Optimization of media components for laccase production by litter dwelling fungal isolate Fusarium incarnatum LD‐3

Laccase production by solid state fermentation (SSF) using an indigenously isolated litter dwelling fungus Fusarium incarnatum LD‐3 was optimized. Fourteen medium components were screened by the initial screening method of Plackett‐Burman. Each of the components was screened on the basis of ‘p’ (probability value) which was above 95% confidence level. Ortho‐dianisidine, thiamine HCl and CuSO₄ · 5 H₂O were identified as significant components for laccase production. The Central Composite Design response surface methodology was then applied to further optimize the laccase production. The optimal concentration of these three medium components for higher laccase production were (g/l): CuSO₄ · 5 H₂O, 0.01; thiamine HCl, 0.0136 and ortho‐dianisidine, 0.388 mM served as an inducer. Wheat straw, 5.0 g was used as a solid substrate. Using this statistical optimization method the laccase production was found to increase from 40 U/g to 650 U/g of wheat straw, which was sixteen times higher than non optimized medium. This is the first report on statistical optimization of laccase production from Fusarium incarnatum LD‐3. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Optimization of laccase production by Pleurotus ostreatus IMI 395545 using the Taguchi DOE methodology

Production of laccase using a submerged culture of Pleurotus orstreatus IMI 395545 was optimized by the Taguchi orthogonal array (OA) design of experiments (DOE) methodology. This approach facilitates the study of the interactions of a large number of variables spanned by factors and their settings, with a small number of experiments, leading to considerable savings in time and cost for process optimization. This methodology optimizes the number of impact factors and enables to calculate their interaction in the production of industrial enzymes. Eight factors, viz. glucose, yeast extract, malt extract, inoculum, mineral solution, inducer (1 mM CuSO₄) and amino acid (l‐asparagine) at three levels and pH at two levels, with an OA layout of L18 (2¹ × 3⁷) were selected for the proposed experimental design. The laccase yield obtained from the 18 sets of fermentation experiments performed with the selected factors and levels was further processed with Qualitek‐4 software. The optimized conditions shared an enhanced laccase expression of 86.8% (from 485.0 to 906.3 U). The combination of factors was further validated for laccase production and reactive blue 221 decolorization. The results revealed an enhanced laccase yield of 32.6% and dye decolorization up to 84.6%. This methodology allows the complete evaluation of main and interaction factors. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Production of laccase from Trametes trogii TEM H2: a newly isolated white‐rot fungus by air sampling

This work represents the first report of isolation of potential laccase producers by air sampling using media supplemented with 2,2‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonate) and guaiacol for laccase production and secretion indicators. Nine fungal isolates showed positive reactions with 2,2‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonate) and guaiacol. The isolate named TEM H2 exhibited the largest and intensive oxidation zones with 2,2‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonate) (85 mm) and guaiacol (66 mm) and therefore it was selected for detailed investigations. The strain was identified as Trametes trogii TEM H2 due to the morphological characteristics and the comparison of internal transcribed spacer ribosomal DNA gene sequences. The laccase production was screened in different liquid cultures. The best laccase production medium was determined as soluble starch yeast extract medium in which laccase production was reached to a maximum level (989.6 U l^–1) on the 8^th day of cultivation. Effects of different initial pH values on laccase production were tested. Optimum pH value for laccase production in soluble starch yeast extract medium was determined as pH 3.0 with 15425.0 U l^–1laccase production at 12^th day of cultivation. In addition, effects of eight inducers (veratryl alcohol, ferulic acid, 1‐Hydroxybenzotriazole, syringic acid, 2,2‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonate), 1 mmol l^–1 CuSO₄, 3% ethanol, guaiacol) were examined. Only cultures with 2,5‐xylidine exhibited 1.9 fold increase in laccase activity reaching to 28890.0 U l^–1. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Production of laccase from Pleurotus florida using agro‐wastes and efficient decolorization of Reactive blue 198

Pleurotus florida NCIM 1243 produced laccase as the dominant lignolytic enzyme during the dye decolorization. Banana peel was the best substrate for extracellular laccase production under solid state fermentation when compared to mandarin peel and cantaloupe peel. The maximum activity of laccase (5.4 U/g) was detected on the 10 day. The ratio of banana peel: mandarin peel: cantaloupe peel (5:2:3) showed increased production of laccase (6.8 U/g). P. florida produced two extracellular laccase isoenzymes (L1 and L2). The half life of laccase at 60 °C was 2 h and at 4 h it retained 25% residual activity. P. florida laccase showed high thermostability and an interesting difference was noticed in the behavior of laccase isoenzymes at different temperature. The L1 isoenzyme of laccase showed remarked thermostability at 60 °C in the native PAGE when compared to L2 isoenzyme. The optimum pH, temperature and enzyme concentration for maximum decolorization was found to be 4.5, 60 °C and 1.2 U/ml, respectively. Partially purified laccase enzyme showed excellent decolorization activity to Reactive blue 198. The maximum decolorization (96%) was observed at lower dye concentrations (50–100 ppm) which decreased markedly when the dye concentration was increased beyond 150 ppm. The thermostable laccase of P. florida could be effectively used to decolorize the synthetic dyes in the textile effluent and other biotechnological applications. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Evaluation of laccase production by Ganoderma lucidum in submerged and solid‐state fermentation using different inducers

Laccases are multicopper oxidases with high potential for industrial applications. Several basidiomycete fungi are natural producers of this enzyme; however, the optimization of production and selection of inducers for increased productivity coupled with low costs is necessary. Lignocellulosic residues are important lignin sources and potential inducers for laccase production. Pinus taeda, a dominant source of wood‐based products, has not been investigated for this purpose yet. The aim of this study was to evaluate the production of laccase by the basidiomycete fungus Ganoderma lucidum in the presence of different inducers in submerged and solid‐state fermentation. The results of submerged fermentation in presence of 5 μM CuSO ₄, 2 mM ferulic acid, 0.1 g/L P. taeda sawdust, or 0.05 g/L Kraft lignin indicated that although all the tested inducers promoted increase in laccase activity in specific periods of time, the presence of 2 mM ferulic acid resulted in the highest value of laccase activity (49 U/L). Considering the submerged fermentation, experimental design following the Plackett–Burman method showed that the concentrations of ferulic acid and P. taeda sawdust had a significant influence on the laccase activity. The highest value of 785 U/L of laccase activity on submerged fermentation was obtained on the seventh day of cultivation. Finally, solid‐state fermentation cultures in P. taeda using ferulic acid or CuSO ₄ as inducers resulted in enzymatic activities of 144.62 and 149.89 U/g, respectively, confirming the potential of this approach for laccase production by G. lucidum.     

Laccase production by Pycnoporus sanguineus under different culture conditions

Pycnoporus sanguineus is a white‐rot fungus that produces ligninolytic enzymes such as laccases. These enzymes can endure temperatures as high as 60 °C and are useful for pulp bleaching, dye decolorization and phenolic degradation. Laccase production by fungi depends not only on the carbon and nitrogen sources but also on the nitrogen concentration of the culture medium. In this work, we examined the effect of four carbon sources (maltose, glucose, fructose and sucrose) and four nitrogen sources (ammonium tartrate, sodium nitrate, asparagine and yeast extract) on the activity of laccase from Pycnoporus sanguineus. All carbon and nitrogen sources exhibited a strong influence on laccase activity, a sucrose–asparagine medium providing the best results (320 mU/ml). Moreover, using an asparagine concentration 5 times higher than the reference level increased laccase activity to 820 mU/ml. Higher asparagine concentrations, however, resulted in no further increase in activity. Consistent with previous results, the carbon and nitrogen sources, and the nitrogen concentration, had a strong impact on laccase activity, the optimum conditions depending on the particular fungus. The conditions of the culture medium had a marked effect on laccase activity, which increased up to 820 mU/ml. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Production of extracellular protease by Aspergillus tamarii

The production of protease by Aspergillus tamarii was investigated in submerged fermentation using different substrates. Low basal proteolytic activity was detected on cultures with casein or gelatin as the only substrate (16–20 U/ml). The enrichment of protein medium with glucose increased the protease production up to 6 times (110 U/ml). The highest proteolytic levels were obtained in cultures supplemented with wheat bran (120 U/ml) and soybean meal (161 U/ml). One major proteolytic band with estimated molecular mass of 48 KDa was detected by SDS‐PAGE. The acetone‐precipitated enzyme was active and stable over a wide range of pH (6–9.5), and temperature (30–55 °C). The enzyme was stable for more than ten hours at temperatures up to 45 °C. The half‐lives at 50 and 55 °C were 90 min and 12 min, respectively. In presence of 5.0 mM CaCl₂, the half‐life of the enzyme at 55 °C was increased to 130 min.     

Laccase from an alkalitolerant basidiomycetes Crinipellis sp. RCK-1: production optimization by response surface methodology

A newly isolated alkalitolerant basidiomycetous fungus, identified as Crinipellis sp. RCK-1 was observed to produce laccase. The effect of different physicochemical factors on laccase production was studied. The identification of the important factors (initial pH of the medium, copper and tryptophan) with simple screening experiment involving optimization using single factor at a time strategy, was followed by application of complex response surface design for further maximizing the laccase production and was helpful in defining the effects and interactions of the physiological and nutritional factors. The statistical optimization by response surface methodology resulted in a 27.0-fold (619.9 U ml(-1)) increase in the production of laccase from Crinipellis sp. RCK-1 when compared to laccase production in unoptimized medium (23.0 U ml(-1)). The results from the response surface curve suggested that there was interaction between tryptophan and copper in a way that might resulted in positive effect on laccase production from Crinipellis sp. RCK-1. The relatively higher laccase production by Crinipellis sp. RCK-1 showed promise of offering great potential in various biotechnological applications.     

Isolation,characterization, and interaction of lignin-degrading bacteria from rumen of buffalo (Bubalus bubalis)

The purpose of this study was to isolate lignin-degrading bacteria from buffalo rumen and to explore their interactions further. Using lignin as the carbon source, three bacteria, B-04 (Ochrobactrum pseudintermedium), B-11 (Klebsiella pneumoniae), and B-45 (Bacillus sonorensis), which have shown lignin degradation potential, were successfully isolated and identified from the rumen fluid of buffalo by colony morphology, 16S ribosomal RNA gene sequencing, and biochemical and physiological analyses. The degradation rates of lignin were determined, and the maximum values were 4.86%, 11.1%, and 7.68% for B-04, B-11, and B-45, respectively. The maximum laccase activities were 0.65, 0.93, and 1.15 U/ml, while the maximum lignin peroxidase activities were 5.72, 8.29, and 18.69 U/ml, respectively. Pairwise interaction studies showed inhibitory interaction between B-04 and B-45, inhibitory interaction between B-04 and B-11, and symbiotic interaction between B-11 and B-45. This is the first report on the lignin degradation ability of bacteria isolated from the buffalo's rumen, which provides a new understanding for revealing the mechanism of roughage tolerance of buffalo.     

Sequential parametric optimization of lipase production by a mutant strain Rhizopus sp. BTNT-2

Lipase production by the mutant strain Rhizopus sp. BTNT-2 was optimized in submerged fermentation. Different chemical and physical parameters such as carbon sources, nitrogen sources, oils, inoculum level, pH, incubation time, incubation temperature and aeration have been extensively studied to increase lipase productivity. Potato starch (1.25% w/v) as a carbon source, corn steep liquor (1.5% w/v) as a nitrogen source and olive oil (0.5% v/v) as lipid source were found to be optimal for lipase production. The optimal levels of other parameters are 4 ml of inoculum (2.6x10(8) spores/ml), initial pH of 5.5, incubation time of 48 hours, incubation temperature of 28 degrees C and aeration rate of 120 rpm. With the optimized parameters, the highest production of lipase was 59.2 U/ml while an yield of only 28.7 U/ml was obtained before optimization resulting in 206% increase in the productivity.     

Facile Route for the Synthesis of Adamantane‐Containing Polypeptides through Polycondensation of Activated Urethane Derivative of α‐Amino Acids

A facile route has been discovered for the synthesis of polypeptides containing adamantane moieties in the side chain, poly(Nδ‐adamantyl‐l ‐glutamine) P1 and poly(Nγ‐adamantyl‐l ‐asparagine) P2 , through polycondensation of an N‐phenoxycarbonyl derivative of the corresponding Nδ‐adamantyl‐l ‐glutamine 1 and Nγ‐adamantyl‐l ‐asparagine 2 , respectively, along with the elimination of phenol and CO₂. The urethane derivatives are readily synthesized by the N‐carbamylation of tetrabutylammonium salts of α‐amino acids with diphenyl carbonate. Polycondensation of the urethane derivative proceeds smoothly upon heating to 60 °C in N,N‐dimethylacetamide using n‐butylamine (n‐BuNH₂) as an initiator. Size exclusion chromatography, ¹H NMR spectra, and matrix‐assisted laser desorption/ionization time‐of flight mass spectrometry analyses reveal that polycondensation of 1 with n‐BuNH₂ yields a well‐defined polypeptide in terms of molecular weight and terminal structure. On the other hand, polycondensation of 2 affords poor molecular weight control and leads to the formation of oligopeptides. Furthermore, using amine‐terminated poly(ethylene‐glycol) in place of n‐BuNH₂ yields a diblock copolymer composed of polyether and polypeptide segments bearing an adamantane moiety through polycondensation of the urethane derivative.

     

Microbial production of docosahexaenoic acid by a low temperature‐adaptive strain Thraustochytriidae sp. Z105: screening and optimization

As an alternative source in addition to fish oil, microbial production of docosahexaenoic acid has been recieved more and more attentions owing to their culture advantage. A unicellular eukaryotic microbe with high DHA production and capable of low temperature‐adaptive growth was isolated from seawater and identified as Thraustochytriidae sp. Z105. The siginificant effect of temperature on cell growth and DHA synthesis by the strain was revealed. It could grow and produce DHA even at 4 °C, but hardly grow above 35 °C. Low temperature (15–25 °C) was favorable for formation of biomass, lipids and DHA, but DHA synthesis was completely blocked above 30 °C. Conditions for high level DHA production by Thraustochytriidae sp. Z105 in flask culture were optimized as follows: medium containing glucose 80 g/l, yeast extract 5.0 g/l, K₂HPO₄ · 3 H₂O 1.0 g/l, MgSO₄ · 7 H₂O 0.5 g/l, seawater crystal 20 g/l, pH 6.0, liquid volume 30 ml/250 ml, temperature 20 °C, agitation speed of 200 r/min, and culture for 120 h. Under the optimal conditions, biomass of 16.72 g/l, total lipids of 5.35 g/l, DHA yield of 1.71 g/l (accounting for 32% of the total lipids) were achieved, respectively. In flask cluture level, the DHA productivity of Thraustochytriidae sp. Z105 was higher than most reported results, which suggested the wild type strain was a potential superior candidate for industrialization of DHA production. Moreover, the strain is an unique and valuable resource for investigation of the low temperature adaptive mechanism related to DHA synthesis. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

L‐methioninase production by Aspergillus flavipes under solid‐state fermentation

Solid‐state fermentation was carried out for the production of extra‐cellular L‐methioninase by Aspergillus flavipes (Bain and Sart.) using nine agro‐industrial residues, namely wheat bran, rice bran, wheat flour, coconut seeds, cotton seeds, ground nut cake, lentil hulls, soya beans and chicken feathers. Chicken feathers were selected as solid substrate for L‐methioninase production by A. flavipes. The maximum L‐methioninase productivity (71.0 U/mg protein) and growth (11 mg protein/ml) of A. flavipes was obtained using alkali pretreated chicken feathers of 50% initial moisture content as substrate supplemented with D‐glucose (1.0% w/v) and L‐methionine (0.2% w/v). External supplementation of the fermentation medium with various vitamin sources has no overinductive effect on L‐methioninase biosynthesis. The partially purified A. flavipes L‐methioninase preparation showed highest activity (181 U/ml) at pH 8.0 with stability over a pH range (pH 6–8) for 2 h. L‐methioninase activity was increased by preincubation of the enzyme for 2 h with Co²⁺, Mn²⁺, Cu²⁺ and Mg²⁺ and strongly inhibited by the presence of EDTA, NaN₃, Li²⁺, Cd²⁺, DMSO and 2‐mercaptoethanol. The enzyme preparation has a broad substrate spectrum showing a higher affinity to deaminate L‐glycine, N ‐acetylglucosamine and glutamic acid, in addition to their proteolytic activity against bovine serum albumin, casein, gelatin and keratin. The partially purified enzyme was found to be glyco‐metalloproteinic in nature as concluded from the analytical and spectroscopic profiles of the enzyme preparation. The demethiolating activity of the enzyme was also visualized chromogenially. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Sustainable use of the spent mushroom substrate of Pleurotus florida for production of lignocellulolytic enzymes

Spent mushroom substrate (SMS), a major byproduct of the mushroom industry, is a lignocellulosic biomass, which contains approximately 57–74.3% of holocellulose fraction. This study was aimed at utilizing SMS of Pleurotus florida for recovery of lignocellulolytic enzymes and sugars and also as a substrate for production of cellulolytic enzymes using different isolates of Trichoderma and Aspergillus under solid-state fermentation (SSF). SMS of P. florida extracts contained significant amounts of laccase (3,015.8 ± 29.5 U/g SMS) and xylanase (1,187.9 ± 12 U/g SMS) activity. Crystallinity pattern and chemical changes in SMS revealed that SMS had a lower crystallinity index (34.2%) as compared with the raw biomass (37.8%), which, in turn, helps in enhancing the accessibility of cellulolytic enzymes to holocellulose. Among the isolates, Trichoderma longibrachiatum A-01 showed maximum activity of endoglucanase (220.4 ± 5.9 U/mg), exoglucanase (78.5 ± 3.2 U/mg) and xylanase (1,550.4 ± 11.6 U/mg) while Aspergillus aculeatus C-08 showed maximum activity of cellobiase (113.9 ± 3.9 U/mg). Extraction with sodium citrate buffer (pH 4.8) showed maximum cellulolytic enzyme activity as compared with other solvents tested. Partial purification of endoglucanase, exoglucanase, xylanase, and cellobiase resulted in 56.3% (1,112.5 U/mg), 48.4% (212.5 U/mg), 44% (4,492.3 U/mg), and 62% (705.0 U/mg) yield with an increase by 5.2-, 4.5-, 4.1-, and 5.0-fold as compared with crude extract. The results reveal that SMS from P. florida could be a potential and cost-effective substrate for production of cellulolytic enzymes from T. longibrachiatum A-01 and A. aculeatus C-08.     

Production of extracellular enzymes during growth and autolysis of Pycnoporus cinnabarinus

Changes in cultural parameters and the production of some extracellular enzymes during growth and autolysis of the white-rot fugus Pycnoporus cinnabarinus have been studied. At the end of the autolytic period (52 days of incubation) the degree of autolysis was greater than 50%. The laccase activity reached its highest value during the growth period, when the level of the carbon source in the medium was still comparatively high. When the fungus was cultivated in the presence of kraft lignin (Indulin AT) 0.05% (w/v), the activity of laccase was 3 times higher than when grown in the absence of lignin. The presence of lignin did not affect the activities of 1,3-β-glucanase, exoxylanase and proteases.     

Water‐soluble red pigments from Isaria farinosa and structural characterization of the main colored component

The present study describes the red pigment synthesized by the filamentous fungi Isaria farinosa under submerged culture conditions. The pigment production was optimal under the following conditions: pH 5, agitation speed 150 rpm, temperature 27 °C, incubation time 192 h, light source total darkness, sucrose and glucose as carbon source, yeast extract, meat peptone and monosodium glutamate at a fixed concentration of 3% as nitrogen source. The addition of 10 mM CaCl₂ to the culture medium increased the biomass and pigment production. Structural elucidation of the pigment using gas chromatography‐mass spectrometry, Fourier transform infrared spectroscopy and ¹H nuclear magnetic resonance spectroscopy revealed that the red pigment contains an anthraquinone‐related compound. In addition, the isolated pigment was water soluble, and was stable when exposed to salt solution (96.1% of stability after treatment with sodium chloride), acid (72.1% with citric acid), heat (86.2% at 60 °C), and sunlight (99.4%). These results are promising to further exploit the fungal culture of Isaria farinosa for producing the red pigment and, subsequently, to considerably increase its yield. The study has commercial importance in the production of Isaria farinosa pigment for industrial application after considerable toxicological examination. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Optimization of different process variables for the production of an indolizidine alkaloid, swainsonine from Metarhizium anisopliae

Swainsonine is a polyhydroxylated indolizidine alkaloid having anticancer, antimetastatic, antiproliferative and immunomodulatory activities and also potential therapeutic applications against AIDS. In the present study, ten isolates of M. anisopliae were screened and enzyme assayed for the production of swainsonine in different media (Complex oatmeal, Czapekdox media with and without lysine (8% w/v) and Sabouraud dextrose broth (SDB)). Among these strains, ARSEF 1724 (UM8) was found to produce highest amount of swainsonine (1.34 μg/l) after 72 h of incubation under shake flask conditions at 180 rpm and 28 °C in complex oatmeal media. In order to maximize the yield of swainsonine the media composition including macro and micronutrients were optimized. The process variables including the chemical factors like carbon sources, nitrogen sources of both organic and inorganic nature and pH with constant inoculum size (1 × 10⁸ spores/ml) were screened using classical one‐factor‐at‐a‐time (OFAT) approach to find their optimum levels. The present study shows that the nutrient requirement is specific for each strain of Metarhizium. Oatmeal extract (6%) was found to be the best supporting media along with nitrogen source, glucose (2%) as best carbon source and pH (～5) as the best for swainsonine production. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Optimization of medium and process parameters for the production of lipase from an oil‐tolerant Aspergillus sp. (RBD‐01)

Extracellular lipase production by Aspergillus sp. (RBD‐01) was monitored by modulating pH of the growth medium, ambient temperature for growth, source of nitrogen and percentage of carbon (virgin cottonseed oil). This strain was observed to be viable and produces lipase even up to 50% oil as a main carbon source. Maximum lipase activity of 21.8 U/ml was obtained with 50% (v/v) oil acting as the main carbon source and peptone (0.5% w/v) as nitrogen source. The optimum pH and temperature for enzymatic activity were observed to be 7.5 and 35 °C, respectively. The observations are of significance due to limited reports on use of 50% of oil as the main carbon source while obtaining significant lipase activity of 21.8 U/ml. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A radiochemical test for phospholipase A2 catalytic activity

Summary A position-specifically labelled phosphatidylcholine is the substrate for the selective determination of Phospholipase A₂ in serum, ascites and tissue samples. Optimal reaction conditions and simplifications of handling are discussed.A control group of human serum samples ranged up to 2.1 U/l. The maximum serum activity in samples of patients with acute pancreatitis was 126 U/l. In human ascites activities up to 380 U/l were measured. The method described here turned out to be a practicable instrument for the determination of phospholipase A₂ activity using only commercially available reagents.Abbreviation PlA₂ Phospholipase A₂     

Expression,purification, and characterization of a novel laccase from Setosphaeria turcica in Eschericha coli

Laccases are multicopper oxidases (E.C. 1.10.3.2) that catalyze the oxidation of many phenolic compounds. In this study, a novel laccase, Stlac4, from Setosphaeria turcica was cloned and expressed in Escherichia coli by insertion into the pET‐30a expression plasmid. The recombinant laccase was purified and visualized on SDS–PAGE as a single band with an apparent molecular weight of 71.5 KDa, and confirmed by Western blot. The maximum activity of the purified laccase was 127.78 U · mg^?1, the optimum temperature and pH value were 60 °C and 4.0 respectively, measured by oxidation of 2,2′‐Azinobis‐(3‐ethylbenzthiazoline‐6‐sulphonate) (ABTS). Purified laccase activity under different metal ions and an inhibitor were tested, revealing that laccase activity increased by approximately 434.8% with Fe³⁺, and 217.4% with Cu²⁺ at 10 mmol · L^?1 concentrations, Mn²⁺ increased the laccase activity only at 5 mmol · L^?1, while Na⁺ increased activity at 1 mmol · L^?1 but inhibited activity at 5 and 10 mmol · L^?1. SDS increased laccase activity at 1 mmol · L^?1, and inhibited activity at 5 and 10 mmol · L^?1.