Stable Laccase Research Articles

Spawn-based pellets of Pleurotus ostreatus as an applied approach for the production of laccase in different types of water.

In recent years, oxidoreductase enzymes such as laccases have received considerable attention for their ability to degrade and eliminate organic micropollutants from contaminated water in a process known as enzyme-based wastewater treatment. Thus, methods to produce high laccase activity in water are a point of focus, with white-rot fungi being highlighted as a tool in this context. This study, therefore, explored the applied approach of direct addition of mushroom spawn of the white-rot fungi Pleurotus ostreatus into water and its potential for laccase production under different conditions. Grain spawn was observed to be preferable to sawdust spawn, resulting in laccase activity of 53.9±5.9U/L and 4.8±0.8U/L, respectively. Laccase activity was induced by adding kraft lignin (4g/L), and an eightfold increase to 446.3±43.1U/L was observed for grain spawn. Lignin accumulated in the spawn over time, resulting in brown pellets composed of spawn, mycelium and lignin. Our results demonstrated that high levels of laccase activity could be obtained in different types of water, including effluent municipal wastewater, using this method. No impact from the addition of inorganic nitrogen (ammonium nitrate, N-levels 14mg/L, 140mg/L) or organic nitrogen sources (urea, yeast extract, wheat bran, N-levels 14mg/L, 140mg/L) was observed for the treatment with grain spawn and lignin, suggesting that stable laccase activity can be expected under these nutritional conditions.

Production and characterization of a highly stable laccase from extreme thermophile Geobacillus stearothermophilus MB600 isolated from hot spring of Gilgit Balitistan (Pakistan)

Extracellular thermophilic multicopper oxidase i.e. laccase was purified, identified and characterized from Geobacillus stearothermophilus MB600. Fast protein liquid chromatography (FPLC) of lacasse showed a prominent peak at 610 nm. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for laccase presented a molecular weight of 59.13 kDa. Analysis via Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) confirmed the presence of laccase similar to in Geobacillus thermoleovorans B23 and Geobacillus sp. LEMMY01. Laccase showed Km 0.532 mM for guaiacol and 0.307 mM for ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)). Its optimum activity was witnessed at 90°C, at pH 5.0. Laccase catalytic activity was completely inhibited by sodium dodecyl-sulfate (SDS), sodium azide (NaN3), and ethylenediaminetetraacetic acid (EDTA). Among the metal salts against Cu, Ca, Cd, and Li enhanced while Mg inhibited the enzyme activity. It was stable against Cl, I and solvents (Ethanol > Methanol > Dimethyl sulfoxide > Acetonitrile). Thermophilic laccase effectively degraded 93% of Remazol brilliant blue R, 92% Bisphenol A diglycidyle ether without mediator.

Optimisation and physicochemical characterisation of a thermo-alkali stable laccase produced by wastewater associated Bacillus sp. NU2

ABSTRACT Laccase is a multicopper enzyme that plays a unique role in bioremediation of environmental pollutants. Bacteria were isolated from hospital wastewater and screened for laccase production. The laccase production process condition was optimised, and the laccase obtained was characterised. The 16S rRNA molecular analysis conducted on the best laccase producer revealed a Bacillus sp. NU2 identified. The process conditions: pH5, 45°C, 100 rpm, 5% inoculum, and growth constituents viz: tangerine peel and wheat bran agro-wastes, beef extract, ammonium persulfate, glucose, galactose, xylose, sorbitol, fructose carbon sources; and 4-aminophenol inducer optimally stimulated laccase production. The Bacillus sp. NU2 laccase was optimal at pH and temperature conditions of 8.0°C and 60°C, with a noteworthy pH and thermal stability observed. Furthermore, NU2 laccase showed a moderate/high tolerance and relative activity effect on various chemical inhibitors, halides and surfactant of triton x-100 (105 ± 0.92%), PMSF (107 ± 0.81%), and NaCl (94 ± 0.81%) at 1, 3, and 6 (mM) concentration. Additionally, NU2 laccase maintained a relative activity of 101%, 104%, and 102% for Mg2+, Zn2+, and Fe3+ at 1, 3, and 6 mM respectively. Acetone and propanol significantly upregulated laccase activity at 114 ± 0.0008% and 118.24 ± 0.35 and also at 30 and 20 (%) concentrations. Conclusively, the tolerant effect of Bacillus sp. NU2 laccase in pH, temperature, inhibitors and organic solvents suggests its potential for biotechnological application and promotion of a greener environment.

Deciphering the alkaline stable mechanism of bacterial laccase from Bacillus pumilus by molecular dynamics simulation can improve the decolorization of textile dyes.

Laccases are considered promising tools for removing synthetic dyes from textile and tannery effluents. However, the alkaline pH in the effluents causes laccase instability, inactivation, and difficulty in its bioremediation. Based on a Bacillus pumilus ZB1 (BpLac) derived alkaline stable laccase, this study aimed to elucidate its alkaline stable mechanism at molecular level using molecular dynamics simulation. The effects of metal ions, organic solvents, and inhibitors on BpLac activity were assessed. BpLac formed more salt bridges and negatively charged surface in alkaline environment. Thereafter, pH-induced conformation changes were analyzed using GROMACS at pH 5.0 and 10.0. Among the identified residues with high fluctuation, the distance between Pro359 and Thr414 was stable at pH 10.0 but highly variable at pH 5.0. DSSP analysis suggested that BpLac formed more β-sheet and less coil at pH 10.0. Principal component analysis and free energy landscape indicated that irregular coils formed at pH 5.0 benefit for activity, while rigid α-helix and β-sheet structures formed at pH 10.0 contributed to alkaline stability. Breaking the α-helix near T1 copper center would not reduce alkaline stability but could improve dye decolorization by BpLac. Overall, these findings would advance the potential application of bacterial laccase in alkaline effluent treatment.

Enzymatically triggered delignification through a novel stable laccase: A mixed in-silico /in-vitro exploration of a complex environmental microbiota

Laccases have been broadly applied as a multitasking biocatalyst in various industries, but their applications tend to be limited by easy deactivation, lack of adequate stability, and susceptibility under complex conditions. Identifying stable laccase as a green-biocatalyst is crucial for developing cost-effective biorefining processes. In this direction, we attempted in-silico screening a stable metagenome-derived laccase (PersiLac1) from tannery wastewater in a complex environment. The laccase exhibited high thermostability, retaining 53.19% activity after 180 min at 70 °C, and it was stable in a wide range of pH (4.0–9.0). After 33 days of storage at 50°C, pH 6.0, the enzyme retained 71.65% of its activity. Various metal ions, inhibitors, and organic solvents showed that PersiLac1 has a stable structure. The stable PersiLac1 could successfully remove lignin and phenolic from quinoa husk and rice straw. In the separate hydrolysis and fermentation process (SHF) after 72 h, hydrolysis was obtained 100% and 73.4% for quinoa husk and rice straw, and fermentation by the S. cerevisiae was be produced 41.46 g/L and 27.75g/L ethanol, respectively. Results signified that the novel lignin-degrading enzyme was confirmed to have great potential for industrial application as a green-biocatalyst based on enzymatically triggered to delignification and detoxify lignocellulosic biomass.

High efficiency of osmotically stable laccase for biotransformation and micro-detoxification of levofloxacin in the urea-containing solution: Catalytic performance and mechanism

Laccase-catalyzed oxidation was applied in the biotransformation of levofloxacin (a potentially environmental antibiotic contamination); however, the enzyme may denature in urea-containing wastewater and lead to the formation of an inactive form followed by decreasing the yield of the bio-removal. In this study, the osmolytes-stabilized laccase was used to eliminate levofloxacin in the urea-containing solution. Sorbitol and proline 100 mM appeared to be the two most efficient laccase protectants against the urea-induced denaturation. In a 1-M urea solution, the maximum velocity (Vmax) of laccase was estimated to be 39.1 μmol min–1 mg–1. This value was improved to 101.7 and 51.8 μmol min–1 mg–1 in the presence of sorbitol and proline, respectively. In optimal conditions for the elimination of levofloxacin, sorbitol- and proline-treated laccase led to 82.9 % and 76.2 % bio-removal of the applied fluoroquinolone in 1 M urea solution, respectively. Biotransformation products of the parent antibiotic were spectroscopically analyzed that assigned to different reaction pathways including demethylation, defluorination, decarboxylation, deamination, and hydroxylation. A micro-toxicity study concerning the growth of some Gram+ and Gram– bacteria exhibited decreasing in inhibition of laccase-treated levofloxacin after a 10-h incubation at 37 °C.

Evaluation of laccase production by two white-rot fungi using solid-state fermentation with different agricultural and forestry residues

Pleurotus ostreatus and a newly isolated Ganoderma lingzhi strain were evaluated for their laccase secretion capacity by solid-state fermentation with different agricultural and forestry residues. There was a significant difference among fungi for biosynthetic potential. In principle, the laccase secretion capacity of P. ostreatus CY 568 was stronger than that from G. lingzhi Han 500. Different species of fungi had a preference for agricultural and forestry residues. The presence of cottonseed hull and Populus beijingensis were helpful for accelerating the rate of laccase enzyme production of P. ostreatus CY 568. Cottonseed hull and corncob were useful for improving the production of laccase from G. lingzhi Han 500. Continuous and stable laccase production was found on cottonseed hull by P. ostreatus CY 568 and G. lingzhi Han 500. Maximum laccase activity obtained from P. ostreatus CY 568 on Toona sinensis, Sophora japonica, Salix babylonica, Populus beijingensis, corncob, cottonseed hull, and straw of Oryza sativa was higher than that from G. lingzhi Han 500, and was nearly 1.16-fold, 1.59-fold, 3.32-fold, 1.39-fold, 1.08-fold, 1.08-fold, and 1.36-fold, respectively. These findings will be helpful for developing new productive strains and expanding more species for industrial application to obtain efficient and low-cost laccase.

A comparative study on the laccase activity of four basidiomycete fungi with different lignocellulosic residues via solid-state fermentation

The laccase producing abilities of four Basidiomycete fungi species were compared using solid-state fermentation using four different lignocellulosic residues. The biosynthetic potential of the Basidiomycetes was highly dependent on the type of fungi. In general, the laccase secreting ability of Cerrena unicolor Han 849 was greater than Lenzites betulinus Han 851, Stropharia rugosoannulata Han 1321, and Auricularia heimuer Han 1333. The maximum laccase production of C. unicolor Han 849 was approximately 11.25, 122.26, and 15.27 times higher than L. betulinus Han 851, S. rugosoannulata Han 1321 and A. heimuer Han 1333, respectively. Different species of fungi had a preference in lignocellulosic residues. The presence of Firmiana platanifolia was conducive to secreting laccase via C. unicolor Han 849 during solid-state fermentation. A continuous and stable laccase production via C. unicolor Han 849 was an obvious advantage of solid-state fermentation with any of the four lignocellulosic residues used. The maximum laccase production of C. unicolor Han 849 using Firmiana platanifolia was approximately 2.12, 1.68, and 6.13 times higher than Populus beijingensis, Sorghum bicolor, and Oryza sativa, respectively. These findings will be helpful for developing new productivity strains in industrial applications and selecting suitable lignocellulosic residues for laccase production.

Partial purification and characterization of a thermophilic and alkali-stable laccase of Phoma herbarum isolate KU4 with dye-decolorization efficiency

Production of an extracellular thermophilic and alkali stable laccase from Phoma herbarum isolate KU4 was reported for the first time, both in submerged fermentation (SmF, highest 1590 U/mL) and solid state fermentation (SSF, highest 2014.21 U/mL) using agro-industrial residues. The laccase was partially purified to 7.93 fold with the apparent molecular weight of 298 kDa. The enzyme had pH optimum at 5.0 and temperature optimum at 50 °C, with maximum stability at pH 8.0. It showed activity towards various phenolic and non-phenolic compounds. The kinetic parameters, Km , Vmax and Kcat of the laccase for DMP were 0.216 mM, 270.27 U/mg and 506.69 s−1, respectively. Laccase activity was inhibited by various metal ions and conventional inhibitors, however, it was slightly increased by Zn2+. The laccase showed good decolorization efficiency towards four industrial dyes, namely, methyl violet (75.66%), methyl green (65%), indigo carmine (58%) and neutral red (42%) within 24 h. FTIR analysis of the decolorized products confirmed the degradation of the dyes. The decolorization efficiency of the enzyme suggests that the partially purified laccase could be used to decolorize synthetic dyes present in industrial effluents and for waste water treatments. The thermophilic and alkali stable laccase may also have wider potential industrial applications.

Lignocellulolytic enzyme activity pattern of three white oyster mushroom (Pleurotus ostreatus (Jacq.) P. Kumm.) strains during mycelial growth and fruiting body development

Pleurotus ostreatus is one of the edible mushrooms that can utilize lignocellulose as substrate because of their ability to secrete the lignocellulolytic enzyme. The purpose of this research is to investigate and compare the lignocellulolytic enzymes production of P. ostreatus InaCC F209, F216 and LIPI on solid-state fermentation using sawdust during 70 days of mycelial growth (vegetative phase) and fruiting body development (reproductive phase). Supernatant of the extracted enzyme solutions were employed to investigate the reducing sugar, soluble protein, and enzyme activities. The results revealed that reducing sugar concentration of the three P. ostreatus strains subjected increased during growth and reached the maximum concentration on the reproductive phase, while the total protein content fluctuated during the growth but reached the maximum concentration on the reproductive phase. Laccase, LiP, and MnP activities of three P. ostreatus strains were higher on the vegetative phase, while the endoxylanase and endoglucanase activities were higher on the reproductive phase. β-glucosidase activity showed different variations between three P. ostreatus strains. Pleurotus ostreatus InaCC F209 produced the highest and most stable laccase, β-glucosidase, endoglucanase, and endoxylanase than two others.

Purification and characterization of a novel white highly thermo stable laccase from a novel Bacillus sp. MSK-01 having potential to be used as anticancer agent

Laccases are multicoopper oxidases catalyzing the oxidation of phenolic as well as non-phenolic compounds. Laccases show typical blue color due to the presence of covalent Type 1 Cu-Cys bond which absorbs at 600 nm. However, recently some white laccases have also been identified which lacks typical spectra of blue laccases and do not show peak at 600 nm. In the present study, a novel white laccase was isolated from Bacillus sp. MSK-01. MSK laccase was purified and characterized in detail and the purified laccase was referred to MSKLAC. It has a molecular weight of 32 KDa. UV–visible spectrum of purified MSKLAC do not show characteristic peak at 600 nm and bend at 330 nm. The enzyme was repressed by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and β-mercaptoethanol. The laccase was highly thermo-stable enzyme having optimum temperature of 75 °C and could treasure more than 50% activity even at 100 °C. The optimum pH for ABTS and guaiacol was 4.5 and 8.0 respectively. MSKLAC was stable in the presence of most of the metal ions and surfactants. The effect of MSKLAC on lung cancer cell line was also assessed. It was observed that MSKLAC is inhibitory to lung cell cancer line. Thus, MSKLAC has potential to be used as an anti-proliferative agent to cancer cells.

Kinetic characterization of purified laccase from Trametes hirsuta: a study on laccase catalyzed biotransformation of 1,4-dioxane.

Laccase is one of the best known biocatalysts which degrade wide varieties of complex molecules that are both non-cyclic and cyclic in structure. The study focused on enzyme kinetics of a purified laccase from Trametes hirsuta L. fungus and its application on biotransformation of a carcinogenic molecule 1,4-dioxane. Laccase was purified from white-rot fungus T. hirsuta L. which showed specific activity of 978.34 U/mg after the purification fold of 54.08. The stable laccase activity (up to 16h) is shown at 4-6 pH and 20-40°C temperature range. The purified enzyme exhibited significant stability for 10 metal ions up to 10mM concentration, except for Fe2+ and Hg2+. The Cu2+ ion induced laccase activity up to 142% higher than the control at 10mM concentration. The laccase enzyme kinetic parameters Km was 20 ± 5µM and 400 ± 60µM, whereas Kcat was 198.29 ± 0.18/s and 80.20 ± 1.59/s for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol respectively. The cyclic ether 1,4-dioxane (100ppm) was completely degraded in presence of purified laccase within 2h of incubation and it was confirmed by HPLC and GC analysis. The oxidation reaction was accelerated by 25, 22, 6 and 19% in presence of 1mM syringaldehyde, vanillin, ABTS and guaiacol mediators respectively. In this study, fungal laccase (a natural biocatalyst) based degradation of synthetic chemical 1,4-dioxane was reported for the first time. This method has added advantages over the multiple methods reported earlier being a natural remedy.

Solid-state fermentation on poplar sawdust and corncob wastes for lignocellulolytic enzymes by different Pleurotus ostreatus strains

Solid state fermentation with different lignocellulolytic materials as inducers was used for lignocellulolytic enzyme production in this study. Pleurotus ostreatus strains were assessed by measuring laccase, CMCase, and xylanase activities. The secretion potential of the lignocellulolytic enzymes by wild and cultivated strains was analyzed for the first time. The wild and cultivated strain showed their unique capacities for secreting lignocellulolytic enzymes on solid-state fermentation with different lignocellulosic materials. The wild P. ostreatus strain preferred corncob for the secretion of laccase and xylanase activity, but the cultivated strain preferred poplar sawdust. The wild strain and cultivated strain showed a consistent preference for poplar sawdust for the secretion of CMCase activity. The wild strain was advantageous because it achieved the maximum hydrolytic enzyme activities within a short time period. Poplar sawdust and corncob were conducive to laccase secretion by the wild or cultivated strains and the rapid accumulation of laccase on solid-state fermentation. Additionally, continuous, stable laccase production was an extremely important advantage by solid-state fermentation of poplar sawdust, particularly in the wild strain. These findings are helpful in selecting the appropriate strain that corresponds to suitable lignocellulosic materials. The optimization of integrated industrial lignocellulolytic enzyme production can also be achieved.

High-level expression of Bacillus amyloliquefaciens laccase and construction of its chimeric variant with improved stability by domain substitution.

Large-scale application of bacterial laccases is usually limited by their low production, and their recombinant expression in Escherichia coli is prone to form inactive aggregates in the cytoplasm. In this work, we optimized the expression conditions of Bacillus amyloliquefaciens laccase (LacA) in E. coli, and obtained high yield for the extracellular production of LacA. The final activity reached 20,255 U/L for LacA, which is among one of the highest activities for recombinant bacterial laccases. Moreover, a chimeric enzyme (Lac3A/S) was designed based on LacA by domain substitution with a stable laccase from B. subtilis. The hybrid laccase could also be secreted into the culture medium with high expression level, and had higher thermal and alkaline stabilities than those of LacA. It was fully active after 10-day incubation at pH 9.0, and retained 47% of its initial activity after incubation at 70°C for 5h. Homology analysis of protein structure indicated Lac3A/S had a more packed structure in the copper-binding sites than LacA, which might lead to an enhancement in stability under harsh conditions.

A highly stable laccase from Bacillus subtilis strain R5: gene cloning and characterization.

The gene encoding copper-dependent laccase from Bacillus subtilis strain R5 was cloned and expressed in Escherichia coli. Initially the recombinant protein was produced in insoluble form as inclusion bodies. Successful attempts were made to produce the recombinant protein in soluble and active form. The laccase activity of the recombinant protein was highly dependent on the presence of copper ions in the growth medium and microaerobic conditions during protein production. The purified enzyme exhibited highest activity at 55 °C and pH 7.0. The recombinant protein was highly thermostable, albeit from a mesophilic source, with a half-life of 150min at 80 °C. Similar to temperature, the recombinant protein was stable in the presence of organic solvents and protein denaturants such as urea. Furthermore, the recombinant protein was successfully utilized for the degradation of various synthetic dyes reflecting its potential use in treatment of wastewater in textile industry. Abbreviations: ABTS,2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid; CBB, Coomassie brilliant blue; SGZ, syringaldazine; DMP, 2,2-dimethoxy phenol.

A highly stable laccase obtained by swapping the second cupredoxin domain

The robustness of a high-redox potential laccase has been enhanced by swapping its second cupredoxin domain with that from another fungal laccase, which introduced a pool of neutral mutations in the protein sequence without affecting enzyme functionality. The new laccase showed outstanding stability to temperature, pH (2–9) and to organic solvents, while maintaining the ability to oxidize high-redox potential substrates. By engineering the signal peptide, enzyme secretion levels in Saccharomyces cerevisiae were increased, which allowed to purify the engineered enzyme for further characterization. The purified domain-swap laccase presented higher activity in the presence of ethanol or methanol, superior half-lives at 50–70 °C, improved stability at acidic pH, and similar catalytic efficiency for DMP albeit a lower one for ABTS (due to a shift in optimum pH). A new N-glycosylation site and a putative new surface salt-bridge were evaluated as possible determinants for the improved stability by site-directed mutagenesis. Although neither seemed to be strictly responsible for the improved thermostability, the new salt bridge was found to notably contribute to the high stability of the swapped enzyme in a broad pH range. Finally, the application potential of the new laccase was demonstrated with the enzymatic treatment of kraft lignin, an industrially relevant lignin stream, at high temperature, neutral pH and short incubation times.

Lyoprotection and stabilization of laccase extract from Coriolus hirsutus, using selected additives

The development of stable lyophilized laccase, obtained from Coriolus hirsutus, using a wide range of temperature treatments and storage conditions, was investigated. Using selected lyoprotectants, including, dextran 6 kDa, sucrose and a mixture of sodium benzoate, potassium sorbate and sorbitol (BKSS) (1.5:1.0:98.5; w/w/w) resulted by 2.4, 1.4 and 1.8-fold increase in laccase activity after lyophilization as compared to the fresh enzyme, respectively, whereas the addition of mannitol preserved 98.2% of its activity. Using 2.5% (w/v) dextran (15–25 kDa) or mannitol appeared to be the most appropriate lyoprotectants for the laccase activity. The laccase stability of the lyophilized enzymatic extract was greatly enhanced with the presence of mannitol, with 96.2, 38.9 and 24.7% of residual activity after 4 weeks of storage at − 80, 4 and 25 °C, respectively. The inactivation constant (kinactivation) value and the amount required to decrease 50% of the laccase activity (C1/2) showed that Carbowax® polyethylene glycol (PEG)-8000 was the most appropriate additive for laccase activity, followed by glycerol and CuSO4. When the enzymatic extract was incubated at 50 °C in the presence of either CuSO4, PEG-8000 or glycerol, the time required to decrease 50% of the laccase initial activity (t50), were 52.9, 54.6, 50.2 h, respectively, as compared to that of the control trial of 38.9 h.

Decolorization of dyes by a novel sodium azide-resistant spore laccase from a halotolerant bacterium, Bacillus safensis sp. strain S31.

The aim of this work was to find a new stable laccase against inhibitors and study the decolorization ability of free and immobilized laccase on different classes of dyes. Spores from a halotolerant bacterium, Bacillus safensis sp. strain S31, isolated from soil samples from a chromite mine in Iran showed laccase activity with maximum activity at 30 °C and pH 5.0 using 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the substrate. The enzyme retained about 60% of its initial activity in the presence of 10% (v v-1) methanol, ethanol, and acetone. In contrast to many other laccases, NaN3, at 0.1 and 1 mM concentrations, showed a slight inhibitory effect on the enzyme activity. Also, the spore laccase (8 U l-1) decolorized malachite green, toluidine blue, and reactive black 5 at acidic pH values; the highest decolorization percent was 75% against reactive black 5. It was observed that addition of ABTS as a redox mediator enhanced the decolorization activity. Furthermore, immobilized spore laccase encased in calcium alginate beads decolorized 95% of reactive black 5 in the absence of mediators. Overall, this isolated spore laccase might be a potent enzyme to decolorize dyes in polluted wastewaters, especially those containing metals, salts, solvents, and sodium azide.

Spore cells from BPA degrading bacteria Bacillus sp. GZB displaying high laccase activity and stability for BPA degradation

Laccase has been applied extensively as a biocatalyst to remove different organic pollutants. This study characterized a spore-laccase from the bisphenol A (BPA)-degrading strain Bacillus sp. GZB. The spore-laccase was encoded with 513 amino acids, containing spore coat protein A (CotA). It showed optimal activity at 70 °C and pH = 7.2 in presence of 2, 6-dimethoxyphenol. At 60 °C, optimal activity was also seen at pH = 3.0 and pH = 6.8 with 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine, respectively. The spore-laccase was stable at high temperature, at acidic to alkaline pH values, and in the presence of different organic solvents. Spore-laccase activity was increased by introducing Cu2+, Mg2+, and Na+, but was strongly inhibited by Fe2+, Ag+, l-cysteine, dithiothreitol, and NaN3. The cotA gene was cloned and expressed in E. coli BL21 (DE3); the purified protein was estimated as having a molecular weight of ~63 kDa. Different synthetic dyes and BPA were effectively decolorized or degraded both by the spore laccase and recombinant laccase. When BPA oxidation was catalyzed using laccase, there was an initial formation of phenoxy radicals and further oxidation or CC bond cleavage of the radicals produced different organic acids. Detailed reaction pathways were developed based on nine identified intermediates. The acute toxicity decreased gradually during BPA degradation by laccase. This study is the first report about a genus of Bacillus that can produce a highly active and stable laccase to degrade BPA.

Optimization and up scaling of ionic liquid tolerant and thermo-alkali stable laccase from a marine Staphylococcus arlettae S1-20 using tea waste

Abstract Laccase are versatile oxidoreductase enzyme having a capability of degrading variety of phenolic and non phenolic compounds. Therefore, in this work cost effective process is developed for the higher production of extracellular, thermo-alkali stable laccase (S1-20LAC) from Staphylococcus arlettae S1-20 using tea waste. To enhance the enzyme yield, multiple sequential statistical tools i.e. RSM and ANN linked GA are employed which led to 16 fold enhancement in enzyme yield. Further, a pilot scale bioprocess is developed using optimum conditions shown by GA i.e., 2.50% tea waste, 4.95 mM NaCl, 37 °C Temperature and 5.65 mM L -DOPA which led to 72 fold (879 nkatml−1) improve in the enzyme yield . The optimum temperature and pH for S1-20LAC is 85 °C and 9.0. S1-20 retained significant amount of activity even in the presence of 20% (v/v) Ionic liquids (ILs). The detailed kinetic and thermodynamic studies revealed that the overall quality of laccase is spontaneous process at high temperature. The outcome of this study shows that high production and remarkable properties of S1-20LAC offers a significant opportunity for the degradation and synthesis of wide range of compounds, which makes it an attractive green biocatalyst for the future aspects.