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Hydrophobin proteins were extracted from Agrocybe cylindracea mycelia, the culture media (potato dextrose broth, PDB), and fruiting bodies. The putative hydrophobins obtained showed approximate sizes ranging from 8.0 to 25.0 kDa, dependent on their source. Multiple hydrophobin protein bands were detected in fruiting bodies. The hydrophobin yielded from aerial mycelia, or fruiting bodies, was approximately 6 mg/g dried weight. The crude extracts were examined for their properties in regards to surface modification, emulsification, and surface activity. Coating of hydrophobic Teflon sheet with crude extract made the surface significantly hydrophilic, whereas exposure of glass surfaces to extracts resulted in enhanced hydrophobicity. Crude extracts from culture media of A. cylindracea displayed emulsifying activity when mixed with hexane and could significantly reduce the surface tension of 60% ethanol and deionised water. The putative hydrophobin protein band from culture media (9.6 kDa), as analysed using LC-MS/MS, contained an amino acid fragment structurally similar to class I hydrophobin proteins from Basidiomycetes.

Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10–30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 °C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements.

To better understand the light regulation of ligninolytic systems in Trametes polyzona KU-RNW027, ligninolytic enzymes-encoding genes were identified and analyzed to determine their transcriptional regulatory elements. Elements of light regulation were investigated in submerged culture. Three ligninolytic enzyme-encoding genes, mnp1, mnp2, and lac1, were found. Cloning of the genes encoding MnP1 and MnP2 revealed distinct deduced amino acid sequences with 90% and 86% similarity to MnPs in Lenzites gibbosa, respectively. These were classified as new members of short-type hybrid MnPs in subfamily A.2 class II fungal secretion heme peroxidase. A light responsive element (LRE), composed of a 5&prime, CCRCCC-3&prime, motif in both mnp promoters, is reported. Light enhanced MnP activity 1.5 times but not laccase activity. The mnp gene expressions under light condition increased 6.5- and 3.8-fold, respectively. Regulation of laccase gene expression by light was inconsistent with the absence of LREs in their promoter. Blue light did not affect gene expressions but impacted their stability. Reductions of MnP and laccase production under blue light were observed. The details of the molecular mechanisms underlying enzyme production in this white-rot fungus provide useful knowledge for wood degradation relative to illumination condition. These novel observations demonstrate the potential of enhancing ligninolytic enzyme production by this fungus for applications with an eco-friendly approach to bioremediation.

A repeated batch of synthetic dye decolorization was efficiently demonstrated in a 5 L airlift bioreactor. A laccase from Ganoderma sp. KU-Alk4, degrading commercial aromatic dyes was selected. The crude enzyme extract expressed laccase activity, and was immobilized under optimal conditions in copper-alginate beads, 3 IU/bead. The immobilized enzyme showed high efficiency in degrading various synthetic dyes under non-buffered conditions, in particular the indigoid dye Indigo Carmine. The immobilized laccase also showed marked increase in stability toward temperature and pH when compared with free enzyme preparation. Immobilization enhanced its temperature stability to maintain initial activity up to 55 °C, ten degrees higher than the free enzyme. The immobilized laccase was stable in the alkaline region up to pH 10.0. The dye decolorization system in 5 L airlift bioreactor was demonstrated with 25 mg/L Indigo Carmine dissolved in tap water and a total immobilized laccase activity of 6 × 104 IU. Airflow rate was the most important factor affecting the number of batch runs and the time for 100% dye degradation. An optimal airflow rate was of 4 L/min. Fourteen batch runs of complete dye degradation were successfully completed with only a single enzyme supplementation, and this could be a feasible system for operation in industry. Total dye degraded by this repeated process at 4 L/min airflow rate was 1.8 g. Isatin sulfonic acid was a metabolic product of Indigo Carmine degradation catalyzed by the immobilized laccase. This development of an effective repeatable bioprocess using enzymes for the treatment of dye-contaminated effluent has potential for implementation on an industrial scale.

Regulation of alkaline-resistant laccase from Perenniporia tephropora KU-Alk4 was proved to be controlled by several factors. One important factor was the initial pH, which drove the fungus to produce different kinds of ligninolytic enzymes. P. tephropora KU-Alk4 could grow at pH 4.5, 7.0, and 8.0. The fungus produced laccase and MnP at pH 7.0, but only laccase at pH 8.0. The specific activity of laccase in the pH 8.0 culture was higher than that in the pH 7.0 culture. At pH 8.0, glucose was the best carbon source for laccase production but growth was better with lactose. Low concentrations of glucose at 0.1% to 1.0% enhanced laccase production, while concentrations over 1% gave contradictory results. Veratryl alcohol induced the production of laccase. A trace concentration of copper ions was required for laccase production. Biomass increased with an increasing rate of aeration of shaking flasks from 100 to 140 rpm; however, shaking at over 120 rpm decreased laccase quantity. Highest amount of laccase produced by KU-Alk4, 360 U/mL, was at pH 8.0 with 1% glucose and 0.2 mM copper sulfate, unshaken for the first 3 days, followed by addition of 0.85 mM veratryl alcohol and shaking at 120 rpm. The crude enzyme was significantly stable in alkaline pH 8.0~10.0 for 24 hr. After treating the pulp mill effluent with the KU-Alk4 system for 3 days, pH decreased from 9.6 to 6.8, with reduction of color and chemical oxygen demand at 83.2% and 81%, respectively. Laccase was detectable during the biotreatment process.

Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10–30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 °C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements.

The biodegradation of three polycyclic aromatic hydrocarbons (PAHs), phenanthrene, fluorene, and pyrene, by a newly isolated thermotolerant white rot fungal strain RYNF13 from Thailand, was investigated. The strain RYNF13 was identified as Trametes polyzona, based on an analysis of its internal transcribed spacer sequence. The strain RYNF13 was superior to most white rot fungi. The fungus showed excellent removal of PAHs at a high concentration of 100 mg·L-1. Complete degradation of phenanthrene in a mineral salt glucose medium culture was observed within 18 days of incubation at 30°C, whereas 90% of fluorene and 52% of pyrene were degraded under the same conditions. At a high temperature of 42°C, the strain RYNF13 was still able to grow, and degraded approximately 68% of phenanthrene, whereas 48% of fluorene and 30% of pyrene were degraded within 32 days. Thus, the strain RYNF13 is a potential fungus for PAH bioremediation, especially in a tropical environment where the temperature can be higher than 40°C. The strain RYNF13 secreted three different ligninolytic enzymes, manganese peroxidase, laccase, and lignin peroxidase, during PAH biodegradation at 30°C. When the incubation temperature was increased from 30°C to 37°C and 42°C, only two ligninolytic enzymes, manganese peroxidase and laccase, were detectable during the biodegradation. Manganese peroxidase was the major enzyme produced by the fungus. In the culture containing phenanthrene, manganese peroxidase showed the highest enzymatic activity at 179 U·mL-1. T. polyzona RYNF13 was determined as a potential thermotolerant white rot fungus, and suitable for application in the treatment of PAH-containing contaminants.

Silver nanoparticles (AgNPs) in chitosan (CS) stabilizer were successfully synthesized using electron beam irradiation. The effects of irradiation dose, molecular weight (MW) of CS stabilizer, concentration of AgNO 3 precursor and addition of tert -butanol on AgNPs production were studied. The stability of the AgNPs under different temperatures and storage times were also investigated. The AgNPs formation in CS was observed using UV–vis, FT-IR and XRD. The characteristic surface plasmon resonance (SPR) of the obtained AgNPs was around 418 nm. The CS stabilizer and its MW, AgNO 3 precursor and irradiation doses are important parameters for the synthesis of AgNPs. The optimum addition of 20% v/v tert -butanol could assist the formation of AgNPs. The AgNPs in CS stabilizer were stable over a period of one year when the samples were kept at 5 °C. The AgNPs observed from TEM images were spherical with an average particle size in the range of 5–20 nm depending on the irradiation doses. The AgNPs in CS solution effectively inhibited the growth of several fungi, i.e., Curvularia lunata , Trichoderma sp., Penicillium sp. and Aspergillus niger , which commonly found on the building surface.

The biodegradation of three polycyclic aromatic hydrocarbons (PAHs), phenanthrene, fluorene, and pyrene, by a newly isolated thermotolerant white rot fungal strain RYNF13 from Thailand, was investigated. The strain RYNF13 was identified as Trametes polyzona, based on an analysis of its internal transcribed spacer sequence. The strain RYNF13 was superior to most white rot fungi. The fungus showed excellent removal of PAHs at a high concentration of 100 mg·L

A novel immobilisation system was developed for dye decolorisation using laccase produced by Ganoderma sp. KU-Alk4. The enzyme showed high efficiency in dye decolorisation when entrapped in Cu–Al and Cu-alginate beads. The former gave the highest activity but the enzyme activity survived longer in the latter. An experimental design of two 3 × 3 Latin Square experiments was applied to evaluate the effects of three different alginate compositions (low, intermediate and high mannuronate), concentration of alginate, (1.5, 3.0 and 4.5% w/v) and concentration of cross-linking agent, CuSO4 (0.075, 0.15 and 0.225 M) on the decolorisation of indigo carmine dye and residual laccase activity in beads. The most significant factor for residual activity was the concentration of the cross-linking agent (P

Physiological regulation of laccase production from Ganoderma sp. KU-Alk4, isolated in Thailand, was controlled by the initial glucose concentration in liquid culture. Different laccase isozymes were produced using different starting concentrations of glucose. With 1% glucose, two isozymes, KULac 1 and 2 were produced, while with 4% glucose, three different isozymes, KULac 3, 4 and 5, were produced. The KULacs differed in their molecular mass, ranging from 53 to 112 kDa. KULac 2 was a new laccase that had a different N-terminal amino acid sequence from other laccases previously reported. All the isozymes had optimum pH at 3.5 and were stable over the wide range of pH, 3.0–10.0, especially in alkaline pH. It is noteworthy that the activities of the four KULacs with 2,6-dimethoxyphenol were extremely high up to 90°C. They retained 100% of their activities at 60°C for 1 h.

To engineer the production of laccase by Ganoderma sp. KU-Alk4, a newly isolated white-rot fungus, a seven-level Box−Behnken factorial design was employed to optimize the culture medium composition. A mathematical model was developed to show the effect of each medium component and their interactions on the production of laccase activity in submerged fermentation. The model estimated the optimal concentrations of glycerol, yeast extract and veratryl alcohol as 40, 0.22 g/l and 0.85 mM, respectively, with the medium pH of 6.0. These predicted conditions were verified by validation experiments. The optimized medium gave laccase activity of 240 U/ml, which is 12 times higher than that produced in non-optimized medium. Thus, this statistical approach enabled rapid identification and integration of key medium parameters for Ganoderma sp. KU-Alk4, resulted the high laccase production.