laccase
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A Curvularia sp. isolated from soil was found to contain laccase activity toward guaiacol as substrate. The organism produced an extracellular laccase in a medium containing yeast extract, peptone and dextrose. Initial medium pH 4.0 and cultivation temperature 30 degrees C were found to be most suitable for maximum enzyme production. The optimum pH and temperature for laccase activity were found to be 5.2 and 50 degrees C, respectively. Under optimum conditions, the enzyme had a Km (guaiacol) of 0.75 mmol/L and a V of 1.50 CU min-1 ml-1. Some divalent metal ions inhibited laccase activity at very low concentrations.
The aim was to determine which specific regions of the visible light spectrum were responsible for the induction or inhibition of laccase in Pycnoporus sanguineus. Cultures were exposed to various bandwidth lights: blue (460 nm), green (525 nm), white (a combination of 460 and 560 nm), red (660 nm), and darkness. The results indicate that short wavelengths strongly inhibit the production of laccase: green (3.76 ± 1.12 U/L), blue (1.94 ± 0.36 U/L), and white (1.05 ± 0.21 U/L) in proportions of 85.8, 92.6, and 96.0%, respectively; whereas long wavelengths inhibit laccase production only partially i.e., red light (14.05 ± 4.79 U/L) in a proportion of 46.8%. Maximum activity was induced in absence of visible light (30 °C, darkness), i.e., 30.76 ± 4.0 U/L. It is concluded that the production of laccase in P. sanguineus responds to light stimuli [measured as wavelengths and lx] and that it does so inversely. This can be explained as an ecological mechanism of environmental recognition, given that P. sanguineus develops inside lignocellulose structures in conditions of darkness. The presence of short wavelength light (460-510 nm) would indicate that the organism finds itself in an external environment, unprovided of lignin, and that it is therefore unnecessary to secrete laccase. This possible new regulation in the laccase production in P. sanguineus has important biotechnological implications, for it would be possible to control the production of laccase using light stimuli.
Laccases have been widely explored for their ligninolytic capability in bioethanol production and bioremediation of industrial effluents. However, low reaction rates have posed a major challenge to commercialization of such processes. This study reports the first evidence of laccase inhibition by two types of lignin degradation intermediates - fungal-solubilized lignin and alkali-treated lignin - thus offering a highly plausible explanation for low reaction rates due to buildup of inhibitors during the actual process. Reversed-phase high-performance liquid chromatography revealed the presence of similar polar compounds in both lignin samples. A detailed kinetic study on laccase, using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as the substrate, was used to calculate the Michaelis constant (Km) and maximum reaction rate (Vmax). With an increase in the concentration of lignin degradation intermediates, Vmax remained nearly constant, while Km increased from 1.3 to 4.0 times that of pure laccase, revealing that the inhibition was competitive in nature. The kinetic studies reported here and the insight gained into the nature of inhibition can help design process strategies to mitigate this effect and improve overall process efficiency. This work is applicable to processes that employ laccase for delignification of biomass, such as second-generation biofuels processes, as well as for industrial effluent treatment in paper and pulp industries.
- MeSH
- biodegradace MeSH
- biokatalýza MeSH
- fungální proteiny chemie MeSH
- kinetika MeSH
- lakasa chemie MeSH
- lignin chemie MeSH
- Trametes chemie enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
Laccase-producing fungus (MY3) was successfully isolated from soil samples collected from Mansoura Governorate, Egypt. This fungal isolate has shown a high laccase production level over other isolated fungi. The identity of this isolate was determined by the molecular technique 18SrRNA as Curvularia lunata MY3. The enzyme purification was performed using ammonium sulfate precipitation followed by Sephacryl S-200 and DEAE-Sepharose column chromatography. The denatured enzyme using SDS-PAGE had a molar mass of 65 kDa. The purified laccase had an optimum temperature at 40 °C for enzyme activity with 57.3 kJ/mol activation energy for 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidation. The enzyme had an optimum pH of 5.0, and it has shown a high stability at the acidic range (4.5 to 5.5). Mn2+ and Mg2+ ions enhanced the enzyme activity, while most of the enzyme activity was inhibited by Hg2+. Some compounds such as 2-mercaptoethanol, L-cysteine, and sodium azide at a concentration of 10 mmol/L had shown a high suppression effect on the enzyme activity. The enzyme strongly oxidized ABTS and syringaldazine and moderately oxidized DMP and guaiacol. The antimicrobial activity of the purified enzyme towards three pathogenic strains (Escherichia coli ATCC-25922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC-10231) was evaluated for the potential use as an antimicrobial therapeutic enzyme.
Pleurotus ostreatus degrades polychlorinated biphenyls (PCBs) with an increase of laccase activity. Laccases are well known for their detoxifying activity. We show, using reverse transcription polymerase chain reaction and a biochemical assay, that reduction in PCBs (di, tri, tetra, and penta) levels are correlated with an increase in laccase activity. P. ostreatus cultures were obtained from 0 to 30 days in the presence or absence of 7,100 mg/L PCBs (from transformer oil) and a surfactant. After each selected time cultures were withdrawn and remaining PCBs were determined, a maximal removal percentage of PCBs was obtained at 20 (63.5 ± 2.0) and 30 days (63.8 ± 4.6) post-induction. Also, the activity of the enzyme was analyzed and it was found to increase at 10 (6.9-fold) and 20 (6.77-fold) days post-induction in the presence of PCBs, as determined by its activity. Taken together, these data suggest that PCBs induce laccase expression and that laccase catalyzes PCBs removal.
- MeSH
- biodegradace MeSH
- fungální proteiny genetika metabolismus MeSH
- lakasa genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- Pleurotus enzymologie genetika růst a vývoj metabolismus MeSH
- polychlorované bifenyly metabolismus MeSH
- sekvence nukleotidů MeSH
- upregulace MeSH
- Publikační typ
- časopisecké články MeSH
The small bacterial laccase from the actinobacterium Streptomyces coelicolor which lacks the second of the three domains of the laccases structurally characterized to date was crystallized. This multi-copper phenol oxidase crystallizes in a primitive tetragonal lattice, with unit-cell parameters a = b = 179.8, c = 175.3 A. The crystals belong to either space group P4(1)2(1)2 or P4(3)2(1)2. The self-rotation function shows the presence of a noncrystallographic threefold axis in the structure. Phases will be determined from the anomalous signal of the natively present copper ions.
The production of ligninolytic enzymes (laccase and Mn-dependent peroxidase) by the white-rot fungus Pleurotus pulmonarius (FR.) Quélet was studied in solid-state cultures using agricultural and food wastes as substrate. The highest activities of laccase were found in wheat bran (2,860 ± 250 U/L), pineapple peel (2,450 ± 230 U/L), and orange bagasse (2,100 ± 270 U/L) cultures, all of them at an initial moisture level of 85 %. The highest activities of Mn peroxidase were obtained in pineapple peel cultures (2,200 ± 205 U/L) at an initial moisture level of 75 %. In general, the condition of high initial moisture level (80-90 %) was the best condition for laccase activity, while the best condition for Mn peroxidase activity was cultivation at low initial moisture (50-70 %). Cultures containing high Mn peroxidase activities were more efficient in the decolorization of the industrial dyes remazol brilliant blue R (RBBR), Congo red, methylene blue, and ethyl violet than those containing high laccase activity. Also, crude enzymatic extracts with high Mn peroxidase activity were more efficient in the in vitro decolorization of methylene blue, ethyl violet, and Congo red. The dye RBBR was efficiently decolorized by both crude extracts, rich in Mn peroxidase activity or rich in laccase activity.
BACKGROUND: Polyphenols and phenolic acid are able to slow down or prevent oxidation processes and are therefore thought to have important effects in malting and brewing. Laccase catalyses the oxidation of a wide variety of substrates, including polyphenols. The aim of this paper was to determine the changes in polyphenol compounds and the relative expression of the HvLac1 gene during malting. RESULTS: The dominant phenolic acid was ferulic acid. The amount of ferulic acid increased, whereas the amount of vanillic acid decreased during malting. The highest levels of expression of the HvLac1 gene were observed during the third air rest period in varieties with the 'Haruna Nijo' (HN) allele, as recommended for the production of beer with the protected geographical indication (PGI) 'Česke pivo' (Czech beer), whereas the highest expression was observed in the first day of germination in varieties with the 'Morex' (M) allele. However, the profiles of HvLac1 gene expression in varieties with alternative alleles during malting were similar, and the level of polyphenol compounds throughout malting was different. CONCLUSION: The polyphenol contents in barley increased several-fold during malting, and the degree of increase differed with variety. The expression of HvLac1 transcript was similar in every barley variety.
- MeSH
- ječmen (rod) enzymologie MeSH
- kyseliny kumarové chemie metabolismus MeSH
- lakasa genetika metabolismus MeSH
- polyfenoly chemie MeSH
- regulace genové exprese enzymů fyziologie MeSH
- regulace genové exprese u rostlin fyziologie MeSH
- semenáček enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Humic substances (HS) in soil are widely distributed in cold environments and account for a significant fraction of soil's organic carbon. Bacterial strains (n = 281) were isolated at 15 °C using medium containing humic acids (HA), a principal component of HS, from a variety of polar soil samples: 217 from the Antarctic and 64 from the Arctic. We identified 73 potential HA-degrading bacteria based on 16S rRNA sequence similarity, and these sequences were affiliated with phyla Proteobacteria (73.9%), Actinobacteria (20.5%), and Bacteroidetes (5.5%). HA-degrading strains were further classified into the genera Pseudomonas (51 strains), Rhodococcus (10 strains), or others (12 strains). Most strains degraded HA between 10 and 25 °C, but not above 30 °C, indicating cold-adapted degradation. Thirty unique laccase-like multicopper oxidase (LMCO) gene fragments were PCR-amplified from 71% of the 73 HA-degrading bacterial strains, all of which included conserved copper-binding regions (CBR) I and II, both essential for laccase activity. Bacterial LMCO sequences differed from known fungal laccases; for example, a cysteine residue between CBR I and CBR II in fungal laccases was not detected in bacterial LMCOs. This suggests a bacterial biomarker role for LMCO to predict changes in HS-degradation rates in tundra regions as global climate changes. Computer-aided molecular modeling showed these LMCOs contain a highly-conserved copper-dependent active site formed by three histidine residues between CBR I and CBR II. Phylogenetic- and modeling-based methods confirmed the wide occurrence of LMCO genes in HA-degrading polar soil bacteria and linked their putative gene functions with initial HS-degradation processes.
The strain Pleurotus ostreatus Florida f6, its 45 basidiospore-derived isolates (both monokaryons and dikaryons prepared in our laboratory), Trametes versicolor strain CCBAS 614 and 22 other T. versicolor isolates obtained from the sporocarps collected in distant localities were successfully preserved for 12 y using perlite and straw cryopreservation protocols. All tested isolates survived a 12-year storage in liquid nitrogen (LN) and their laccase production and Poly B411 decolorization capacity was preserved. Also mycelium extension rate and the types of colony appearance of individual isolates remained unchanged. Different cryopreservation techniques were also tested for the short time (24 h) and the long time (6 m) storage of the culture liquid with extracellular laccase produced by T. versicolor strain CCBAS 614. The results showed that 10 % glycerol was the most suitable cryopreservant. The absence of the cryopreservant did not cause high loss of laccase activity in the samples; the presence of DMSO (5 or 10 %) in LN-stored samples caused mostly a decrease of laccase activity. For the preservation of laccase activity in the liquid culture the storage in the freezer at -80 °C is more convenient than the storage in liquid nitrogen.
- MeSH
- fungální proteiny analýza metabolismus MeSH
- kryoprezervace metody MeSH
- lakasa analýza metabolismus MeSH
- mikrobiální viabilita MeSH
- Pleurotus chemie enzymologie růst a vývoj izolace a purifikace MeSH
- rostliny mikrobiologie MeSH
- Trametes chemie enzymologie růst a vývoj izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
