-06 for maximum decolorization of Solar brilliant red 80 dye. The effects of inexpensive carbon and nitrogen sources were also investigated. Percent dye decolorization was determined by a reduction in optical density at the wavelength of maximum absorbance (λ_max, 590 nm). Under optimum conditions, the S. commune IBL-06 completely decolorized (100%) the Solar brilliant red 80 dye using maltose and ammonium sulfate as inexpensive carbon and nitrogen sources, respectively in 3 days. S. commune IBL-06 produced the three major ligninolytic enzymes lignin peroxidase (LiP), manganase peroxidase (MnP) and lacaase (Lac) during the decolorization of Solar brilliant red 80. LiP was the major enzyme (944 U/mL) secreted by S. commune IBL-06 along with comparatively lower activities of MnP and Laccase.     

Treatment of high-strength wastewater in tropical vertical flow constructed wetlands planted with Typha angustifolia and Cyperus involucratus

The ability of vertical flow (VF) constructed wetland systems to treat high-strength (ca. 300 mg L^?1 of COD and ca. 300 mg L^?1 total-nitrogen) wastewater under tropical climatic conditions was studied during a 5-month period. Nine 0.8-m diameter experimental VF units (depth 0.6 m) were used: three units were planted with Typha angustifolia L., another three units were planted with Cyperus involucratus Rottb and three units were unplanted. Each set of units were operated at hydraulic loading rates (HLRs) of 20, 50 and 80 mm d^?1. Cyperus produced more shoots and biomass than the Typha, which was probably stressed because of lack of water. The high evapotranspirative water loss from the Cyperus systems resulted in higher effluent concentrations of COD and total-P, but the mass removal of COD did not differ significantly between planted and unplanted systems. Average mass removal rates of COD, TKN and total-P at a HLR of 80 mm d^?1 were 17.8, 15.4 and 0.69 g m^?2 d^?1. The first-order removal rate constants at a HLR of 80 mm d^?1 for COD, TKN and total-P were 49.8, 30.1 and 13.5 m year^?1, respectively, which is in the higher range of k-values reported in the literature. The oxygen transfer rates were ca. 80 g m^?2 d^?1 in the planted systems as opposed to ca. 60 g m^?2 d^?1 in the unplanted systems. The number of Nitrosomonas was two to three orders of magnitude higher in the planted systems compared to the unplanted systems. Planted systems thus had significantly higher removal rates of nitrogen and phosphorus, higher oxygen transfer rates, and higher quantities of ammonia-oxidizing bacteria. None of the systems did, however, fully nitrify the wastewater, even at low loading rates. The vertical filters did not provide sufficient contact time between the wastewater and the biofilm on the gravel medium of the filters probably because of the shallow bed depth (0.6 m) and the coarse texture of the gravel. It is concluded that vertical flow constructed wetland systems have a high capacity to treat high-strength wastewater in tropical climates. The gravel and sand matrix of the vertical filter must, however, be designed in a way so that the pulse-loaded wastewater can pass through the filter medium at a speed that will allow the water to drain before the next dose arrives whilst at the same time holding the water back long enough to allow sufficient contact with the biofilm on the filter medium.     

Robust polyfunctional T-helper 1 responses to multiple fungal antigens from a cell population generated using an environmental strain of Aspergillus fumigatus

Background and aimsAspergillus fumigatus infections are the leading cause of invasive fungal infection-related deaths in stem cell transplant patients, and may be amenable to correction with adoptive immunotherapy providing T lymphocytes specific for A. fumigatus. However, a clinically usable source of antigen and a reliable procedure for the generation of large numbers of Aspergillus-specific T lymphocytes to clinical-grade standards is not available.MethodsAn environmental strain of A. fumigatus (WMAfES) was isolated and cultured using materials and reagents suitable for clinical manufacture. Water-soluble lysate from germinated conidia of WMAfES was used as the antigen source. Peripheral blood mononuclear cells were stimulated with antigen-pulsed autologous dendritic cells on days 0 and 7. Cells were expanded with a cocktail of interleukin (IL)-2, IL-7 and IL-15 from days 7 to 21.ResultsWe obtained a mean 32.8-fold increase in cell numbers over 21 days of culture (n = 8). Resultant cultures were predominantly effector and central memory CD4⁺ T cells, which produced T-helper (h)1 and Th17 cytokines when restimulated with A. fumigatus antigen derived from environmental or clinically isolated A. fumigatus. Cultured cells exhibited a high level of specific expansion and chemokine production when restimulated. Moreover, cultured cells cross-reacted with antigens from other fungi, including Penicillium, Candida albicans and other non-fumigatus Aspergillus species.ConclusionsWe describe a simple, robust, reproducible and clinically applicable procedure using a clinically appropriate antigen preparation for the expansion of polyfunctional A. fumigatus-specific T cells from normal donors of varying HLA types.     

Enzymatic decolorization of sulfonphthalein dyes

The white rot fungus (WRF) Pleurotus ostreatus produced manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities during solid state fermentation of wheat straw, a natural lignocellulosic substrate. Most of the sulfonphthalein (SP) dyes were decolorized by MnP at pH 4.0. The higher K_m for meta-cresol purple (40 μM) and lower K_m for ortho-cresol red (26 μM) for MnP activities explained the preference for the position of methyl group at ortho than at meta on chromophore. Bromophenol blue decolorizing activity was higher at pH 3.5 and decreased as the concentration of Mn^II was increased. SP-decolorizing activity was associated not only with MnP but also with MIP. Additional bromine group along with the methyl group on SP chromophores decreases the rate of decolorization. Bromination of sulfonphthalein chromophore makes them the poorer substrate for MnP. This is evident from the higher K_m for bromocresol green (117 μM) when compared to bromocresol purple (36 μM) and bromophenol blue (78 μM). The order of preference for the SP dyes as substrate for the MnP-catalyzed decolorizing activity is phenol red > ortho-cresol red > meta-cresol purple > bromophenol red > bromocresol purple > bromophenol blue > bromocresol green and the order of preference for the SP dyes as substrate for the MIP-catalyzed decolorizing activity is bromocresol green > bromophenol blue > bromocresol purple > bromophenol red > meta-cresol purple > ortho-cresol red > phenol red. Inhibition of PR decolorizing activity by NaN₃ provided the evidence of decolorizing activity as an oxidative process.     

Evaluation of fungal contamination and mycotoxin production in maize silage

Agricultural activities involve daily use of maize silage as feed for livestock, which can be contaminated by mycotoxigenic molds. To evaluate fungal contamination, and the production of mycotoxins in maize silage we propose a multi-disciplinary approach utilizing PCR methods with genes of the aflatoxin (ver-1, omt-1 and apa-2), fumonisin (FUM1) and trichothecene (TRI6) biosynthesis pathways. To detect Aspergillus fumigatus, a 26S/intergenic spacer region of the rDNA complex was amplified. These specific PCR assays allowed three major groups of toxigenic fungi-like aflatoxin-producing Aspergilli, fumonisin and trichothecene-producing Fusaria, and the ubiquitous mold A. fumigatus, to be targeted. A multimycotoxin method is also proposed to simultaneously quantify seven mycotoxins (i.e., aflatoxin B₁, citrinin, deoxynivalenol, fumonisin B₁, gliotoxin, ochratoxin A, zearalenone) in maize silage by high-performance liquid chromatography coupled to mass spectrometry (HPLC–MS). These microbiological and analytical tools revealed three potentially toxigenic groups of fungi and A. fumigatus grown from mature maize silage (11 month old) that was collected in Normandy (France) and the mycotoxins aflatoxin B₁ (7.0–51.3 μg/kg), citrinin (10.1–14.2 μg/kg), deoxynivalenol (128.0–181.0 μg/kg) and gliotoxin (6.6–11.9 μg/kg). Results indicate that the combination of PCR and HPLC–MS can be used to assess fungal quality of maize silages.     

Identification of lactic acid bacteria and yeast from boza

Boza is a low-alcohol beverage produced from the fermentation of barley, oats, millet, maize, wheat or rice. The number of lactic acid bacteria isolated from three boza samples ranged from 9 × 10⁶ to 5 × 10⁷ CFU/mL. Carbohydrate fermentation reactions and PCR with species-specific primers classified the isolates as Lactobacillus paracasei subsp. paracasei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus rhamnosus and Lactobacillus fermentum. No filamentous fungi were isolated. Yeasts were isolated from two of the three boza samples, with cell numbers ranging from 1.3 × 10² to 1.8 × 10³ CFU/mL. Results obtained from sequencing of the D1/D2 rDNA region identified the yeasts as Candida diversa, Candida inconspicua, Candida pararugosa, Issatchenkia orientalis, Pichia fermentans, Pichia guillliermondii, Pichia norvegensis, Rhodotorula mucilaginosa and Torulaspora delbrueckii. C. inconspicua has been isolated from human sputum and tongue and is an opportunistic pathogen. R. mucilaginosa is also an opportunistic pathogen implicated in fungaemia, endocarditis and meningitis. P. norvegensis has been associated with septicaemia in humans. Saccharomyces cerevisiae, commonly associated with fermented beverages, has not been detected in any of the boza samples, despite enrichment.     

Synthesis and antimicrobial activity of novel amphiphilic aromatic amino alcohols

We report in this work the preparation and in vitro antimicrobial evaluation of novel amphiphilic aromatic amino alcohols synthesized by reductive amination of 4-alkyloxybenzaldehyde with 2-amino-2-hydroxymethyl-propane-1,3-diol. The antibacterial activity was determined against four standard strains (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa) and 21 clinical isolates of methicillin-resistant Staphylococcus aureus. The antifungal activity was evaluated against four yeast (Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis). The results obtained showed a strong positive correlation between the lipophilicity and the antibiotic activity of the tested compounds. The best activities were obtained against the Gram-positive bacteria (MIC = 2–16 μg ml^?1) for the five compounds bearing longer alkyl chains (4c–g; 8–14 carbons), which were also the most active against Candida (MIC = 2–64 μg ml^?1). Compound 4e exhibited the highest levels of inhibitory activity (MIC = 2–16 μg ml^?1) against clinical isolates of MRSA. A concentration of twice the MIC resulted in bactericidal activity of 4d against 19 of the 21 clinical isolates.     

Characterization and kinetic properties of the purified Trematosphaeria mangrovei laccase enzyme

The properties of Trematosphaeria mangrovei laccase enzyme purified on Sephadex G-100 column were investigated. SDS–PAGE of the purified laccase enzyme showed a single band at 48 kDa. The pure laccase reached its maximal activity at temperature 65 °C, pH 4.0 with K_m equal 1.4 mM and V_max equal 184.84 U/mg protein. The substrate specificity of the purified laccase was greatly influenced by the nature and position of the substituted groups in the phenolic ring. The pure laccase was tested with some metal ions and inhibitors, FeSO₄ completely inhibited laccase enzyme and also highly affected by (NaN₃) at a concentration of 1 mM. Amino acid composition of the pure enzyme was also determined. Carbohydrate content of purified laccase enzyme was 23% of the enzyme sample. The UV absorption spectra of the purified laccase enzyme showed a single peak at 260–280 nm.     

Overproduction of Trametes versicolor laccase by making glucose starvation using yeast

In the present paper, overproduction of laccase by microbe interaction was studied. When Trametes versicolor was co-cultured with Candida sp. HSD07A in submerged fermentation, laccase activity could be improved significantly and reached 10500 ± 160 U/l, 11.8 times more than that of the contrast group. Fermentation tests of the yeast indicated that it could produce amylase and cellulase, but couldn’t excrete laccase and the overproductive laccase was produced by T. versicolor; the interaction mechanism between T. versicolor and Candida sp. HSD07A was investigated and the results showed that amylase and cellulose could hydrolyze cell walls of T. versicolor; however, the degree of hydrolysis was at a very low level, could not lead to overproduction of laccase; glucose starvation state made by the yeast was the real reason why T. versicolor could overproduce laccase; moreover, this study also proved that making glucose starvation using the yeast was a novel and effective method.     

Purification and characterization of yellow laccase from Trametes hirsuta MTCC-1171 and its application in synthesis of aromatic aldehydes

A yellow laccase from the culture filtrate of Trametes hirsuta MTCC-1171 has been purified. The purification methods involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on diethylaminoethyl cellulose. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 55.0 kDa. Using 2,6-dimethoxyphenol, 2,2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] and 3,5-dimethoxy-4-hydroxybenzaldehyde azine as the substrates, the K_m, k_cat and k_cat/K_m values of the laccase were found to be 420 μM, 13.04 s⁻¹, 3.11 × 10⁴ M⁻¹ s⁻¹, 225 μM, 13.03 s⁻¹, 1.3 × 10⁵ M⁻¹ s⁻¹ and 100 μM, 13.04 s⁻¹, 5.8 × 10⁴ M⁻¹ s⁻¹, respectively. The pH and temperature optima were 4.5 and 60 °C, respectively while pH and temperature stabilities were pH 4.5 and 50 °C. The activation energy for thermal denaturation of the enzyme was 18.6 kJ/mol/K. The purified laccase has yellow colour and does not show absorption band around 610 nm like blue laccases. The purified laccase transforms toluene, 3-nitrotoluene, 4-nitrotoluene, 3-chlorotoluene, 4-chlorotoluene and 3,4-dimethoxytoluene to benzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, 3-chlorobenzaldehyde, 4-chlorobenzaldehyde and 3,4-dimethoxybenzaldehyde in the absence of mediator molecules in high yields.