Laccase/caffeic acid-catalyzed crosslinking coupled with galactomannan alters the conformational structure of ovalbumin and alleviates Th2-mediated allergic asthma

Ovalbumin(OVA)is the major allergenic protein that can induce T helper 2(Th2)-allergic reactions,for which current treatment options are inadequate.In this study,we developed a polymerized hypoallergenic OVA product via laccase/caffeic acid(Lac/CA)-catalyzed crosslinking in conjunction with galactomannan(Man).The formation of high molecular weight crosslinked polymers and the Ig G-binding were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting.The study indicated that Lac/CA-catalyzed crosslinking plus Man conjugation substantially altered secondary and tertiary structures of OVA along with the variation in surface hydrophobicity.Gastrointestinal digestion stability assay indicated that crosslinked OVA exhibited less resistance in simulated gastric fluid(SGF)and simulated intestinal fluid(SIF).Mouse model study indicated that Lac-Man/OVA ameliorated eosinophilic airway inflammatory response and efficiently downregulated the expression of Th2-related cytokines(interleukin(IL)-4,IL-5,and IL-13),and upregulated IFN-γand IL-10 expression.Stimulation of bone marrow-derived dendritic cells with Lac-Man/OVA suppressed the expression of phenotypic maturation markers(CD80 and CD86)and MHC class II molecules,and suppressed the expression levels of proinflammatory cytokines.The knowledge obtained in the present study offers an effective way to acquire a hypoallergenic OVA product that can have a therapeutic effect in alleviating OVA-induced allergic asthma.

Analysis of the electron transfer pathway in small laccase by EPR and UV-vis spectroscopy coupled with redox titration

Bacterial small laccases(SLAC) are promising industrial biocatalysts due to their ability to oxidize a broad range of substrates with exceptional thermostability and tolerance for alkaline p H. Electron transfer between substrate, copper centers, and O2is one of the key steps in the catalytic turnover of SLAC. However, limited research has been conducted on the electron transfer pathway of SLAC and SLAC-catalyzed reactions, hindering further engineering of SLAC to produce tunable biocatalysts for novel applications. Herein, the combinational use of electron paramagnetic resonance(EPR) and ultraviolet-visible(UV-vis) spectroscopic methods coupled with redox titration were employed to monitor the electron transfer processes and obtain further insights into the electron transfer pathway in SLAC. The reduction potentials for type 1 copper(T1Cu), type 2 copper(T2Cu) and type 3copper(T3Cu) were determined to be 367 ± 2 mV, 378 ± 5 m V and 403 ± 2 mV,respectively. Moreover, the reduction potential of a selected substrate of SLAC, hydroquinone(HQ), was determined to be 288 mV using cyclic voltammetry(CV). In this way, an electron transfer pathway was identified based on the reduction potentials. Specifically,electrons are transferred from HQ to T1Cu, then to T2Cu and T3Cu, and finally to O2.Furthermore, superhyperfine splitting observed via EPR during redox titration indicated a modification in the covalency of T2Cu upon electron uptake, suggesting a conformational alteration in the protein environment surrounding the copper sites, which could potentially influence the reduction potential of the copper sites during catalytic processes. The results presented here not only provide a comprehensive method for analyzing the electron transfer pathway in metalloenzymes through reduction potential measurements, but also offer valuable insights for further engineering and directed evolution studies of SLAC in the aim for biotechnological and industrial applications.

Rhus laccase catalysis and product characterization of 1,2-dimethoxyphenol in organic solutions

Dimethoxyphenol was a widely used substrate in determination of laccases activity. It was surprised, however, that the products of it had not been completely determined until now. Studies were thus conducted on Rhus laccase （RL） and immobilized Rhus laccase （IRL）-catalyzed oxidation of 2,6-dimethoxyphenol （DMP） in water-organic solvent systems. Only one product, 3,3′,5,5′- tetramethoxy-1 ,1′-biphenyl-4,4′-diol （TMBP）, was produced by RL catalysis, and it was thoroughly characterized by FT-IR, NMR and GC-MS, etc.

DNA去甲基化酶对拟南芥Laccase家族基因甲基化及转录模式的影响

植物Laccase家族蛋白,是铜蓝氧化酶超家族的重要成员,具有包括抗病性在内的多种生物学功能。利用拟南芥野生型及ros1、rdd和dme突变体的全基因组甲基化测序(BS-SEQ)数据,分析了拟南芥Laccase蛋白家族基因(AtLACs)的DNA甲基化模式。结果表明:在野生型和不同DNA去甲基化酶突变体中,该家族少数成员在基因上游区和转录区存在较高水平的甲基化修饰现象。转录组测序(RNA-SEQ)数据分析结果显示,在野生型和不同突变体中,ALAC家族基因的转录水平普遍较低。而AT5G48100基因在拟南芥胚乳细胞中剧烈表达,DME突变后,其表达水平进一步剧烈增强。综合而言,在野生型和不同DNA去甲基化酶突变体中,ALAC基因家族大部分成员的DNA甲基化修饰与转录调控关联不强,但有些基因的DNA甲基化程度与转录水平之间有密切联系。

Decolorization of reactive dyes by laccase immobilized in alginate/gelatin blent with PEG

To achieve effective decolorization of reactive dyes,laccase immobilization was investigated.Laccase 0.2%(m/V)(Denilite IIS) was trapped in beads of alginate/gelatin blent with polyethylene glycol(PEG),and then the supporters were activated by cross-linking with glutaraldehyde.The results of repeated batch decolorization showed that gelatin and appropriate concentration of glutaraldehyde accelerated the decolorization of Reactive Red B-3BF(RRB);PEG had a positive effect on enzyme stability and led to an inc...

Enhancement of Laccase Activity by Marine-derived Deuteromycete Pestalotiopsis sp. J63 with Agricultural Residues and Inducers

Pestalotiopsis sp. J63, producing a high activity of laccase, is a new marine-derived fungus isolated from the oceanic sediment of the East China Sea. Since the marine environment is oligotrophic nutrient, marine derived fungi may use small amount of nutrients to grow and produce laccases. Agricultural residues that are mainly composed of lignin, cellulose and hemicellulose are difficult to be degraded and few microbes can take them as substrates, so they are considered as oligotrophic nutrient and have the potential to be used to produce value added products. In this study, the ability of Pestalotiopsis sp. J63 to use agricultural residues to produce laccases was tested in the submerged fermentation. The combination of 3 g·L 1maltose and 20 g·L 1rice straw was the best carbon sources and 8 g·L 1ammonium sulfate was the best nitrogen source under the condition without inducers. The effects of five inducers, the feeding time and concentration of inducer on laccase production were investigated.Adding 0.09 mmol·L 1phenol after 24 h of incubation led to high laccase activity(5089 U·L 1), while with 0.09mmol·L 1phenol in the medium and wheat bran as the nitrogen source, the laccase activity could reach 5791.7U·L 1. Native-PAGE results showed that two laccase isozymes were present in the cultures. One existed in both induced and non-induced culture filtrates, while the other was only found in the fermentation with the addition of phenol, guaiacol and veratryl alcohol.

Biodegradation Kinetics of DDT in Soil under Different Environmental Conditions by Laccase Extract from White Rot Fungi

Biodegradation of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane(DDT)in soil by laccase extract from white rot fungi under different experimental conditions was investigated.DDTs,which stands for the sum of p,p′-DDE,o,p′-DDT,p,p′-DDD and p,p′-DDT in soil was degraded efficiently,and the residue decreased rapidly during the first 15 days and then slowly during the period of 16-25 days.The biodegradation of DDTs in soil fitted the pseudo-first-order kinetics.For 5,10,15 and 25 days of incubation with laccase,the residue of DDTs in soil under different atmospheres was decreased by 20%-33%,34%-52%,41%-61%and 41%-69%respectively,under different flooding conditions that was decreased by 12%-17%,17%-30%,30%-45%and 35%-52%respectively, and for different soils that was decreased by 25%-34%,39%-53%,44%-58%and 47%-62%respectively.The half-life of DDTs in soil ranged from 15.07 to 32.95 days under O2,air or N2 atmospheres,23.07 to 40.71 days under different flooding conditions,and 18.78 to 28.88 days for different soils.Laccase is an efficient and safe agent for bioremediation of DDT-contaminated soil.

Production of laccase by Coriolus versicolor and its application in decolorization of dyestuffs: (Ⅱ) Decolorization of dyes by laccase containing fermentation broth with or without self-immobilized mycelia

The capability of decolorization for commercial dyes by Coriolus versicolor fermentation broth containing laccase with or without immobilized mycelium was evaluated. With cell free fermentation broth containing laccase, high decolorization ratio was achieved for acid orange 7, but not for the other dyes concerned. The immobilized mycelium was proved to be more efficient than the cell free system. All the four dyestuffs studied were found being decolourized with certain extent by immobilized mycelium. The repeated batch decolorization was carried out with satisfactory results. The experimental data showed that the continuous decolorization of wastewater from a printing and dyeing industry was possible by using the self immobilized C. Versicolor.

Effects of nonionic surfactant Triton X-100 on the laccase-catalyzed conversion of bisphenol A

The laccase-catalyzed conversion of bisphenol A （BPA） in aqueous solutions was studied in the absence and presence of nonionic surfactant Triton X-100. It was found that the addition of Triton X-100 into the reaction system increased the conversion of BPA, especially near the critical micelle concentration of Triton X-100. Also it was found that the stability of laccase was greatly improved in the presence of TritonX-100. Studies on the endogenous fluorescence emission of laccase indicated that there existed an interaction between Triton X-100 and laccase, which was beneficial to folding and stabilizating of laccase. The binding of Triton X-100 to the laccase surface also mitigated the inactivation effect caused by the free radicals and polymerization products. Under otherwise identical conditions, a lower dosage of laccase was needed for the higher conversion of BPA in the presence of Triton X-100.

Decolorization on Indigo Dyeing Wastewater by Laccase from Coriolus versicolor

[ Object] The study aimed to discuss the decolorization on indigo dyeing wastewater by laccase from Coriolus versicolor. [ Method ] Firstly, the effects of temperature, pH, indigo concentration, HBT concentration, laccase dosage on the decolorization of indigo dyeing wastewater by laccase/HBT, and then the synergism of laccase and acid cellulase was analyzed. [Result] Using ABTS as the substrate, the kinetic parame- ters, K,, and Vmax, were 0.318 mmol/L and 0.035 5 mmol/（ L . min） respectively. The decolorization rate of indigo reached 96.5% when the lacca- se acted on indigo for 40 min with HBT as an introducer at temperature 50 ℃, pH =4.5, indigo concentration 100 mg/L, HBT concentration 0.1% and laccase dosage 100 lU/L. Due to the synergism of laccase and acid cellulase during the bio-finishing of blue jeans, the backstaining degree of blue jeans reduced by 85% when the amount of laccase added was 15 000 IU/kg. Menawhile, the synergism of the laccase and acid cellulase de- creased indigo concentration in wastewater by 83.8%. [ Conclusion ] The laccase from Coriolus versicolor had a good prospect in the bio-finishing of blue jeans and the decolorization of indigo dyeing wastewater.

Preparation of Magnetic Chitosan Nanoparticles and Immobilization of Laccase

The magnetic chitosan nanoparticles were prepared by reversed-phase suspension method using Span-80 as an emulsifier, glutaraldehyde as cross-linking reagent. And the nanoparticles were characterized by TEM, FT-IR and hysteresis loop. The results show that the nanoparticles are spherical and almost superparamagnetic. The laccase was immobilized on nanoparticles by adsorption and subsequently by cross-linking with glutaraldehyde. The immobilization conditions and charac-terizations of the immobilized laccase were investigated. The optimal immobilization conditions were as follows： 10 mL of phosphate buffer （0.1 M, pH 7.0） containing 50 mg of magnetic chitosan nanoparticles, 1.0 mg·mL-1 of laccase and 1% （v/v） glutaraldehyde, immobilization temperature of 4 ℃ and immobilization time of 4 h. The immobilized laccase exhibited an appreciable catalytic capability （480 units·g-1 support） and had good storage stability and operation stability. The Km of immobilized and free laccase for ABTS were 140.6 and 31.1 μM in phosphate buffer （0.1 M, pH 3.0） at 37 ℃, respectively. The immobilized laccase is a good candidate for the research and development of biosensors based on laccase catalysis.

Cloning,expression in Escherichia coli,and enzymatic properties of laccase from Aeromonas hydrophila WL-11

A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology （more than 60%） with bacterial laccases in the genome and protein databases and the highest degree of similarity （61% identity） was observed with the multicopper oxidase of KlebsieUa sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS （2,2＇-azino-bis（3-ethylbenzthiazolinesulfonic acid） and DMP （2,6-dimethoxyphenol）, respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.

Effects of Treating with Laccase on Properties of Dyed Cotton Fabric

A laccase （Denilite IIS） was used to treat different cotton fabrics dyed with 0.2 g · L^-1 of vat dyes or reactive dyes. The results indicated that the laccase could remove the loosely adhering, unfixed or hydrolyzed dyes from the dyed fabric efficiently, which led to obvious improvements of color fastness. Furthermore, the wavelength of maximum absorbance of the residual solution of dyeing laccase-treated was different from that of the detergent-treated, which implied the laccase could accelerate structural changes of the adhering or hydrolyzed dyes from fabric in treating, resulting in obvious color changes of the residual solution. In addition, excessive laccase also could decolorize a few fixed reactive dyes from the dyed fabric, with a decrease of color strength and less further improvements of color fastness.

Immobilized laccase on magnetic nanoparticles for enhanced lignin model compounds degradation

As a natural aromatic polymer,lignin has great potential but limited industrial application due to its complex chemical structure.Among strategies for lignin conversion,biodegradation has attracted promising interest recently in term of efficiency,selectivity and mild condition.In order to overcome the issues of poor stability and non-reusability of enzyme in the biodegradation of lignin,this work explored a protocol of immobilized laccase on magnetic nanoparticles(MNPs)with rough surfaces for enhanced lignin model compounds degradation.Scanning electron microscope with energy dispersive spectrometer(SEM-EDS),flourier transformation infrared spectroscopy(FTIR)and thermal gravimetric analysis(TGA)were utilized to characterize the immobilization of laccase.The results showed a maximum activity recovery of 64.7%towards laccase when it was incubated with MNPs and glutaraldehyde(GA)with concentrations of 6 mg·ml^-1and 7.5 mg·ml^-1for 5 h,respectively.The immobilized laccase showed improved thermal stability and pH tolerance compared with free laccase,and remained more than 80%of its initial activity after 20 days of storage at 4℃.In addition,about 40%residual activity of the laccase remained after 8 times cycles.Gas chromatography–mass spectrometry(GC–MS)was utilized to characterize the products of lignin model compound degradation and activation,and the efficiency of immobilized laccase was calculated to be 1–5 times that of free laccase.It was proposed that the synergistic effect between MNPs and laccase displays an important role in the enhancement of stability and activity in lignin model compound biodegradation.

Efficient elimination and detection of phenolic compounds in juice using laccase mimicking nanozymes

Residual phenols in the juice can cause turbidity and affect its sensory quality.Laccase is used to remove phenolic compounds from fruit juice s.In order to overcome the shortcomings of natural laccase instability and high cost,in this work,we prepared a laccase mimic enzyme based on copper ion and adenosine monophosphate(AMP-Cu nanozymes).At the same mass concentration(1 mg·ml^(-1)), the catalytic activity of the nanozyme is about 15 times that of laccase.The AMP-Cu nanozymes had a higher V_(max) and a lower Km than laccase.The laccase mimic enzyme had a good stability under the condition of 30-90 ℃ and pH> 6.It also maintained high catalytic activity at high salt concentrations and 9 days storage time.Furthermore,the AMP-Cu nanozyme s maintained an initial catalytic activity of about 80% after six consecutive cycles of reaction.The linear range of detection of phenolic compounds by AMP-Cu nanozymes was 0.1-100 μmol·L^(-1) with a detection limit of 0.033 μmol·L^(-1).The phenol removal rate of AMP-Cu nanozymes was much higher than that of laccase under different reaction times.When the reaction was performed for 5 h,the phenol removal rate of the fruit juice by AMP-Cu nanozymes was about 65%.The efficient removal of phenolic compounds from different juices by AMP-Cu nanozymes indicate s that they have good application prospect in the food juice industry.

Organic Solvents Enhanced Spectrofluorimetric Method for Determination of Laccase Activity

A novel fluorimetric method for determination of laccase activity in organic solvents is proposed, based on the oxidation ofo-phenylenediamine (1,2-diaminobenzene, OPDA) catalyzed by laccase yielding 2,3-diaminophenazine. The optimal conditions for laccase in organic media areT=55°C, pH=6.5, 1.0×10?2mol/L OPDA, 1.25 mL ethanol, 1.25 mL 1,4-dioxane and 1.25 mL acetone. The linear range of the method proposed in ethanol, 1,4-dioxane and acetone media were 0.44–19.33, 0.11–20.85, 0.38–21.05 U with the detection limit of 0.088, 0.022, 0.076 U, respectively. The proposed method has been applied to the analysis of laccase activity of real samples with more accurate and sensitive than that of the previous method reported.

Bonding Mechanism of Bamboo Particleboards Made by Laccase Treatment

Using 1–8 years bamboo as materials,the content of different chemical constituent was tested,and the reactive oxygen species(ROS)free radicals produced from laccase treated bamboo were detected by electron spin-resonance(ESR)spectroscope.The wet-process particleboard was made from laccase-treated bamboo by hot pressing and board mechanical properties including internal bond strength(IB),modulus of rupture(MOR)and thickness swelling(TS)after 2-hours water absorption were tested under different conditions.Results showed that laccase mainly catalyze the bamboo components and improved the bonding strength of laccase-treated boards.By ESR measurement on each single component such as milled bamboo lignin,xylan and pore cotton treated with laccase,it was proved that laccase helped the degradation of bamboo lignin to produce ROS free radicals and could not catalyze the oxidation of cellulose and hemicelluloses.A logarithmic function relationship was found between board mechanical properties and ROS free radical level.It is optimal to using 5-year-old bamboo for high efficient utilization.The laccase treatment improves the activity of bamboo particles participating in self-adhesion reaction.

Properties of crude laccase from Trametes versicolor produced by solid-substrate fermentation

A low-cost process for the production of laccases is necessary for a sustainable enzymatic wastewater treatment. Therefore, it is necessary to establish an easy and low-cost procedure for the production of laccase. In the present study the properties of crude laccase from Trametes versicolor produced by solid-substrate fermentation is investigated. The application of the enzyme for dye decolorization is also studied. Crude laccase from the studied culture established maximal activity at 45ºC. The enzyme retained over 90% of its activity in the temperature range 40- 47ºC and pH 4.5. The kinetic constants of the crude enzyme was also determined. In the presence of KCl, NaCl, CaCl2, MnSO4 and MgSO4, laccase demonstra- ted high stability—over 50% of its initial activity was still retained after 4-month incubation. Complete loss of enzymatic activity was observed in the presence of CuCl2, FeCl2, FeCl3 and NaN3 after 30 min of incubation. 100% decolorization by investigated crude laccase was completed in the case of Indigo Carmine for 4 h, Remazol Brilliant Blue R—for 6 h, Orange II— for 48 h and Congo Red—for 13 d.

Cloning of multicopper oxidase gene from <i>Ochrobactrum sp</i>. 531 and characterization of its alkaline laccase activity towards phenolic substrates

A 1602 bp fragment was cloned from a soil bacterium Ochrobactrum sp. 531. It contained an open reading frame (ORF) of 1092 bp which was identified as a multicopper oxidase (MCO) with potential laccase activity. After inserting the cloned gene into the expression vector pET23a, it was expressed in E. coli BL21(DE3)pLysS, and its product was purified to homogeneity through chromatography. The Ochrobactrum sp. 531 MCO, consisting of 533 amino acids with a molecular mass of 57.8 kDa, was quite stable in neutral pH and showed laccase-like activity oxidizing 2,6-dimethoxyphenol (DMP), 2,2’-azino-bis(3-ethylbe- nzthiazolinesulfonic acid) (ABTS), and syringaldazine (SGZ). The enzyme showed optimum activity towards DMP, ABTS, and SGZ at the pH 8.0, 3.6, and 7.5 respectively. Kinetic studies gave this enzyme Km, kcat and kcat//Km values of: 0.09 mM, 7.94 s–1, and 88.22 s–1?mM–1 for DMP;0.072 mM, 2.95 s–1, and 40.97 s–1.mM–1 for ABTS;and 0.015 mM, 2.4 s–1, and 160 s–1.mM–1 for SGZ. Our results demonstrate that Ochrobactrum sp. 531 MCO is a bacterial laccase which oxidized phenolic substrates DMP and SGZ effectively under alkaline conditions. These unusual properties make the enzyme an interesting biocatalyst in applications for which classical laccases are unsuitable.

Isolation,expression and single nucleotide polymorphisms(SNPs) analysis of LACCASE gene(LkLAC8) from Japanese larch(Larix kaempferi)

Nucleotide diversity (pi) and linkage disequilibrium (LD) analysis based on SNP marker could provide a sound basis for choosing an association analysis method. Japanese larch (Larix kaempferi) is an important timber coniferous tree species for pulping and papermaking, but its high lignin content has significantly restricted it application potential. In this study, the LACCASE gene, that plays an important regulatory role for lignin biosynthesis, was selected as research target. The full-length cDNA and genomic sequences of the encoding LkLAC8 gene were isolated from the LACCASE expressed sequence tags of the Japanese larch transcriptome database using the rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA was determined to be 1940 bp, with an open reading frame (ORF, 1734 bp) that encoded a protein of 577 AA. This protein contains four highly specific Cu2+ binding sites and 11 glycosylation sites, thus belonging to the LACCASE family. The deduced protein sequence shared an 89% identity with the PtaLAC from Pinus taeda. A real-time PCR analysis showed that the LkLAC8 transcript was expressed predominantly in mature xylem, with moderate levels in the immature xylem, cambium and mature leaves, the lowest in the roots. Lastly, the genomic sequences of LkLAC8 in 40 individuals from six naturally distributed populations of Japanese larch were amplified, and a total of 201 SNPs (103 and 98 mutation types of transition and transversion, respectively) were detected; the frequency of the SNPs was 1/19 bp. Nucleotide diversity among the six populations ranged from 0.0034 to 0.0053, which suggested that there were no significant differences among the populations. The LD analysis showed that the LD level decayed rapidly within the increasing length of the LkLAC8 gene. These results implied that LD mapping and association analysis based on candidate gene may be feasible for the marker-assisted breeding of new germplasms with low lignin in Japanese larch.