A protein from Pleurotus eryngii var. tuoliensis C.J. Mou with strong removal activity against the natural steroid hormone,estriol: Purification,characterization, and identification as a laccase

A protein with strong removal activity against the natural estrogen estriol was purified from a culture supernatant of Pleurotus eryngii var. tuoliensis C.J. Mou. The protein was characterized as a laccase and had a molecular mass of 60 kDa on SDS-PAGE. The enzyme was most active at pH 7.0 and 50 °C. The partial N-terminal amino acid sequence of the enzyme showed homology with laccases from mushrooms, such as Pleurotus ostreatus, Coriolus versicolor (current name: Trametes versicolor), Pycnoporus cinnabarinus, and P. eryngii. A recombinant yeast assay confirmed that laccase treatment was very efficient for removing the estrogenic activity of steroid estrogens. Our results suggest that the enzyme may be applicable as a potential factor for removing natural steroid hormones.     

Trametes versicolor: Potential for atrazine bioremediation in calcareous clay soil,under low water availability conditions

This study examined the feasibility of Trametes versicolor to actively degrade atrazine (0.5 μg g^?1) in non-sterile calcareous clay soil (Algarve, Portugal) microcosms for up to 24 weeks (20 °C), under low water availability (soil water potentials of ?0.7 and ?2.8 MPa). Soil respiration, laccase activity, and atrazine quantification by high-performance liquid chromatography (HPLC) were assessed. Respiration was significantly (p < 0.05) enhanced in soil containing the inoculant, particularly in the presence of atrazine, indicating that it remained metabolically active throughout the study. Furthermore, up to 98% and 85% (at ?0.7 and ?2.8 MPa, respectively) of atrazine was degraded in soil containing both the atrazine and the inoculant, compared to 96% and 50% in soil containing atrazine only. The contribution of T. versicolor to atrazine degradation was only significant (p < 0.005) under the driest soil treatment conditions. The strategies used for enhancing colonisation and biodegradation capabilities of the inoculant, as well as the selection of sawdust as carrier, were thus effective. However, there were no differences (p > 0.05) in quantified laccase activity in soil containing the inoculant and the control. Overall, this study demonstrated that T. versicolor was a strong candidate for atrazine bioremediation in soil with low moisture and organic matter contents, such as that found in semi-arid and Mediterranean-like ecosystems.     

Decolorization of synthetic dyes by white rot fungi,involving laccase enzyme in the process

In this study, decolorization of Remazol Brillant Blue Royal (RBBR) and Drimaren Blue CL-BR (DB) was investigated using three white rot fungi named as Pleurotus ostreatus (P. ostreatus), Coriolus versicolor (C. versicolor) and Funalia trogii (F. trogii). Decolorization studies were continued for 48 h under static conditions at 30 °C and pH 5.0. The degree of pH, dry mycelium weight (DMW), dye concentration, laccase activity and protein content were analyzed; the enzyme responsible for decolorization was detected for both dyes. Maximum and minimum decolorizations were obtained by F. trogii and P. ostreatus, respectively. Both dyes at all concentrations were found to be toxic for P. ostreatus growth, whereas only DB above 60 mg/L was found to be toxic for C. versicolor growth. Maximum and minimum laccase activities were detected in decolorization media of F. trogii and P. ostreatus, respectively. Results of activity staining following SDS-PAGE showed that laccase is the only enzyme that is responsible for decolorization of DB and RBBR.     

Immobilization of fungal (Trametes versicolor) laccase onto Amberlite IR-120 H beads: Optimization and characterization

Laccase from Trametes versicolor was immobilized on Amberlite IR-120 H beads. Maximum immobilization obtained was 78.7% at pH = 4.5 and temperature T = 45 °C. Kinetic parameters, K_m and V_max values, were determined respectively as 0.051 mM and 2.77 × 10^?2 mM/s for free and 4.70 mM and 5.27 × 10^?3 mM/s for immobilized laccase. The Amberlite–laccase system showed a 30% residual activity after 7 cycles. On the other hand, the loss of activity for free laccase after 7 days of storage at 4 °C was 18.5% in comparison to Amberlite–laccase system with a loss of 1.4%, during the same period. Improved operational, thermal and storage stabilities of the immobilized laccase were obtained compared to the free counterpart. Therefore, the use of low-cost matrices, like Amberlite for enzyme immobilization represents a promising product for enzymatic industrial applications.     

Laccase mediator system for activation of agarose gel: Application for immobilization of proteins

Cross-linked Sepharose beads were treated with laccase–TEMPO system for oxidation of the primary alcohol groups on the sugar moieties. Optimal activation conditions using Trametes versicolor laccase were at pH 5 and 22 °C, giving an aldehyde content of 55 μmol g⁻¹ Sepharose with 28 units g⁻¹ of laccase and 12.5 mM TEMPO. The activated Sepharose was used for immobilization of trypsin as model protein. Highest degree of immobilization was obtained at pH 10.5 but the activity yield was only 31% of that loaded on the gel. The yield of gel bound trypsin activity was increased to 76% (corresponding to about 43 U g⁻¹ Sepharose) when the immobilization was performed in the presence of trypsin inhibitor, benzamidine. The immobilization yields were comparable to that obtained on the matrix activated using sodium periodate (containing 72 μmol aldehyde per g Sepharose). Recycling and storage of the immobilized trypsin preparations showed high stability of the enzyme bound to laccase–TEMPO activated gel.     

Separation and purification of laccases from two different fungi using aqueous two-phase extraction

Selective purification still poses a challenge in the downstream processing of biomolecules such as proteins and especially enzymes. In this study a polyethylene glycol 3000 (PEG 3000)–phosphate aqueous two-phase system at 25 °C and pH 7 was successfully used for laccase purification and separation. Initially, the effect of phase forming components on enzyme activities in homogenous systems was studied. In the course of the extraction experiments tie lines, enzyme source, initial enzyme activities, phase ratio and sodium chloride concentrations were varied and their influence on the activity partitioning was determined. Partitioning results were validated using clear-native-PAGE and isoelectric focusing. Based on these results, the separation of laccases from Trametes versicolor and Pleurotus sapidus was investigated using the principle of superposition. Sodium chloride was used to adjust laccase partitioning in the applied aqueous two-phase system (ATPS). Finally, two modes of operation are proposed depending on the aim of the purification task. One mode with 0.133 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and without sodium chloride separates P. sapidus laccases from T. versicolor laccases with clearance factors of 5.23 and 6.45, respectively. The other mode of operation with 0.124 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and 0.013 g g⁻¹ of sodium chloride enables a partitioning of both laccases into the bottom phase of the ATPS resulting in a purification factor of 2.74 and 96% activity recovery.     

Detection,quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry

This study presents a combined method to analyze extracellular fungal laccases using a new anti-laccase antibody together with the identification of tryptic laccase peptides by mass spectrometry (nanoLC–ESI–MS/MS). The polyclonal anti-laccase antibody LccCbr2 was raised against peptides designed from the copper binding region II of fungal laccases using in silico data obtained from GenBank database. As a consequence, detection requires denaturation of the enzymes due to the stable conformation of the copper binding region II. The specificity of the antibody was shown with denatured laccase Lcc1 of Coprinopsis cinerea and laccase of Hypholoma fasciculare. LccCbr2 detected amounts as low as 5 ng of highly purified laccase, indicating a possible use of the antibody for quantification of laccase proteins. Denatured extracellular laccases from culture supernatants of the basidiomycetes C. cinerea, H. fasciculare, Lentinula edodes, Mycena sp., Piriformospora indica, Pleurotus cornucopiae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor and furthermore the ascomycete Verpa conica were detected with apparent molecular masses between 60 and 70 kDa by LccCbr2. The identity of extracellular laccases from C. cinerea, H. fasciculare, P. ostreatus, P. cinnabarinus and T. versicolor were verified by tryptic peptides using nanoLC–ESI–MS/MS.     

Extraction and purification of laccase by employing a novel rhamnolipid reversed micellar system

Biosurfactant-based reversed micellar extraction (RME) is an innovative method for separation and purification of biomolecules. In this study, rhamnolipid (RL), a kind of biosurfactant, was firstly adopted to form a novel reversed micellar system for extracting and purifying laccase from Coriolus versicolor crude extract. Several significant factors affecting both forward and backward extraction processes were studied. The appropriate conditions for forward extraction process were: 3.3 mM RL, 50 mM KCl, pH 5.5 and extracting time 40 min. As regards backward extraction process, 250 mM KCl, pH 7.0 and extracting time 40 min were suggested. The corresponding activity recovery (AR) and purification fold (PF) were 92.7% and 4.79, respectively. Electrophoresis analysis indicated that the laccase was successfully purified. After this reversed micellar system was reused three times, the AR and PF declined to 70.8% and 4.35, respectively, indicating that the reversed micellar system could be reused. Comparisons results of synthetic surfactant-based RME and RL-based RME further verified the superiority of RL.     

Cyanobacterial biomass as N-supplement to agro-waste for hyper-production of laccase from Pleurotus ostreatus in solid state fermentation

The potential application of dry biomass of a cyanobacterium Anacystis nidulans as a supplement in SSF for the production of laccase from Pleurotus ostreatus was evaluated. Experiments were carried out in solid culture using groundnut shell as a basic substrate supplemented with four independent nitrogen sources (ammonium sulphate, urea, yeast extract and dry powder of cyanobacteria). All the four supplements enhanced the enzyme yield, and yeast extract showed precedence over inorganic nitrogenous sources. However, when dry biomass of A. nidulans was used as an additive to groundnut shell (agricultural residues), it supported maximum cell growth (56.83 ± 5.56 mg/g dry substrate) and laccase production (49.21 ± 4.89 U/g dry substrate). Addition of 1 mM copper salt in the medium containing groundnut shell supplemented with yeast extract gave laccase activity of 32.64 ± 3.4 U/g dry substrate. When dry powder of cyanobacterial biomass was used as N-supplement, laccase production enhanced to 65.42 ± 6.48 U/g dry substrate. In addition to the enhancement to enzyme production inhibitory effects of high concentrations of copper was also diminished in the medium having dry cyanobacterial biomass. This study, forms the first report on the potential application of cyanobacterial biomass as an additive for production of laccase by Pleurotus ostraetus MTCC 1804 in solid state fermentation and has relevance in scale-up production of this fungal enzyme of commercial significance.     

A novel membrane-surface liquid co-culture to improve the production of laccase from Ganoderma lucidum

In this work, a laccase producer, Ganoderma lucidum, was separated and identified according to its morphological characteristics and phylogenetic data. A 4000 U/l and 8500 U/l of laccase activity was obtained in 500 ml flask by submerged culture and biomembrane-surface liquid culture (BSLC), respectively. Furthermore, the novel biomembrane-surface liquid co-culture (BSLCc) was developed by adding Saccharomyces cerevisiae to reactor in order to shorten the fermentation period and improve laccase production. Laccase activity obtained by BSLCc, 23 000 U/l, is 5.8 and 2.7 times of that obtained by submerged culture and BSLC, respectively. In addition, laccase production by BSLCc was successfully scaled-up to 100 l reactor, and 38 000 U/l of laccase activity was obtained on day 8. The mechanism of overproducing laccase by BSLCc was investigated by metabolism pathway analysis of glucose. The results show glucose limitation in fermentation broth induces the secretion of laccase. The addition of S. cerevisiae, on one hand, leads to an earlier occurrence of glucose limitation state, and thus shortens the fermentation time; on the other hand, it also results in the appearance of a series of metabolites of the yeast including organic acids, ethanol, glycerol and so forth in fermentation broth, and both polyacrylamide gel electrophoresis analysis and enzyme activity detection of laccase show that these metabolites contribute to the improvement of laccase activity.     

Screening of yeast strains for transfructosylating activity

Fructooligosaccharides (FOSs) are functional food ingredients with prebiotic properties, and a recent increase in the use of oligosaccharides in the food industry has led to the search for “new” microorganisms and enzymes for the production of oligosaccharides. This paper focuses on the screening of yeasts obtained from fruits and flowers (from Brazilian tropical forests), and capable of secreting extra-cellular enzymes with high fructosyl transferase activity (FTA). The screening and isolation procedures resulted in four potentially interesting yeast strains: Candida sp. (LEB-I3), Rhodotorula sp. (LEB-U5.), Cryptococcus sp. (LEB-V2) and Rhodotorula sp. LEB-V10. All were able to produce more then 100 g l⁻¹ of FOS from a 500 g l⁻¹ sucrose solution, but only the last one, (LEB-V10), showed no hydrolytic activity with respect to the FOS produced, giving a continuous increase in FOS content up to the end of the reaction, when it was about 50% of the total carbohydrates.     

Purification and characterization of a new laccase from Shiraia sp.SUPER-H168

A new laccase from Shiraia sp.SUPER-H168 was purified by ion exchange column chromatography and gel permeation chromatography and the apparent molecular mass of this enzyme was 70.78 kDa, as determined by MALDI/TOF-MS. The optimum pH value of the purified laccase was 4, 6, 5.5 and 3 with 2,6-dimethoxyphenol (DMP), syringaldazine, guaiacol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as substrates, respectively. The optimum temperature of the purified laccase was 50 °C using DMP, syringaldazine and guaiacol as substrates, but 60 °C for ABTS. Inhibitors and metal ions of SDS, NaN₃, Ag⁺ and Fe³⁺ showed inhibition on enzyme activity of 10.22%, 7.86%, 8.13% and 67.50%, respectively. Fe²⁺ completely inhibited the purified laccase. The K_cat/K_m values of the purified laccase toward DMP, ABTS guaiacol and syringaldazine were 3.99 × 10⁶, 3.74 × 10⁷, 8.01 × 10⁴ and 2.35 × 10⁷ mol^?1 L S^?1, respectively. The N-terminal amino acid sequence of the purified laccase showed 36.4% similarity to Pleurotus ostrestus. Approximately 66% of the Acid Blue 129 (100 mg L^?1) was decolorized by 2.5 U of the purified laccase after a 120 min incubation at 50 °C. Acid Red 1 (20 mg L^?1) and Reactive Black 5 (50 mg L^?1) were decolorized by the purified laccase after the addition of Acid Blue 129 (100 mg L^?1).     

Chromatographic profiles and antimicrobial activity of the essential oils obtained from some species and cultivars of the Mentheae tribe (Lamiaceae)

The present study was focused on the chemical composition and antimicrobial activity of the essential oils (EsO) obtained from five Lamiaceae representatives grown in the south of Ukraine. Among them are Salvia sclarea L., Monarda didyma (cultivar ‘Cambridge Scarlet’), Thymus pulegioides (cultivar ‘2/6-07’), Thymus vulgaris (cultivar ‘Jalos’), and Thymus serpyllum L. The component analysis of the EsO was carried out by gas chromatography method coupled with mass spectrometry (GC–MS). The antimicrobial properties of the EsO were determined using the agar diffusion test against widespread pathogenic bacterial strains (Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Streptococcus pyogenes) and opportunistic yeast Candida albicans. The EsO of Thymus serpyllum and Thymus vulgaris (cultivar ‘Jalos’) displayed noteworthy antibacterial properties against a wide spectrum of the microorganisms. These antimicrobial properties could be attributed to the high content of aromatic monoterpenoid thymol (52.56% and 47.33%, respectively). The EsO of Salvia sclarea with the dominance of linalyl acetate (45.51%) and linalool (38.98%) as well as Thymus pulegioides (cultivar ‘2/6-07’) containing α-citral (27.10%) and β-citral (17.11%) demonstrated the strongest antimicrobial effects on typical and clinical strains of Staphylococcus aureus with the inhibition zones in the range of 24.0–31.0 mm. The Salvia sclarea EsO demonstrated the most significant effect against clinical strains of Candida albicans. In conclusion, the present study revealed the chemical composition of five Lamiaceae species and cultivars grown in the south of Ukraine and considerable antimicrobial activity of the tested EsO, especially against the typical and clinical strains of Staphylococcus aureus and Candida albicans. The obtained results could be perspective for applying in the pharmaceutical industry and for the conservation of food and cosmetic products.     

An efficient in vitro refolding of recombinant bacterial laccase in Escherichia coli

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are important multicopper enzymes that are used in many biotechnological processes. A recombinant form of laccase from Bacillus sp. HR03 was overexpressed in Escherichia coli BL-21(DE3). Inclusion body (IB) formation happens quite often during recombinant protein production. Hence, developing a protocol for efficient refolding of proteins from inclusion bodies to provide large amounts of active protein could be advantageous for structural and functional studies. Here, we have tried to find an efficient method of refolding for this bacterial enzyme. Solubilization of inclusion bodies was carried out in phosphate buffer pH 7, containing 8 M urea and 4 mM β-mercaptoethanol and refolding was performed using the dilution method. The effect of different additives was investigated on the refolding procedure of denaturated laccase. Mix buffer (phosphate buffer and citrate buffer, 100 mM) containing 4 mM ZnSO₄ and 100 mM sorbitol was selected as an optimized refolding buffer. Also Kinetic parameters of soluble and refolded laccase were analyzed.     

Psychrophilic α-amylase from Aeromonas veronii NS07 isolated from farm soils

Among 120 isolates examined in this study, three isolates were selected for amylase production on starch agar plates following incubation at 10 °C. Identification by 16SrRNA on selected bacterium disclosed the highest similarity for protean regions of this gene as Aeromonas veronii NS07. A 63 kDa psychrophilic amylase enzyme from NS07 strain was purified by two-steps chromatography. The enzyme had the highest specific activity at pH 4 and was active at the range of temperatures from 0 to 50 °C, although the optimum temperature for enzyme activity was found at 10 °C. Analysis of the N-terminal amino acid sequencing disclosed 20 amino acids from purified amylase which had no similarity with other known α-amylases, indicating that the presented enzyme was novel. Amylase activity was enhanced in relation to optimum activity with the presence of sodium sulphate (161%), MnCl₂ (298%), CaCl₂ (175%), FeCl₂ (182%), MgCl₂ (237%), ZnCl₂ (169%), NiCl₂ (139%), NaCl (158%), each at 5 mM, while EDTA, phenylmethane sulphonylfluoride (PMSF) (3 mM), urea (8 M) and SDS (1%) inhibited the enzyme up to 5%, 2%, 80% and 18%, respectively. NS07 strain seems to be suitable as biocatalyst for practical use in liquefaction of starch at low temperatures, detergent and textile industries.     

Purification and characterization of an extracellular laccase from a Pseudomonas sp. LBC1 and its application for the removal of bisphenol A

The Pseudomonas sp. LBC1 produced extracellular laccase when grown in the nutrient broth. The enzyme was purified using acetone precipitation and an anion-exchange chromatography. The molecular weight of the purified laccase was estimated as 70 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An enzyme showed maximum substrate specificity towards o-tolidine than other substrates of laccase including 2,2′-azinobis, 3-ethylbenzothiazoline-6-sulfonic acid, hydroquinone, N,N′-dimethyl phenylene diamine, syringic acid and veratryl alcohol. The optimum pH and temperature for the laccase activity were 4.0 and 40 °C, respectively. Cyclic voltammogram revealed the redox potential of purified enzyme as 0.30 V. The laccase was stable up to 40 °C and within pH range 6.0–8.0. Sodium azide and EDTA strongly inhibited laccase activity. The purified laccase completely degraded the higher concentration of bisphenol A within 5 h. Biodegradation metabolites of bisphenol A were characterized by using FTIR, HPLC and GC–MS.     

Characterization and kinetic properties of the purified Trematosphaeria mangrovei laccase enzyme

The properties of Trematosphaeria mangrovei laccase enzyme purified on Sephadex G-100 column were investigated. SDS–PAGE of the purified laccase enzyme showed a single band at 48 kDa. The pure laccase reached its maximal activity at temperature 65 °C, pH 4.0 with K_m equal 1.4 mM and V_max equal 184.84 U/mg protein. The substrate specificity of the purified laccase was greatly influenced by the nature and position of the substituted groups in the phenolic ring. The pure laccase was tested with some metal ions and inhibitors, FeSO₄ completely inhibited laccase enzyme and also highly affected by (NaN₃) at a concentration of 1 mM. Amino acid composition of the pure enzyme was also determined. Carbohydrate content of purified laccase enzyme was 23% of the enzyme sample. The UV absorption spectra of the purified laccase enzyme showed a single peak at 260–280 nm.     

Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from Trametes versicolor

The hydrolysis of phenolic compounds using an immobilized and highly active and stable derivative of laccase from Trametes versicolor is presented. The enzyme was immobilized on aldehyde supports. For this, the enzyme was enriched in amino groups by chemical modification of its carboxyl groups. The aminated enzyme was immobilized with a high recovered activity (over 60%). Aldehyde derivatives were more stable than soluble or aminated-soluble enzyme and the reference derivatives after incubation in different inactivating conditions (high temperatures, different pH values or presence of organic cosolvents). The most stable derivative was obtained immobilizing the chemically aminated enzyme at pH 10 on aldehyde supports with a stabilization factor approximately 280 fold after incubation at pH 7 and 55 °C. In addition, it was possible to prepare immobilized derivatives with a maximal enzyme loading of 60 mg g^?1 of support. This derivative could be reused for 10 reaction cycles with negligible lost of activity.     

Forming nanoparticles of α-amylase and embedding them into solid surfaces

In the current work nanoparticles (NPs) of α-amylase were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on polyethylene (PE) films, or polycarbonate (PC) plates, or on microscope glass slides. The α-amylase NPs coated on the solid surfaces have been characterized by ESEM, TEM, FTIR, XPS and AFM. The substrates immobilized with α-amylase were used for hydrolyzing soluble potato starch to maltose. The amount of enzyme introduced in the substrates, leaching properties, and the catalytic activity of the immobilized enzyme were compared. The catalytic activity of the amylase deposited on the three solid surfaces was compared to that of the same amount of free enzyme at different pHs and temperatures. α-Amylase coated on PE showed the best catalytic activity in all the examined parameters when compared to native amylase, especially at high temperatures. When immobilized on glass, α-amylase showed better activity than the native enzyme over all pH and temperature values studied. However, the immobilization on PC did not improve the enzyme activity at any pH and any temperature compared to the free amylase. The kinetic parameters, K_m and V_max were also calculated. The amylase coated PE showed the most favorable kinetic parameters (K_m = 5 g L⁻¹ and V_max = 5E−07 mol mL⁻¹ min⁻¹). In contrast, the anchored enzyme-PC exhibited unfavorable kinetic parameters (K_m = 16 g L⁻¹, V_max = 4.2E−07 mol mL⁻¹ min⁻¹). The corresponding values for amylase-glass were K_m = 7 g L⁻¹, V_max = 1.8E−07 mol mL⁻¹ min⁻¹, relative to those obtained for the free enzyme (K_m = 6.6 g L⁻¹, V_max = 3.3E−07 mol mL⁻¹ min⁻¹).     

Characterization of a laccase gene from the white-rot fungi Trametes sp. 5930 isolated from Shennongjia Nature Reserve in China and studying on the capability of decolorization of different synthetic dyes

Laccase belongs to a family of multi-copper oxidases which is especially useful for biotechnological and industrial applications. A laccase-producing white-rot fungi strain designated as Trametes sp. 5930 was nearly isolated from Shennongjia Nature Reserve in China. Trametes sp. 5930 had the high yield of laccase and was capable of decolorizing different dyes efficiently. Laccase played a very important role in the decolorization of different dyes by this fungus. The laccase gene lac5930-1 and its corresponding full-length cDNA were then cloned and characterized from Trametes sp. 5930. The 1563 bp full-length cDNA of lac5930-1 encoded a mature laccase protein consisting of 499 amino acids preceded by a signal peptide of 21 amino acids. lac5930-1 gene was successfully expressed in Pichia pastoris, which verified the function of lac5930-1 encoding active laccase by means of gene expression. The recombinant laccase produced by the yeast transformant in which lac5930-1 was efficiently expressed, conferred the ability to decolorize different dyes. The capability of decolorizing different dyes was positively related to the laccase activity, which provided strong evidence for the important function of laccase used in decolorizing industrial dyes.