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The continuous requirement for pre-bleaching processes on kraft pulp, employing a range of compatible enzymes, aims to mitigate the pollution caused by chemical bleaching agents. In the present study, the laccase-producing bacterium Bacillus licheniformis BK-1 was isolated from the Bakreshwar hot spring in India and tested for laccase production using different lignocellulosic substrates. The isolate was found to produce maximum laccase (8.25 IU/mL) in the presence of rice bran as a substrate, followed by 5.14 IU/mL using sawdust over a 48 h period. Laccase production doubled when medium parameters were optimized using a central composite design. The bleaching of rice straw pulp was accomplished using a laccase, xylanase (previously extracted from the same bacteria), and laccase–xylanase mixture. The mix-wood kraft pulp treated with the enzyme mixture at pH 7.0 and 50 °C temperature for up to 180 min reduced the chlorine amount by 50% compared to the control. The results also revealed that the enzyme mixture improved the pulp’s optical (brightness 10.39%) and physical (tear index 39.77%, burst index 22.82%, and tensile strength 14.28%) properties with 50% chlorine dose. These exceptional properties underscore the enzyme mixture’s suitability for pulp pre-bleaching in paper manufacturing, offering a safer and more environmentally friendly process.

Laccases are copper-containing enzymes that can oxidize a wide variety of substrates. Thanks to this feature of laccase, some dyes that cause environmental pollution can be decolorized. Some bacteria, such as Bacillus licheniformis, naturally produce the enzyme laccase. A new affinity column was tested in this study. For this purpose, the extracellular laccase sepharose 4B-L-tyrosine-ρ-aminobenzoic acid produced by bacteria grown in suitable media was isolated by affinity chromatography method. Its purity was checked by SDS-PAGE method. The decolorization effect of some dyestable in textile wastewater of laccase isolated from B. lichenisformis O12 by affinity column was investigated. No mediator was used in this procedure. .As a result, laccase was purified 4.82-fold purification with a yield of 38.3% respectively, The molecular weight of the purified enzyme was determined as ~70 kDa by the SDS-PAGE method. The enzyme showed optimum activity at pH 4.0 and temperature 92°C. The enzyme was found to retain 100% activity even after 12 hours of incubation at 60°C and 92°C. The kinetic parameters were determined with laccase substrates such as ABTS, 2,6-DMP, and guaiacol. The purified laccase was decolorized with varied efficiencies such as 35% of Reactive black, 31% of Acid black 1, 28% of Methylene blue, and 15% of Acid red 27 without the use of any redox mediators. These properties of B. licheniformis O12 laccase enzyme make it a potential candidate enzyme for use in various biotechnological and industrial applications. [ABSTRACT FROM AUTHOR]

Laccases can catalyze the remediation of hazardous synthetic dyes in an eco-friendly manner, and thermostable laccases are advantageous to treat high-temperature dyeing wastewater. A novel laccase from Geothermobacter hydrogeniphilus (Ghlac) was cloned and expressed in Escherichia coli. Ghlac containing 263 residues was characterized as a functional laccase of the DUF152 family. By structural and biochemical analyses, the conserved residues H78, C119, and H136 were identified to bind with one copper atom to fulfill the laccase activity. In order to make it more suitable for industrial use, Ghlac variant Mut2 with enhanced thermostability was designed. The half-lives of Mut2 at 50 °C and 60 °C were 80.6 h and 9.8 h, respectively. Mut2 was stable at pH values ranging from 4.0 to 8.0 and showed a high tolerance for organic solvents such as ethanol, acetone, and dimethyl sulfoxide. In addition, Mut2 decolorized approximately 100% of 100 mg/L of malachite green dye in 3 h at 70 °C. Furthermore, Mut2 eliminated the toxicity of malachite green to bacteria and Zea mays. In summary, the thermostable laccase Ghlac Mut2 could effectively decolorize and detoxify malachite green at high temperatures, showing great potential to remediate the dyeing wastewater.

The continuous requirement for pre-bleaching processes on kraft pulp, employing a range of compatible enzymes, aims to mitigate the pollution caused by chemical bleaching agents. In the present study, the laccase-producing bacterium Bacillus licheniformis BK-1 was isolated from the Bakreshwar hot spring in India and tested for laccase production using different lignocellulosic substrates. The isolate was found to produce maximum laccase (8.25 IU/mL) in the presence of rice bran as a substrate, followed by 5.14 IU/mL using sawdust over a 48 h period. Laccase production doubled when medium parameters were optimized using a central composite design. The bleaching of rice straw pulp was accomplished using a laccase, xylanase (previously extracted from the same bacteria), and laccase–xylanase mixture. The mix-wood kraft pulp treated with the enzyme mixture at pH 7.0 and 50 °C temperature for up to 180 min reduced the chlorine amount by 50% compared to the control. The results also revealed that the enzyme mixture improved the pulp's optical (brightness 10.39%) and physical (tear index 39.77%, burst index 22.82%, and tensile strength 14.28%) properties with 50% chlorine dose. These exceptional properties underscore the enzyme mixture's suitability for pulp pre-bleaching in paper manufacturing, offering a safer and more environmentally friendly process. [ABSTRACT FROM AUTHOR]

Background: Laccase has been considered important for the degradation of lignocellulose by wood rot fungi. The properties and functions of laccase in white rot fungi have been investigated extensively, but those from brown rot fungi remain largely unknown. In this paper, a laccase isoform Pplcc2 from the brown rot fungus Postia placenta MAD-698-R was expressed heterologously in Pichia pastoris GS115, purified and the properties of the enzyme were determined. Results: The molecular weight of the protein was determined to be 67 kDa using SDS-PAGE. It cannot oxidize syringaldazine (SGZ), but it can oxidize 2,2′-azino-di-(3-ethylbenzothialozin-6-Sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP). Specific activity for ABTS was 1960 ± 19 Unit/mg. The catalytic constant (kcat) was 1213 ± 18.3 s-1 for ABTS and 293.2 ± 21.9 s-1 for DMP. Km was 22.08 μM for ABTS and 11.62 μM for DMP. The optimal pH for the oxidation of ABTS and DMP was 3.5 and 5.0 respectively. The optimal temperature for the oxidation of ABTS and DMP was 60°C. Conclusions: This is the first identified thermo activated and thermostable laccase in brown rot fungi. This investigation will contribute to understanding the roles played by laccases in brown rot fungi.

Abstract Laccases are multicopper oxidases that are being studied for their potential application in pretreatment strategies of lignocellulosic feedstocks for bioethanol production. Here, we report the expression and characterization of a predicted laccase (LAC_2.9) from the thermophilic bacterial strain Thermus sp. 2.9 and investigate its capacity to delignify lignocellulosic biomass. The purified enzyme displayed a blue color typical of laccases, showed strict copper dependence and retained 80% of its activity after 16 h at 70 °C. At 60 °C, the enzyme oxidized 2,2′-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) and 2,6-dimethoxyphenol (DMP) at optimal pH of 5 and 6, respectively. LAC_2.9 had higher substrate specificity (k cat/K M) for DMP with a calculated value that accounts for one of the highest reported for laccases. Further, the enzyme oxidized a phenolic lignin model dimer. The incubation of steam-exploded eucalyptus biomass with LAC_2.9 and 1-hydroxybenzotriazole (HBT) as mediator changed the structural properties of the lignocellulose as evidenced by Fourier transform infrared (FTIR) spectroscopy and thermo-gravimetric analysis (TGA). However, this did not increase the yield of sugars released by enzymatic saccharification. In conclusion, LAC_2.9 is a thermostable laccase with potential application in the delignification of lignocellulosic biomass.

A laccase producing new bacterial strain (Alcaligenes faecalis XF1) was isolated from green site of Chandigarh (India) by standard screening method. Nutrient broth medium containing 0.2 mM CuSO4 was used for the production of laccase. Maximum production (110 U/ml) was achieved after four days of incubation. The extracellular laccase from the medium was purified by simple salt precipitation and ion exchange technique to get 3.8 fold purified protein with 1637.8 U/mg specific activity. Purified laccase (named as LAC1*) revealed its optimum activity at pH 8.0 and 80 °C temperature, and displayed remarkable stability in the range of 70–90 °C and in the pH range (pH 7.0–9.0). The single bands on SDS-PAGE represents the purity of LAC1* with molecular weight of ∼71 kDa. The kinetic parameters for 2,6-DMP oxidation was Km, Vmax and kcat were 480 μM, 110 U/mL and 1375 s−1. Enzyme activity of the LAC1* was significantly enhanced by Cu2+, Mg2+, Mn2+, SDS and NaCl, and was slightly inhibited in the presence of conventional inhibitors like cysteine, EDTA and sodium azide. Extracellular nature and significant stability of LAC1* under extreme conditions of temperature, pH, heavy metals, halides and detergents confined its suitability for various biotechnological and industrial applications which required these qualities of laccase. So after recognizing all these properties the purified laccase was studied for its application in decolorization of industrial dyes. Keywords: Laccase, Alcaligenes faecalis, Decolorization

Synthetic dyes are widely used in many industries, but they cause serious environmental problems due to their carcinogenic and mutagenic properties. In contrast to traditional physical and chemical treatments, biodegradation is generally considered an environmental-friendly, efficient, and inexpensive way to eliminate dye contaminants. Here, a novel laccase-like enzyme Lac1326 was cloned from a marine metagenomic library. It showed a maximum activity at 60°C, and it retained more than 40% of its maximal activity at 10°C and more than 50% at 20–70°C. Interestingly, the laccase behaved stably below 50°C, even in commonly used water-miscible organic solvents. The enzyme decolorized all tested dyes with high decolorization efficiency. This thermostable enzyme with high decolorization activity and excellent tolerance of organic solvents and salt has remarkable potential for bioremediation of dye wastewater. It is thus proposed as an industrial enzyme.

Laccases can catalyze the remediation of hazardous synthetic dyes in an eco-friendly manner, and thermostable laccases are advantageous to treat high-temperature dyeing wastewater. A novel laccase from Geothermobacter hydrogeniphilus (Ghlac) was cloned and expressed in Escherichia coli . Ghlac containing 263 residues was characterized as a functional laccase of the DUF152 family. By structural and biochemical analyses, the conserved residues H78, C119, and H136 were identified to bind with one copper atom to fulfill the laccase activity. In order to make it more suitable for industrial use, Ghlac variant Mut2 with enhanced thermostability was designed. The half-lives of Mut2 at 50 °C and 60 °C were 80.6 h and 9.8 h, respectively. Mut2 was stable at pH values ranging from 4.0 to 8.0 and showed a high tolerance for organic solvents such as ethanol, acetone, and dimethyl sulfoxide. In addition, Mut2 decolorized approximately 100% of 100 mg/L of malachite green dye in 3 h at 70 °C. Furthermore, Mut2 eliminated the toxicity of malachite green to bacteria and Zea mays . In summary, the thermostable laccase Ghlac Mut2 could effectively decolorize and detoxify malachite green at high temperatures, showing great potential to remediate the dyeing wastewater.

Laccase, an important oxidoreductase, is widely distributed in various organisms. Termites are known to decompose lignocellulose efficiently with the aid of gut microorganisms. However, few laccases have been characterized from termite or its gut microbes. We aimed to screen the strain capable of degrading lignocellulose from fungus-growing termites. In this study, Bacillus stratosphericus BCMC2 with lignocellulolytic activity was firstly isolated from the hindgut of fungus-growing termite Macrotermes barneyi. The laccase gene (BaCotA) was cloned both from the BCMC2 strain and termite intestinal metagenomic DNA. BaCotA was overexpressed in E. coli, and the recombinant BaCotA showed high specific activity (554.1 U/mg). BaCotA was thermostable with an optimum temperature of 70 °C, pH 5.0. Furthermore, BaCotA was resistant to alkali and organic solvents. The enzyme remained more than 70% residual activity at pH 8.0 for 120 min; and the organic solvents such as methanol, ethanol and acetone (10%) had no inhibitory effect on laccase activity. Additionally, BaCotA exhibited efficient decolorization ability towards indigo and crystal violet. The multiple enzymatic properties suggested the presented laccase as a potential candidate for industrial applications. Moreover, this study highlighted that termite intestine is a good resource for either new strains or enzymes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A putative laccase gene (lacG) from Geobacillus sp. JS12 was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli BL21 (DE3) cells, and the protein was primarily found in inclusion bodies. The resulting insoluble proteins were solubilized with 6 M guanidine HCl and refolded using an on-column refolding procedure. Ni-chelation affinity chromatography found the laccase to be a 30 kDa monomeric protein. Spectrophotometry and electron paramagnetic resonance (EPR) analysis indicated LacG as a multi-copper oxidase, with the usual laccase copper sites, Type 1, 2, and 3 Cu(II). The optimum pH for enzymatic activity was 3.0, 6.0, and 6.5 with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The recombinant protein displayed high thermostability, with a heat inactivation half-life of approximately 2 h at 95 °C, and an optimum temperature of 80 °C with 2,6-DMP. Catalytic efficiency (k cat /K m ) showed that guaiacol and 2,6-DMP were highly oxidized by the enzyme. The enzymatic reaction was significantly enhanced by Co 2+ and Mn 2+ , while activity was strongly inhibited in the presence of Fe 2+ , Zn 2+ , and thiol compounds. LacG decolorized 43% of Congo red and 14% of Malachite green, and the addition of ABTS as a redox mediator dramatically increased the dye decolorization efficiency., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A laccase producing new bacterial strain (Alcaligenes faecalis XF1 ) was isolated from green site of Chandigarh (India) by standard screening method. Nutrient broth medium containing 0.2 mM CuSO 4 was used for the production of laccase. Maximum production (110 U/ml) was achieved after four days of incubation. The extracellular laccase from the medium was purified by simple salt precipitation and ion exchange technique to get 3.8 fold purified protein with 1637.8 U/mg specific activity. Purified laccase (named as LAC1*) revealed its optimum activity at pH 8.0 and 80 °C temperature, and displayed remarkable stability in the range of 70-90 °C and in the pH range (pH 7.0-9.0). The single bands on SDS-PAGE represents the purity of LAC1* with molecular weight of ∼71 kDa. The kinetic parameters for 2,6-DMP oxidation was K m , V max and k cat were 480 μM, 110 U/mL and 1375 s -1 . Enzyme activity of the LAC1* was significantly enhanced by Cu 2+ , Mg 2+ , Mn 2+, SDS and NaCl, and was slightly inhibited in the presence of conventional inhibitors like cysteine, EDTA and sodium azide. Extracellular nature and significant stability of LAC1* under extreme conditions of temperature, pH, heavy metals, halides and detergents confined its suitability for various biotechnological and industrial applications which required these qualities of laccase. So after recognizing all these properties the purified laccase was studied for its application in decolorization of industrial dyes., Competing Interests: The authors with listed names declare no conflict of interest to disclose., (© 2019 The Authors. Published by Elsevier B.V.)