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1.
Sci Rep ; 14(1): 13371, 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38862560

ABSTRACT

Broad-spectrum biocatalysts enzymes, Laccases, have been implicated in the complete degradation of harmful pollutants into less-toxic compounds. In this study, two extracellularly produced Laccases were purified to homogeneity from two different Ascomycetes spp. Trichoderma lixii FLU1 (TlFLU1) and Talaromyces pinophilus FLU12 (TpFLU12). The purified enzymes are monomeric units, with a molecular mass of 44 kDa and 68.7 kDa for TlFLU1 and TpFLU12, respectively, on SDS-PAGE and zymogram. It reveals distinct properties beyond classic protein absorption at 270-280 nm, with TlFLU1's peak at 270 nm aligning with this typical range of type II Cu site (white Laccase), while TpFLU12's unique 600 nm peak signifies a type I Cu2+ site (blue Laccase), highlighting the diverse spectral fingerprints within the Laccase family. The Km and kcat values revealed that ABTS is the most suitable substrate as compared to 2,6-dimethoxyphenol, caffeic acid and guaiacol for both Laccases. The bioinformatics analysis revealed critical His, Ile, and Arg residues for copper binding at active sites, deviating from the traditional two His and a Cys motif in some Laccases. The predicted biological functions of the Laccases include oxidation-reduction, lignin metabolism, cellular metal ion homeostasis, phenylpropanoid catabolism, aromatic compound metabolism, cellulose metabolism, and biological adhesion. Additionally, investigation of degradation of polycyclic aromatic hydrocarbons (PAHs) by purified Laccases show significant reductions in residual concentrations of fluoranthene and anthracene after a 96-h incubation period. TlFLU1 Laccase achieved 39.0% and 44.9% transformation of fluoranthene and anthracene, respectively, while TpFLU12 Laccase achieved 47.2% and 50.0% transformation, respectively. The enzyme structure-function relationship study provided insights into the catalytic mechanism of these Laccases for possible biotechnological and industrial applications.


Subject(s)
Laccase , Talaromyces , Trichoderma , Talaromyces/enzymology , Laccase/metabolism , Laccase/chemistry , Laccase/isolation & purification , Laccase/genetics , Trichoderma/enzymology , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Fungal Proteins/isolation & purification , Fungal Proteins/genetics , Substrate Specificity , Copper/metabolism , Kinetics , Oxidoreductases/metabolism , Oxidoreductases/chemistry , Oxidoreductases/isolation & purification , Catalytic Domain
2.
Microb Cell Fact ; 23(1): 150, 2024 May 24.
Article in English | MEDLINE | ID: mdl-38790055

ABSTRACT

BACKGROUND: Azo dyes represent a common textile dye preferred for its high stability on fabrics in various harsh conditions. Although these dyes pose high-risk levels for all biological forms, fungal laccase is known as a green catalyst for its ability to oxidize numerous dyes. METHODS: Trichoderma isolates were identified and tested for laccase production. Laccase production was optimized using Plackett-Burman Design. Laccase molecular weight and the kinetic properties of the enzyme, including Km and Vmax, pH, temperature, and ionic strength, were detected. Azo dye removal efficiency by laccase enzyme was detected for Congo red, methylene blue, and methyl orange. RESULTS: Eight out of nine Trichoderma isolates were laccase producers. Laccase production efficiency was optimized by the superior strain T. harzianum PP389612, increasing production from 1.6 to 2.89 U/ml. In SDS-PAGE, purified laccases appear as a single protein band with a molecular weight of 41.00 kDa. Km and Vmax values were 146.12 µmol guaiacol and 3.82 µmol guaiacol/min. Its activity was stable in the pH range of 5-7, with an optimum temperature range of 40 to 50 °C, optimum ionic strength of 50 mM NaCl, and thermostability properties up to 90 °C. The decolorization efficiency of laccase was increased by increasing the time and reached its maximum after 72 h. The highest efficiency was achieved in Congo red decolorization, which reached 99% after 72 h, followed by methylene blue at 72%, while methyl orange decolorization efficiency was 68.5%. CONCLUSION: Trichoderma laccase can be used as an effective natural bio-agent for dye removal because it is stable and removes colors very well.


Subject(s)
Azo Compounds , Coloring Agents , Laccase , Temperature , Laccase/metabolism , Laccase/chemistry , Laccase/isolation & purification , Azo Compounds/metabolism , Coloring Agents/metabolism , Coloring Agents/chemistry , Kinetics , Hydrogen-Ion Concentration , Congo Red/metabolism , Osmolar Concentration , Hypocreales/enzymology , Hypocreales/metabolism , Biodegradation, Environmental , Fungal Proteins/metabolism , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification
3.
Microbiol Spectr ; 12(6): e0401323, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38712938

ABSTRACT

Laccases (EC 1.10.3.2) are oxidoreductases that belong to the multicopper oxidase subfamily and are classified as yellow/white or blue according to their absorption spectrum. Yellow laccases are more useful for industrial processes since they oxidize nonphenolic compounds in the absence of a redox mediator and stand out for being more stable and functional under extreme conditions. This study aimed to characterize a new laccase that was predicted to be present in the genome of Chitinophaga sp. CB10 - Lac_CB10. Lac_CB10, with a molecular mass of 100.06 kDa, was purified and characterized via biochemical assays using guaiacol as a substrate. The enzyme demonstrated extremophilic characteristics, exhibiting relative activity under alkaline conditions (CAPS buffer pH 10.5) and thermophilic conditions (80-90°C), as well as maintaining its activity above 50% for 5 h at 80°C and 90°C. Furthermore, Lac_CB10 presented a spectral profile typical of yellow laccases, exhibiting only one absorbance peak at 300 nm (at the T2/T3 site) and no peak at 600 nm (at the T1 site). When lignin was degraded using copper as an inducer, 52.27% of the material was degraded within 32 h. These results highlight the potential of this enzyme, which is a novel yellow laccase with thermophilic and alkaline activity and the ability to act on lignin. This enzyme could be a valuable addition to the biorefinery process. In addition, this approach has high potential for industrial application and in the bioremediation of contaminated environments since these processes often occur at extreme temperatures and pH values. IMPORTANCE: The characterization of the novel yellow laccase, Lac_CB10, derived from Chitinophaga sp. CB10, represents a significant advancement with broad implications. This enzyme displays exceptional stability and functionality under extreme conditions, operating effectively under both alkaline (pH 10.5) and thermophilic (80-90°C) environments. Its capability to maintain considerable activity over extended periods, even at high temperatures, showcases its potential for various industrial applications. Moreover, its distinctive ability to efficiently degrade lignin-demonstrated by a significant 52.27% degradation within 32 h-signifies a promising avenue for biorefinery processes. This newfound laccase's characteristics position it as a crucial asset in the realm of bioremediation, particularly in scenarios involving contamination at extreme pH and temperature levels. The study's findings highlight the enzyme's capacity to address challenges in industrial processes and environmental cleanup, signifying its vital role in advancing biotechnological solutions.


Subject(s)
Enzyme Stability , Laccase , Lignin , Laccase/metabolism , Laccase/genetics , Laccase/isolation & purification , Laccase/chemistry , Lignin/metabolism , Hydrogen-Ion Concentration , Bacteroidetes/enzymology , Bacteroidetes/genetics , Substrate Specificity , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Temperature , Biodegradation, Environmental , Guaiacol/metabolism , Copper/metabolism
4.
Braz J Microbiol ; 55(2): 1529-1543, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38340257

ABSTRACT

Multiple copper oxidase (MCO) like laccase is widely distributed in higher plant, fungi and bacteria. This study identified MCO like laccase producing bacterium isolated from a wastewater treatment plant based on 16S rRNA sequence analysis, and they were further confirmed by phylogenetic reconstruction. Biochemical and gene characterization of MCO like laccase from Stenotrophomonas sp. YBX1 is presented. Purification of MCO like laccase was carried out by ion exchange HQ Trap column and followed by gel filtration spheracryl S-100 column. The purified MCO like laccase from Stenotrophomonas sp. YBX1 shows a total activity of 1252 units and specific activity 391.2 U/mg and protein concentration 0.32 mg/mL. In SDS PAGE, the approximate molecular mass was found at 66 kDa and further confirmed from an MS spectrum of MALDI-TOF. The purified MCO like laccase is capable of degradation of antibiotics such as tetracycline completely, whereas oxytetracycline (78%) and ampicillin (62%) degraded within 96 min without any redox mediators at pH 5 and 30 ºC. Its degradation pathway was based on identification of metabolites by LC-MS spectrum. The enzymatic degradation may be used in advanced treatment of antibiotics containing wastewater.


Subject(s)
Ampicillin , Anti-Bacterial Agents , Laccase , Oxytetracycline , Phylogeny , Stenotrophomonas , Tetracycline , Laccase/metabolism , Laccase/genetics , Laccase/chemistry , Laccase/isolation & purification , Anti-Bacterial Agents/metabolism , Oxytetracycline/metabolism , Ampicillin/metabolism , Tetracycline/metabolism , Stenotrophomonas/genetics , Stenotrophomonas/metabolism , Stenotrophomonas/enzymology , Stenotrophomonas/isolation & purification , RNA, Ribosomal, 16S/genetics , Wastewater/microbiology , Oxidoreductases/metabolism , Oxidoreductases/genetics , Oxidoreductases/chemistry , Biodegradation, Environmental
5.
Braz J Microbiol ; 54(3): 1935-1942, 2023 Sep.
Article in English | MEDLINE | ID: mdl-37581711

ABSTRACT

A new Lysinibacillus fusiformis strain with abundant laccase activity was isolated from soil under forest rotted leaf and identified as L. fusiformis W11 based on its 16S rRNA gene sequence and physiological characteristics. The laccase LfuLac was purified and characterized. The optimum temperature and pH of LfuLac on guaiacol were 45 °C and pH 9, respectively. LfuLac kept 78%, 88%, 92%, 74%, and 47% of activity at pH 7-11, respectively, suggesting the alkali resistance of the enzyme. The effects of various metal ions on LfuLac showed that Cu2+, Mg2+, and Na+ were beneficial to laccase activity and 10 mM Cu2+ increased the activity of LfuLac to 216%. LfuLac showed about 90% activity at 5% organic solvents and more than 60% activity at 20%, indicating its resistance to organic solvents. In addition, LfuLac decolorized different kinds of dyes. This study enriched our knowledge about laccase from L. fusiformis W11 and its potential industrial applications.


Subject(s)
Bacillaceae , Coloring Agents , Laccase , Alkalies , Coloring Agents/chemistry , Hydrogen-Ion Concentration , Laccase/chemistry , Laccase/genetics , Laccase/isolation & purification , RNA, Ribosomal, 16S/genetics , Solvents , Temperature , Bacillaceae/enzymology
6.
Sci Rep ; 12(1): 2416, 2022 02 14.
Article in English | MEDLINE | ID: mdl-35165332

ABSTRACT

The aim of this work to study an efficient laccase producing fungus Ganoderma leucocontextum, which was identified by ITS regions of DNA and phylogenetic tree was constructed. This study showed the laccase first-time from G. leucocontextum by using medium containing guaiacol. The growth cultural (pH, temperature, incubation days, rpm) and nutritional (carbon and nitrogen sources) conditions were optimized, which enhanced the enzyme production up to 4.5-folds. Laccase production increased 855 U/L at 40 °C. The pH 5.0 was suitable for laccase secretion (2517 U/L) on the 7th day of incubation at 100 rpm (698.3 U/L). Glucose and sucrose were good carbon source to enhance the laccase synthesis. The 10 g/L beef (4671 U/L) and yeast extract (5776 U/L) were the best nitrogen source for laccase secretion from G. leucocontextum. The laccase was purified from the 80% ammonium sulphate precipitations of protein identified by nucleotides sequence. The molecular weight (65.0 kDa) of purified laccase was identified through SDS and native PAGE entitled as Glacc110. The Glacc110 was characterized under different parameters. It retained > 90% of its activity for 16 min incubation at 60 °C in acidic medium (pH 4.0). This enzyme exerted its optimal activity at pH 3.0 and temperature 70 °C with guaiacol substrate. The catalytic parameters Km and Vmax was 1.658 (mM) and 2.452 (mM/min), respectively. The thermo stability of the laccase produced by submerged fermentation of G. leucocontextum has potential for industrial and biotechnology applications. The results remarked the G. leucocontextum is a good source for laccase production.


Subject(s)
Fungal Proteins/chemistry , Fungal Proteins/metabolism , Ganoderma/enzymology , Laccase/chemistry , Laccase/metabolism , Phylogeny , Base Sequence , Chemical Precipitation , Enzyme Stability , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Ganoderma/chemistry , Ganoderma/classification , Ganoderma/genetics , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Laccase/genetics , Laccase/isolation & purification
7.
Int J Biol Macromol ; 192: 219-231, 2021 Dec 01.
Article in English | MEDLINE | ID: mdl-34624382

ABSTRACT

Trichoderma harzianum S7113 as an efficient fungal isolate for laccase production was identified using the 18S rRNA sequencing. T. harzianum S7113 attained its maximal laccase production level on the 14th day of static incubation at 28 °C and pH 5.0 using the inoculum size of 5 discs (14 mm), according to the one factor per time (OFT) method. The most appropriate carbon, organic and inorganic nitrogen sources to promote maximal laccase synthesis were glucose (15 g/L), beef extract (5 g/L), and ammonium chloride (4 g/L), respectively. Results of Response Surface Methodology (RSM) revealed that glucose, meat extract, and ammonium chloride concentrations of 17.54, 7.17, and 4.36 g/L respectively, at a pH value of 6.74 are the favorite conditions for high titer production. The ANOVA analysis highlighted an excellent match between the actual experimental results and the model predicted laccase production levels. The biodegradation of hydroquinone (HQ) by T. harzianum S7113 laccase was most efficient in the pH range of 5.0 to 6.5. The increase in laccase concentration led to a significant increase in the HQ conversion to get a biodegradation rate of 92 ± 2.6% with a laccase concentration of 0.75 U/mL after 3 h of reaction.


Subject(s)
Fermentation , Hydroquinones/metabolism , Hypocreales/metabolism , Laccase/biosynthesis , Biodegradation, Environmental , Carbon/metabolism , Culture Media , Glucose/metabolism , Hydrogen-Ion Concentration , Hypocreales/classification , Hypocreales/genetics , Hypocreales/isolation & purification , Laccase/isolation & purification , Metabolic Engineering , Nitrogen/metabolism , Phylogeny
8.
Int J Biol Macromol ; 190: 574-584, 2021 Nov 01.
Article in English | MEDLINE | ID: mdl-34506861

ABSTRACT

Laccases are multi­copper oxidases that possess the potential for industrial wastewater treatments. In this study, a putative laccase from Sulfitobacter indolifex was recombinantly produced and characterised. The enzyme was found to be stable and active at low to ambient temperature and across a range of pH conditions. The ability of the putative bacterial laccase to catalyse the decolourisation of seven common industrial dyes was also examined. Our results showed that the putative laccase could efficiently decolourise Indigo Carmine, Coomassie Brilliant Blue R-250, Congo Red, Malachite Green and Alizarin in the presence of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a redox mediator. Furthermore, the use of enzyme immobilisation technology to improve the operational stability and reusability of the putative laccase was also investigated. We found that immobilising the enzyme through the cross-linked enzyme aggregate method significantly improved its tolerance towards extreme pH as well as the presence of organic solvents. This work expands the arsenal of bacterial laccases available for the bioremediation of dye-containing wastewater.


Subject(s)
Coloring Agents/isolation & purification , Laccase/metabolism , Organic Chemicals/isolation & purification , Rhodobacteraceae/enzymology , Amino Acid Sequence , Color , Copper/metabolism , Cross-Linking Reagents/chemistry , Enzyme Assays , Enzyme Stability , Enzymes, Immobilized/metabolism , Hydrogen-Ion Concentration , Kinetics , Laccase/chemistry , Laccase/isolation & purification , Recombinant Proteins/isolation & purification , Salts/chemistry , Solvents/chemistry , Temperature
9.
Appl Environ Microbiol ; 87(23): e0135521, 2021 11 10.
Article in English | MEDLINE | ID: mdl-34524901

ABSTRACT

Lignin is a complex natural organic polymer and is one of the primary components of lignocellulose. The efficient utilization of lignocellulose is limited because it is difficult to degrade lignin. In this study, we screened a lacz1 gene fragment encoding laccase from the macrotranscriptome data of a microbial consortium WSC-6, which can efficiently degrade lignocellulose. The reverse transcription-quantitative PCR (RT-qPCR) results demonstrated that the expression level of the lacz1 gene during the peak period of lignocellulose degradation by WSC-6 increased by 30.63 times compared to the initial degradation period. Phylogenetic tree analysis demonstrated that the complete lacz1 gene is derived from a Bacillus sp. and encoded laccase. The corresponding protein, LacZ1, was expressed and purified by Ni-chelating affinity chromatography. The optimum temperature was 75°C, the optimum pH was 4.5, and the highest enzyme activity reached 16.39 U/mg. We found that Cu2+ was an important cofactor needed for LacZ1 to have enzyme activity. The molecular weight distribution of lignin was determined by gel permeation chromatography (GPC), and changes in the lignin structure were determined by 1H nuclear magnetic resonance (1H NMR) spectra. The degradation products of lignin by LacZ1 were determined by gas chromatography and mass spectrometry (GC-MS), and three lignin degradation pathways (the gentian acid pathway, benzoic acid pathway, and protocatechuic acid pathway) were proposed. This study provides insight into the degradation of lignin and new insights into high-temperature bacterial laccase. IMPORTANCE Lignin is a natural aromatic polymer that is not easily degraded, hindering the efficient use of lignocellulose-rich biomass resources, such as straw. Biodegradation is a method of decomposing lignin that has recently received increasing attention. In this study, we screened a gene encoding laccase from the lignocellulose-degrading microbial consortium WSC-6, purified the corresponding protein LacZ1, characterized the enzymatic properties of laccase LacZ1, and speculated that the degradation pathway of LacZ1 degrades lignin. This study identified a new, high-temperature bacterial laccase that can degrade lignin, providing insight into lignin degradation by this laccase.


Subject(s)
Bacillus/enzymology , Laccase , Lignin , Bacillus/genetics , Laccase/genetics , Laccase/isolation & purification , Lignin/metabolism , Phylogeny
10.
Int J Biol Macromol ; 188: 983-992, 2021 Oct 01.
Article in English | MEDLINE | ID: mdl-34403677

ABSTRACT

Plant laccases have been proposed to participate in lignin biosynthesis. However, there is no direct evidence that individual laccases in Populus can polymerize lignin monomers and alter cell wall structure. Here, a Populus laccase, PtrLAC16, was expressed and purified in a eukaryotic system. Enzymatic analysis of PtrLAC16 showed that it could polymerize lignin monomers in vitro. PtrLAC16 preferred sinapyl alcohol, and this preference is associated with an altered S/G ratio in transgenic Populus lines. PtrLAC16 was localized exclusively in the cell walls of stem vascular tissue, and a reduction in PtrLAC16 expression led to a significant decrease in lignin content and altered cell wall structure. There was a direct correlation between the inhibition of PtrLAC16 expression and structural changes in the stem cell wall of Populus. This study provides direct evidence that PtrLAC16 plays a key role in the polymerization of lignin monomers, especially for sinapyl lignin, and affects the formation of xylem cell walls in Populus.


Subject(s)
Biocatalysis , Cell Wall/enzymology , Laccase/metabolism , Lignin/metabolism , Plant Proteins/metabolism , Polymerization , Populus/enzymology , Xylem/enzymology , Gene Expression Regulation, Plant , Kinetics , Laccase/isolation & purification , Organ Specificity , Phylogeny , Plant Proteins/genetics , Plant Proteins/isolation & purification , Plant Vascular Bundle/metabolism , Plants, Genetically Modified , Populus/genetics , Protein Transport , Spectrum Analysis, Raman , Subcellular Fractions/metabolism , Nicotiana , Xylem/ultrastructure
11.
Biomolecules ; 11(6)2021 06 02.
Article in English | MEDLINE | ID: mdl-34199365

ABSTRACT

Psychrophilic laccases catalyzing the bond formation in mild, environmentally friendly conditions are one of the biocatalysts at the focus of green chemistry. Screening of 41 cold-adapted strains of yeast and yeast-like fungi revealed a new laccase-producing strain, which was identified as Kabatiella bupleuri G3 IBMiP according to the morphological characteristics and analysis of sequences of the D1/D2 regions of 26S rDNA domain and the ITS1-5,8S-ITS2 region. The extracellular activity of laccase in reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at the optimal pH 3.5 was 215 U/L after 15 days of growth in a medium with waste material and 126 U/L after 25 days of cultivation in a defined medium. Copper (II) ions (0.4 mM), Tween 80 (1.0 mM) and ascorbic acid (5.0 mM) increased the production of laccase. The optimum temperature for enzyme operation is in the range of 30-40 °C and retains over 60% of the maximum activity at 10 °C. New laccase shows high thermolability-half-life at 40 °C was only 60 min. Enzyme degradation of synthetic dyes was the highest for crystal violet, i.e., 48.6% after 1-h reaction with ABTS as a mediator. Outcomes of this study present the K. bupleuri laccase as a potential psychrozyme for environmental and industrial applications.


Subject(s)
Ascomycota/enzymology , Coloring Agents/chemistry , Fungal Proteins , Gentian Violet/chemistry , Laccase , Enzyme Stability , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Hot Temperature , Laccase/chemistry , Laccase/isolation & purification
12.
Int J Mol Sci ; 22(9)2021 May 01.
Article in English | MEDLINE | ID: mdl-34062799

ABSTRACT

In recent years, researchers have focused on developing simple and efficient methods based on electrochemical biosensors to determine hydroxycinnamic acids from various real samples (wine, beer, propolis, tea, and coffee). Enzymatic biosensors represent a promising, low-cost technology for the direct monitoring of these biologically important compounds, which implies a fast response and simple sample processing procedures. The present review aims at highlighting the structural features of this class of compounds and the importance of hydroxycinnamic acids for the human body, as well as presenting a series of enzymatic biosensors commonly used to quantify these phenolic compounds. Enzyme immobilization techniques on support electrodes are very important for their stability and for obtaining adequate results. The following sections of this review will briefly describe some of the laccase (Lac) and tyrosinase (Tyr) biosensors used for determining the main hydroxycinnamic acids of interest in the food or cosmetics industry. Considering relevant studies in the field, the fact has been noticed that there is a greater number of studies on laccase-based biosensors as compared to those based on tyrosinase for the detection of hydroxycinnamic acids. Significant progress has been made in relation to using the synergy of nanomaterials and nanocomposites for more stable and efficient enzyme immobilization. These nanomaterials are mainly carbon- and/or polymer-based nanostructures and metallic nanoparticles which provide a suitable environment for maintaining the biocatalytic activity of the enzyme and for increasing the rate of electron transport.


Subject(s)
Biosensing Techniques , Coumaric Acids/isolation & purification , Laccase/isolation & purification , Monophenol Monooxygenase/isolation & purification , Carbon/chemistry , Coumaric Acids/chemistry , Electrochemical Techniques , Enzymes, Immobilized/chemistry , Humans , Laccase/chemistry , Metal Nanoparticles/chemistry , Monophenol Monooxygenase/chemistry
13.
Prep Biochem Biotechnol ; 51(9): 901-918, 2021.
Article in English | MEDLINE | ID: mdl-33586595

ABSTRACT

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.


Subject(s)
Coloring Agents/chemistry , Fungal Proteins , Hot Temperature , Laccase , Phoma/enzymology , Enzyme Stability , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Hydrogen-Ion Concentration , Laccase/chemistry , Laccase/isolation & purification
14.
Int J Biol Macromol ; 176: 394-403, 2021 Apr 15.
Article in English | MEDLINE | ID: mdl-33548319

ABSTRACT

This study reports an efficient and fast procedure for the purification of laccase (PaL) obtained from the resin of Pistacia atlantica Desf. It was purified by one-step affinity chromatography and showed the specific activity of 393 U/mg with 81.9-fold purification. The molecular weight of PaL was estimated to be approximately 60 kDa using gel electrophoresis SDS-PAGE. Moreover, it depicted diphenolase activity and high affinity towards 2,6-dimethoxy phenol (Km = 10.01 ± 0.5 mM) and syringaldazine (Km = 6.57 ± 0.2 mM) comparing with plant-origin polyphenol oxidases reported in the literature. It should be noted that PaL possessed optimal activity at pH 7.5 and 45 °C. It also remained stable under different conditions of pH (6.5-8.0), temperature (25-45 °C), and when it was exposed to several metal ions. The MTT and flow cytometry assays demonstrated that the enzyme treatment significantly affected growth of HeLa, HepG2, and MDA-MB-231 cells with LC50 values of 4.83 ± 0.02, 61 ± 0.31, and 26.83 ± 0.11 µM after 72 h, respectively. NOVELTY STATEMENT: This is the first attempt to isolate and characterize a new oxidoreductase from the resin of Pistacia atlantica Desf., native species of Iran, to recruit it in cytotoxicity researches. In the purification process by an efficient affinity column (SBA-NH2-GA), the enzyme was eluted promptly with a satisfied yield. The purified laccase exerted higher affinity to diphenolic compounds and pH-thermal stability compared to other plant-derived polyphenol oxidases. The purified enzyme was found to show anti-oxidant capacity and significantly inhibited the growth of cancerous cells in vitro. PaL showed more cytotoxic activity towards HeLa and MDA-MB-231 cells by induction of apoptosis. The cytotoxic activity of the laccase was measured by flow cytometry.


Subject(s)
Cytotoxins , Laccase , Pistacia/chemistry , Plant Proteins , Resins, Plant/chemistry , Catalysis , Cytotoxins/chemistry , Cytotoxins/isolation & purification , Cytotoxins/pharmacology , HeLa Cells , Hep G2 Cells , Humans , Laccase/chemistry , Laccase/isolation & purification , Laccase/pharmacology , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Plant Proteins/pharmacology
15.
Int J Biol Macromol ; 172: 270-280, 2021 Mar 01.
Article in English | MEDLINE | ID: mdl-33418049

ABSTRACT

Enzyme immobilization can increase enzyme reusability to reduce cost of industrial production. Ginkgo biloba leaf extract is commonly used for medical purposes, but it contains ginkgolic acid, which has negative effects on human health. Here, we report a novel approach to solve the problem by degrading the ginkgolic acid with immobilized-laccase, where core/shell composite nanoparticles prepared by coaxial electrospraying might be first applied to enzyme immobilization. The core/shell Fe3O4/nylon 6,6 composite nanoparticles (FNCNs) were prepared using one-step coaxial electrospraying and can be simply recovered by magnetic force. The glutaraldehyde-treated FNCNs (FNGCNs) were used to immobilize laccase. As a result, thermal stability of the free laccase was significantly improved in the range of 60-90 °C after immobilization. The laccase-immobilized FNGCNs (L-FNGCNs) were applied to degrade the ginkgolic acids, and the rate constants (k) and times (τ50) were ~0.02 min-1 and lower than 39 min, respectively, showing good catalytic performance. Furthermore, the L-FNGCNs exhibited a relative activity higher than 0.5 after being stored for 21 days or reused for 5 cycles, showing good storage stability and reusability. Therefore, the FNGCNs carrier was a promising enzyme immobilization system and its further development and applications were of interest.


Subject(s)
Ferrosoferric Oxide/chemistry , Fungal Proteins/chemistry , Ginkgo biloba/chemistry , Laccase/chemistry , Magnetite Nanoparticles/chemistry , Salicylates/chemistry , Cross-Linking Reagents/chemistry , Electrochemical Techniques , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/isolation & purification , Equipment Reuse , Fungal Proteins/isolation & purification , Glutaral/chemistry , Hydrolysis , Kinetics , Laccase/isolation & purification , Magnetite Nanoparticles/ultrastructure , Nylons/chemistry , Plant Extracts/chemistry , Plant Leaves/chemistry , Polyporaceae/chemistry , Polyporaceae/enzymology
16.
Int J Biol Macromol ; 173: 99-108, 2021 Mar 15.
Article in English | MEDLINE | ID: mdl-33460660

ABSTRACT

The present investigation reports an in-vitro study using combination of laccase and an enhancer capable of inhibiting the growth of pathogenic microorganisms, preventing biofilm formation, and whitening teeth. Laccase-cinnamic acid system remarkably inhibited the growth of Aggregatibacter actinomycetemcomitans, Candida albicans, S. aureus, and Streptococcus mutans whilst showed no significant effects on Gram-negative bacteria. Data presented that cinnamic acid (10 mM) with laccase (0.125 U ml-1) led to a maximum decrease of about 90%, in S. mutans biofilm formation. The confocal laser scanning microscopy showed considerable detachment of S. mutans cells from glass substratum. The combined laccase-cinnamic acid system could remove teeth discoloration caused by coffee. SEM of the teeth surface exhibited no damages such as surface cracking or fracture. Liquid chromatography-tandem mass spectrometry (LC-MS) and cyclic voltammetry (CV) studies showed that laccase can catalyze the one-electron oxidation of cinnamic acid to the respective radical. This radical can then undergo several fates, including recombination with another radical to form a dimeric species, dismutation of the radical back to cinnamic acid or decarboxylation to give various reduced oxygen species. Therefore, the redox potential values of phenolic monomers/oligomers are related with their biological activities.


Subject(s)
Aggregatibacter actinomycetemcomitans/drug effects , Anti-Bacterial Agents/pharmacology , Cinnamates/pharmacology , Fungal Proteins/pharmacology , Hericium/chemistry , Laccase/pharmacology , Aggregatibacter actinomycetemcomitans/growth & development , Biofilms/drug effects , Biofilms/growth & development , Caffeic Acids/pharmacology , Candida albicans/drug effects , Candida albicans/growth & development , Catechols/pharmacology , Drug Synergism , Escherichia coli/drug effects , Escherichia coli/growth & development , Fungal Proteins/isolation & purification , Gallic Acid/pharmacology , Hericium/enzymology , Hydroquinones/pharmacology , Laccase/isolation & purification , Lactobacillus/drug effects , Lactobacillus/growth & development , Microbial Sensitivity Tests , Oxidation-Reduction , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/growth & development , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Streptococcus mutans/drug effects , Streptococcus mutans/growth & development , Tooth Bleaching Agents/pharmacology
17.
Int J Biol Macromol ; 170: 298-306, 2021 Feb 15.
Article in English | MEDLINE | ID: mdl-33347931

ABSTRACT

Laccases are enzymes able to catalyze the oxidation of a wide array of phenolic and non-phenolic compounds using oxygen as co-substrate and releasing water as by-product. They are well known to have wide substrate specificity and in recent years, have gained great biotechnological importance. To date, most well studied laccases are from fungal and mesophilic origin, however, enzymes from extremophiles possess an even greater potential to withstand the extreme conditions present in many industrial processes. This research work presents the heterologous production and characterization of a novel laccase from a thermoalkaliphilic bacterium isolated from a hot spring in a geothermal site. This recombinant enzyme exhibits remarkably high specific activity (>450,000 U/mg) at 70 °C, pH 6.0, using syringaldazine substrate, it is active in a wide range of temperature (20-90 °C) and maintains over 60% of its activity after 2 h at 60 °C. Furthermore, this novel spore-coat laccase is able to biodecolorize eight structurally different recalcitrant synthetic dyes (Congo red, methyl orange, methyl red, Coomassie brilliant blue R250, bromophenol blue, malachite green, crystal violet and Remazol brilliant blue R), in just 30 min at 40 °C in the presence of the natural redox mediator acetosyringone.


Subject(s)
Coloring Agents/chemistry , Laccase/chemistry , Laccase/isolation & purification , Anthraquinones/chemistry , Azo Compounds/chemistry , Bacillus/enzymology , Bacillus/metabolism , Bacteria/metabolism , Biodegradation, Environmental , Hydrogen-Ion Concentration , Laccase/metabolism , Oxidation-Reduction , Spores/metabolism , Wastewater/chemistry
18.
Int J Biol Macromol ; 170: 232-238, 2021 Feb 15.
Article in English | MEDLINE | ID: mdl-33340630

ABSTRACT

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 ß-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.


Subject(s)
Bacillus/enzymology , Laccase/chemistry , Laccase/isolation & purification , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Bacillus/metabolism , Color , Coloring Agents/metabolism , Hydrogen-Ion Concentration , Laccase/metabolism , Oxidation-Reduction , Substrate Specificity , Temperature
19.
Biotechnol Appl Biochem ; 68(2): 297-306, 2021 Apr.
Article in English | MEDLINE | ID: mdl-32282952

ABSTRACT

A homogeneous monomeric laccase (ASL) from Agaricus sinodeliciosus, with a molecular mass of 65 kDa, was isolated using ion-exchange chromatography (CM-cellulose and Q-Sepharose) and gel-filtration chromatography (Superdex 75). This laccase exhibited maximum activity at 50 °C and pH 5.0. Hg2+ and Cd2+ significantly inhibited its activity. The laccase displayed a Km value of 0.9 mM toward 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS). In addition to ABTS, ASL exhibited higher affinity toward o-toluidine and benzidine than other substrates. ASL is able to decolorize malachite green and Eriochrome black T.


Subject(s)
Agaricus/enzymology , Fungal Proteins , Laccase , Cadmium/chemistry , Enzyme Stability , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Hot Temperature , Hydrogen-Ion Concentration , Laccase/chemistry , Laccase/isolation & purification , Mercury/chemistry
20.
Cell Biol Int ; 45(3): 536-548, 2021 Mar.
Article in English | MEDLINE | ID: mdl-32052524

ABSTRACT

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5-5.0 and 3.0-5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics.


Subject(s)
Ascomycota/enzymology , Extracellular Space/enzymology , Laccase/isolation & purification , Laccase/metabolism , Ascomycota/drug effects , Ascomycota/growth & development , Benzhydryl Compounds/metabolism , Benzothiazoles/metabolism , Biodegradation, Environmental/drug effects , Bioreactors/microbiology , Calibration , Carbon/pharmacology , Diclofenac/metabolism , Electrochemistry , Electrodes , Kinetics , Nitrogen/pharmacology , Phenols/metabolism , Salts/pharmacology , Sulfonic Acids/metabolism , Xenobiotics/metabolism
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