127 results about "Laccase activity" patented technology

The invention discloses a preparation method of bacillus subtilis LS02 (CGMCC No.4261) spore laccase, the catalytic property of the spore laccase and application of the spore laccase to decoloring of a dye. The invention also relates to a nucleotide sequence for encoding the spore laccase. The method comprises the following steps of: fermenting bacillus subtilis LS02 on a solid-state sporulation medium at the temperature of 37 DEG C for 5 days; flushing spores with deionized water; and performing a series of treatment such as lysozyme treatment to obtain a spore suspension with laccase activity. The spore laccase has high catalyst activity under alkaline and high temperature conditions, has good decoloring effects on synthetic dyes with different structures under the action of a mediator,and can be applied to treatment of industrial dye waste water.

The invention discloses a laccase biosensor based on magnetic carbon nano tube and chitosan / silicon dioxide gel, comprising a magnetic carbon paste electrode, the electrode surface of the magnetic carbon past electrode is modified with a magnetic carbon nano tube layer, the surface of the magnetic carbon nano tube layer is absorbed with laccase, the laccase is wrapped by a chitosan-carbon dioxide composite film; the preparation method of the laccase biosensor includes the steps of material preparation, magnetic carbon nano tube modification, laccase absorption and laccase wrapping. The method for detecting pyrocatechol by applying the laccase biosensor includes the following steps: the laccase biosensor is taken as a working electrode, a three electrode system is established, the three electrode system is connected with an electrochemical work station, and reduction current amount is detected to quantitatively indicate the concentration of pyrocatechol in solution to be detected. The sensor of the invention has the advantages of low cost, simple manufacturing, long service life, high laccase activity, high detection accuracy and strong anti-interference.

The invention belongs to the field of biotechnology, and discloses a laccase gene as well as an encoded protein and an application thereof. The open reading frame sequence of the laccase gene fmb-L103 is shown in SEQ ID NO. 1. The amino acid sequence of the protein encoded by the laccase gene fmb-L103 disclosed by the invention is shown in SEQ ID NO. 2. The laccase gene fmb-L103 disclosed by the invention is applied to decoloration for triphenlmethane dyes. The inventor clones from the selected dead bacillus vallismortis fmb-L103 with a spore laccase activity to obtain a novel prokaryotic laccase gene fmb-L103, and realizes the heterologous efficient expression of escherichia colli expression host bacteria. The recombinase fmb-L103 can be used for efficiently catalyzing the degradation of the triphenlmethane dyes of malachite green, brilliant green and aniline blue.

The invention provides hypsizigus marmoreus and a method for establishing laccase transfer system in breeding thereof, belongs to the technical field of the hypsizigus marmoreus, and solves the problems that the hypsizigus marmoreus does not have laccase activity, has slow growing speed, longer production period and weak bacterial resistance in the prior art. The Hypsizigus marmoreus new strain Jinshan No.1 has the laccase activity, and is reserved by China General Microbiological Culture Collection Center on 12 June, 2009 with the accession number of CGMCC No. 3120. Pleurotus pulmonarius withthe laccase activity and the hypsizigus marmoreus are subjected to protoplast fusion to establish the laccase transfer system in the hypsizigus marmoreus. The hypsizigus marmoreus new strain culturedby the method has excellent properties of high laccase activity, high growing speed, short production period and the like.

The invention discloses a laccase mutant and an encoding gene and an application thereof. The laccase mutant disclosed by the invention is any one of the following protein in a) to d): a) protein as shown in a sequence 1; b) protein as shown in a sequence 3; c) protein as shown in a sequence 5; d) protein with laccase activity and obtained by substituting and / or deleting and / or adding one or multiple amino acid residues in the amino acid sequences of the proteins limited by any one of a) to c). As shown in experiments, compared with wild laccase, the laccase mutant disclosed by the invention has the advantages of high activity, wide pH tolerance range and the like. The laccase mutant disclosed by the invention is expected to be applied in such fields as wastewater treatment in pulp bleaching and dyeing and printing industry, biological detection in medicine field, food industry, organic synthesis, organic pesticide degradation, etc.

The invention discloses preparation, a catalytic property and use in dye decolorizing of spore laccase from Bacillus licheniformis LS04 (CGMCC No.4263). The invention also relates to a nucleotide sequence coding the spore laccase. The invention also relates to a nucleotide sequence for coding the spore laccase. In the invention, Bacillus licheniformis LS04 ferments at 37 DEG C for 5 days on a solid sporulation culture medium, spores are washed off by deionized water, and pore suspension with laccase activity can be obtained by series measures such as lysozyme treatment. The spore laccase has high catalytic activity under alkaline and high-temperature conditions, can maintain high enzyme activity in high-concentration organic solvent and salt solution, has good decolorizing effect on synthesized dyes of different structures under action of an amboceptor and can be used for treating industrial dye waste water.

The invention discloses a fermentation production method of fungal laccase and an application of the laccase. The method comprises the following steps: preparing Panus conchatus suspension; inoculating the suspension into a fermentation tank containing 3-5L of fermentation medium and culturing for 10-15 days, thus obtaining fermentation liquor; and finally, filtering or centrifuging the fermentation liquor, thus obtaining a laccase preparation. The method of the invention is simple and convenient in subsequent treatment of the fermentation liquor and low in production cost, and the activity of the Panus conchatus laccase obtained through fermentation is over 200IU / mL. Compared with other domestic and overseas laccase production methods in a fermentation tank scale, the fermentation production method of the fungal laccase, which is disclosed by the invention, has the advantages that the total laccase yield and the laccase activity reach a relatively high level, and the prepared laccase preparation has a good pulp bleaching effect.

The invention discloses a carbon nano tube immobilized laccase, which is prepared by the following steps of carrying out grafting of succinimido and grafting of a nitrilotriacetic acid functional group on a carbon nano tube; carrying out surface functionalization treatment through copper ion chelating and modifying, and carrying out immobilized reaction with the laccase with copper ion removal. The laccase active center is directly connected with the copper ions of the carbon nano tube surface functional group, so that the real meaning direct electron transfer can be realized, and the shielding effect produced by a laccase protein shell on the direct electron transfer process from the active center to the carbon nano tube is overcome. The carbon nano tube immobilized laccase provided by the invention solves the problem of limitation of the electron transfer rate during the carbon nano tube immobilized laccase catalytic process, the sensitivity of the immobilized laccase in biological detection can be improved, the lowest detection limit during phenol determination is reduced by 85 percent to 96 percent, and the current density in biological fuel cell application is improved by 80 percent to 120 percent.

The invention relates to a decomposition agent for branch composting treatment. The microbial agent is a solid biological microbial agent which is obtained by inoculating a composite bacterial liquidcontaining active ingredients of bacillus velezensis, bacillus arabinodoxae, bacillus megaterium, bacillus kaempferiae and bacillus megatherium on a solid fermentation substrate and fermenting, wherein the effective viable count of the microbial agent is not less than 1.0 * 10 < 10 > cfu / g. The cellulase activity, the laccase activity, the manganese peroxidase activity and the lignin peroxidase activity in the solid biological agent are not lower than 100.0 U / g, 1.0 U / g, 1.0 U / g and 1.5 U / g respectively, and when the solid biological agent is used for composting treatment, the degradation efficiency of lignin and cellulose can be improved, and the composting rate and quality can be improved.

The invention discloses a preparation method of bacillus amyloliquefaciens LC02 (CGMCC No.4258) spore laccase, the catalysis capability and the application in the decoloring the dye. The invention further relates to a nucleotide sequence which codes the spore laccase. The bacillus amyloliquefaciens LC02 is fermented on a solid state spore generating culture medium at 37 DEG C for 5 days, and the spore is washed by the deionized water to obtain the spore suspension liquid with the laccase activity by a series of measures such as the lysozyme. The spore laccase is better in catalytic activity under the alkaline and high-temperature conditions, is better in a decoloring effect to the synthetic dyes with different structures under the action of a medium, and is used for treating the industrial dye waste water.

The invention discloses a laccase generation cerrena and an application thereof. The laccase generation cerrena is Cerrena unicolor GSM-01, and the registration serial number of the Cerrena unicolor at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms is CGMCC No.7713. As shown in experiments, the Cerrena unicolor GSM-01CGMCC No.7713 of the invention is shake cultivated in a PD culture medium for 6 days at 25 DEG C and at a rotating speed of 180rpm (the rotating radius is 20mm), the laccase activity of per ml fermentation broth is 965.42+ / -13.71U, and the laccase activity of each mg (wet weight) of mycelium is 2822.71+ / -312.33U.

The invention discloses a fungal laccase gene Lac1. The nucleotide sequence of the fungal laccase gene Lac1 is shown in SEQ ID NO:1. The invention further discloses a construction method of a laccaserecombinant strain. The construction method comprises the following steps: connecting the fungal laccase gene Lac1 with an expression vector pPIC9K, performing linearization on the obtained recombinant plasmid pPIC9K / Lac1, converting pichia pastoris GS115, and constructing a pichia pastoris recombinant strain GS115 / pPIC9K-Lac1; and performing induced fermentation on the pichia pastoris recombinantstrain, and screening the pichia pastoris recombinant strain with laccase activity as a laccase recombinant strain. The invention further provides a method for preparing recombinant laccase by usingthe laccase recombinant strain. The recombinant laccase is applied to dye decoloring.

The invention discloses a system for co-immobilizing laccase and a mediator on amino silanized magnetic oxidized graphene nano particles and a preparation method thereof. The system is obtained by co-immobilizing the laccase and the mediator on the amino silanized magnetic oxidized graphene nano particles, 0.3-0.6 mmol of mediator is fixed in every 1 g of laccase and mediator co-immobilizing system, and the laccase activity after co-immobilization is 120-300 U / g. The laccase and the mediator are co-immobilized on the magnetic oxidized graphene carrier for the first time and can be recovered and recycled through an external magnetic field, the electron transfer efficiency between the laccase and a substrate is improved through graphite oxide, accordingly the laccase utilization rate is improved, and the cost is reduced.

The invention relates to bacillus subtilis and a culture method and application thereof. The bacillus subtilis ASD05 is preserved in the General Microbiology Center of the China Committee for CultureCollection of Microorganisms on May 15, 2019, the preservation address is No.3, No.1 Courtyard, Beichen West Road, Chaoyang District, Beijing City, and the preservation number is CGMCC No.17807. The invention further relates to the culture method and application of the bacillus subtilis ASD05. It is found for the first time that the bacillus subtilis ASD05 has the effect of adhering microorganismsrich in straw degradation effect to the surface of straw, and meanwhile has the capacity of high yield of xylanase and pectinase, cellulase activity is low, and laccase activity is avoided.

The quick screening method for high yield laccase fungus includes the culture of the spawn with PDA culture medium containing guaiacol and the combined culture test after the culture medium becomes iron red. It includes the following steps: designing several PB culture medium combination based on the key factors for raising enzyme generating activity and culturing spawn; culturing for 7 days while measuring the laccase activity once every day; obtaining highest laccase activity and performing variance analysis to obtain the p values of all the factors; and estimating the effects of all factors on the laccase generation based on the p values. The present invention has greatly reduced test times, can determine the laccase producing capacity under the action of different factors, and obtain high yield laccase fungus through comparing the laccase yield in optimized condition.

This invention discloses a method for producing laccase by fungi co-culture. The method comprises: inoculating the suspension of Trametes sp. AH28-2 into a fermentation culture medium, culturing at 22-37 deg.C for 1-4 days, adding the suspension of Trichoderma sp. ZH1, culturing for 5-10 days, and separating to obtain laccase agent with living laccase content 6,000 U / L. The culture medium is liquid culture medium, and mainly comprises certain amounts of carbon source, nitrogen source, phosphates of potassium and sodium, sulfates of copper and iron, adenine and vitamin B. The method avoids aromatic compounds and heavy metal ions to induce, thus is safe and environmentally friendly, and has high and stable laccase activity.

The invention provides protein of bacterial laccase laclK. An amino sequence of the protein is uniform with SEQ ID NO:1 by at least 90%, and the protein has laccase activity. The invention further provides gene of the bacterial laccase laclK. The gene codes the protein of the bacterial laccase laclK. The invention further provides a decoloring method. The decoloring method includes: enabling a to-be-decolored material to contact with the protein of the bacterial laccase laclK under enzymatic reaction. The invention further provides application of the protein of the bacterial laccase laclK in detection analysis of dye and treatment of printing and dyeing wastewater. The bacterial laccase laclK has high long-time stability and heat resistance and can be applied in a wider application range.

The invention relates to a preparation method and an application for bionic laccase based on dipeptide. The bionic laccase based on the dipeptide takes metal ions as a catalytic active center and dipeptide as an organic framework, and has laccase-like activity; and the bionic laccase based on the dipeptide is a dipeptide-crosslinking agent-metal compound. The bionic laccase prepared by using the method provided by the invention has the following advantages: the preparation process is simple, and the preparation conditions are mild; the prepared bionic laccase has the catalytic activity of simulating natural laccase; the catalytic efficiency of the bionic laccase is far higher than the catalytic efficiency of natural laccase; and the bionic laccase has high stability and good biocompatibility. The preparation method for the series of bionic laccase based on the dipeptide has potential application in development of industrial applications of wastewater treatment, textile bleaching, foodsafety and the like.

The invention discloses armillariella tabescens laccase and a gene thereof. The inventor constructs recombined pichia pastoris engineering bacteria according to armillariella tabescens laccase and the gene thereof. By using the engineering bacteria, efficient fermentation can be achieved for obtaining recombined armillariella tabescens laccase. On the basis of pichia pastoris heterologous expression, the inventor carries out purification and enzymology activity study on zymoprotein. The result shows that laccase has laccase activity, armillariella tabescens laccase or recombined armillariella tabescens laccase can catalyze 2,4-dichlorophenol degradation effectively, metallic copper ions have a function of promoting laccase activity, while manganese ions, zinc ions, magnesium ions, calcium ions, silver ions and SDS have a function of inhibiting activity. Stability analysis of prepared recombined laccase shows that stability of recombined laccase is good, acid environment tolerance is high, and application value is high.

The invention discloses a thermus thermophilus laccase (benzenediol: oxygen oxidoreductases), engineering bacteria, a recombinant laccase and a use of the recombinant laccase. The thermus thermophilus laccase has an amino acid sequence shown in the formula of Seq No.2. The DNA sequence for coding the above laccase is shown in the formula of Seq No.1. The DNA can be used in construction of a Pichia pastoris expression system. Through expression vector pHKFA1-lacTT linearization and then Pichia pastoris conversion by the expression vector, recombinant Pichia pastoris engineering bacteria are obtained. The recombinant laccase is obtained by fermentation of the recombinant Pichia pastoris engineering bacteria. The recombinant laccase has laccase activity and high temperature resistance, can be used in dye decoloring and especially in azo dye and anthraquinone dye decoloring, and has a decolourization ratio of 100%.

The invention provides a modification method on the basis of crosslinking reaction between laccase amino acid residue terminal carboxyl group (-COOH) and amino group of methyl L-phenylalaninate hydrochloride to enhance activity and stability of the laccase, which comprises the following step: mixing laccase, methyl L-phenylalaninate hydrochloride and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride at a certain temperature to react to obtain the modified laccase. Compared with the unmodified laccase, the modified laccase has higher activity and stability. The modified laccase can be used for aftergrowth fiber modification, and can save the laccase consumption on the premise of enhancing the strength property of the paper sheets made from aftergrowth fibers.

The invention discloses a culture medium for fermenting panus rudis to produce laccase and a laccase production method. A culture medium formula comprises 10.04g / L of soluble starch, 0.2g / L of peptone, 0.50g / L of K2HPO4, 0.50g / L of KH2PO4, 0.01g / L of ZnSO4.7H2O, 0.5g / L of MgSO4.7H2O, 0.01g / L of CuSO4.7H2O, 0.005g / L of FeSO4.7H2O, 0.05g / L of MnSO4, 0.08g / L of CaCl2, 0.1g / L of VB1, 0.4% of tween-40, and 0.05g / L of veratryl alcohol; the initial pH value of the culture medium is 6.8. A fermentation seed solution is inoculated in the culture medium so as to culture and produce the laccase at the temperature of 31 DEG C and the rotating speed of 160r / min. The culture medium for fermenting the panus rudis to produce the laccase, provided by the invention, has the advantages of high laccase productivity, high laccase activity and the like; the culture method is short in period and mild in conditions.

The invention discloses a preparation method for Trichoderma harzianum degrading p-toluenesulfonic acid. A solid fermentation method is used for preparing the Trichoderma harzianum, and the preparation method includes the steps that straw powder is added into a fermentation vat, water and a nitrogen source are added and stirred sufficiently and evenly, and the mixture is inoculated with Trichoderma harzianum strains and make full contact with the straw powder; fermentation is carried out under the natural condition, and the temperature of a fermentation substrate is controlled not to exceed 50 DEG C. When laccase activity in the substrate is not increased, fermentation is stopped, and the obtained fermentation substrate is the Trichoderma harzianum used for treating industrial waste water. An industrial waste water treatment method with the Trichoderma harzianum includes the step that the additive amount of the Trichoderma harzianum in industrial waste water accounts for 0.1-5% of the waste water amount; the pH value of the industrial waste water is 3.0-6.0, the treatment temperature is 20 DEG C-50 DEG C, and the treatment time is 2-10 h.

The invention discloses a preparation method and application of bionic laccase functionalized imine covalent organic framework nano-enzyme, and the method comprises the following steps: firstly dissolving an organic ligand for synthesizing imine COFs in an organic mixed solution (solution A), and then ultrasonically dissolving a laccase active ligand in an acetic acid aqueous solution (solution B); and then, adding the solution B into the solution A to induce an amine-aldehyde condensation reaction, so as to obtain the nano-enzyme precursor for in-situ modification of the laccase active ligand on the imine COFs skeleton. On the basis of an amine-aldehyde condensation reaction, defect amino groups on the imine COFs are connected with amino acid-like molecules by utilizing glutaraldehyde, and then are coordinated with copper ions, so that the bionic laccase nano-enzyme can be obtained. The bionic laccase nano-enzyme is synthesized by taking COF-OMe as a representative, and the material has relatively high specific surface area, good structural stability and rich laccase-like active sites, and has excellent detection performance and degradation effect on phenolic compounds such as 2, 4-dichlorophenol, hydroquinone, epinephrine and the like.

The invention belongs to the technical field of fluorescent biological detection, and discloses a mercury ion detection method based on an enzymatic reaction double-emission fluorescent probe. The method comprises: laccase can catalytically oxidize 2, 3-diaminophenazine (DAP) generated by o-phenylenediamine (OPD), and an internal filtration effect existing between DAP and gold nanoclusters (AuNCs), so that the dual-emission fluorescent probe is obtained; using the characteristic that mercury ions have an inhibiting effect on laccase activity, and adding the mercury ions to reduce the laccase activity, so that the amount of DAP generated by catalysis is reduced, and sensitive detection of the mercury ions in an actual sample is realized. The response range of the ratiometric fluorescence sensing method based on enzymatic reaction to mercury ions is 8.0 * 10<-7>-35 * 10<-6> mol.L<-1>, the detection limit is 2.7 * 10<-7> mol.L<-1>, and a novel enzyme-based ratiometric fluorescence sensingmethod is provided for measuring mercury ions in an actual sample.

The invention discloses immobilized laccase and a preparation method thereof, and application of the immobilized laccase in antibiotic degradation. The preparation method comprises the following steps: adding laccase into a solution containing copper ions, carrying out uniform mixing, then adding a trimesic acid solution, carrying out uniform mixing, performing centrifuging to remove a supernatant, conducting cleaning with a cleaning solution, and carrying out drying in vacuum to obtain the immobilized laccase. The method is simple to operate; the laccase can be coated in a carrier; and an organic framework structure provides shell protection for the laccase, and damage to the structure of the laccase due to external factors such as friction and shearing force in the use process is avoided. According to the invention, reduction in enzyme activity avoided, and the enzyme activity is greatly improved (the enzyme activity of the immobilized laccase obtained by the immobilization method is1.5-30 times of the activity of equivalent free laccase). The immobilized laccase disclosed by the invention has efficient catalytic degradation capability on antibiotics such as tetracycline, ampicillin, tetracycline derivatives and derivatives of ampicillin, can achieve an effect close to complete degradation in an extremely short time, and is good in reusability and free of secondary pollution.

This application relates to laccase variants and uses thereof as eco-friendly biocatalysts in various industrial processes. More in particular, the disclosure relates to a polypeptide with laccase activity comprising an amino acid sequence that is more than 80% identical to the amino acid sequence according to SEQ ID NO: 1, wherein the polypeptide comprises a non-polar amino acid, preferably an amino acid residue selected from the group consisting of proline, alanine, glycine and valine at a position corresponding to amino acid 113 of SEQ ID NO: 1.

The invention relates to a fungal fermentation culture medium and process. More particularly, the invention relates to a culture medium and method for producing laccase by Ascheronia placenta fermentation. The raw materials of the culture medium comprise 1-4wt% of glucose, 1-4wt% of (NH4)2SO4, 0.5*10<-4>-4*10<-4> mol / L of Zn<2+>, and 1*10<-4>-1*10<-3> wt% of VB4; and the initial pH of the culture medium is 3.8-4.4. According to the method, a fermented seed solution is inoculated to the culture medium, the liquid containing quantity of a 250 mL triangular flask is 50 mL, the inoculum size is 1mL, the number of glass beads is zero, and laccase is cultured at 25 DEG C at the rotational speed of 160 r / min. The fermentation method is short in fermentation period, mild in condition, and easy to control; and the culture medium provided for producing laccase by Ascheronia placenta fermentation has the advantages that the laccase yield is high, the laccase activity is high, and the like.

The invention discloses an immobilized laccase. Laccase is immobilized on a carrier through absorption and chelation by using chelate resin as the carrier, and the chelate resin is bonded with an active functional group containing carboxyl. The immobilized laccase can be applied to treating wastewater containing phenols pollutants, and has the characteristics of simple production process, low cost, high laccase absorption rate, little laccase activity, stable property, and the like.

The invention provides quick-drying hydrophobic lacquer and a preparation method thereof. The preparation method comprises the following steps: performing aldehyde-ammonia condensation on 4-formaldehyde-2,2'-bipyridine and aminated nano silicon dioxide, adding a copper salt, performing a reaction to obtain a nano silicon dioxide supported bipyridyl Cu(II) complex, adding the nano silicon dioxide supported bipyridyl Cu(II) complex into raw lacquer, and performing uniform stirring to prepare a raw lacquer composite film. According to the invention, the natural raw lacquer is utilized as a raw material, the nano silicon dioxide supported bipyridyl Cu(II) complex is added, so that laccase activity is improved, the drying time of a lacquer film is reduced, and roughness of the surface of the lacquer film is increased to make a contact angle of the surface of the lacquer film is increased; therefore the quick-drying hydrophobic lacquer with a fast drying speed and good hydrophobic performance is obtained. The surface of the lacquer film can be dried in half an hour, and the contact angle reaches up to 100 degrees; and time cost in production is greatly saved, corrosion resistance of thelacquer film to moist atmosphere, acid rain, seawater and salt spray is improved, and foundation of industrialization of raw lacquer is laid.