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An extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4, with a potential to biobleach softwood pulp.

ABSTRACT: Degradation of residual lignin in kraft pulp by chemical bleaching is implicated in causing environmental pollution. The use of thermo- and alkali-tolerant bacterial laccases is considered to be important biological alternative to chemical processing. Laccases from Bacillus species have shown promise in this respect but their intracellular/spore bound presence make their industrial application economically unfeasible. We report here on a novel extracellular active thermo-alkali-stable laccase (SN4 laccase) which is active at 90 °C and pH 8.0 using 2,6-dimethoxyphenol as substrate from Bacillus tequilensis SN4. SN4 laccase retained 27 % activity for 5 min at 100 °C and more than 80 % activity for 24 h at 70 °C. The enzyme is also stable at a higher pH (9.0-10.0). Enzyme production was optimized by submerged fermentation. Relatively high yields (18,356 nkats ml^-1) of SN4 laccase was obtained in a medium containing 650 μM MnSO₄, 350 μM FeSO₄, and 3.5 % ethanol. A 764-fold increase in laccase activity was observed under optimal conditions. In addition, reduction in kappa number and increase in brightness of softwood pulp by 28 and 7.6 %, respectively, were observed after treatment with SN4 laccase without a mediator. When N-hydroxybenzotriazole was used as a mediator, the kappa number was decreased to 47 % and brightness was increased to 12 %.

SUBMITTER: Sondhi S

PROVIDER: S-EPMC4362739 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Purification and characterization of an extracellular, thermo-alkali-stable, metal tolerant laccase from Bacillus tequilensis SN4.

Project description:A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (kcat/Km) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and β-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2'-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu2+, Co2+, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.

| S-EPMC4037180 | biostudies-literature

Properties of the newly isolated extracellular thermo-alkali-stable laccase from thermophilic actinomycetes, Thermobifida fusca and its application in dye intermediates oxidation.

Project description:Laccases are diphenol oxidases that have numerous applications to biotechnological processes. In this study, the laccase was produced from the thermophilic actinomycetes, Thermobifida fusca BCRC 19214. After 36 h of fermentation in a 5-liter fermentor, the culture broth accumulated 4.96 U/ml laccase activity. The laccase was purified 4.64-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Q-Sepharose FF and Sephacryl™ S-200 column chromatography. The overall yield of the purified enzyme was 7.49%. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sephacryl™ S-200 was found to be 73.3 kDa and 24.7 kDa, respectively, indicating that the laccase from T. fusca BCRC 19214 is a trimer. The internal amino acid sequences of the purified laccase, as determined by LC-MS/MS, had high homology with a superoxide dismutase from T. fusca YX. Approximately 95% of the original activity remained after treatment at 50°C for 3 h. and approximately 75% of the original activity remained after treatment at pH 10.0 for 24 h. This laccase could oxidize dye intermediates, especially 2,6-dimethylphenylalanine and p-aminophenol, to produce coloring. This is the first report on laccase properties from thermophilic actinomycetes. These properties suggest that this newly isolated laccase has potential for specific industrial applications.

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Introducing a Thermo-Alkali-Stable, Metallic Ion-Tolerant Laccase Purified From White Rot Fungus Trametes hirsuta.

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A thermo-alkali stable and detergent compatible processive β-1,4-glucanase from Himalayan Bacillus sp. PCH94.

Project description:Present study reports a novel and robust GH9 processive endoglucanase β-1,4-glucanase from Bacillus sp. PCH94 (EGaseBL) with thermo-alkali stable properties. The EGaseBL gene was cloned in pET-28b(+) and expressed in Escherichia coli BL21(DE3) cells. The recombinant protein was purified 94-fold with a yield of 67.8%. The biochemical characterization revealed an active enzyme at a wide pH (4.0-10.0) and temperature (4-100°C). It showed a Km and Vmax of 1.10 mg/ml and 208.24 IU/mg, respectively, using β-glucan as a substrate. The EGaseBL showed dual activities for endoglucanase (134.17 IU/mg) and exoglucanase (28.76 IU/mg), assayed using substrates β-glucan and Avicel, respectively. The enzyme is highly stable in neutral and alkaline pH and showed a half-life of 11.29 h, and 8.31 h in pH 7.0 and 9.0, respectively. The enzyme is also compatible with commercial detergents (Tide, Surf, Ghadi, Raj, and Healing tree) of the Indian market and retained > 85% enzyme activity. Concisely, robustness, extreme functionality, and detergent compatibility endorse EGaseBL as a potential bioresource for the detergent industry, in addition to its implications for the bioethanol industry. Highlights - Cloning, expression, and purification of putative novel GH9 family β-1,4-glucanase.- Processive endoglucanase with CBM3 domain and bi-functional (endo/exo) activity.- Broad pH-temperature active and stable enzyme.- Compatible with commercial detergent powders.

| S-EPMC9682278 | biostudies-literature

Bleach enhancement of mixed wood pulp by mixture of thermo-alkali-stable xylanase and mannanase derived through co-culturing of Alkalophilic Bacillus sp. NG-27 and Bacillus nealsonii PN-11.

Project description:The Application of a combination of enzymes is the best alternative to reduce the use of chemicals in the paper industry. Bacillus sp. NG-27 and Bacillus nealsonii PN-11 are known to produce thermoalkali stable xylanse (X) and mannanase (M) respectively having potential for pulp biobleaching. The Present study, reports the production of a mixture of X + M by co-culturing of strains in SSF and standardizing its application for pulp biobleaching. Production of enzymes by co-cultivation in SSF was optimized by statistical methods. Substantial increase in the yield of enzymes; 3.61 fold of xylanase and 37.71 fold of mannanase was achieved. Application of enzyme cocktail for pulp biobleaching resulted in a 45.64% reduction of kappa number with 55 IU g-1odp of enzyme dose (xylanase:mannanase; 3:1) at pH 8.0 in 1h at 65 °C along with significant increase in brightness (11%) and whiteness (75%). The Same quality of paper as made up from chemical treated pulp can be made from enzyme-treated pulp with 30% less use of chlorine. Structural analysis of enzyme-treated pulp showed dissolution of hemicellulose as indicated by pores, cracks and increased roughness all over the surface. Cocktail of X + M produced economically in a single fermentation having all the requisite characteristics for pulp biobleaching is a highly suitable candidate for application in the pulp and paper industry.

| S-EPMC7855340 | biostudies-literature

Biocontrol Potential of Bacillus subtilis and Bacillus tequilensis against Four Fusarium Species.

Project description:The use of biological control agents as opposed to synthetic agrochemicals to control plant pathogens has gained momentum, considering their numerous advantages. The aim of this study is to investigate the biocontrol potential of plant bacterial isolates against Fusarium oxysporum, Fusarium proliferatum, Fusarium culmorum, and Fusarium verticillioides. Isolation, identification, characterization, and in vitro biocontrol antagonistic assays of these isolates against Fusarium species were carried out following standard protocols. The bacterial endophytes were isolated from Glycine max. L leaves (B1), Brassica napus. L seeds (B2), Vigna unguiculata seeds (B3), and Glycine max. L seeds (B4). The bacterial isolates were identified using 16S rRNA PCR sequencing. A phylogenetic analysis shows that the bacterial isolates are closely related to Bacillus subtilis (B1) and Bacillus tequilensis (B2-B4), with an identity score above 98%. All the bacterial isolates produced a significant amount (p < 0.05) of indole acetic acid (IAA), siderophores, and protease activity. In vitro antagonistic assays of these isolates show a significant (p < 0.05) growth inhibition of the fungal mycelia in the following order: F. proliferatum > F. culmorum > F. verticillioides > F. oxysporum, compared to the control. The results suggest that these bacterial isolates are good biocontrol candidates against the selected Fusarium species.

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Characterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii.

Project description:The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ~0.5-2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (K(M)) but to pH dependence of catalytic turnover: The k(cat) of B. clausii cotA was 1 s⁻¹ at pH 6 and 5 s⁻¹ at pH 8 in contrast to 6 s⁻¹ at pH 6 and 2 s⁻¹ at pH 8 for of B. subtilis cotA. Overall, k(cat)/K(M) was 10-fold higher for B. subtilis cotA at pH(opt). While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500-700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ~20 minutes half-life at 80°C, less than the ~50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH~8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization.

| S-EPMC4051777 | biostudies-literature

Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum.

Project description:BackgroundLaccases are multi-copper oxidases that catalyze the one electron oxidation of a broad range of compounds. Laccase substrates include substituted phenols, arylamines and aromatic thiols. Such compounds are activated by the enzyme to the corresponding radicals. Owing to their broad substrate range laccases are considered to be versatile biocatalysts which are capable of oxidizing natural and non-natural industrial compounds, with water as sole by-product.ResultsA novel CotA-type laccase from Bacillus pumilus was cloned, expressed and purified and its biochemical characteristics are presented here. The molecular weight of the purified laccase was estimated to be 58 kDa and the enzyme was found to be associated with four copper atoms. Its catalytic activity towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine (SGZ) was investigated. The kinetic parameters KM and kcat for ABTS were 80 ± 4 ?M and 291 ± 2.7 s(-1), for 2,6-DMP 680 ± 27 ?M and 11 ± 0.1 s(-1) and for SGZ only kcat could be estimated to be 66 ± 1.5 s(-1). The pH optimum for ABTS was 4, for 2,6-DMP 7 and for SGZ 6.5 and temperature optima for ABTS and 2,6-DMP were found to be around 70°C. The screening of 37 natural and non-natural compounds as substrates for B. pumilus laccase revealed 18 suitable compounds. Three of them served as redox mediators in the laccase-catalyzed decolorization of the dye indigocarmine (IC), thus assessing the new enzyme's biotechnological potential.ConclusionsThe fully copper loaded, thermostable CotA laccase from Bacillus pumilus is a versatile laccase with potential applications as an industrial biocatalyst.

| S-EPMC3041658 | biostudies-literature

Laccase-Catalyzed Surface Modification of Thermo-Mechanical Pulp (TMP) for the Production of Wood Fiber Insulation Boards Using Industrial Process Water.

Project description:Low-density wood fiber insulation boards are traditionally manufactured in a wet process using a closed water circuit (process water). The water of these industrial processes contains natural phenolic extractives, aside from small amounts of admixtures (e.g., binders and paraffin). The suitability of two fungal laccases and one bacterial laccase was determined by biochemical characterization considering stability and substrate spectra. In a series of laboratory scale experiments, the selected commercial laccase from Myceliophtora thermophila was used to catalyze the surface modification of thermo-mechanical pulp (TMP) using process water. The laccase catalyzed the covalent binding of the phenolic compounds of the process water onto the wood fiber surface and led to change of the surface chemistry directly via crosslinking of lignin moieties. Although a complete substitution of the binder was not accomplished by laccase, the combined use of laccase and latex significantly improved the mechanical strength properties of wood fiber boards. The enzymatically-treated TMP showed better interactions with the synthetic binder, as shown by FTIR-analysis. Moreover, the enzyme is extensively stable in the process water and the approach requires no fresh water as well as no cost-intensive mediator. By applying a second-order polynomial model in combination with the genetic algorithm (GA), the required amount of laccase and synthetic latex could be optimized enabling the reduction of the binder by 40%.

| S-EPMC4457874 | biostudies-literature