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 v...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.展开更多
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 su...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 increase of color removal.While the beads contained 0.2%,2.0%,2.0%,and 0.5%(m/V)of laccase,alginate,gelatin,and PEG,respectively.The dye of 50 mg/L initial concentration of RRB was decolorized down to 50%during the tenth repeated batch.As far as the decolorization mechanism was concerned,the thermal and pH stabilities of the immobilized laccase were also investigated and were both appreciably improved.The study indicates that the immobilized laccase can be potential candidate for utilization in biodecolorization processes.展开更多
Nucleotide diversity(π) 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 conif...Nucleotide diversity(π) 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 c DNA and genomic sequences of the encoding Lk LAC8 gene were isolated from the LACCASE expressed sequence tags of the Japanese larch transcriptome database using the rapid amplification of c DNA ends-polymerase chain reaction(RACE-PCR).The c DNA 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 Cu^(2+) binding sites and 11 glycosylation sites,thus belonging to the LACCASE family.The deduced protein sequence shared an 89% identity with the Pta LAC from Pinus taeda.A real-time PCR analysis showed that the Lk LAC8 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 Lk LAC8 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 Lk LAC8 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.展开更多
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...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.展开更多
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 ...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.展开更多
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...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.展开更多
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 ...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.展开更多
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-reson...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.展开更多
In this work, a new immobilization method based on dopamine(DA) self-polymerization was developed for laccase immobilization on magnetic nanoparticles(Fe_3O_4 NPs). To optimize the immobilization condition including r...In this work, a new immobilization method based on dopamine(DA) self-polymerization was developed for laccase immobilization on magnetic nanoparticles(Fe_3O_4 NPs). To optimize the immobilization condition including reaction pH, DA concentration and enzyme concentration, a central composite response surface method was applied. The optimal condition was determined as p H value of 5.92, laccase concentration of 0.25 mg mL^(-1) and DA concentration of 12.74 mg mL^(-1), under which a high enzyme activity recovery of 88.17% was obtained.By comparing with free laccase, the stabilities of immobilized laccase towards p H, thermostability, storage were enhanced significantly.Approximately 60% of relative activity for immobilized laccase was remained after being incubated for 6 h at 50℃, but the free laccase only remained 25%. After 40 days of storage at 4℃, the laccase immobilized by DA kept about 89% of its original activity, but the free laccase only retained 48%. After recycled 10 times, the relative activity of immobilized laccase still retained 70%. The immobilized laccase was then applied to catalyze the degradation of 4-chlorophenol(4-CP), 86% percentage of 4-CP was removed within 2 h. After degraded 10 times, the relative activity of immobilized laccase still remained 64% of its initial activity, which exhibits an excellent reusability and operational stability.展开更多
Laccases are blue multicopper enzymes, capable of oxidizing diverse aromatic and non-aromatic compounds of industrial interest, concomitantly with reduction of molecular oxygen to water. Tolerance to extreme condition...Laccases are blue multicopper enzymes, capable of oxidizing diverse aromatic and non-aromatic compounds of industrial interest, concomitantly with reduction of molecular oxygen to water. Tolerance to extreme conditions, such as high temperature, salinity or extreme pH, is required for practical industrial applications. Here we focus on bacterial laccases from the phylum Actinobacteria, notably the order Actinomycetales. Currently, less than 10 enzymes have been properly characterized, all belonging to genus Streptomyces, but it is noteworthy that all of them have exhibited industrially important properties. Furthermore, studies with enzymes from this phylum revealed a novel molecular structure of laccases, providing the basis for a distinct family, the two-domain laccases. The relevant traits of actinomycetes laccases emphasize the need for more studies involving the isolation of this bacterial group from lignin-rich environmental samples, detection of their laccase activity and thereafter, characterization of the proteins and related genes. The nonhomogeneous responses of actinomycetes laccases to traditional inhibitors, substrates or metal ions have challenged the currently accepted “laccase concept”. Finally, considering that distinguishing laccase activity in vitro from other ligninolytic enzymes becomes a difficult task due to overlaps in catalytical properties of the enzymes, we proposed a simple flow chart to help experimental assays.展开更多
White rot fungi were optimized to cultivate highly active laccase. The characteristics of laccase incubated by continuous culture were compared with those of direct culture. The enzyme activity of laccase incubated by...White rot fungi were optimized to cultivate highly active laccase. The characteristics of laccase incubated by continuous culture were compared with those of direct culture. The enzyme activity of laccase incubated by continuous culture technology reached a higher value on the fifth day of the growth. The optimization incubation time of high activity laccase was the eleventh day. A large amount of highly active laccase can be obtained in a relatively short time by continuous culture to replace traditional laccase. After laccase treatment, the lignin composition of wood fibers were oxidation-catalyzed by laccase. The number of chemical-bonding points between the wood fibers was increased. The wood fibers treated by laccase were fabricated into boards and their mechanical properties improved with the laccase-incubation times.Compared with the fiberboards made from fibers that were pre-treated by laccase of incubation 5 days, the static bending strength of those that were pre-treated by laccase of incubation 11 days was increased by 18.95%, the elastic modulus was increased by 35.49%, and the internal bond strength was increased by 44.11%.展开更多
It is studied that Rhus vernicifera laccase cata ly zed oxidation of 26 disubstituted ferrocene and its metal complexes, π-arene- π-cyclopentadienyli ron derivatives and metal meso-(tetra-4-sulfanatophenyl)porphyrin...It is studied that Rhus vernicifera laccase cata ly zed oxidation of 26 disubstituted ferrocene and its metal complexes, π-arene- π-cyclopentadienyli ron derivatives and metal meso-(tetra-4-sulfanatophenyl)porphyrin in the di ethylene glycol monobutyl ether (DGBE)/phosphate buffer (V/V,1/5).It is foun d th at 1,1’-bishydroxylmethy1 ferrocene etc 16 compounds are new substrates of lacc ase. The relation of structure and function of substrates is discussed. T he affective factors of laccase-catalyzed oxidation of 1,1’-bishydroxylmethyl ferrocene——pH, temperature, substrate concentration, laccase quantity and surf actant were investigated further.展开更多
The molar reation enthalpy,the Michaelis constant and the observed rate constant of the reaction between the Rhus vernicifera laccase and p-phenylenediamine have been determined at 298. 15 K by LKB-2107 microcalorimet...The molar reation enthalpy,the Michaelis constant and the observed rate constant of the reaction between the Rhus vernicifera laccase and p-phenylenediamine have been determined at 298. 15 K by LKB-2107 microcalorimetry system in 0.1 mol/L phosphate salt buffer (pH7. 4) to be △rHm=-136.36±0. 36kJ/mol, Km= 5. 58×10-3 mol/L and k1 =8. 63×10-3s-1, respectively. The catalyst activity of laccase withp-phenylenediamine as substrate has been determined to be EA=0. 045 IU in the experimental condition.The observed activation energy of non-enzymic step of the reaction, the Gibbs binding energy of the combination process of laccase and substrate have been also calculated. The physical significance of the determined parameters were discussed for different step of the reaction.展开更多
Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants. In Setosphaeria turcica, 9 laccase-like multicopper ...Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants. In Setosphaeria turcica, 9 laccase-like multicopper oxidases have been identified, and StLAC2 is involved in the synthesis of the melanin that accumulates in the cell wall. The function of another major laccase gene, StLAC6, was studied here. The knockout of StLAC6 had no effect on the growth, morphology or invasion ability of S. turcica, but the morphology and function of peroxisomes of knockout mutants were abnormal. The knockout of the StLAC6 gene resulted in increased contents of phenolic compounds and melanin and the sensitivity to fungicides increased compared with wild type strains. In the mutants of StLAC6, there is a significant change of the expression levels of other laccase genes. This study provides a new insight into laccase functions and the relationship of the laccase gene family in plant pathogenic fungi.展开更多
This work is focused on immobilization of laccase from Myceliophthora thermophila expressed in Aspergillus oryzae(Novozym 51003?laccase)on amino modified fumed nano-silica(AFNS)and the possible use in bioremediation.H...This work is focused on immobilization of laccase from Myceliophthora thermophila expressed in Aspergillus oryzae(Novozym 51003?laccase)on amino modified fumed nano-silica(AFNS)and the possible use in bioremediation.Hereby,for the first time,factors affecting the immobilization of Novozym 51003?laccase on AFNS were investigated for defining the immobilization mechanism and optimizing the utilization of AFNS as support for laccase immobilization.The highest specific activity(13.1 IU·mg-1 proteins)was achieved at offered 160 mg per g of AFNS and for the same offered protein concentration the highest activity immobilization yield,reaching68.3%after the equilibrium time,at optimum pH 5.0,was obtained.Laccase immobilization occurs by adsorption as monolayer enzyme binding in 40 min,following pseudo-first-order kinetics.The possible use of obtained immobilized preparation was investigated in degradation of pesticide lindane.Within 24 h,lindane concentration was reduced to 56.8%of initial concentration and after seven repeated reuses it retained 70%of the original activity.展开更多
The continuous use of chemical dyes in various industries,and their discharge into industrial effluents,results in severe problems to human life and water pollution.Laccases have the ability to decolorize dyes and tox...The continuous use of chemical dyes in various industries,and their discharge into industrial effluents,results in severe problems to human life and water pollution.Laccases have the ability to decolorize dyes and toxic chemicals in industrial effluents as green biocatalysts.Their possible industrial applications have been limited by poor reusability,low stability,and loss of free laccase action.In this research,lac-case was immobilized on zeolitic imidazolate framework coated multi-walled carbon nanotubes(Laccase@ZIF-8@MWCNTs)via metal affinity adsorption to develop an easy separable and stable enzyme.The optimum reaction conditions for immobilized laccase are at a pH of 3.0 and a temperature of 60℃.The immobilized laccase was enhanced in storage and thermal stability.The results indicated that Laccase@ZIF-8@MWCNTs still maintained 68%of its original activity after 10 times of repeated use.Most importantly,the biocatalytic system was applied for decolorization of different dyes(20 mg·L^(-1))without a mediator,and up to 97.4%for Eriochrome black T and 95.6%Acid red 88 was achieved in 25 min.Biocatalysts with these properties may be used in a variety of environmental and industrial applications.展开更多
基金supported by the Guangdong Basic and Applied Basic Research Foundation(2021B15151300042021B1515140021)+2 种基金the Scientific Research Start-up Funding of Guangdong Medical University(1026/4SG21229G)China Postdoctoral Science Foundation(2021M702781)Guangdong Medical University Post-doctoral Research Funding(2BH19006P)。
文摘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.
基金supported by the National Hi-Tech Research and Development Program(863)of China(No.2007AA02Z218)the Open Project Program of Key Lab-oratory of Eco-Textiles,Jiangnan University,Ministry of Education,China(No.KLET0625) the Youth Fundof Jiangnan University(No.2006LQN002).
文摘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 increase of color removal.While the beads contained 0.2%,2.0%,2.0%,and 0.5%(m/V)of laccase,alginate,gelatin,and PEG,respectively.The dye of 50 mg/L initial concentration of RRB was decolorized down to 50%during the tenth repeated batch.As far as the decolorization mechanism was concerned,the thermal and pH stabilities of the immobilized laccase were also investigated and were both appreciably improved.The study indicates that the immobilized laccase can be potential candidate for utilization in biodecolorization processes.
基金financially supported by the Fundamental Research Funds for the Central Non-profit Research Institution of CAF(RIF2014-06)the Forestry Industry Research special funds for Public Welfare Projects(201504104)
文摘Nucleotide diversity(π) 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 c DNA and genomic sequences of the encoding Lk LAC8 gene were isolated from the LACCASE expressed sequence tags of the Japanese larch transcriptome database using the rapid amplification of c DNA ends-polymerase chain reaction(RACE-PCR).The c DNA 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 Cu^(2+) binding sites and 11 glycosylation sites,thus belonging to the LACCASE family.The deduced protein sequence shared an 89% identity with the Pta LAC from Pinus taeda.A real-time PCR analysis showed that the Lk LAC8 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 Lk LAC8 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 Lk LAC8 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.
文摘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.
文摘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.
基金financially supported by the National Natural Science Foundation of China (31772058)the Science and Technology Development Project of Jilin Province, China (20190302088GX and 20190701079GH)+1 种基金the Jilin Provincial Strategic Economic Infrastructure Adjustment fund (2019C043-5 and 2020C023-5)Fundamental Research Funds for the Central Universities。
文摘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.
基金supported by the Startup Foundation of Beijing Institute of Technology,China(3160011181808)。
文摘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.
基金The authors thank Zhejiang Provincial Science and Technology Department Project(2019F1065-4)&Zhejiang Provincial Natural Science Foundation(LY19C160004)&Public Welfare Technology Research Agricultural Project of Zhejiang Province(2013C32104)for their financial contributions.
文摘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.
基金support from the National Natural Science Foundation of China(Grant Nos.51378487,51425405,21376249,21336010)Youth Innovation Promotion Association,CAS(2014037)973 Program(2013CB733604)
文摘In this work, a new immobilization method based on dopamine(DA) self-polymerization was developed for laccase immobilization on magnetic nanoparticles(Fe_3O_4 NPs). To optimize the immobilization condition including reaction pH, DA concentration and enzyme concentration, a central composite response surface method was applied. The optimal condition was determined as p H value of 5.92, laccase concentration of 0.25 mg mL^(-1) and DA concentration of 12.74 mg mL^(-1), under which a high enzyme activity recovery of 88.17% was obtained.By comparing with free laccase, the stabilities of immobilized laccase towards p H, thermostability, storage were enhanced significantly.Approximately 60% of relative activity for immobilized laccase was remained after being incubated for 6 h at 50℃, but the free laccase only remained 25%. After 40 days of storage at 4℃, the laccase immobilized by DA kept about 89% of its original activity, but the free laccase only retained 48%. After recycled 10 times, the relative activity of immobilized laccase still retained 70%. The immobilized laccase was then applied to catalyze the degradation of 4-chlorophenol(4-CP), 86% percentage of 4-CP was removed within 2 h. After degraded 10 times, the relative activity of immobilized laccase still remained 64% of its initial activity, which exhibits an excellent reusability and operational stability.
文摘Laccases are blue multicopper enzymes, capable of oxidizing diverse aromatic and non-aromatic compounds of industrial interest, concomitantly with reduction of molecular oxygen to water. Tolerance to extreme conditions, such as high temperature, salinity or extreme pH, is required for practical industrial applications. Here we focus on bacterial laccases from the phylum Actinobacteria, notably the order Actinomycetales. Currently, less than 10 enzymes have been properly characterized, all belonging to genus Streptomyces, but it is noteworthy that all of them have exhibited industrially important properties. Furthermore, studies with enzymes from this phylum revealed a novel molecular structure of laccases, providing the basis for a distinct family, the two-domain laccases. The relevant traits of actinomycetes laccases emphasize the need for more studies involving the isolation of this bacterial group from lignin-rich environmental samples, detection of their laccase activity and thereafter, characterization of the proteins and related genes. The nonhomogeneous responses of actinomycetes laccases to traditional inhibitors, substrates or metal ions have challenged the currently accepted “laccase concept”. Finally, considering that distinguishing laccase activity in vitro from other ligninolytic enzymes becomes a difficult task due to overlaps in catalytical properties of the enzymes, we proposed a simple flow chart to help experimental assays.
基金funded by National Natural Science Foundation of China(31170515)Specialized Research Fund for the Doctoral Program of Higher Education of China(20130062110012)Undergraduate Training Program for Innovation and Entrepreneurship of Northeast Forestry University,China(201410225157)
文摘White rot fungi were optimized to cultivate highly active laccase. The characteristics of laccase incubated by continuous culture were compared with those of direct culture. The enzyme activity of laccase incubated by continuous culture technology reached a higher value on the fifth day of the growth. The optimization incubation time of high activity laccase was the eleventh day. A large amount of highly active laccase can be obtained in a relatively short time by continuous culture to replace traditional laccase. After laccase treatment, the lignin composition of wood fibers were oxidation-catalyzed by laccase. The number of chemical-bonding points between the wood fibers was increased. The wood fibers treated by laccase were fabricated into boards and their mechanical properties improved with the laccase-incubation times.Compared with the fiberboards made from fibers that were pre-treated by laccase of incubation 5 days, the static bending strength of those that were pre-treated by laccase of incubation 11 days was increased by 18.95%, the elastic modulus was increased by 35.49%, and the internal bond strength was increased by 44.11%.
基金Supported by the National Natural Science Foundation of China(39170 2 0 3)
文摘It is studied that Rhus vernicifera laccase cata ly zed oxidation of 26 disubstituted ferrocene and its metal complexes, π-arene- π-cyclopentadienyli ron derivatives and metal meso-(tetra-4-sulfanatophenyl)porphyrin in the di ethylene glycol monobutyl ether (DGBE)/phosphate buffer (V/V,1/5).It is foun d th at 1,1’-bishydroxylmethy1 ferrocene etc 16 compounds are new substrates of lacc ase. The relation of structure and function of substrates is discussed. T he affective factors of laccase-catalyzed oxidation of 1,1’-bishydroxylmethyl ferrocene——pH, temperature, substrate concentration, laccase quantity and surf actant were investigated further.
文摘The molar reation enthalpy,the Michaelis constant and the observed rate constant of the reaction between the Rhus vernicifera laccase and p-phenylenediamine have been determined at 298. 15 K by LKB-2107 microcalorimetry system in 0.1 mol/L phosphate salt buffer (pH7. 4) to be △rHm=-136.36±0. 36kJ/mol, Km= 5. 58×10-3 mol/L and k1 =8. 63×10-3s-1, respectively. The catalyst activity of laccase withp-phenylenediamine as substrate has been determined to be EA=0. 045 IU in the experimental condition.The observed activation energy of non-enzymic step of the reaction, the Gibbs binding energy of the combination process of laccase and substrate have been also calculated. The physical significance of the determined parameters were discussed for different step of the reaction.
基金supported by the grants from the National Natural Science Foundation of China(31901827)the China Agriculture Research System(CARS-02)+2 种基金the Natural Science Foundation of Hebei Province,China(C2020204039)the Key Research and Development Projects of Hebei Province(20326510D)the Hebei Province Projects of Overseas Foundation(C20190508)。
文摘Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants. In Setosphaeria turcica, 9 laccase-like multicopper oxidases have been identified, and StLAC2 is involved in the synthesis of the melanin that accumulates in the cell wall. The function of another major laccase gene, StLAC6, was studied here. The knockout of StLAC6 had no effect on the growth, morphology or invasion ability of S. turcica, but the morphology and function of peroxisomes of knockout mutants were abnormal. The knockout of the StLAC6 gene resulted in increased contents of phenolic compounds and melanin and the sensitivity to fungicides increased compared with wild type strains. In the mutants of StLAC6, there is a significant change of the expression levels of other laccase genes. This study provides a new insight into laccase functions and the relationship of the laccase gene family in plant pathogenic fungi.
基金the Ministry of Education,Science and Technological Development,Republic of Serbia,within projectsⅢ46010,Ⅲ45019 and TR31035 for the financialDirectorate of Measures and Precious Metals,Ministry of Economy,Republic of Serbia for the technical support。
文摘This work is focused on immobilization of laccase from Myceliophthora thermophila expressed in Aspergillus oryzae(Novozym 51003?laccase)on amino modified fumed nano-silica(AFNS)and the possible use in bioremediation.Hereby,for the first time,factors affecting the immobilization of Novozym 51003?laccase on AFNS were investigated for defining the immobilization mechanism and optimizing the utilization of AFNS as support for laccase immobilization.The highest specific activity(13.1 IU·mg-1 proteins)was achieved at offered 160 mg per g of AFNS and for the same offered protein concentration the highest activity immobilization yield,reaching68.3%after the equilibrium time,at optimum pH 5.0,was obtained.Laccase immobilization occurs by adsorption as monolayer enzyme binding in 40 min,following pseudo-first-order kinetics.The possible use of obtained immobilized preparation was investigated in degradation of pesticide lindane.Within 24 h,lindane concentration was reduced to 56.8%of initial concentration and after seven repeated reuses it retained 70%of the original activity.
基金supported by the National Natural Science Foundation of China(Nos.21576068,21276060,21276062,and 21306039)the Natural Science Foundation of Tianjin City(16JCY-BJC19800)+3 种基金the Natural Science Foundation of Hebei Province(B2015202082,B2016202027,and B2020202036)the Science and Technology Program Project of Tianjin(20YDTPJC00260)the Program for Top 100 Innovative Talents in Colleges and Universities of Hebei Province(SLRC2017029)Hebei High level personnel of support program(A2016002027).
文摘The continuous use of chemical dyes in various industries,and their discharge into industrial effluents,results in severe problems to human life and water pollution.Laccases have the ability to decolorize dyes and toxic chemicals in industrial effluents as green biocatalysts.Their possible industrial applications have been limited by poor reusability,low stability,and loss of free laccase action.In this research,lac-case was immobilized on zeolitic imidazolate framework coated multi-walled carbon nanotubes(Laccase@ZIF-8@MWCNTs)via metal affinity adsorption to develop an easy separable and stable enzyme.The optimum reaction conditions for immobilized laccase are at a pH of 3.0 and a temperature of 60℃.The immobilized laccase was enhanced in storage and thermal stability.The results indicated that Laccase@ZIF-8@MWCNTs still maintained 68%of its original activity after 10 times of repeated use.Most importantly,the biocatalytic system was applied for decolorization of different dyes(20 mg·L^(-1))without a mediator,and up to 97.4%for Eriochrome black T and 95.6%Acid red 88 was achieved in 25 min.Biocatalysts with these properties may be used in a variety of environmental and industrial applications.
