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.

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.

Abstract16 A repeated batch of synthetic dye decolorization was efficiently demonstrated in a 5 L airlift 17 bioreactor. A laccase from Ganoderma sp. KU-Alk4, degrading commercial aromatic dyes was 18 selected. The crude enzyme extract expressed laccase activity, and was immobilized under optimal 19 conditions in copper-alginate beads, 3 IU/bead. The immobilized enzyme showed high efficiency 20 in degrading various synthetic dyes under non-buffered conditions, in particular the indigoid dye 21 Indigo Carmine. The immobilized laccase also showed marked increase in stability toward 22 temperature and pH when compared with free enzyme preparation. Immobilization enhanced its 23 temperature stability to maintain initial activity up to 55 °C, ten degrees higher than the free 24 enzyme. The immobilized laccase was stable in the alkaline region up to pH 10.0. The dye 25 decolorization system in 5 L airlift bioreactor was demonstrated with 25 mg/L Indigo Carmine 26 dissolved in tap water and a total immobilized laccase activity of 6x10 4 IU. Airflow rate was the 27 most important factor affecting the number of batch runs and the time for 100% dye degradation. 28 An optimal airflow rate was of 4 L/min. Fourteen batch runs of complete dye degradation were 29 successfully completed with only a single enzyme supplementation, and this could be a feasible 30 system for operation in industry. Total dye degraded by this repeated process at 4 L/min airflow 31 rate was 1.8 g. Isatin sulfonic acid was a metabolic product of Indigo Carmine degradation 32 catalyzed by the immobilized laccase. This development of an effective repeatable bioprocess 33 using enzymes for the treatment of dye-contaminated effluent has potential for implementation on 34 an industrial scale.

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.

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 9 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, CuSO 4 (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 \ 0.05) followed by alginate composition (P \ 0.1). Increasing the alginate concentration resulted in only small increase in the dye decolorisation. However, higher laccase activity remained in 3.0% w/v alginate beads. Maximal dye decolorisation was achieved when 3.6% w/v low mannuronate alginate and 0.15 M CuSO 4 was used. Optimal conditions were confirmed in an extended experimental run. Results are presented from 9 successive batch runs over 12 days, reaching 96% removal of the dye (216 mg/l).